Journal of Chromatography A, 1430 (2016) 16–78

Contents lists available at ScienceDirect

Journal of Chromatography A
journal homepage:www.elsevier.com/locate/chroma

Review article

Recent advances and trends in the liquid-chromatography–mass

spectrometry analysis of flavonoids
∗
André de Villiers , Pieter Venter, Harald Pasch
Department of Chemistry and Polymer Science, Stellenbosch University, Private Bag X1, Matieland 7602, South Africa

article info abstract

Article history: Flavonoids have elicited significant attention as a result of their importance in plants, their influence on the properties of
Received 14 September 2015 Accepted natural-product derived commodities and especially as a consequence of their pur-ported health benefits. Research in all of
25 November 2015 Available online 28 these fields relies heavily on accurate analytical data, and in this LC–MS has come to play an influential role by allowing
November 2015 relatively fast tentative identification and accurate quantification of low levels of flavonoids in a variety of matrices. The field
has undergone rapid expansion in the last decade due to important developments in both HPLC and MS instrumentation,
Keywords: which nowadays allow much faster and more accurate analysis of flavonoids. This contribution aims to provide an overview
Comprehensive two-dimensional LC
of these developments and their application in flavonoid analysis since 2009. The discussion is focussed first on
(LC × LC)
methodologies which provide improved LC separation of flavonoids in terms of speed and/or resolution, including ultra high
Flavonoids
Liquid-chromatography–mass pressure LC (UHPLC), monolithic and superficially porous phases, high temperature LC (HTLC) and comprehensive two-
spectrometry (LC–MS) dimensional LC (LC × LC). The fun-damental background relevant to each of these will be briefly outlined, as well as the
MS/MS implications and promise of their hyphenation to MS. Secondly, the possibilities and limitations of a range of the latest MS
Superficially porous phases instruments available in combination with advanced LC analysis will be discussed, including ion trap, triple quadrupole, time-
Ultra high pressure liquid chromatography of-flight, Orbitrap, ion mobility and various hybrid instruments. Examples from the latest literature will be used to illustrate
(UHPLC) the performance gains achievable in flavonoid analysis by the hyphenation of advanced LC separation and high-end MS
instrumentation.

© 2015 Elsevier B.V. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
2. LC–MS analysis of flavonoids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
2.1. General aspects
of LC separation of flavonoids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
2.2. Detectors used in the LC analysis of flavonoids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
2.3. General aspects
(n)
of LC–MS analysis of flavonoids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
2.3.1. Flavonoid glycosides . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
2.3.2. Flavonoid
aglycones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
3. Advances in HPLC separation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
3.1. One-dimensional
HPLC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
3.1.1. Ultra high pressure liquid
chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
3.1.2. Monolithic columns . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
3.1.3. Superficially porous stationary phases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
3.1.4. High temperature liquid chromatography (HTLC) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34 3.2.
Comprehensive multi-dimensional LC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
Fundamental aspects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 4.
Advances in LC–MS analysis of flavonoids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40

∗
Corresponding author.
E-mail address: ajdevill@sun.ac.za (A. de Villiers).
http://dx.doi.org/10.1016/j.chroma.2015.11.077
0021-9673/© 2015 Elsevier B.V. All rights reserved.
 A. de Villiers et al. / J. Chromatogr. A 1430 (2016) 16–78 17

4.1. Ionisation modes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41

4.2. Mass analysers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
4.2.1. Nominal mass instruments: ion trap (IT) and triple quadrupole (QqQ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
4.2.2. Time-of-flight (TOF) instruments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
4.2.3. Fourier transform ion cyclotron resonance (FT-ICR) MS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
4.2.4.
Orbitrap instruments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
4.2.5.
Ion mobility spectrometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
4.2.6. Overview of mass spectrometers used in flavonoid analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64 5. Conclusions and perspectives .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65 Symbols and abbreviations . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66 Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67

1. Introduction mechanisms, including protection of DNA and low density lipo-proteins,

inhibition of enzymes and of platelet aggregation. The antioxidant activity and
Flavonoids are secondary plant metabolites defined by a diphenylpropane metal chelating properties of flavonoids are considered important properties in
(C6 C3 C6) skeleton. Classification of flavonoids is based on the oxidation determining their contri-bution to human health [22]. More recent
state and degree of unsaturation of the central heterocyclic ring (or the lack epidemiological studies however report more ambiguous findings regarding
thereof in the case of chalcones), according to which the major families of the beneficial properties ubiquitously ascribed to flavonoids [23,24],
flavones, isoflavones, flavanones, flavanonols, flavanols, flavonols, antho- especially since knowledge on the metabolism of these compounds is still
cyanidins and chalcones can be distinguished (Fig. 1). Oligomeric and limited [24,25]. Much current flavonoid research is therefore focussed on the
polymeric flavan-3-ols collectively comprise the proantho-cyanidin family metabolism of flavonoids in vivo [26,27].
[1], while additional bi-, tri-, tetra-, penta- and hexa-flavonoids originate
mainly, although not exclusively, from the oxidative coupling of constituents The second major development that has propelled flavonoid research
of the other flavonoid classes [2]. Within each of these chemical classes, involves improvements in the analytical techniques used for their
structural diversity stems from different substitution patterns of primarily the characterisation. In early research, scientists relied on techniques such as thin
A- and B-aromatic rings, as well as extensive conjugation including glyco- layer chromatography (TLC) and UV/vis spectrophotometry, which lack
sides (both C- and O-glycosylated flavonoids occur in nature) and acylated sufficient specificity and sensi-tivity. As analytical techniques applicable to
glycosides. The number of identified flavonoids was esti-mated to exceed flavonoids have developed, progressively more information about the
4000 in 1994 [3]; by 2005 the number was more than 7000 [4] and has numbers of compounds and their structural properties has become available,
certainly grown significantly in the interim due to extensive research focussed thereby supporting more detailed research in the field. Influential
on these compounds. developments in this regard include the application of gas chro-matography
(GC, although mainly used for sugar determination, the requirement of
derivatisation to render flavonoids volatile means that the technique is rarely
1
Flavonoids are biosynthesized via condensation of phenylalanine-derived used in flavonoid analysis nowadays) and H nuclear magnetic resonance
hydroxycinnamic acids and malonoyl-moieties originating from (NMR) spectroscopy in the late 1960s, followed by C NMR in the 1970s
13
phenylpropanoid metabolism and the shikimate and arogenate pathways, and subsequent advanced two-dimensional NMR techniques from this middle
respectively, which ultimately form the B- and A-rings of flavonoids [6]. Only of this decade. High performance liquid chromatography (HPLC) was first
plants are capa-ble of flavonoid synthesis, and therefore the consumption of applied for flavonoid analysis in the late 1970s [28–30] and liquid
plant-derived products provides the most important source of flavonoids in chromatography–mass spectrometry (LC–MS) from the 1980s. Since the last
the human diet. Flavonoids have been shown to fulfil several important roles decade of the 20th century the application of capillary electrophoresis (CE,
in plants, including attraction of insects, protection against abiotic and biotic including various electrophoretic separation modes) has also found application
stress factors such as UV radiation, harmful insects and animals as well as in flavonoid analy-sis [31–33], while countercurrent separation techniques
regulation of physiological and metabolic processes [3,7,8]. such as countercurrent chromatography (CCC) and centrifugal partition
chromatography (CPC) have more recently found application pri-marily as
Initially thought to be metabolic waste products and of interest only for purification methods for natural products [33–36].
taxonomic reasons, interest in flavonoids has grown almost exponentially
since the late 1960s, supported by two key develop-ments. In the first
instance, recognition of the biological activities of these compounds prompted
substantive research (interestingly, one of the first studies to provoke such From a structural elucidation perspective, NMR is indispens-able in
interest reported ‘Vita-min P’ in oranges [9], although the name was later flavonoid research as the technique allows the complete assignment of
revoked when the compound was identified as the flavonol rutin). Especially flavonoid structures, including the aglycone identity, stereochemistry, nature
at the end of the 20th century, a large body of evidence pointed to several and position of sugars and acylated sugars, etc. [37]. NMR is however
beneficial physiological effects associated with flavonoid-rich diets [10–13]. hampered by cost and throughput limita-tions and is not suited to all
Recognition of the contribution of phenolics to the ‘French Paradox’ [14], applications, especially because of the requirement of sufficient
where the French population was found to exhibit low incidences of concentrations of relatively pure analytes. The hyphenation of HPLC with
cardiovascular mortality despite a high fat diet – a phenomenon explained in NMR has been demonstrated to be a powerful method for flavonoid
part by the relatively high consumption of red wine by the French – proved characterisation, circumventing the need for exhaustive isolation of
influential in stimulating further research in flavonoids and their contri-bution compounds prior to their iden-tification [38–40]. However, this technology is,
to the human diet. Subsequent studies have highlighted several potentially due instrumental complexity and cost, not in widespread use.
important roles of flavonoids in vitro, including their protective effects
against cardiovascular diseases and some forms of cancer [15–21], amongst It is fair to say that the hyphenation of LC with mass spectrom-etry (MS),
others, through a variety of especially once successfully attained using atmospheric pressure ionisation
sources, has revolutionised the analysis of com-plex flavonoid fractions; LC–
MS has become an obligatory tool in flavonoid characterisation as a
complementary technique to
18 A. de Villiers et al. / J. Chromatogr. A 1430 (2016) 16–78
Fig. 1. Structures of the main classes of flavonoids, with numbering of rings and carbons indicated. Note the difference in the numbering scheme for chalcones [5]. Substituents on the backbones are
mostly hydroxyl and methoxy groups, while additional mono- to oligosaccharides with varying degrees of acylation are also common.
NMR. Whereas NMR provides average compositional information on
complex mixtures, MS addresses the composition of single molecules.
Developments in commercial mass analysers over the last decade and a half
now routinely allow determination of molec-ular formulae and molecular
fragmentation using collision induced dissociation (CID) on single or multi-
stage MS systems, and are largely responsible for the fact that LC–MS has
assumed its current position of eminence in the field of flavonoid analysis.
The field of LC–MS has experienced exceptional growth during this period
due to the commercial availability of a range of high resolution and
hyphenated MS detectors based on atmospheric pressure ionisa-tion (API)
ionisation sources [41,42].
The combination of chromatographic resolution provided by HPLC and
n (n)
structural information provided by MS enables rou-tine separation and
tentative identification of complex mixtures of flavonoids spanning several
orders of magnitude in concentra-tion, something that was not possible 25
years ago. Furthermore, important advances in LC technology have been
realised in the last decade. Significant developments include the use of ultra
high pressures and sub-2 m stationary phases (ultra high pressure liq-uid
chromatography, UHPLC), alternative stationary phases such as monoliths
and superficially porous phases, high temperature HPLC [43,44] and
multidimensional HPLC [45–49].
As a consequence of the intensive research activity involving flavonoids,
a wealth of information may be obtained regarding important aspects such as
their analysis, biochemistry, health prop-erties and recently identified
compounds. Especially a number of comprehensive book series serve as
excellent reference works that provide detailed overviews of the field and are
updated periodi-cally [3,4,50–52]. More dedicated information on the analysis
of flavonoids may be found in books such as [53,54], while landmark reports
provide comprehensive overviews of particular aspects such as the analytical
chemistry of flavonoids in food [55,56], extraction and analysis of phenolics
[57,58], the role of MS in the structural analysis of flavonoids [59,60], also in
biological samples
[27] and wine chemistry [61], separation and detection methods for
flavonoids [62] and advanced separation methods for selected flavonoids
[33]. In light of the spectacular developments in the field during the last 5–10
years, however, these seminal reference-works are somewhat outdated.
Several more recent review papers have addressed particular areas of the
HPLC or LC–MS analy-sis of flavonoids, such as developments in the HPLC
separation of phenolic compounds [63,64], analysis of food polyphenols by
ultra high pressure LC (UHPLC) combined with MS [65], chromato-graphic
analysis of flavonoids and metabolites [66], developments in the analysis of
natural product [67] and food phenolics [68] and metabolites [69–73]. None
of these works comprehensively address the fundamental and instrumental
aspects pertinent to
the current state-of-the-art in flavonoid analysis by LC–MS; in this work we
will endeavour to fill this gap.
The emphasis of the current report will be on the most impor-tant
advances in the HPLC separation and MS instrumentation and their
application in the LC–MS analysis of flavonoids in a variety of matrices.
Considering the very large number of liter-ature reports appearing annually in
this field, we will limit the scope of this review to the last 7 years (since
2009). We will fur-ther limit the scope to UHPLC separation (defined here as
HPLC separations performed on columns packed with sub-2 m phases and/or
◦
operated at pressures above 400 bar), the use of elevated temperatures (≥50
C), alternative stationary phase morphologies (including monoliths,
superficially porous phases), comprehensive two-dimensional LC separations
1
(LC × LC) and multi-stage and/or high resolution MS. The fundamental
background of advanced HPLC methods and MS instrumentation and their
application to flavonoid analysis will be addressed briefly, and the benefits of
these developments will be highlighted on the hand of important literature
reports. We will attempt to highlight the most important contemporary trends
in the LC–MS analysis of flavonoids based on these reports.
Note that sample preparation and extraction of flavonoids, although
essential in all analytical workflows for flavonoids and especially so in the
case of LC–MS analysis to reduce matrix effects, will not be addressed in
detail in the current work. This aspect has been covered in detail by several
reviews [16,33,56–59,62,65,66,74,75] and developments in the field are less
dramatic than those involving the analysis step.
Finally, a caveat should be added: because of the very large number of
papers falling within the scope of this review, a com-prehensive list of all
relevant reports is not possible in this format; neither will we attempt to
discuss each report in detail. Rather, tables will be used to summarise the
information, and the discus-sion will focus on important contributions and
trends deduced from these works.
2. LC–MS analysis of flavonoids
2.1. General aspects of LC separation of flavonoids
Contemporary HPLC separation of flavonoids is almost exclu-sively
performed using reversed phase liquid chromatography
1 This of course implies that a large number of excellent research papers published in the last 7 years will not be
included due to the criteria defined for the scope of the current work. This was however important to limit the number of
papers to manageable numbers.
 A. de Villiers et al. / J. Chromatogr. A 1430 (2016) 16–78
(RP-LC), with the notable exceptions being normal phase liq-uid
chromatography (NP-LC) for oligomeric proanthocyanins [76], and the recent
increasing application of hydrophilic interaction chromatography (HILIC).
Other modes of separation which have been applied to flavonoid separation
include mixed-mode ion-exchange-reversed phase [77–79] separation of
anthocyanins, size exclusion chromatography (SEC) analysis of flavonol
glycosides [80,81] and theaflavins and proanthocyanidins [81]. Because of the
relatively sparse use of the latter modes, however, they will not be discussed
in detail in the current work (except when used in LC × LC), which will focus
primarily on RP-LC in accordance with the scope and preponderance of this
mode in flavonoid literature.
RP-LC has demonstrated its suitability for the separation of flavonoids
based on the nature of the aglycone (including the oxidation state, substitution
patterns and stereochemistry), the nature and degree of glycosylation as well
as the nature and degree of acylation. By far the majority of RP-LC
separations are performed using C18 octadecyl-silica (ODS) phases, although
C8 [82,83], C12 [84], phenyl or phenyl-hexyl [76,85–90], pentafluo-rophenyl
(PFP) [90–95] and polar embedded RP phases [88,96–98] as well as
polymeric RP-LC phases [99] have also found appli-cation in flavonoid
analysis [100–102]. Mobile phases typically comprise aqueous/organic phases
containing methanol, acetoni-trile and less commonly tetrahydrofuran [103],
isopropanol [101] or ethanol [99] and acidic modifiers such as acetic acid,
formic acid, ammonium acetate or trifluoroacetic acid (TFA) [101]
(phosphoric, citric or perchloric acids have also been used in combination
with UV detection, although these are not suited to hyphenation with MS).
For anthocyanins, highly acidic mobile phases (>4–10% formic acid, 0.1–
0.6% TFA) [104–107] are used to ensure prevalence of the flavylium cationic
species in solution and therefore improve chromatographic efficiency
[33,100,101,105].
Retention of flavonoids in RP-LC is based on hydrophobicity; some
selectivity differences may be observed based on the nature of the stationary
phase, endcapping, carbon loading, polar embedded groups as well as the
nature of the silica phase (e.g. organo-silanes) [108,109]. However, the
general elution order of flavonoids on RP-LC columns is mostly consistent in
the following sequence for flavonoid aglycones: flavanols < flavanones <
flavonols < flavones [4,59,110,111] (anthocyanins elute close to flavanols
using ‘generic’ (i.e. not highly acidic) mobile phases [111,112]). For each of
these classes, retention decreases with the number of hydroxyl groups on the
flavonoid backbone, and increases with the number of methoxy substituents.
Glycosylation decreases RP-LC retention, whereas acylation of glycosyl
groups increases retention relative to the corresponding glycosylated
derivatives. Common sugars encountered in glycosylated flavonoids include
glucose, galac-tose, rhamnose, xylose and arabinose, with mannose, fructose
and glucoronic and galactoronic acids being rarer. The general elution order
of glycosylated flavonoids in RP-LC is -galactosides < - glucosides <
-arabinosides < -xylosides < -rhamnosides [101]. Di-and higher oligomeric
saccharides are common in some prod-ucts, with the most common
disaccharides being rutinoside (rhamnosyl-( 1→6)-glucose) and neo-
hesperidose (rhamnosyl-(-( 1→2)-glucose) [59]. The most common aliphatic
and aromatic acids involved in acylation include (in rough order of their RP-
LC elution sequence) malic, acetic, malonic, succinic, gallic, proto-catechuic,
hydroxybenzoic, vanillic, caffeic, syringic, p-coumaric, ferulic and synaptic
acids [101]. For more detailed informa-tion regarding the relative elution
orders of different classes of flavonoids in RP-LC, the reader is referred to
[101].
As a rule, NP-LC is not suited to flavonoid analysis due to the lim-ited
solubility of these compounds in typical NP-LC mobile phases, the exception
being apolar flavonoids such as polymethoxylated flavones [113] and
acetylated flavonoids [114]. One notable appli-cation of NP-LC is however
the separation of proanthocyanins
19
according to molecular weight (MW), first reported in 1979 [76] (and based
on previous findings on silica gel TLC [115]) and sub-sequently adapted to
HPLC format and refined by several groups [116,117]. This type of separation
is typically performed in adsorp-tive mode on bare silica stationary phases and
mobile phases containing dichloromethane/methanol/acidified water
[116,117], hexane/acetone or hexane/methanol/ethyl acetate [118]. Proan-
thocyanins elute in order of their degree of polymerisation (DP) under these
conditions, with isomeric compounds of the same DP co-eluting.
A more recent trend is the application of HILIC for flavonoid anal-ysis.
HILIC can be considered an aqueous variant of NP-LC, where retention is
primarily governed by partitioning of polar compounds into an aqueous-rich
layer immobilised on the surface of polar bonded phases [119,120]. The first
application of HILIC to flavonoid analysis, and one of the first applications of
this mode of separa-tion [121] (although the term and a description of its
separation mechanism was to follow only much later), was reported by Lea
[76], who used a pellicular polyamide phase and a mobile phase comprising
methanol, acetic acid and water for the separation of epicatechin and
phloridzin. Similar retention of proanthocyanins is observed in HILIC
compared to NP-LC [122,123]. However, the more polar mobile phases used
in HILIC, typically comprising binary mixtures of acetonitrile and acidified
water, makes the technique more suitable for flavonoid analysis than NP-LC
and improves ion-isation efficiency for these compounds compared to RP-LC
due to higher organic content of the mobile phase [124]. Indeed, the appli-
cation of HILIC for the analysis of flavones and flavonols [125–127],
dihydrochalcones and flavanones [127], anthocyanins [128] and phlorotannins
[129] has been demonstrated recently. HILIC sta-tionary phases used in
flavonoid analysis include silica [130], diol [122,131], polyethylene glycol
(PEG) [131], cyclodextrin (CD) [130], ZIC-HILIC [132] and amide [128,130]
phases. One of the major driv-ing forces behind the interest in HILIC for
flavonoid analysis is the alternative separation mechanism to RP-LC offered
by this separa-tion mode [133], which typically results in class–type
separations of flavonoids according to the degree of glycosylation and/or acy-
lation. As a consequence, HILIC has found increasing application in the LC ×
LC separation of flavonoids in combination with RP-LC (see Table 2).
2.2. Detectors used in the LC analysis of flavonoids
A wide range of detectors can and have been used in combi-nation with
HPLC separation to detect and/or identify flavonoids. These include
electrochemical detection (ED) [134,135], fluores-cence (FL) [62,136], UV–
vis, diode array [101,137], NMR [38–40] and of course MS detectors
[59,60,62,110,138]. Of these, by far the most common nowadays are diode
array and MS detectors, which will be discussed briefly in this and the next
section, respectively.
The conjugated aromatic character of flavonoids proved to be a significant
benefit especially in early flavonoid research: absorption at relatively long
wavelengths increases the selectivity of qual-itative and quantitative
spectrophotometric methods, while the characteristic spectra of different
classes of flavonoids allow dis-tinction between them. These characteristics
are equally useful when UV–vis detection is performed in combination with
HPLC separation.
Flavonoids display two UV–vis absorption maxima: an absorp-tion band
in the region of 240–285 nm (Band II) ascribed to the A-ring, and a second
band at 300–550 nm due to the B-ring (Band I). Of these, the latter is more
useful because it provides more selective information, since all flavonoids
absorb in the region of 240–285 nm. Flavanols exhibit only Band II
absorption (269–279 nm) due to a lack of conjugation between A- and B-
rings; the same applies to flavanones, dihydroflavonols and isoflavones.
20 A. de Villiers et al. / J. Chromatogr. A 1430 (2016) 16–78

Fig. 2. Examples of UV–vis spectra of different classes of flavonoids: (A) anthocyanins, (B) flavanols, (C) flavanones, (D) flavones, (E) flavonols and (F) isoflavones.
Source: Reproduced with permission from [56].

Flavonols and flavones show Band I absorption between 300 and 380 nm,
whereas anthocyanidins are easily distinguished by their Band I absorption
between 460 and 550 nm in the visible range. Typical examples of the UV–vis
absorbance spectra of the principal classes of flavonoids are presented in Fig.
2.
As a rule, increasing hydroxylation of A- and B-rings leads to
bathochromic shifts in the wavelength of maximum absorption, max, of Bands
II and I; this effect is somewhat offset by methylation of the hydroxyl groups.
Glycosylation does not have a significant effect on the absorbance spectra of
most flavonoids, and the nature of the sugar moiety has no effect. Exceptions
are glycosylation at C-3, which causes a hypsochromic shift of 15–20 nm in
absorption Band I for flavonols and flavones, C-glycosylation which results in
a bathochromic shift of Band II, and the absence of a shoul-der at 440 nm for
anthocyanidin-3,5-di-O-glycosides compared to anthocyanidin-3-O-
diglycoside [101,139]. Acylation involving
aromatic acids leads to increased absorbance in the UV region close to Band
II (260–290 nm), whereas for hydroxycinnamic acids increased absorbance is
observed in the region 305–325 nm. In the interpretation of UV spectra
obtained using on-line diode array detection, it is important to consider the
effect of the solvent (both the nature of mobile phase constituents and their
composition at the point of elution) when comparing with reference spectra,
since this can have a significant effect on flavonoid spectra. For more detailed
information on the UV–vis absorption characteristics of flavonoids, the reader
is referred a range of excellent dedicated texts available [101,110,140].
While the structural analysis of flavonoids based on their UV–vis spectra
has in recent years largely been surpassed by more pow-erful tools such as
MS and NMR, useful information can easily be obtained based on UV–vis
spectral properties, the most impor-tant being the chemical class of
flavonoids. For this reason UV–vis
 A. de Villiers et al. / J. Chromatogr. A 1430 (2016) 16–78
detection remains a very useful technique in combination with HPLC and MS
for flavonoid analysis. Because UV–vis detection is non-destructive, the
detector can be connected before other detec-tors such as MS and NMR, is
compatible with high mobile phase flow rates and provides extremely reliable
quantification (provided that co-elution of compounds with similar
absorbance properties can be avoided) [137]. The sensitivity of UV detection
depends on the flavonoid target and instrument used, but is generally in the
region of 0.02–10 ng injected mass [94,95,103,141,142], which is sufficient
for most natural product and food applications. Further-more, UV–vis spectra
can be used to distinguish between different classes of flavonoids as outlined
above. While this may sound trivial, this information in many cases may
prove pivotal. For example, sev-eral glycosylated flavonoids exhibit identical
MS behaviour due to identical molecular formulae and fragmentation, yet
belong to dif-ferent flavonoid families [143]. As an example, even accurate
mass MS cannot distinguish between the delphinidin and cyanidin gly-cosides
(anthocyanins) and quercetin and kaempferol glycosides (flavonols),
respectively, unless high-collision energy CID exper-iments are performed.
However, UV–vis spectra of anthocyanins and flavonols are clearly distinct
(Fig. 2) and therefore allow facile assignment of the chemical class without
additional experiments. Furthermore, UV absorbance at ∼500 nm can be used
for selec-tive detection and accurate quantification of anthocyanins, even in
cases where co-elution occurs with flavonols. In light of the fact that UV
detectors are relatively cheap and commonly available, it is no surprise that
on-line hyphenation of HPLC separation with UV and MS detection in
sequence has become the norm in flavonoid analysis, especially in profiling
methods where the aim is to obtain maximum information [144].
Post-column addition of UV shift reagents has been used to study
flavonoid substitution patterns, and post-column or pre-column derivatisation
(for example fluorescent labelling to exploit the benefits of this mode of
detection) has also been used in flavonoid analysis, although these are less
common nowadays [145,146].
Fluorescence detection is used much less often in the LC analysis of
flavonoids, a consequence of the fact that only few flavonoid classes exhibit
fluorescent behaviour: isoflavones, methoxylated flavones and flavonoids
with an OH substituent at carbon 3 (Fig. 1) [62]. Significantly, this includes
flavanols such as (epi)catechin and the procyanidins, which makes
fluorescence detection extremely useful for the selective and sensitive
analysis of these compounds, and the technique has continued to find
application in research focussed on these classes [147–150].
NMR clearly remains the most important spectroscopic tech-nique for
flavonoid analysis, although the technique is still relatively rarely used on-line
in combination with HPLC separation due to the challenges associated with
this hyphenation. In LC-NMR, two approaches are possible: direct coupling
using a flow probe for the NMR, or stop-flow NMR measurements. In the
former case, deuterated solvents are used in combination with pulse
sequences aimed at suppressing background signals. Stop-flow NMR involves
the use of a valve triggered from an upstream detector to park the peak of
interest in the flow cell, and provides much higher sensitiv-ity as a
consequence. The power of LC-NMR for natural products has been
demonstrated admirably by Wolfender and co-workers; since the topic falls
outside the scope of this work, readers are referred to several important
overviews of the field for further information [38–40].
(n)
2.3. General aspects of LC–MS analysis of flavonoids
In this section, a brief overview of the background infor-mation relevant
to the MS detection and MS/MS structural elucidation of flavonoids will be
presented, with the emphasis
21
on general ionisation and fragmentation processes. The contem-porary
importance of LC–MS in flavonoid analysis is reflected by the large number
of excellent review articles reported in the last decade on this topic
[59,60,110], and the reader is referred to these for more in-depth information
on flavonoid identification by LC–MS. Furthermore, much more detailed
infor-mation may be obtained regarding specific classes of flavonoids such as
dihydroflavonols [151], isoflavones [152], flavonoid-
[153] and flavone-di-C-glycosides [154], flavonoid aglycones [155,156],
flavonoid-O-glycosides [157,158] and flavonoid glyco-sides [108,159,160] in
dedicated research reports.
The following discussion will be limited to the currently most relevant
LC–MS ionisation sources, the API sources electrospray ionisation (ESI),
atmospheric pressure chemical ionisation (APCI) and atmospheric pressure
photoionisation (APPI). Fortunately, the general ionisation and fragmentation
behaviour of flavonoids is largely independent of the ionisation source and
instrument used [60,62,161,162], although relative ion intensities may vary
some-what [60,153,163] and some differences have been noted between CID-
2 3
MS/MS and in-source CID spectra [164] and between MS and pseudo MS
spectra on IT and Q-TOF instruments [162]. Never-theless, the following brief
overview can be considered generally applicable to most instruments.
For all API sources, the composition of the mobile phase plays a critical
role in ionisation, since analytes should either be ionised in solution (ESI) or
in the gas phase after mobile phase evaporation (APCI and APPI). In fact,
divergent conclusions have been drawn regarding the best mobile phase
composition for flavonoid analysis. For negative mode ESI ionisation, optimal
mobile phases reported in literature include 0.1% formic acid [108], 10 mM
ammonium acetate (pH 4) [165] or 0.5% acetic acid [156], whereas for posi-
tive mode, 0.2–0.75% formic acid [108,165] is preferred; although
trifluoroacetic acid has been used, this additive has also been shown to reduce
ESI-MS sensitivity due to its ion-pairing prop-erties [108,165]. For
anthocyanins, highly acidic mobile phases containing 5–10% formic acid are
commonly used, and since the anthocyanidins occur as flavylium cations in
such mobile phases, positive ionisation is almost exclusively used [138]. The
two most common organic modifiers used in the RP-LC anal-ysis of
flavonoids, methanol and acetonitrile, have little direct effect on the ionisation
of molecules, although nebulisation effi-ciency will be affected by the %
organic modifier in the mobile phase (generally low percentages of
acetonitrile are detrimental)
[108].
Negative ionisation has been shown to be more sensitive than positive
ionisation for all ionisation sources [165], with ESI being the most sensitive
[62]. This is not so much due to ionisation effi-ciency, but is rather a
consequence of increased chemical noise in positive ionisation mode
[108,165]. Positive ionisation generally results in more extensive
fragmentation than negative ionisation [59,108,165], and remains popular due
to the amount of struc-tural information that may be obtained using this mode
[59,62,110], especially for the analysis of flavonoid-C-glycosides [153].
In first-order MS spectra of flavonoids, with low cone voltages, protonated
+ −
[M+H] and de-protonated [M−H] ions are most com-mon, with little
fragmentation present. Depending on the mobile phase composition, however,
− − −
adducts such as [M−H+AcONa] , [M+HSO4] , [M+AcO] and
−
[M−H+AcONa+MeOH] may be detected, in addition to molecular
− + +
complexes such as [2M−H] , [2M+H] and [2M+Na−H] [110,153].
Complexation with metals and chelating ligands (added post-column in the
case of LC–MS) has also been used in the MS/MS study of flavonoids
[37,60].
For most of the fragmentation pathways outlined below, tan-dem mass
spectrometers operated under conditions of relatively high collision energies
(10–47 eV) are used to generate second-order mass spectra. However, single
stage mass spectrometers may
22 A. de Villiers et al. / J. Chromatogr. A 1430 (2016) 16–78
be used to obtain fragmentation information by adaptation of the cone voltage
to attain in-source CID [110,166].
2.3.1. Flavonoid glycosides
The nomenclature proposed by Domon and Costello [167] has found
widespread acceptance for the fragmentation of glycosylated flavonoids, and
is illustrated for flavonoid O- and C-glycosides in Fig. 3A and B, respectively.
(Note that the ionisation mode is spec-ified by the superscript + or − in this
nomenclature [62]). When the charge is located on the aglycone fragment,
k,l ± ± ±
ions are labelled X j , Y j and Z j , where the subscript j denotes the
number of the interglycosydic bond cleaved (the aglycone–glycosidic bond is
numbered 0) and superscripts k and l indicate cleavages within the
carbohydrate rings. When more than one glycosylation pos-itions are
involved, an additional superscript m is used to denote the position of
k,l ±m ±m
aglycone substitution, e.g. ions X j , Y j . For charged carbohydrate
k,l ± ± ±
fragments, ions are designated A i , B i and C i , where the subscript i
(≥1) represents the number of the glycosidic bond cleaved, counting from the
non-reducing end numbered 1.
Flavonoids commonly occur in nature as glycosidic species, with two
types distinguished based on the nature of the flavonoid–saccharide linkage.
Flavonoid-O-glycosides are more common (and exclusively occur in the case
of anthocyanins), sugars are attached to one or more hydroxyl groups through
an acid-labile C O bond; hydroxyl groups at C-3 and C-7 are common
glycosylation sites (C-3 and C-5 for anthocyanidins). Flavonoid-C-glycosides
contain acid-resistant C C bonds linking the sugar directly to the flavonoid
nucleus (Fig. 3B). C-glycosidic bonds occur virtually exclusively at carbons
C-6 and C-8 of the aglycone [108].
The labile nature of the O-glycosidic bond ensures that the bond is
cleaved under low collision energy conditions, lead-ing to the loss of neutral
sugar residues (loss of 162, 146 and 132 amu for hexosides, deoxyhexosides
± Similar to flavonoid-di-O-glycosides, flavonoid-di-C-glycosides containing
and pentosides, respec-tively) and the corresponding Y 0 aglycone ion in the
case of monoglycosides [59,60,110]. The mechanism for this fragmenta-tion
has been shown to entail rearrangement involving hydrogen migration [169].
± iden-tify flavone-di-C-glycosides based on negative ionisation MS/MS spectra
In some cases the corresponding saccharide ions, B 1, are observed at m/z
163 or 147 for hexoses and pentoses, respectively [59,110]. In negative
• −
ionisation mode, a radical agly-cone ion, [Y 0−H] , resulting from the
homolytic cleavage of the glycosidic bond, may also be observed [59,170].
Flavonoid-O-diglycosides, where a disaccharide is attached to the aglycone,
± ±
show Y 1 and Y 0 fragments, with higher collision energies favouring the
± the O-glycosidic bond, which typically occurs at lower collision energies, and
formation of the Y 0 aglycone ion. An interesting exception is observed for
flavonol- and flavanone-O-rutinosides (rhamnosyl-( 1→6)-glucose) and
flavanone-7-O-neohesperidoses
+ studies for this class of compounds have been reported.
(rhamnosyl-( 1→2)-glucose), where an Y * ion is observed. This ion results
+
from the loss of the inner glucose residue ([M+H−162] ) through hydrogen
migration from the C-5 hydroxyl of the agly-cone to the terminal rhamnose,
± 2.3.2. Flavonoid aglycones
followed by a rearrangement reaction [59,110,171]. The ratio of the Y 1and
±
Y 0 fragments in positive ionisation mode can be used to assign the class of
flavonoid-O-disaccharide, whereas negative or positive ionisation second
order spectra can be used to distinguish between iso-meric flavonoid-O-
neohesperidosides and -O-rutinosides, which differ in terms of their
interglycosidic linkage (1 → 2 and 1 → 6, respectively) [157,158,168]. This
approach can be extended to di- to penta-glycosylated flavonoids to allow
characterisation of the interglycosidic linkage and to differentiate positional
isomers based on negative ionisation MS/MS spectra [172]. Especially the
− − − 7−
relative abundance of Y 1, Z 1, Y 0 and Y 0 ions are determina-tive in
this regard. The stereochemical assignment of the terminal monosaccharide
units in flavonoid-O-glycosides using ESI and
FAB-MS/MS following acetylation has been reported [173], although this
approach has found limited application since.
Flavonoid-di-O-glycosides, where two monosaccharides are linked to
±3 ±7
different aglycone hydroxyls, show Y 0, Y 0 fragments due to the cleavage
±3 ±5
of one of the glycosidic bonds (for anthocyanins these will be Y 0, Y 0). In
±3 ±7
some cases Y 0Y 0 fragments corre-sponding to cleavage of both sugar
linkages are also observed [110]. The susceptibility of the sugar–aglycone
bond to cleavage varies according to position, with fragmentation
preferentially occurring at the C-3 position compared to the C-7 position
[174]. This allows assignment of glycan substitution for flavonoid-di-O-
glycosides containing sugars of different molar masses.
The relative stability of the C-glycosidic bond means that high collision
energies are required for fragmentation, which mainly involves cross-ring
cleavages of the saccharide residue and the loss of water molecules. Typical
fragmentation patterns are pre-sented in Fig. 3B, with the main fragment ions
0,2 ± 0,1 ± 0,4 ±
corresponding to X 0, X 0, and X 0, often coinciding with the loss
0,3 ±
of water or CH2O fragments [175,176]. The X 0 ion is only present at
0,3 ± 0,2 ±
noticeable levels for flavonoid-C-8-glycosides [110]. X 0, X 0, and
0,1 ±
X 0 fragment ions correspond to losses of 90, 120 and 150 amu,
respectively, for flavonoid-hexosides. The corresponding fragments for
flavonoid-pentosides result in losses of 60, 90 and 120 amu [59,110].
C-6 and C-8 positional isomers can be distinguished based on more
0,2 ± 0,3 +
extensive fragmentation of X 0 fragments for the former; the X 0 and
+
[M+H−4H2O] ions have been proposed as being diagnostic for the
differentiation of C-8 and C-6 isomers in posi-tive ESI-MS/MS [153]. Waridel
et al. [162] compared Q-TOF and IT instruments in positive and negative
mode APCI, and found loss of water to be characteristic for the C-6
0,2 +
glycosides, whereas MS/MS of the X ion in positive mode allowed
distinction if the iso-mers based on the losses of small molecules (and the
1,3 +
presence of a A ion observed in Q-TOF spectra only). A more complete set
of guidelines has been reported in both positive and negative ionisation [177].
sugar residues of different mass can be assigned based on the more extensive
fragmentation of the C-6-glycoside moiety [59]. More recently, an approach to
has been reported [154] (refer to Section 4.2.1 for further details).
Flavonoid-O,C-diglycosides can contain the O-glycoside moiety linked
either to a hydroxyl group of the aglycone or to a hydroxyl group of the C-
bound glycoside. Fragmentation of these compounds entails both cleavage of
cross-ring cleavage of the C-bound saccharide at higher collision energies
[59,110].
Differentiation of isomeric acylated flavonoid-glycosides is also possible
to some extent based on MS/MS spectra [178], although limited systematic
According to the nomenclature proposed by Ma et al. [168] (improved
from that developed by Mabry et al. [140]) for flavonoid aglycones, ions are
designated i,jA± 0+ and i,jB± 0 for fragment ions containing intact A- and B-
rings, respectively. The superscripts i and j indicate the cleaved C-ring bonds,
while the subscript 0 dis-tinguishes these ions from carbohydrate fragments
k,l ±
A i and B± i (i ≥ 1). In general, the fragmentation pathways discussed below
are applicable to both positive and negative ionisation, although the former
mode is more commonly used in the structural analysis of flavonoids
(exclusively in the case of anthocyanidins). Nega-tive ionisation requires
higher collision energies and may lack some diagnostic ions in product spectra
[59,62,179]. Fragment ions
 A. de Villiers et al. / J. Chromatogr. A 1430 (2016) 16–78
Fig. 3. General fragmentation scheme and illustration of nomenclature used for the MS
formed from cleavage of the C-ring bonds are very informative, as they
provide information on the number and nature of substituents on the A- and
B-rings. The fragmentation pathways entail retro-Diels–Alder (RDA)
reactions involving the C-ring, typically at bond positions 1/2, 0/2, 0/3, 0/4 or
2/4 [56,59,62,110,155] (Fig. 3C).
The relative prevalence of each fragmentation pathway depends on the
1,3 ±
class and substitution pattern of the flavonoid. The A 0 ion, observed for
all flavonoid subclasses in positive and neg-ative modes, is most readily
formed and often constitutes the base peak ion in second-order mass spectra
[59,62]. For flavones and flavonols, cleavage of the 0/2 bonds is common,
0,2 ±
leading to the formation of the B 0 ion, while the corresponding
ion is characteristic for flavonols [155]. Further losses of 18 amu ( H2O), 28
amu ( CO), 42 amu ( C2H2O) and in the case of nega-tive ionisation 34 amu
( CO2) or successive losses of these groups are also commonly observed in
MS/MS spectra. In the case of O-methylated flavonoids the loss of 15 amu
± ± •
( CH3) is prominent, with the [M H−CH3] radical ion generally
constituting the base peak. Direct cleavage of the bond between the B- and C-
ring can be observed for some ions in negative ionisation mode and antho-
±
cyanidins in positive mode [180], resulting in an [M−B-ring]
[179].
Chalcones and dihydrochalcones differ from the other flavonoid
subclasses in that they do not possess a heterocyclic C-ring (Fig. 1). In the
case of dihydrochalcones, the saturated 3-carbon chain frag-ments easily at
low collision energies in positive ionisation mode. Cleavage of the bond
between the A-ring and the carbon atom from the keto group, followed by
+ +
loss of a ketone, yields the product ions [C9H9O2] and [C7H7O] ,
respectively. Other ions characteristic for the dihydrochalcones include
+ +
[C8H9O4] and [C6H7O3] , produced from the rupture of the and carbons
adjacent to the carbonyl [110]. Chalcones on the other hand have been shown
to fragment according to two pathways, depending on the number and
position of phenolic substituents on the A-ring. The first reaction, prevalent
for compounds with a C -2 hydroxyl, involves formation of the cor-
responding flavanone isomer, which then fragments according to a RDA
mechanism as outlined above. For compounds without a C -2 hydroxyl,
cleavage of the bonds on either side of the carbonyl group is common [143].
Proanthocyanidins show relatively distinct fragmentation reac-tions,
illustrated schematically in Fig. 4 for positive ionisation. The first of these
involves elimination of a B-ring as a result of
23
(n)
analysis of (a) flavonoid-O-glycosides (b) flavonoid-C-glycosides and (c) flavonoid aglycones [167,168].
0,2 ±
RDA fission of ring C (in essence resulting in the A fragment of the
relevant proanthocyanidin moiety) [181–183]; this path-way is energetically
favoured in the top unit of proanthocyanidins
[184] and is commonly followed by the loss of an additional water molecule.
The second fragmentation pathway, designated het-erolytic ring fission (HRF)
[184], involves loss of the A-ring, again primarily of the top unit (this
1,4 ±
corresponds to the B fragment). The loss of a 126 amu neutral fragment is
indicative of a 1,3,5-trihydroxybenzene structure of the A-ring. The third
fragmentation pathway involves quinone methide (QM) cleavage of the inter-
flavan bond [181–183]. This pathway is useful in the assignment of the
0,2 ±
A 0 position of A-type interflavan bonds in higher oligomers [181,184]. A fourth
mechanism, benzofuran-forming (BFF) fission, follows a similar mechanism
to HRF to produce a C-ring benzofuran derivative [185]. Further loss of water
molecules is also common in MS/MS spectra of proanthocyanins, which may
further complicate the spectra of high molecular weight compounds. Li and
Deinzer
[185] proposed a set of rules that may be used to identify the composition of
procyanidins based on MS/MS spectra and the four fragmentation pathways,
fragment demonstrating the applicability thereof by assigning molecules up to a DP of
six. A similar approach has also been used in the characterisation of A-type
proanthocyanins [186]. It is important to note though that assignment of
stereoisomers is not possible based on MS/MS data.
Finally, it is worth pointing out that a range of electronic databases
containing MS/MS and HR-MS(/MS) data for metabo-lites, phenolics or
flavonoids are nowadays available [54,187,188], or may be compiled in-house
[189], and these are finding increasing application in the screening and
identification of plant flavonoids [190–193].
3. Advances in HPLC separation
Like all forms of chromatographic separation, the performance of HPLC
methods is inherently constrained by the properties of the target analytes, the
stationary and mobile phases as well as instru-mental limits. This also has
clear implications for the analysis of flavonoids by HPLC. The two main
driving forces behind improve-ments in HPLC in general apply equally in the
HPLC analysis of flavonoids: the need for decreased analysis times and
increased resolution. The former applies mainly in routine environments,
where large numbers of samples need to be analysed daily, but is
24 A. de Villiers et al. / J. Chromatogr. A 1430 (2016) 16–78

OH HO O -H O
2 HO O
HO O OH BFF m/z 287
OH RDA OH OH
OH HO O HO
OH OH -H2O O
OH OH
HO O OH OH
OH
OH HRF m/z 427
m/z 563 O
O
QM BFF
OH OH OH
-H2O
OH
HO O
HO O
OH
O O
OH m/z 297
OH
OH
OH RDA
OH O
m/z 273 m/z 437
OH
OH OH m/z 285
HO O
HO O
OH
OH
m/z 291 OH

Fig. 4. General fragmentation scheme for procyanidins illustrated for a procyanidin dimer in positive ionisation mode. The fragmentation pathways involving retro-Diels–Alder (RDA), heterolytic ring
fission (HRF), quinone methide (QM) and benzofuran-forming fission (BFF) are demonstrated.
Source: Adapted from [185].

also important from a cost-saving perspective and in cases where analytical
data is required within a short time. The latter require-ment is important for
the analysis of complex samples, especially where numerous chemically
related species which cannot be dis-tinguished by the detector used occur in
the same sample. This is clearly the case for a wide range of flavonoid-
containing natu-ral products [4] and therefore explains the incentive for
improving HPLC separation in flavonoid analysis.
As a measure of the separation performance of chromatographic systems,
the concept of peak capacity has gained popularity as an alternative to more
conventional measures such as efficiency and resolution, which are limited to
isocratic analyses and selected peak pairs, respectively. The peak capacity of a
separation represents the number of compounds that can theoretically be
separated under defined experimental conditions (column, gradient, flow rate,
tem-perature, etc.) [194]. It is important to note though that no real-life
separation can resolve a number of components equal to the peak capacity of
that separation, a consequence of irregular distribu-tion of analyte peaks
across the separation window. Davis and Giddings [195] used the statistical
theory of component overlap to demonstrate that to improve the probability of
obtaining pure chromatographic peaks, the peak capacity should exceed the
num-ber of components to be separated by a significant factor. To use an oft-
quoted example, to resolve with 98% certainty n randomly dis-tributed peaks,
the peak capacity should be equal to 100 × n [196]. For relatively simple
samples containing few analytes of interest, the experienced chromatographer
can select experimental condi-tions so as to improve the likelihood of
obtaining separation (for example through the careful selection of stationary
phase, mobile phase composition and gradient, temperature, etc.). However,
for highly complex samples, this is no longer possible for all analyte
peaks – in this case, the sample components approximate a ran-dom
distribution and Davis and Giddings’ findings emphasise the importance of
improved chromatographic performance.
For gradient separations, the peak capacity, n c, can be deter-mined
according to [197]:
(1)
n =1+
c t g
(1/n) 1w
n b
where tg refers to the gradient time (i.e. the separation window) and w b the
baseline width of analyte peaks averaged for n peaks. Typical peak capacity
values for RP-LC vary between ∼50 for fast separations and in the region of a
few hundred for highly opti-mised separations. Taking cognisance of the
findings of Davis and Giddings, and considering that many natural products
contain in excess of 100 flavonoids, it is clear that continued efforts to
improve the performance of LC separations are of critical importance also in
the field of flavonoid analysis.
3.1. One-dimensional HPLC
In the last 15 years, a number of significant developments in the field have
extended the performance limits of (uni)-dimensional HPLC. Several of these
have gained widespread acceptance also in the area of flavonoid analysis,
notably the use of UHPLC, alternative stationary phase morphologies such as
monolithic and superficially porous phases, comprehensive multidimensional
separations and to a lesser extent the used of elevated temperature. In the
following sections, the fundamental background for each of these approaches
will be outlined briefly prior to presenting an overview of their application in
the analysis of flavonoids during the last 7 years.
 A. de Villiers et al. / J. Chromatogr. A 1430 (2016) 16–78 25

3.1.1. Ultra high pressure liquid chromatography

One of the oldest and best-known ways to increase performance in HPLC
is by reducing the particle size of the packing material. In fact, the evolution of
HPLC coincides with a continuous decrease in the size of the packing materials
used, dp. This decline was rel-atively gradual until the late 1990s, when 3–3.5
m particles had begun to replace more conventional 5 m phases. Further reduc-
tion in particle size was not considered practical due to the high pressures
required to operate such columns. In a series of landmark papers from 1997,
Jorgenson and co-workers [198] demonstrated the feasibility of using sub-2 m
phases on dedicated high pres-sure instruments, and showed the benefits of this
approach for fast, high-efficiency separations in what has come to be termed
ultra high pressure liquid chromatography (UHPLC). Today this term is
considered to refer to HPLC separations performed at pressures above 400 bar
(the pressure limit of conventional instruments). Commercial instruments
capable of routine operation at pressures up to 1250 bar (∼18400 psi) and a
range of columns packed with particle sizes ranging from 1.3 to 2.5 m have
been introduced since 2005, and in the last decade UHPLC has gained
widespread accep-tance by the separations community. Indeed, UHPLC has
also found extensive application in the analysis of flavonoids, as summarised
in a number of recent reviews [63–65].

The benefits of UHPLC compared to conventional HPLC are com-monly

represented in terms of van Deemter curves depicting the variation in plate
height (H, defined as L/N where L is column length and N the number of
theoretical plates) and the linear velocity of the mobile phase (u0, related to the
flow rate via the column diameter). According to chromatographic theory, a
reduction in particle size results in lower A (eddy dispersion) and C (resistance
to mass trans-fer) terms of the van Deemter equation. The combined effect of
this is a lower minimum plate height (i.e. higher efficiency for a given column
length) and a higher optimal linear velocity. A typical exam-ple of the effect of
reduction in particle size is shown for flavanols in Fig. 5 [199], where van
Deemter curves are compared for 1.7 and 5 m C18 phases (the former operated
at pressures up to 1000 bar). The benefits of the 1.7 m phase are evident: the
minimum value for H is much lower (4.2 compared to 11.2 m, meaning that for
the same column length, 2.7× higher efficiency can be obtained), and an
increase in the optimal mobile phase linear velocity (u opt) from 0.6 to 1.3 mm/s
(i.e. separations will be 2.2× faster for the same column length). Plate height
curves such as these are com-mon for most flavonoids on such columns,
implying that UHPLC offers two principal advantages compared to
conventional HPLC: increased speed, achieved by a combination of shorter
columns and higher flow rates, and increased efficiency, achieved by
maintaining column lengths.

While the benefits of UHPLC are well documented, the ulti-mate

implications are less well appreciated. Inspection of plate height curves such as
presented in Fig. 5a elicits the conclusion that columns packed with smaller
particle sizes will always out-perform those with large particles. This is
however not true in all cases. For example, if very high efficiencies are
required, very long columns should be used (N is directly proportional to L,
while res-olution is proportional to the square root of N). Column length is
however limited by the maximum instrumental pressure available, and the
pressure drop across the column ( P) is determined by its permeability (K v0)
according to Darcy’s law:

where ε and εT are the external and total porosities, respectively. It is clear that a reduction in d p coincides

where u0 is the linear velocity and the viscosity of the mobile phase. K v0 is related
to the particle size via [200,201]:
u · ·L with a significant decrease in column permeability. The somewhat counter-intuitive result of this is that
Kv0 = 0
higher overall efficiencies are attained with an increase in particle size, a result of the relative importance
P of column per-meability and (current) instrumental pressure constraints.
2 3
dp · ε In this context, plate height curves such as the van Deemter provide limited information due to the fact
Kv0 = 2 that column perme-ability, and by extension the maximum column length, are not taken into account (H is
180 · (1 − ε) · εT
independent of L). A more accurate rep-resentation of the ultimate performance of column/instrument
combinations can be obtained using so-called kinetic plots, the origins of which can be traced to Giddings
[202], but which have been adapted by allied band broadening due to the establish-ment of a radial temperature
various authors [203–205]. In essence, these gradient inside the column [211,212]; this has also been confirmed
plots combine plate height data with experimentally [213]. This effect can be minimised by reducing the pressure
column permeability and pressure (i.e. the flow rate or column length), or more practically by reducing the
constraints to provide an accurate reflection internal diameter of the column (which coincides with a reduction in
of the performance of specific volumetric flow rate, thereby further reducing the effects of frictional
column/instrument combination as a heating). It is for this reason that most UHPLC separations are performed on
function of analy-sis time. The group of columns
Desmet developed a family of kinetic plots
[200,205–207] which allow facile
comparison of the performance of different
columns as a function of a range of
parameters; this approach has also been (2) of 2.1 mm internal diameter (i.d.) or lower. From the perspective of
extended to gradient separations, where hyphenation to MS, this is clearly beneficial. Optimal flow rates on these
systems can be compared in terms of peak columns (taking into account the plate height behaviour of these phases, cf.
Fig. 5a) are in the region of 0.2–0.5 mL/min and are therefore ideally
capacities obtainable as a function of compatible with ESI-MS. The much narrower peak
analysis time [208]. (3) widths typically obtained do however place severe demands on the acquisition
rate of MS detectors. While 4.6 mm i.d. UHPLC columns

From the kinetic plot curves presented

for catechin in Fig. 5b it is clear that
UHPLC provides considerable advantages
in terms of separation speed compared to
conventional HPLC methods for the same
efficiency (peak capacity). UHPLC also
offers the option to increase
chromatographic efficiency for
conventional analysis times (moving
horizontally from 5 to 1.7 m curves for a
given analysis time in Fig. 5b), although
this gain is ultimately limited by pressure
constraints. The region of most interest to
researchers in flavonoid analysis coincides
with that where UHPLC clearly out-
performs HPLC. In light of the above, it is
no surprise that UHPLC has been applied
extensively as a means to speed up conven-
tional methods for HPLC analysis (see
below); exploitation of the improved
efficiency benefit of UHPLC is somewhat
less common in this field.

It is important to note that the

conclusions drawn from plate height and
kinetic plot representations are analyte-
dependant [209], and therefore caution
should be exercised when applying
conclusions drawn for other analytes to
flavonoids. Limited kinetic data for
flavonoids has been reported in literature,
but several studies have indicated that
flavonols, flavones and dihydrochal-cones
[210], procyanidins [199] and especially
anthocyanins [105] are characterised by
much lower optimal linear velocities and
increased C-term contributions to plate
height than (neutral) small molecules.
Practically this means that gains in terms of
analysis speed are less pronounced than for
such small molecules, but also that the
benefits of UHPLC are extended to higher
efficiencies in the case of flavonoids.

Operation at
elevated
pressures is
associated with
the risk of
frictional
heating and
26 A. de Villiers et al. / J. Chromatogr. A 1430 (2016) 16–78

25 5 µm porous 1000000 5 µm porous, 400 bar

20 a b
100000 1.7 µm porous, 1000 bar
1.7 µm porous
2.6 µm superficially
15 2.6 µm superficially porous
10000 porous, 600 bar

H (µm)

t R (s)
10 1000

5 100

0 10

0 1 2 3 4 5 6 7 10 100 1000
6 u0 (mm/s) 1000000 nc
tetrameric procyanidin, 25°C tetrameric procyanidin, 25°C
tetrameric procyanidin, 50°C 100000 tetrameric procyanidin, 50°C
catechin, 25°C c catechin, 25°C d
4
catechin, 50°C 10000 catechin, 50°C

(s)
1000
H (µm)

Rt
2
100

0 10 1.7 µm porous, 1000 bar

0 1 2 3 4 5 6 7 10 100 1000
u0 (mm/s) nc
Fig. 5. (a) Comparison of plate height curves obtained for the flavanol catechin on a 5 m porous phase ( Pmax 400 bar, blue diamonds), a 1.7 m porous phase ( Pmax 1000 bar, yellow circles) and a 2.6
m superficially porous phase ( Pmax 600 bar, brown squares). (b) shows the corresponding peak capacity (n c ) as a function of analysis time for catechin (as determined using the corresponding
◦
retention factors). (c) illustrates the plate height curves for catechin (MW 290) and a tetrameric procyanidin (MW 1155) at 25 and 50 C on a 1.7 m porous phase ( P max 1000 bar), and (d) illustrates
the corresponding retention time vs nc plot. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Source: Data adapted from [199].

are commercially available, and have also found extensive use in flavonoid
analysis, optimal flow rates on these columns (typically 1.2–2.5 mL/min,
depending on the analytes) require flow split-ting prior to MS detection.
Furthermore, frictional heating effects become a concern on such column
dimensions (something which is not sufficiently recognised by many
practitioners in flavonoid analysis).
Finally, the impact of typical UHPLC column dimensions should be
considered in the context of extra-column band-broadening. For the short, 2.1
mm i.d. columns which are the norm in UHPLC, peak volumes are especially
small because of the reduction in flow rate and the high column efficiency. In
these circumstances, dispersion in extra-column volume can significantly
affect the measured peak width. For example, Gritti and Guiochon showed
that even the most sophisticated modern instruments (excluding capillary and
nano-systems) lead to losses of 15–25% of column efficiency in the case of
the latest generation high-efficiency columns in isocratic mode [214]. Fekete
et al. [215] reached similar conclusions for sub-2 m porous, superficially
porous and monolithic columns. While the effect of extra-column band
broadening is significantly reduced in gradient separations due to focusing on
the column, dispersion in connection tubing at the outlet of the column can
still have a pro-found effect on the measured peak width. This is often evident
from comparison of peak widths in UV and MS chromatograms obtained by
UHPLC-DAD-MS, where much broader peaks are observed in the latter. This
may be ascribed in part to the size of most MS instru-ments, which requires
relatively long connection tubing between the exit of the UV cell and the
ionisation chamber. However, small changes in tubing length and/or internal
diameter can produce much better MS chromatograms.
UHPLC technology has matured rapidly in the last decade, and has found
widespread acceptance in the separation community – to the extent that the
technique has in many fields replaced conven-tional HPLC methods for
routine analysis. That this is also the case in flavonoid analysis is confirmed
by the large number of applications reported in Tables 1, 2 and 4–7, also
evidenced by the observa-tion that many of the papers cited in this work
report method
validation results, which amply demonstrate the suitability of columns and
instrumentation for routine analysis. We will not attempt to address each of
these, but rather to highlight some trends concerning the application of
UHPLC in this field.
The principal application of UHPLC technology, in flavonoid analysis as
in HPLC in general, is to attain faster separation and higher throughput. This
makes sense, since 100 and 50 mm columns packed with sub-2 m particles
provide roughly equiv-alent efficiencies to 250 and 150 mm columns packed
with 5 m particles, respectively. This performance is obtained in much shorter
analysis times for UHPLC columns because (i) the column is shorter and (ii)
the optimal mobile phase linear velocity is higher. The practical consequence
is that similar separation is attained as for conventional HPLC, with
commensurate sensitivity, robustness etc., but at analysis times 2–9× shorter.
A typical example is the speeding up of routine methods on conventional
HPLC systems to UHPLC columns and instrumentation [107]. Another
application area is in the second dimension of LC × LC separations, where
sub-2 m phases show clear benefits for maximising performance in the
shortest possible analysis time – here the reduced C-term band broadening on
these phases is exploited on short columns operated at high flow rates.
A second approach is to use longer columns packed with sub-2 m phases,
in this case utilising the increased pressure capa-bilities of UHPLC columns
and instrumentation to obtain higher resolution for complex flavonoid
mixtures. Examples are found in the analysis of rooibos tea [216] and
strawberry [217] phenolics and red wine anthocyanins [218] on 200 mm
UHPLC columns. These columns provide isocratic efficiencies ∼2.4× higher
than standard 250 mm 5 m columns, which translates into an increase in reso-
lution by a factor of about 1.5.
3.1.2. Monolithic columns
An alternative approach to overcome the
permeability/pressure limitations of HPLC is to use monolithic
columns. Monolithic stationary phases are cast as single
continuous phases with interconnected skeletons and
through-pores. The higher external
 A. de Villiers et al. / J. Chromatogr. A 1430 (2016) 16–78 27

Table 1
Applications of advanced 1-dimensional LC methods without MS detection to flavonoid analysis (2009–2015).

Sample Flavonoids Detector(s) Stationary phases (column Mobile Reference

dimensions), flow rates, temperature, phase(s)
analysis times
Rhus coriaria L. Myricetin, quercetin and UV C18 monolith (100 mm × 4.6 mm), d
ACN /10 mM [229]
kaempferol c ◦
FR = 4.0 mL/min, 40 C, <1 min KH2 PO4 (38/62,
C18 monolith (100 mm × 4.6 mm), v/v, isocratic)
Tomato Flavonol and flavanone UV d [228]
2 mM FA /ACN
aglycones and glycosides b FR = 1.0 mL/min, 15 min (75/25 (v/v),
LIT (ESI−)
(6 flavonoids) isocratic)
Cranberry juice Proanthocyanidins (9 UV C18 monolith d [186]
A: 1% AA , B:
flavonoids) b (2 mm × 100 mm × 4.6 mm), 1% AA in ACN
QIT (ESI+)
◦
TOF FR = 3 mL/min, 30 C, 16 min
Soy Isoflavone aglycones and UV c A: 1% AA, B: [300]
C18 (100 mm × 4.6 mm, 2.6 m dp SP ),
◦
glycosides (12 flavonoids) FR = 2.7 mL/min, 50 C, 12 min ACN
Tea Flavanols, flavonol UV C18 (100 mm × 2.1 mm, 1.7 m dp ), A: 0.1% FA, B: [301]
aglycones and glycosides, ◦ d
FR = 0.42 mL/min, 25 C, 20 min MeOH
flavanones and flavones
(16 flavonoids) C18, monolith (100 mm × 4.6 mm),
Orange juice Flavanone aglycones and b A: 150 mM AA, [146]
FL
glycosides and flavonols (5 FR = 0.7 mL/min, 25 min B: ACN C:
flavonoids) C18 monolith (100 mm × 4.6 mm), MeOH
Grapes Catechin and epicatechin UV A: 2% AA in [149]
◦
FL FR = 2.5 mL/min, 25 C, 7.25 min 90/10
H2 O/MeOH, B:
2% AA in 10/90
C18 (50 mm × 3.0 mm, 1.8 m dp ), H2 O/MeOH
Reseda luteola (weld) Luteolin-7,3 -O- UV − A: 160 mM FA, [302]
◦
diglucoside, LIT (ESI ) FR = 0.9 mL/min, 35 C, 5 min 40 mM
luteolin-7-O-glucoside and ammonium
luteolin formate,
0.04 mM
d
EDTA , pH 3, B:
C18 (50 mm × 2.1 mm, 1.7 m dp ), MeOH
Wine Flavanols, flavonol UV A: 0.1% FA, B: [303]
◦
aglycones and glycosides FR = 0.25 mL/min, 40 C, 8 min ACN
(6 flavonoids) C18 (100 mm × 4.6 mm, 2.6 m dp SP),
Soy protein sample Isoflavones aglycones and UV A: 1% AA, B: [264]
◦
glycosides (12 flavonoids) FR = 1.2 mL/min, 25 C, 50 min ACN
Wine Flavan-3-ols, flavonol UV C18 (100 mm × 4.6 mm, 2.6 m dp SP), A: 0.1% FA, B: [304]
aglycones and glycosides, FR = 1.3 mL/min, 20 min ACN
flavones and flavanonol (10
flavonoids) C18 (100 mm × 4.6 mm, 2.7 m dp SP),
Apple juice Flavonols and flavones (8 UV H2 O/ACN [305]
◦
flavonoids) FR = 0.8 mL/min, 27 C, 25 min (60/40, v/v,
C18 (100 mm × 4.6 mm, 1.8 m dp ), isocratic)
Rooibos tea (Aspalathus Dihydrochalcones, flavones UV A: 2% AA, B: 2% [306]
◦
linearis) and flavonols (14 FR = 1.0 mL/min, 37 C, 50 min AA in ACN
flavonoids) C18 (50 mm × 4.6 mm, 2.6 m dp SP),
Cocoa Flavanols and UV A: 0.1% FA, B: [199]
◦
proanthocyanidins (9 FR = 0.4 mL/min, 50 C, 10–18 min ACN
flavonoids) C18 (150 mm × 4.6 mm, 2.7 m dp SP),
Plant extracts Flavonols and flavones (10 UV A: phosphoric [103]
◦
flavonoids) FR = 1.0 mL/min, 30 C, 45 min acid (pH 2), B:
d
C18 (100 mm × 4.6 mm, 2.6 m dp SP), ACN C: THF
Red and white grape Flavonol aglycones and UV A: 0.5% H3 PO4 , [95]
◦
juice and wine, teas glycosides, flavanone and FR = 2.0 mL/min, 45 and 30 C, B: 0.5% H3 PO4
and plant extracts anthocyanins (10 18–23.5 min in MeOH
flavonoids) c
PFP (100 mm × 2.1 mm, 2.6 m dp SP),
◦
FR = 0.2 mL/min, 45 C, 40 min
Tomato, broccoli, Catechin, epicatechin, rutin UV C18 (100 mm × 2.1 mm, 1.8 m dp ), A: 0.1% FA, B: [307]
◦
onion, green and red and kaempferol FR = 0.25 mL/min, 40 C, 14 min MeOH
pepper and beetroot C18 monolith (100 mm × 4.6 mm)
Jattropha gossypifolia Flavone glycosides (6 UV A: 0.1% AA, B: [234]
flavonoids) b ◦ 0.1% AA in
Q-TOF FR = 3.0 mL/min, 25 C, 40 min
(ESI+) C18 (150 mm × 2.1 mm, 2.6 m dp SP) MeOH
Mouse plasma and Genistein UV 10 mM [308]
◦
tissue FR = 0.25 mL/min, 40 C, 13 min NaH2 PO4 /MeOH
(55/45, v/v,
Amide (100 mm × 3.0 mm, 2.7 m dp isocratic)
Food supplements Rutin, troxerutin, diosmin UV ACN/H2 O [98]
and hesperidin SP), FR = 1.0 mL/min containing AA
pH 3 (30:70,
v/v, isocratic)
28 A. de Villiers et al. / J. Chromatogr. A 1430 (2016) 16–78
Table 1 (Continued )

Sample Flavonoids Detector(s) Stationary phases (column Mobile Reference

dimensions), flow rates, temperature, phase(s)
analysis times

Staghorn sumac fruit Quercetin-3-rhamnoside, UV C18 (100 mm × 2.1 mm, 1.7 m dp ), A: [309]
◦
(Rhus hirta L.) quercetin and peonidin-3- QIT FR = 0.35 mL/min, 30 C, 14 min MeOH/H2 O/FA,
O-galloyl-hexose (ESI+/−) 95:2:3 (v/v/v),
B:
MeOH/H2 O/FA,
C18 (100 mm × 2.1 mm, 1.7 m dp ), 2:95:3 (v/v/v)
Hops (Humulus lupulus Xanthohumol and UV A: 0.1% FA, B: [310]
◦
L.) isoxanthohu-mol FR = 0.25 mL/min, 30 C, 13 min ACN
‘Copaíba’, ‘copaiva’ or Quercitrin and afzelin UV C18 monolith (100 mm × 4.6 mm), A: H2 O, B: ACN [226]
‘paú-de-óleo’ FR = 1.0 mL/min, 60 min
(Copaifera Anthocyanins, flavonols, UV C18 (100 mm × 4.6 mm, 1.8 m dp ), A: 0.5% TFA, B: [107]
langsdorffii)
Juices of strawberry,
◦ d
American cranberry, flavanones, FL FR = 1.0 mL/min, 25 C, 40 min TFA /ACN/H2 O
dihydrochalcones and b (0.5/50/49.5,
bilberry, sour cherry, Q (ESI+)
black grape, orange and flavan-3-ols (48 flavonoids) v/v/v)
apple Apigenin, luteolin and UV C18 (150 mm × 2.1 mm, 1.7 m dp ), A: H2 O/MeOH [266]
Natural dyestuffs
◦
genistein FR = 0.2 mL/min, 40 C, 40 min (10/90, v/v), B:
MeOH C: 0.1%
Q (ESI+/−) PFP (50 mm × 2.1 mm, 1.7 m dp SP), FA
Chokeberry (Aronia Anthocyanins, flavonol A: 5% FA, B: [94]
melanocarpa, Aronia glycosides (7 flavonoids) FL FR = 0.2 mL/min, 18 min MeOH/H2 O
arbutifolia and Aronia UV (30/70, v/v)
prunifolia) Catechins (8 compounds) UV C18 monolith (100 mm × 4.6 mm), H2 O/ACN/MeOH [227]
Green, oolong and
black teas FR = 1.4 mL/min, 7 min (83/6/11, v/v/v,
C18 (50 mm × 2.1 mm, 1.7 m dp ) isocratic)
Red, white, black, and Flavan-3-ol derivatives and UV A: 1% FA, B: [150]
◦
green teas, cocoa quercitin (8 flavonoids) FL FR = 0.5 mL/min, 35 C, 2 min MeOH
LIT-
Orbitrap
(ESI+) C18 (50 mm × 2.1 mm, 1.8 m dp ),
Barringtonia racemosa Rutin, quercetin and UV A: 0.1% TFA, B: [142]
kaempferol FR = 0.6 mL/min, 13.4 min ACN
Standards Flavones, isoflavones, UV C18 (50 mm × 3.0 mm, 2.6 m dp SP), A: 0.05 mM [90]
flavanones, flavan-3-ols FR = 3.0 mL/min, 2 min ammonium
and flavonols (15 acetate, B: ACN
flavonoids) C18 (50 mm × 2.1 mm, 1.8 m dp ),
Chia seeds (Salvia Isoflavones (5 flavonoids) UV A: 2% AA, B: [141]
hispanica L.) FR = 0.4 mL/min, 18 min H2 O/ACN/AA
C18 monolith (50 mm × 2.0 mm), (68/30/2, v/v/v)
Herbal dietary Flavonol, chalcone and UV A: 0.05% TFA [311]
◦
supplements flavanone aglycones and LIT FR = 1.2–1.3 mL/min, 20 C, 5 min (UV) or 0.1% FA
glycosides and flavan-3-ols (MS), B: ACN
(14 flavonoids) Q (ESI−) C18 (150 mm × 4.6 mm, 2.6 m dp SP),
Zhi-Zi-Da-Huang Isoflavone and flavanone A: 0.1% FA, B: [312]
decoction (TCM )
a glycones and glycosides, FR = 0.8 mL/min (0.2 mL/min to MS), ACN
flavonol glycoside and ◦
35 C, 55 min
flava-3-ols (13 flavonoids) Q (ESI−) PFP (100 mm × 4.6 mm, 2.6 m dp SP),
Soy Isoflavones (20 A: 1% AA, B: [93]
◦
compounds) UV FR = 0.8 mL/min, 25 C, 37 min ACN
Forsythia flowers Rutin UV UV: C18 (75 mm × 4.6 mm, 2.7 m dp A: [313]
◦
QIT (ESI−) SP), FR = 1.4 mL/min, 30 C, 22 min H2 O/orthophosphoric
QIT: C18 (150 mm × 2.1 mm, 1.9 m acid (99.5:0.5,
◦
dp ), FR = 0.2 mL/min, 25 C, 68 min v/w), B: ACN
A:
H2 O/ACN/FA,
95:5:0.1
(v/v/v), B: 0.1%
C18 (100 mm × 3.0 mm, 2.6 m dp SP) FA in ACN
Grape pomaces Flavonols and flavan-3-ols UV A: 0.1% FA, B: [314]
◦
(8 flavonoids) FR = 0.8 mL/min, 35 C, 20 min ACN
Soy bean, black bean, Isoflavones (14 UV C18 (100 mm × 2.0 mm, 2 m dp ), A: 0.5% FA, B: [315]
compounds) ◦ ACN
red bean and three FR = 0.3 mL/min, 40 C, 25 min
soybean pastes
a
TCM, traditional Chinese medicine.
c
b LIT, linear ion trap; QIT, quadrupole ion trap; FL, fluorescence detection; Q, (single) quadrupole; Q-TOF, quadrupole-time-of-flight. FR, flow rate;
SP, superficially porous; PFP, pentafluorophenyl.
d
ACN, acetonitrile; FA, formic acid; AA, acetic acid; MeOH, methanol; EDTA, ethylenediaminetetraacetic acid; THF, tetrahudrofuran; TFA, trifluoroacetic acid.
 A. de Villiers et al. / J. Chromatogr. A 1430 (2016) 16–78 29
Table 2

Applications of comprehensive 2-dimensional LC to flavonoid analysis (2009–2015).

Sample(s) Flavonoids Hyphenation Stationary phases (column Mobile phases Detector(s) Reference(s)
mode(s), dimensions), flow rates,
separation temperature, analysis times
modes,
sampling
time(s)
Apple and cocoa Procyanidins and Off-line, 1 1 d UV [147]
D: Diol (250 mm × 1 mm, ( D) A: 1% AA in
c d e
flavonol glycosides HILIC × RP-LC, 5 m dp ), FR = 0.05 mL/min, ACN , B: FL
1 b d e
(41 compounds) ts = 1 min 70 min MeOH /H2 O/AA Q-TOF (ESI−)
2 (94.05/4.95/1
D: C18 ((2×)
50 mm × 4.6 mm, 1.8 m (v/v/v))
◦ 2 d
dp ), FR = 1.8 mL/min, 50 C, ( D) A: 0.1% FA ,
4/30 min B: ACN
Mate extracts Flavonol glycosides On-line, 1 1 UV [367]
D: RP-Amide ( D) A: H2 O (pH
e
(3 compounds) RP-LC × RP-LC, (250 mm × 1.0 mm, 5 m 3), B: ACN (pH 3) IT -TOF
1 ◦ 2 (ESI+/−)
ts = 2 min dp ), FR = 0.01 mL/min, 25 C, ( D) A: H2 O (pH
70 min 3), B: ACN (pH 3)
2
D: C18 (30 mm × 4.6 mm,
c
2.7 m dp , SP ),
◦
FR = 4 mL/min, 45 C
1 c 1
Longdan Xiegan Flavone and On-line, D: ILC (150 mm × 4.6 mm), ( D) 10 mM UV [368]
a b e
Decoction (TCM ) isoflavone ILC × RP-LC, FR = 0.05 mL/min, 400 min ammonium APCI-Q-MS
aglycones and 1 * 2 acetate (pH 6.8, (−)
ts = 10 min D: C18 (150 mm × 4.6 mm),
glycosides, FR = 2.0 mL/min (0.8 mL/min isocratic)
flavanone to MS) 2
( D) A: 0.1% AA, B:
glycoside (8 ACN
flavonoids) 1 c
D: PEG (150 mm × 2.1 mm,
Standards Flavan-3-ols, On-line, 1 2 UV [357]
( D, D) A: 10 mM
flavones, flavanone RP-LC × RP-LC, 5 m dp ), FR = 0.25 mL/min, ammonium
1 ◦
aglycones and ts = 0.58 min 40 C, 35 min, acetate, B: ACN
glycosides, flavonol 2
D: C18 (30 mm × 3 mm,
aglycones and 2.7 m dp SP), FR = 4 mL/min
◦
glycosides, (0.2 mL/min to MS), 60 C
isoflavone (13
flavonoids) 1
D: C8 (250 mm × 4.6 mm,
Fructus aurantii (TCM) Polymethoxylated Off-line, 1 2 UV [374]
( D, D) A: water,
flavones (42 RP-LC × RP-LC, 5 m dp ), FR = 1 mL/min, B: ACN IT-MS (ESI+)
1
flavonoids) ts = 1 min 30 min
2
D: C18 (250 mm × 4.6 mm,
5 m dp ), FR = 1 mL/min,
30 min
Red wines Flavan-3-ols and On-line, 1 1 UV [344]
D: Phenyl (250 mm × 1 mm, ( D) A: H2 O (pH
flavonol glycosides RP-LC × RP-LC, 5 m dp ), FR = 0.01 mL/min, 3), B: ACN (pH 3)
1 ◦ 2
(7 flavonoids) ts = 2 min 25 C, 80 min ( D) A: H2 O (pH
2 3), B: ACN (pH 3)
D: C18 (30 mm × 4.6 mm,
2.7 m, dp SP),
FR = 4 mL/min (0.2 mL/min
◦
to MS), 45 C
Red wine Flavan-3-ols, On-line, 1 1 2 UV [355]
D: PEG (150 mm × 2.1 mm, ( D, D) A: 10 mM
flavones, flavonols, RP-LC × RP-LC, 5 m dp ) FR = 40, 70, and ammonium
flavonol glycosides, 1 240 L/min, 35, 110, and acetate, B: ACN
ts = 1.5 min
◦
flavanone 140 min, 40 C
aglycones and 2
D: C18 (50 mm × 3.0 mm,
glycosides, 2.7 m dp SP), FR = 3.3 and
isoflavones (14 4.8 mL/min, 0.42, 1.4,
◦
flavonoids) 2.0 min, 50 C
Green tea Flavan-3-ols, Off-line, 1 1 UV [126]
D: Diol (250 mm × 1 mm, ( D) A: 1% AA in
proanthocyanidins, HILIC × RP-LC, 5 m dp ), FR = 0.05 mL/min, ACN, B: FL
1
flavonol aglycones ts = 1 min 70 min MeOH/H2 O/AA Q-TOF (ESI−)
and glycosides and 2 (94.05/4.95/1
D: C18 (50 mm × 4.6 mm,
flavone glycosides 1.8 m dp ), FR = 0.8 mL/min, (v/v/v))
(48 flavonoids) ◦ 2
50 C, 30 min ( D) 0.1% FA, B:
ACN
Stevia rebaudiana Flavone and On-line, 1 c 1 UV [366]
D: PA -G ( D) A: ACN (pH
extracts flavonol glycosides HILIC × RP-LC, (250 mm × 1.0 mm, 5 m 3), B: H2 O (pH 3)
(5 flavonoids) 1 2
ts = 20 s dp ), FR = 0.02 mL/min, ( D) A: H2 O (pH
101 min 3), B: ACN (pH 3)
2
D: C18 (30 mm × 2.1 mm,
1.8 m dp ), FR = 3.4 mL/min,
◦
70 C
Citrus juices Flavanones and On-line, 1 1 2 UV [343]
D: PEG (250 mm × 2.1 mm, ( D, D) A: 0.1%
flavones (11 RP-LC × RP-LC, 5 m dp ), FR = 50 L/min, FA, B: H2 O/ACN/ Q-MS (ESI−)
1
flavonoids) ts = 1 min 80 min isopropanol/FA
2 (39.9:20:40:0.1
D: C18 (50 mm × 4.6 mm,
2.7 m dp SP), (v/v))
◦
FR = 3.0 mL/min, 40 C
30 A. de Villiers et al. / J. Chromatogr. A 1430 (2016) 16–78
Table 2 (Continued )

Sample(s) Flavonoids Hyphenation Stationary phases (column Mobile phases Detector(s) Reference(s)
mode(s), dimensions), flow rates,
separation temperature, analysis
modes, times
sampling
time(s)
Scutellaria barbata D. Flavone and Off-line, HILIC × HILIC: HILIC × HILIC: Q-TOF (ESI+) [375]
Don (TCM) flavanone HILIC × HILIC, 1 1 2 UV
D: Silica ( D, D) A:
aglycones and HILIC × RP–LC, (250 mm × 4.6 mm, 5 m ACN/water (95:5
1
glycosides (22 ts = 1 min dp ), FR = 1.0 mL/min, 30 min (v/v)) with 5 mM
flavonoids) 2 ammomium
D: Amide
(150 mm × 4.6 mm, 5 m formate, B: 5 mM
dp ), FR = 1.0 mL/min, 30 min ammonium
HILIC × RP: formate
1
D: Amide HILIC × RP:
(150 mm × 4.6 mm, 5 m 1 2
( D, D) A: 0.1% FA,
dp ), FR = 1.0 mL/min, 30 min B: 0.1% FA in ACN
2
D: C18 (150 mm × 2.1 mm,
5 m dp ), FR = 0.2 mL/min,
50 min
Standards Flavan-3-ols, On-line, 1 1 UV [362]
D: DioEDMA, monolith ( D) A: ACN, B:
flavones, flavonol HILIC × RP-LC, (180 mm × 0.53 mm), 10 mM
1 ◦
and flavanone ts = 1.5 min FR = 0.01 mL/min, 40 C ammonium
2
aglycones and D: RP-Amide, C18 and acetate (pH 3.1)
glycosides, phenyl hexyl (50 and 2
( D) A: 10 mM
isoflavone (13 30 mm × 3.0 mm, 2.6 and ammonium
flavonoids) 2.7 m dp SP), acetate (pH 3.1),
◦
FR = 3–4 mL/min, 50 C, B: ACN
1.5 min
Green and black teas Flavonol- Off-line, 1 c 1 e [81]
D: SEC ( D) A: H2 O, B: ELSD
(Camellia sinenis) glycosides, SEC × RP-LC, (300 mm × 7.8 mm, ACN UV
flavone-glycosides, 1 3 2 e
ts = 1 min 5 × 10 Da size exclusion), ( D) A: 0.1% FA, B: QqQ (ESI−)
◦
flavan-3-ol FR = 1.0 mL/min, 60 C, MeOH
derivatives (54 60 min
flavonoids) 2
D: C18 (50 mm × 2.1 mm,
1.7 m dp ), FR = 1.0 mL/min
◦
(0.1 mL/min to MS), 80 C,
10 min
Rooibos tea (Aspalathus Dihydrochalcones, Off-line/on- 1 1 UV [127]
D: Diol (250 mm × 1 mm, ( D) A: 2% AA in
linearis) flavanones, line, 5 m dp ), FR = 0.05 mL/min, ACN, B: Q-TOF (ESI+/−)
flavones, and HILIC × RP-LC, 100 min MeOH/H2 O/AA
flavonols (20 1 2 (93.05/3.95/2
ts = 1/2 min D: C18 (50 mm × 4.6 mm,
flavonoids) (off-line/on- 1.8 m dp ), (v/v/v))
line) ◦ 2
FR = 1.0 mL/min, 48 C, ( D) A: 1% AA, B:
29 min ACN
Six TCMs Isoflavone, flavone Off-line, 1 c 1 Q-TOF (ESI+) [376]
D: Click CD D) A: H2 O, B:
and flavanone HILIC × RP-LC, (150 mm × 4.6 mm, 5 m ACN, C: 100 mM UV
1 *
aglycones and ts = 2 min dp ), FR = 1.0 mL/min, 30 min ammonium
2
glycosides (25 D: C18 (150 mm × 2.1 mm, formate
2
flavonoids) 5 m dp ), FR = 0.3 mL/min, ( D) A: 0.2% FA, B:
c 0.2% FA in ACN
15 min or Click OEG
(150 mm × 2.1 mm, 5 m
dp ), FR = 1 mL/min, 30 min TOF (ESI−)
Qingkailing (TCM) Baicalin and On-line, 1 1 [350]
D: SEC (20 mm × 2.1 mm), ( D) A: 10 mM
◦
wogonoside SEC × RP-LC, FR = 0.02 mL/min, 25 C ammonium
1 ** 2
ts = 5 min D: C18 (100 mm × 2.1 mm, formate (isocratic)
2
1.7 m dp ), ( D) A: 0.1% FA, B:
◦
FR = 0.7 mL/min, 80 C ACN
Standards Flavan-3-ols, Online, 1 1 2 UV [377]
D: PEG ( D, D) A: 10 mM
flavones, flavonols RP-LC × RP-LC, (150 mm × 2.1 mm, 5 m ammonium
and flavanones (14 1 acetate (pH 3.1),
ts = 1.5 min dp ), FR = 0.066 mL/min,
◦
flavonoids) 40 C, 75 min B: ACN
2 c
D: PFP (50 mm × 3.0 mm,
2.6 m dp SP),
◦
FR = 2.5 mL/min, 40 C
Standards Flavones, flavonols, On-line, 1 1 2 UV [231]
D: Zwitterionic monolith ( D, D) A: 0.01 M
flavanones, HILIC × RP-LC, (210 mm × 0.53 mm), FR = 2 ammonium
flavan-3-ols and 1 ◦ acetate, B: 0.01 M
ts = 1.5 min and 3 L/min, 60 C, 60,
isoflavones (15 100 and 140 min ammonium
flavonoids) 2 acetate in ACN
D: C18 (30 mm × 3.0 mm)
◦
FR = 4.5 mL/min, 50 C, 1.0
and 1.5 min
 A. de Villiers et al. / J. Chromatogr. A 1430 (2016) 16–78 31
Table 2 (Continued )

Sample(s) Flavonoids Hyphenation Stationary phases (column Mobile phases Detector(s) Reference(s)
mode(s), dimensions), flow rates,
separation temperature, analysis times
modes,
sampling
time(s)
Apple Procyanidins, On-line, 1 1 IT-MS (ESI−) [369]
D: Diol (150 mm × 1.0 mm, ( D) A: ACN/AA
dihydrochalcones HILIC × RP-LC, 5 m dp ), FR = 21 L/min, (98/2 (v/v)), B: UV
1
and flavonols (64 ts = 78 s 50 min MeOH/H2 O/AA
2
flavonoids) D: C18 (50 mm × 4.6 mm, (95/3/2 (v/v/v))
2
2.7 m dp SP), ( D) A: 0.1% FA, B:
FR = 3.0 mL/min ACN and A: 0.1%
FA, B: ACN/MeOH
1 (50/50 (v/v))
D: Diol (250 mm × 1 mm,
Cocoa Procyanidins (38 On-line, 1 UV [340,342]
( D) A: 1% AA in
compounds) off-line, 5 m dp ), FR = 0.025 mL/min ACN, B: FL
stop-flow, (on-line) and 0.05 mL/min MeOH/H2 O/AA Q-TOF (ESI−)
HILIC × RP-LC, (stop-flow and off-line), (94.05/4.95/1
1
ts = 1 min 50 min (stop-flow and (v/v/v))
(off-line and off-line) and 100 min 2
( D) A: 0.1% FA, B:
stop flow), (on-line) ACN
1 2
ts = 2/3 min D: C18 column
(on-line) (50 mm × 4.6 mm, 1.8 m
◦
dp ), FR = 1.5 mL/min, 50 C,
12.6 min Q-TOF (ESI−)
Grape seed Procyanidins (78 On-line, 1 1 [148]
D: Diol (250 mm × 1 mm, ( D) A: 1% AA in
single peaks, HILIC × RP-LC, 5 m dp ), FR = 0.025 mL/min, ACN, B: FL
2
species of DP up to ts = 2 min 100 min MeOH/H2 O/AA
16 detected) 2 (94.05/4.95/1
D: C18 (50 mm × 4.6 mm,
2.6 m dp ), FR = 1.5 mL/min, (v/v/v))
◦ 2
50 C, 2 min ( D) 0.1% FA, B:
1 ACN
D: Diol (150 mm × 1.0 mm,
Grape seeds Procyanidins (43 On-line, 1 UV [230]
( D) A: 2% AA in
flavonoids) HILIC × RP-LC, 5 m dp ), FR = 0.015 mL/min, ACN, B: IT-MS (ESI−)
1
ts = 1.3 min 85 min MeOH/H2 O/AA
2
D: C18 (50 mm × 4.6 mm, (95/3/2 (v/v/v))
2
2.7 m dp SP), FR = 3 mL/min ( D) A: 0.1% FA, B:
(0.6 mL/min to MS), 1.3 min ACN/MeOH (50/50
2 (v/v))
D: C18 monolith
(100 mm × 4.6 mm), 1.3 min 2
( D) A:0.1% FA, B:
MeOH or
ACN/MeOH (50/50
1 (v/v))
D Diol (250 mm × 1 mm,
Cocoa and red grape Proanthocyanidins Off-line/on- 1 UV [363]
( D) A: 1% AA in
seeds (42 compounds) line, 5 m dp ), ACN, B: Q-TOF (ESI−)
e
HILIC × RP-LC, FR = 0.05–0.025 mL/min MeOH/H2 O/AA ABTS assay
1 2 (94.05/4.95/1
ts = 1 min D C18 (50 mm × 4.6 mm,
(off-line), 2 min 2.6 m dp SP), (v/v/v))
(on-line) ◦ 2
FR = 1.5 mL/min, 50 C, ( D) A: 0.1% FA, B:
15/2 min (off-line/on-line) ACN
Fenugreek seeds Flavone glycosides Off-line 1 1 d UV [378]
D: C18/propylphenyl ( D) A: 0.1% TFA ,
(15 flavonoids) (heart-cutting) (100 mm × 1 mm, 5 m dp ), B: MeOH/H2 O/TFA Q-MS (ESI+/−)
◦
and on-line, FR = 0.017 mL/min, 20 C (50/50/0.1 (v/v/v))
2 2
RP-LC × RP-LC, D: C18 (100 mm × 2.1 mm, ( D) A: 0.1% TFA,
1
ts = 1.5–4 min 2.6 m dp SP) (off-line) or B: ACN/H2 O/TFA
* C18 (75 mm × 2.10 mm, (33/4/8/0.05
(on-line)
3 m dp ) (on-line), (v/v/v))
FR = 2.3 mL/min (1:1 split
◦
before MS), 20 C
Blueberries, black Anthocyanins (73 Off-line, 1 1 UV [248]
D: Amide ( D) A: 0.4% TFA in
beans, red radish, red compounds) HILIC × RP-LC, (150 mm × 4.6 mm, 2.5 m ACN, B: 0.4% TFA Q-TOF (ESI+)
grape skins, red 1 ◦ 2
ts = 1 min dp ), FR = 0.2 mL/min, 50 C, ( D) A: 7.5% FA, B:
cabbage. 79–113 min 7.5% FA in ACN
2
D: C18 (50 mm × 4.6 mm,
2.6 m dp SP),
◦
FR = 0.5 mL/min, 50 C,
40 min
Ge-Gen (kudzu root, Isoflavones (19 Heart-cutting, 1 c 1 UV [364]
D: CSH C18 ( D) A: 0.1% FA, B:
TCM) flavonoids) on-line, (100 mm × 2.1 mm, 1.7 m MeOH IT-MS (ESI−)
◦ 2
RP-LC × RP-LC, dp ), FR = 0.1 mL/min, 50 C, ( D) A: 0.1% FA, B:
1
ts = 0.5 min 35 min ACN
2
D: Phenyl-hexyl
(50 mm × 3.0 mm, 2.7 m dp
SP), FR = 2.5 mL/min
(0.25 mL/min to MS), 50 ◦ C
32 A. de Villiers et al. / J. Chromatogr. A 1430 (2016) 16–78
Table 2 (Continued )

Sample(s) Flavonoids Hyphenation Stationary phases (column Mobile phases Detector(s) Reference(s)
mode(s), dimensions), flow rates,
separation temperature, analysis
modes, times
sampling
time(s)
Sugarcane (Saccharum Flavones (15 On-line, 1 c 1 UV [356]
D: CN (250 mm × 1 mm, ( D) A: 0.1% AA, B:
d
spp.) flavonoids) RP-LC × RP-LC, 5 m dp ), FR = 0.02 mL/min, 0.1% AA in EtOH Q-MS (ESI+/−)
1 2
ts = 2 min 172 min ( D) A: 0.1% AA, B:
2 0.1% AA in MeOH
D: C18 (30 mm × 4.6 mm,
2.7 m dp SP),
FR = 3 mL/min (0.4 mL/min
to MS)
Radix Astragali (TCM) Isoflavones (34 Off-line, 1 1 UV [370]
D: SEC ( D) A: H2 O, B:
flavonoids) SEC × RP-LC, (300 mm × 4.6 mm), ACN Orbitrap (ESI+)
1 2
ts = 2 min FR = 1 mL/min, 30 min ( D) A: 0.1% AA, B: IT-MS (ESI+)
2 0.1% AA in ACN
D: C18 (250 mm × 4.6 mm,
3 m dp ), FR = 0.8 mL/min
(0.2 mL/min to MS)
Licorice (Glycyrrhiza Flavanone On-line, 1 c 1 UV [379]
D: CSH C18 ( D) A: 0.1% FA, B:
uralensis) aglycones and RP-LC × RP-LC, (100 mm × 2.1 mm, 1.7 m MeOH IT-MS (ESI−)
glycosides, 1 ◦ 2
ts = 0.5 min dp ), FR = 0.1 mL/min, 50 C, ( D) A: 0.1% FA, B:
chalcone 40 min ACN
glycosides, 2
D: Phenyl-hexyl
isoflavones, (50 mm × 3.0 mm, 2.7 m
flavonols and dp SP), FR = 2 mL/min
◦
flavones (39 (0.2 mL/min to MS), 50 C
flavonoids) 1
D: CN (150 mm × 2.0 mm,
Hedyotis diffusa (TCM) Flavonol glycosides On-line, 1 UV [365]
( D) A: H2 O, B:
(10 flavonoids) RP-LC × RP-LC, 3 m dp ), MeOH Q-TOF (ESI+)
1 ◦ 2
ts = 1 min FR = 0.025 mL/min, 50 C, ( D) A: H2 O, B:
150 min 0.05% FA in ACN
2
D: C18 (50 mm × 3.0 mm,
2.6 m dp SP),
FR = 2.0 mL/min
◦
(0.25 mL/min to MS), 50 C
a TCM, traditional Chinese medicine.
1
b ts , sampling time; ILC, immobilised liposome chromatography.
c
FR, flow rate; ILC, immobilised liposome chromatography; PEG, polyethylene glycol; PA, polyamine; SEC, size exclusion chromatography; CD, cyclodextrin; OEG, oligo(ethylene glycol); PFP,
pentafluorophenyl; SP, superficially porous; CN, cyanopropyl; CSH, charged surface hybrid.
d
AA, acetic acid; ACN, acetonitrile; FA, formic acid; MeOH, methanol; EDTA, ethylenediaminetetraacetic acid; TFA, trifluoroacetic acid; EtOH, ethanol.
e
ABTS, 2,2 -azino-bis(3-ethylbenzothiazoline)-6 sulphonic acid; ELSD, evaporative light scattering detector; FL, fluorescence detector; Q, (single) quadrupole; IT, ion trap; TOF, time-of-flight;
QqQ triple quadrupole.
* **
Not truly comprehensive due to the use of excessive sampling times. Not truly
comprehensive due to fraction transfer protocol.

porosity of these phases, resulting from the large through-pores, is responsible
for the higher permeability of monolithic columns (cf. Eq. (3), where ε values
are ∼0.5–7 for monoliths compared to 0.4 for silica packings).
Two types of monolithic columns can be distinguished, namely polymeric
and silica monoliths [219]. The former are produced by in situ polymerisation
using a suitable monomer (such as polymethacrylates, polystyrene-
divinylbenzene, etc.), free radical initiator and porogenic solvent [220]. In
contrast, silica monoliths are produced using sol–gel processes involving
polycondensation of tetra-alkyloxysilanes with polyethylene glycol (PEG) as
poro-gen, followed by derivatisation of the phase [221]. Control of the silica
skeleton and macro- and meso-pore sizes is possible through the selection of
the silane starting material and PEG concentration [222].
The increased permeability of monolithic phases may be exploited in one
of two ways. First, very high flow rates can be used, which in combination
with the favourable mass transfer proper-ties of monoliths allows for fast
analyses. This is the tactic most often used in flavonoid analysis. Fast
analyses using monolithic columns have also been used in the second
dimension of LC × LC separations [223]. Alternatively, high-permeability
silica mono-liths can be used as very long columns to provide high efficiency
[224]; this approach has not been utilised in flavonoid analysis to date.
Comparison of the kinetic performance of monolithic columns with
porous silica phases is challenging due to the wide range of monolithic phases
of different properties available. Neverthe-less, kinetic studies on silica
monoliths, which are mostly used in flavonoid analysis, generally show the
benefit of these phases to primarily lie in the high-efficiency regime, with
conventional phases outperforming monoliths for fast analyses [200,222]. A
®
second generation of commercial silica monoliths (Chromolith High-
Resolution) with macropores of 1.2 m and mesopores of 15 nm was
introduced in 2011. These columns have closed the gap to porous phases in
terms of minimum plate heights of ∼7 m [225]. However, this performance
comes at the cost of lower perme-ability; the net result being that their optimal
kinetic performance shifts to lower efficiencies and faster analysis, yet these
columns are still outperformed by sub-2 m and superficially porous phases in
this region [44]. Increasing the maximum pressure above the cur-rent 200 bar
dictated by the column cladding might improve the competitiveness of
monolithic columns [44].
Monoliths have found some application in flavonoid analysis, primarily in
two areas. The first is for the fast separation of a limited number of target
flavonoids, where typically commercial (Chromolith ® ) C18 silica monoliths
of 100 mm length (or two of these coupled in series [186,226]) are used at
high flow rates to increase analysis speed. For simple mixtures, isocratic
elution is often used [149,227–229]. The second main application
area of
 A. de Villiers et al. / J. Chromatogr. A 1430 (2016) 16–78
monoliths is in comprehensive 2-dimensional LC analyses (refer to Section
3.2), where the high-flow compatibility of these phases are beneficial for fast
second dimension analyses [230], or alterna-tive retention mechanisms of
polymeric monoliths are exploited in the first dimension [231].
Considering that monolithic columns have been around since the late
1980s [232,233], they have found relatively limited appli-cation in the LC–
MS analysis of flavonoids, especially compared to UHPLC (only 5% of the
applications listed in the tables, refer to Fig. 7). One reason for this may lie in
the fact that the very high flow rates typically used for fast analyses on
commercially available monolithic column dimensions of i.d.’s 4.6 or 3 mm
are a serious drawback from the perspective of solvent consumption and
hyphenation to MS. For example, in typical studies utilising mono-liths, flow
rates between 2.5 and 4 mL/min [149,229,234] were used, with flow splitting
required prior to MS detection [186]. Cer-tainly the familiarity and experience
with conventional phases of most practitioners, coupled with the range of
high-performance silica-based columns available nowadays, also plays a role
in this regard, reflected also in the relatively sparse use of monoliths in
general.
3.1.3. Superficially porous stationary phases
Superficially porous (also referred to as core–shell, shell or fused-core)
phases were originally introduced in the 1960s [235,236], but it has only been
with the introduction of second gen-eration superficially phases since 2006
[237,238] that an upsurge in interest in these phases has occurred. This is a
consequence of the remarkable performance of these columns: in contrast to
conventional porous silica phases, minimum plate heights (H min) as low as
1.1–1.8dp have been measured for superficially porous phases, where for
years Hmin = 2dp was considered a practical limit for HPLC columns. Initially
marketed as providing reduced diffusion distances due to the smaller porous
layer surrounding a solid core, and therefore a reduced stationary phase mass
transfer term, exten-sive subsequent fundamental research has demonstrated
that the improved performance of these phases is mostly due to reduced A-
term [239–242] (ascribed to a narrower particle size distribu-tion [238,239]
and/or better packing efficiency [243]) and B-term [239,242,244–247]
contributions to plate height, in addition to a lower C-term [242,247]. The
latter advantage in terms of the C-term behaviour of superficially porous
phases is particularly pronounced for high MW compounds due to the slow
diffusion of these com-pounds [238,241,242]. From the perspective of
flavonoid analysis, this is pertinent in that it implies particular advantages of
these phases for the analysis of oligomeric proanthocyanidins [199] and
highly glycosylated and acylated flavonoids such as anthocyanins [128,248].
The primary benefit of superficially porous phases is therefore that
performance similar to fully porous phases of smaller d p can be obtained,
however at lower pressures due to the larger particles (cf. Eqs. (2) and (3))
[247] – the same applies for larger particle size superficially porous phases
[201]. This reduces the need for ultra high pressure operation and the risk of
frictional heating (super-ficially porous phases have also been shown to be
characterised by higher heat conductivity, further reducing this risk [249]).
The only potential drawback of these phases is reduced loadability asso-ciated
with the increase in the phase ratio [250], although this remains a point of
discussion and is more relevant in the case of ionisable analytes [251–253]
than for flavonoids. Several recent reviews on superficially porous phases can
be consulted for details on the theoretical aspects, applications, stationary
phase manufac-turing and available chemistries [254–257].
Fig. 5a and b illustrates the advantages of superficially porous phases for
flavanols: for a 2.6 m superficially porous phase, H min and uopt values of 3.9
m and 1.2 mm/s were measured for
33
catechin, compared to 4.24 m and 1.27 mm/s for a fully porous 1.7 m phase
[199]. In fact, this column outperforms fully porous phases of various particle
sizes across the entire range of practical efficiencies (Fig. 5b).
Since 2006 a range of superficially porous columns of different (mostly
RP-LC) chemistries, column dimensions and particle sizes varying between
1.3 and 5 m have become commercially avail-able from several manufacturers
[257]. HILIC superficially porous phases are nowadays also available [251],
although to the best of our knowledge these have not to date been applied in
the LC–MS analysis of flavonoids.
Mirroring the contemporary situation in HPLC, application of these phases
has grown rapidly in both one- and multi-dimensional HPLC analysis of
flavonoids during the past seven years (Tables 1, 2 and 4–7). These phases are
increasingly being used as alternatives to sub-2 m porous phases, since they
gen-erally provide similar performance at lower operating pressures.
Accordingly, superficially porous phases are also utilised to achieve two
principal goals: to obtain faster separations, and, to a lesser extent, to
maximise resolution for complex mixtures. The major-ity of applications
therefore use relatively short columns to benefit from speed gains, although
column lengths up to 150 mm have been used to provide maximum resolution
[96,124,258–263] – these provide much higher resolution than conventional
columns of 150 or 250 mm length and packed with 5 m phases.
Several studies have compared the performance of core–shell and fully
porous phases. For example, Ruiz et al. [260] compared
a conventional (250 mm × 4.6 mm, 5 m), a superficially porous (150 mm ×
4.6 mm, 2.6 m) and 2.2 m (150 mm × 2.0 mm) C18 phases for the analysis of
glycosylated flavonols, and found the superficially porous phase to be
superior for this application. For the analysis of polyphenols in lentil seed
coats, two types of core–shell 2.6 m columns (Kinetex C18 and PFP) where
compared to a fully porous 4 m phase. The core–shell columns outperformed
the fully porous particles, with the PFP phase providing better separation of
flavone and flavonol glycosides than the C18 core–shell col-umn [91]. This
phase has also been found to be ideally suited for the analysis of hydrophobic
isoflavones [92,93] and some antho-cyanins [95]. Manchón et al. similarly
reported better performance for a 2.6 m superficially porous C18 phase
compared to 3.5 m fully porous C18 phase and a monolithic column for the
separa-tion of isoflavones [264]. Zhang et al. [265] found a 150 mm 1.6 m
core–shell column to provide better peak capacity and symmetry than two 100
mm sub-2 m phases for the separation of rhubarb constituents. An example of
the performance obtained on this col-umn in gradient mode is presented in
Fig. 6. Note that this column should, based on a minimum plate height of
1.8dp, provide an iso-cratic efficiency of 52,000 plates – the increased
resolving power is clearly evident from Fig. 6. This performance is only
attainable on UHPLC instrumentation though. It should be noted that
compari-son between column performance in gradient mode for complex
samples is far from straightforward due to the effect of differ-ent particle
sizes, column dimensions and selectivity differences between phases. For
example, Serrano et al. found a polar embed-ded 1.7 m fully porous phase to
be better suited than superficially porous phases for the separation of natural
flavonoid dyes [266]. In line with the characteristics of superficially porous
phases, they have also found growing application in LC × LC separations (see
Section 3.2).
It is worth pointing out that although the reduced pressure-drop for
superficially porous columns allows their utilisation on con-ventional HPLC
instruments – a benefit which is widely heralded by manufacturers and in
literature – special care should be taken regarding extra-column band
broadening on such instrumentation. Most conventional HPLC instruments
are characterised by large delay- and extra-column volumes, and even
allowing for careful
34 A. de Villiers et al. / J. Chromatogr. A 1430 (2016) 16–78
Fig. 6. Representative RP-LC–TOF-MS chromatogram illustrating 104 common peaks identified in rhubarb using a 150 mm × 2.1 mm 1.6
◦
Analysis conditions: 0.1% formic acid/ACN gradient; 0.3 mL/min; 40 C; detection: ±ESI-Q-TOF-MS.
Source: Reproduced with permission from [265].
Fig. 7. Overview of the sub-2 m porous, superficially porous and monolithic phases used in the
advanced LC–MS analysis of flavonoids (data based on references listed in Tables 1, 2, 4–7),
and grouped according to particle size (SP indicates superficially porous phases). Less than 2%
of the total number are HILIC phases, the rest are RP-LC phases.
modification to reduce these volumes does not allow attainment of the
potential performance of short, narrow-bore high-efficiency superficially
porous columns [215,253,257].
A graphical representation of the stationary phases used in the reports
listed in Tables 1, 2 and 4–7 is presented in Fig. 7. It is clear that fully porous
sub-2 m phases are still used in the bulk of advanced LC separations of
flavonoids (75% of all analyses cited here), although superficially porous
phases have found growing application especially in the last few years.
Considering the perfor-mance of these phases, this trend is likely to continue.
Furthermore, the fact that superficially porous phases with pressure
capabilities of 1000 bar or above and particle sizes of 1.3–1.6 m have recently
been introduced will translate into increased use of these phases on UHPLC
instrumentation to provide further speed and efficiency gains [267].
3.1.4. High temperature liquid chromatography (HTLC)
Somewhat analogously to superficially porous phases, the first use of high
temperature in HPLC was demonstrated many years ago [268], although the
technique has been largely neglected until the last decade. In contrast to core–
shell particles, however, HTLC currently still finds relatively limited
application in mainstream literature (there are some exceptions, such as for
example HTLC analysis of polymers). The term HTLC is somewhat vaguely
defined
m superficially porous phase.
as any LC separation performed at temperatures above room tem-perature,
◦
typically spanning the range of 40–200 C [269]; in this work we will define
◦
high temperature applications as those per-formed above 50 C.
The primary effect of an increase in temperature in HPLC is to reduce the
viscosity of the mobile phase, with an important con-comitant increase in
analyte diffusion in the mobile phase. The former effect of elevated
temperature results in lower pressures when the same column lengths and
flow rates are used (Eq. (2), reduction in ); this effect can be exploited to
increase efficiency by using longer columns, provided that separation is not
performed under pressure-constrained conditions (i.e. when operating at the
maximum pressure of the column/instrument) [270].
At elevated temperature, an increase in the diffusion coefficient of the
analyte in the mobile phase, Dm, leads to higher B-term and lower C-term
contributions to the plate height, and therefore a shift in u opt to higher values
(illustrated in Fig. 5c and d for the RP-LC separation of flavonols). There is
also an important reduction in the slope of the plate height curve at elevated
temperature (due to a lower C-term), which is clearly beneficial from the
perspective of fast analyses. Under pressure-constrained conditions the
increase in optimal mobile phase flow rate roughly cancels the reduction in
pressure due to lower mobile phase viscosity, and the primary benefit of
elevated temperature operation is therefore an increase in analysis speed (in
fact, the maximum efficiency obtainable on a given stationary
phase/instrument combination decreases slightly with an increase in
temperature [269]). An added benefit is that the use of high flow rates allowed
by elevated temperature operation significantly reduces the time required to
re-equilibrate the column in gradient analysis [271–275], thereby further
increasing through-put. This is especially beneficial in LC × LC
[271,273,275,276]. Further benefits of HTLC include a reduction in solvent
consump-tion due to the fact that less organic modifier or even pure water can
be used at elevated temperatures [277], and potential increases in selectivity
[278].
Despite the theoretical benefits of HTLC, the technique has found limited
application in general [43], and in the analysis of flavonoids in particular. One
reason for this is the lack of commercial instru-ments and columns suitable for
high temperature operation. Most silica-based columns are susceptible to
thermal degradation above 60 ◦ C (although there are some notable
exceptions), and many dedicated HTLC columns show relatively poor
chromatographic performance. Furthermore, effective HTLC operation
requires ded-icated ovens, efficient mobile phase pre-heating and for most
detectors post-column cooling devices [279]; these require-ments are not met
by most commercial instruments – dedicated
 A. de Villiers et al. / J. Chromatogr. A 1430 (2016) 16–78
HTLC ovens have only relatively recently become commercially available.
In the case of flavonoid analysis, the well-known thermal instability of
these molecules raises concern about the risk of on-column degradation
occurring, and is likely the main reason for the restricted application of HTLC
in the field. However, to assess the risk of on-column degradation it is
important to consider the resi-dence time of the analyte in the heated column
relative to the rate of its thermal degradation at this temperature [268,272].
Simply put, if the analyte spends less time in the heated column than is
required for its thermal degradation, HTLC at that temperature is a viable
option. For example, anthocyanins are notoriously thermally labile [280,281].
◦
Yet, it has been demonstrated that operation at tempera-tures up to 80 C are
feasible for anthocyanin analysis provided that analysis times are kept to less
than an hour [105,282]. Grata et al.
[283] similarly reported that no thermal degradation was observed for
metabolites, including flavonoids, in Aridopsis thaliana based on a detailed
◦
analysis of TOF-MS data for separations performed at 30 and 90 C.
From Tables 1, 2 and 4–7, it is clear that the use of temperatures above 40
◦ be discussed below).
C in flavonoid analysis is relatively rare. Nevertheless, a number of studies
◦ ◦
employed column temperatures of 50 C [98,143,180,199,218,284–293], 60
◦ ◦ ◦
C [80,217,294], 70 C [282,295], 80 C [81,296] or 90 C [283] in one- and
two-dimensional analy-ses. Qi et al. [297] implemented a temperature
◦
gradient between 22 and 35 C for the analysis of flavones and flavonols in
traditional Chinese medicine (TCM) in order to limit pressure restraints on a
1.8 m RP column operated on conventional instrumentation.
Two particular applications of HTLC are worth mentioning. Reichelt et al.
◦ cutting methods, where only selected part(s) of a sample are subjected to
[99] used a polymeric PRP-1 column operated at 120 C for the fractionation
of natural products prior to the sen-sory evaluation of the fractions (an
approach termed LC Taste). The mobile phase contained only water and
ethanol, and fractions could be directly tasted after dilution with a basic
tastant solution. High temperature operation is essential to accommodate the
high vis-cosity of the ethanol-based mobile phase. The authors confirmed the
flavour modulating activities of several flavonoids using this approach [99].
High temperature operation also provides a particular benefit in instances
where on-column conversion between multiple species of an analyte occurs
[298,299]. A pertinent example is the case of anthocyanins, where several
different chemical forms may occur in the mobile phase. It has been shown
[105] that the inter-conversion reaction between the flavylium cation and
carbinol pseudoba-sic species is directly responsible for additional band
broadening in the separation of these compounds under conventional con-
ditions (flow rate and temperature). An increase in the analysis temperature
results in faster kinetics of the relevant equilibria, and therefore a reduction in
band broadening [105]. Operation at elevated temperature and optimal flow
rates result in significant increases in chromatographic performance for
anthocyanin analy-sis, without the risk of analyte degradation [105,218].
◦ is however more chal-lenging in practice. Two particular aspects should be
Even a modest increase in temperature to 80 C can have a large impact
on mobile phase viscosity and therefore chromatographic behaviour. For pure
water for example, the viscosity will decrease by about 60% compared to
room temperature (the correspond-ing values are about 40% and 35% for
methanol and acetonitrile, respectively). This implies that the benefits of
elevated temperature operation can be exploited with even a modest increase
in tem-perature in the range where commercial column ovens operate. It has
to be stressed though that for high-flow operation (for example 4.6 mm i.d.
columns and above), efficient mobile phase pre-heating is critical even if the
column can effectively be thermostatted at these temperatures.
While the use of reasonably high temperatures in flavonoid analysis will
likely remain limited to certain niche fields such as
35
anthocyanin analysis and LC × LC separations, increasing aware-ness of the
benefits of elevated temperature and the means to assess the risk of analyte
degradation, coupled to the commercial avail-ability of growing numbers of
columns and instruments suitable for HTLC operation may see a concomitant
increase in the number of reports utilising modest increases in temperature (in
◦
the range 30–80 C) for improved flavonoid analysis in the future.
Guillarme et al. [43] provided an informative comparison of the kinetic
benefits of UHPLC, monolithic columns, superficially porous phases, HTLC,
and combinations of these, compared to conventional HPLC for the flavonol
rutin (MW 610). Fig. 8 presents a graphical overview of their findings, where
the x-axis shows the highest pos-sible peak capacity (P in this figure) for a
gradient time of 3 h, and the y-axis the time required for a peak capacity of
100. The benefits of these technologies for both high throughput and high
resolution separations are evident, as is the complementary nature of UHPLC
and HTLC approaches.
Table 1 lists the applications of UHPLC, monolithic and superfi-cially
porous columns without MS detection to flavonoid analysis during the last 7
years (applications with MS detection are summ-arised in Tables 4–7 and will
3.2. Comprehensive multi-dimensional LC
Fundamental aspects
Multidimensional liquid chromatography (MD-LC) provides a powerful
means to significantly improve the performance of LC separations. This is
achieved by combining the separation power and selectivity of multiple 1-
dimensional HPLC methods. MD-LC approaches can be divided in heart-
multiple separations, and comprehensive separations, where all constituents of
the sam-ple are analysed in several dimensions. In this report, we will limit
our attention to comprehensive MD-LC separations, as these are more generic
in nature and provide much higher resolving power. (As an aside, although
comprehensive systems where the number of separation dimensions exceeds
two have been demonstrated [316], this has not yet been achieved for
flavonoid analysis). The subsequent discussion will therefore focus on
comprehensive 2-dimensional liquid chromatography (LC × LC) only. For
detailed overviews of the field, the reader is referred to several dedicated LC
× LC reviews [45–49,317–320], as well as more specific reviews covering
hyphenation of LC × LC with MS [321], column selectiv-ity in LC × LC
[322] and multidimensional chromatography in food analysis [323].
The incentive behind the growing interest in LC × LC lies in the
exceptional performance gains that may be realised when combin-ing two
HPLC separations under ideal conditions: the peak capacity of such a
separation is theoretically the product of peak capacities of both 1-
dimensional separations [324–328]. LC × LC is therefore capable of providing
separation performance an order of magnitude higher than obtainable by even
highly optimised 1-dimensional HPLC methods. Attaining such performance
addressed to meet the criteria for methods to be considered comprehensive
[329] and exploit the potential of these separations.
The first pertains to the requirement that different separa-tion mechanisms
be used in both dimensions. This is essential to effectively utilise the two-
dimensional separation space pro-vided by LC × LC. The degree to which two
separations provide complementary information is referred to as their
‘orthogonality’. A combination of highly orthogonal separations will result in
the spread of analytes across the two-dimensional space, and therefore
maximise resolution, whereas weakly orthogonal separations (i.e. where
retention of sample constituents between the two separa-tions are correlated to
a significant degree) provide limited benefit.
 A. de Villiers et al. / J. Chromatogr. A 1430 (2016) 16–78

Fig. 8. Comparison of the performance achievable (x-axis: peak capacity, P, for a gradient time of 3 h, y-axis: gradient time required for a peak capacity of 100) by HPLC, UHPLC, monolithic
phases, HTLC and combinations of these. Data obtained for the flavonol rutin.
Source: Reproduced with permission from [43].

Deriving a commonly accepted means of quantifying orthogonality, as is

required to determine the practical peak capacity of an LC × LC separation, has
however proven difficult. Nevertheless, in recent years the so-called surface
coverage metrics, which are based on determining the fraction of the two-
dimensional space accessi-ble to sample components, has gained popularity. A
variety of approaches can be used to determine the fractional surface cov-erage
of the two-dimensional space, fc [330,331]. This value can then be used to
quantitatively account for lack of orthogonality in determining the practical
peak capacity (Eq. (4)).

The second important criterion addresses the requirement that the

1
separation attained in the first dimension ( D) should be essen-tially
2
maintained during the modulation or transfer of fractions to the D column.
This implies that fraction ‘collection’ times, called the first dimension sampling
1
time ( ts), should be less than the first dimension peak width (typically 1/3rd
[332]) – failure to do so would result in the recombination of peaks (partially)
separated in the first dimension, and therefore sacrifice performance. The effect
of this so-called under-sampling can (and should) also be taken into account
when the practical 2-dimensional peak capac-ity is determined by using the
under-sampling correction factor, ˇ
[276,333,334]:
2
· nc · fc
nc,2D ˇ

1 2
where nc and nc are the peak capacities of the first- and second-dimension
separations, respectively, and nc,2D the practical peak capacity of the LC × LC
separation. From this equation it is clear that the potential performance gains of
LC × LC are heavily reliant on both the degree of orthogonality and under-
sampling.
In practice, LC × LC can be performed in one of three ways. The first, and
1
most simple, is the off-line approach, where fractions elu-ting from the D
column are collected (manually or automatically). These can then be
concentrated, dissolved in a suitable solvent or directly injected onto the
second dimension column. Off-line LC × LC can therefore be performed on a
single HPLC instrument, since fractions can be injected after their collection.

On-line LC × LC involves the direct transfer of fractions to the second

2
dimension ( D) column, typically using a switching valve equipped with loops.
2
Analysis in D is then performed while the next fraction is collected – an
important consequence is therefore
1

n
c
 them next to each other in a matrix format which can be viewed as a surface
plot or more commonly as a contour plot (see for example Figs. 11–13).
Quantification can also be performed in LC × LC by combining the
2
that the D analyses must be very fast to meet first-
dimension sampling criteria. Alternative
configurations are also possible, such as for example
using trapping columns and an added make-up flow
to trap analytes eluting from the first dimension. This
approach has the benefit of essentially removing all
or the majority of the first-dimension mobile phase
prior to injection on the second dimension, and can
be used to minimise dilution (see below) and remove
unwanted mobile phase components such as salts (the
approach is often used in ion exchange (IEC) × RP-
2
LC-MS for this reason). Furthermore, multiple D
columns can be used [335,336] to effectively provide
longer second dimension analysis times and therefore
increase the overall resolution, at the cost of
increased instrumental and method complexity
[337,338].

The third mode, stop-flow LC × LC, entails direct

2
transfer of frac-tions to the D column, followed by
1
stopping of the D flow while separation of the
transferred fraction is concluded; this process is
1
continued until the D separation is completed. Both
on-line and stop-flow LC × LC separations have as
minimum instrumen-tal requirements one complete
HPLC instrument, a transfer device such as a valve
2
and a second pump to provide the D flow. Examples
of the different experimental configurations used are
presented in Fig. 9 [48].

Off-line operation is the simplest form of LC × LC

and provides

(4) the best performance, but requires very long

analysis times (in the order of a few hours up
to a few days). In contrast, analysis times in
on-line LC × LC are typically commensurate
with 1-dimensional HPLC, and the
performance is lower than off-line operation
due to very fast second dimension analyses.
Stop-flow provides similar performance
(provided that first-dimension band
broadening dur-ing stop-flow periods is not
excessive [339,340]) and analysis times to
off-line LC × LC [340–342].
LC × LC raw data is typically acquired
either as a series of sepa-rate 2D
chromatograms (in the case of off-line or
stop-flow LC × LC), or in the form of a 1-
dimensional chromatogram (i.e. detector
signal versus time) comprising a string of
sequential 2D chromatograms in which the
1
D separation information is implicitly
contained (on-line LC × LC). Visualisation is
then performed by slicing the raw data into
consecutive 2D chromatograms and placing
 A. de Villiers et al. / J. Chromatogr. A 1430 (2016) 16–78 37

Fig. 9. Schematic representations of instrumental configurations used for on-line LC × LC using (a) empty storage loops, (b) trapping columns and (c) multiple 2D columns; and (d) for stop-flow LC
× LC.
Source: Reproduced with permission from [48].
38 A. de Villiers et al. / J. Chromatogr. A 1430 (2016) 16–78
2 1
contribution of each individual D ‘slice’ of a D chromatographic
peak, or by using more advanced algorithms [343,344]. Advanced
data analysis techniques can also be applied to LC × LC data [345],
although multivariate data analysis is necessarily more compli-
cated than in 1-dimensional separations since alignment of data
in two dimensions present several challenges [346].
Compared to one-dimensional HPLC, method development and
optimisation is much more complex in LC × LC [347–349]. This topic
is much too complex to be addressed fully in the context of this con-
tribution, and we will therefore focus on providing some general
guidelines for parameter selection in LC and the implications for
performance and hyphenation to MS. The first choice involved in
LC × LC method development is the selection of separation modes,
where orthogonality, performance (speed and efficiency) and com-
patibility with the detection strategy to be used are important
criteria. Considering the chemical properties of flavonoids, LC × LC
methods for their analysis have almost exclusively involved RP-LC
and/or HILIC (with a few notable exceptions [81,296,350]). RP-LC
is most often used in the second dimension because of compatibil-
ity with MS and the fact that good performance is obtained in this
mode for fast separations [351], although HILIC is in principle also
2 1
suited to fast D analysis [352,353] prior to MS detection and has
been used for this purpose in proteomics research. The combination
of HILIC and RP-LC promises higher orthogonality than RP-LC × RP-
LC, although hyphenation of the former modes is more complicated
due to the relative elution strengths of the mobile phase used in
each dimension (see below). The orthogonality of RP-LC × RP-LC
systems can be improved through judicious selection of the mobile
phase composition (typically the organic modifier used, since pH in
the range used for flavonoid analysis has little effect on the selec-
tivity) and especially the stationary phase chemistries used in both
dimensions. Furthermore, in the case where gradient separation is
performed in both dimensions – the most common approach which
2
provides this highest overall performance [347] – the D gradi-
1 to a far greater extent following passage through two columns than
ent can be varied throughout the D separation to maximise the
utilisation of the 2-dimensional space [354–356]. This may require
2 the detector requirements in LC × LC.
correction of D retention times when multiple slices of the same
compound elute in consecutive gradients [357].
The choice of hyphenation mode, assuming availability of suf-
ficient instrumentation, depends primarily on the performance
requirements and time considerations: for maximum resolution
off-line or stop-flow approaches should be used, whereas on-line
is preferable for faster separations. Regarding column dimensions,
the selection is governed largely by mobile phase considerations.
RP-LC and HILIC mobile phases are perfectly miscible, but the
solvent composition of most fractions generally implies high
elution strength in the second dimension. In HILIC × RP-LC (or RP-
LC × HILIC) this is because of the disparate percentages of organic
modifier used in each of these modes, whereas in RP-LC × RP-LC
1 unique and complementary retention order obtained in this mode
this follows from the fact that compounds are eluted from the D
column in a solvent composition where their retentivity is low. (It is
for this reason that RP-LC columns with lower retention are mostly
1
used in D for RP-LC × RP-LC). The practical implication of this is
2 cyanidins, where HILIC separation according to size is extensively
that the injection volume on the D column should be minimised
to avoid injection band broadening. The volume of each 1st dimen-
1 1
sion fraction (Vfrac) is related to the D flow rate, F, and sampling
1 LC × LC (31%); stop-flow HILIC × RP-LC has also only been used in
time, ts, according to:
1 1
Vfrac = ts × F
(5)
The most common approach to minimise injection effects in on-line LC ×
1
LC is therefore to reduce the i.d. of the D column, leading to a concomitant
1 temperature has found extensive use to facilitate fast separations in the second
reduction in F. Other options to address this lim-itation include the use of
trapping columns [358] or incorporating a split between the two dimensions
[342,359]. The latter approach is simpler, but reduces overall sensitivity,
whereas the former has
1
the effect of effectively de-coupling fraction volumes from the D
flow rate. In off-line LC × LC, this limitation can be circumvented
by injection of a portion of each fraction, diluting fractions (both
resulting in lower sensitivity) or by evaporating the solvent fol-
lowed by dissolution in a weaker injection solvent.
Second dimension column stationary phases are selected based
on performance for fast separations – technologies such as UHPLC
[360], superficially porous phases [357] and HTLC [147] have there-
fore all found application in the second dimension of LC × LC
systems. For the same reason, relatively short columns are gen-
erally used, typically of i.d. 2.1–4.6 mm to increase capacity and
minimise injection problems. While monolithic columns are ide-
ally suited for fast analyses at high flow rates, the fact that splitting
is required prior to MS detection represents a significant drawback.
Finally, the dilution of analytes occurring in both dimensions
should be considered. For passive modulation (i.e. where analytes
1
are not trapped and focussed after the D separation), such as
mostly used in LC × LC, total dilution is generally much higher than
is the case for 1-D HPLC. Dilution in each dimension, and the total
dilution in LC × LC, can be determined according to [347,361]:
√ 1
× F
1
(6)
DF 2 1
= Vinj
√ 2
F×
2
× SR
2
DF 2
(7)
= 1 1
F × ts
2D 1 2
DF = DF × DF (8)
where DF is the dilution factor, the standard deviation in time
units and F the flow rate for each dimension, as specified by the
1 1
superscript, Vinj is the D injection volume and SR is the ‘split ratio’
to accommodate cases where only a portion of each fraction may
be injected. Eqs. (6)–(8) clearly illustrate that analytes are diluted
is the case for 1-dimensional HPLC; this has clear implications for
LC × LC has found considerable and growing application in
flavonoid analysis, especially during the last five years (Table 2).
An overview of the applications listed in this table is pre-
sented in Fig. 10. Several different combinations of separation
modes have been reported, including RP-LC × RP-LC, HILIC × RP-LC,
HILIC × HILIC and SEC × RP-LC. HILIC × RP-LC has found the most
widespread application in the LC × LC analysis of flavonoids (47%
of applications listed in Table 2), followed by RP-LC × RP-LC (38%)
(Fig. 10a). Compared to LC × LC in general, where according to Li
et al. [351] RP-LC × RP-LC is the most common combination of
modes (32% vs. 22% for NP-LC/HILIC × RP-LC), this relative prefer-
ence for HILIC in flavonoid analysis is likely associated with the
compared to RP-LC. Noteworthy examples include glycosylated
flavonoids, where compounds are separated according to their
degree of glycosylation and/or acylation [126,248], and proantho-
used to study this family [147]. Most reports (two thirds during
the last 7 years, Fig. 10b) use on-line LC × LC, followed by off-line
two reports for the separation of procyanidins [340,342]. For high
speed separations in the second dimension, it is noteworthy that
the use of superficially porous phases exceeds that of fully porous UHPLC
phases; monoliths have found limited application in LC × LC separation of
flavonoids (with some exceptions [230,231,362]). Furthermore, the elevated
dimension, with temper-atures of 50 ◦ C [147,231,248,340,342,362–365], 60 ◦
C [357], 70 ◦ C [366] and 80 ◦ C [81,350] being used.
 A. de Villiers et al. / J. Chromatogr. A 1430 (2016) 16–78
Fig. 10. Overview of the LC × LC analysis of flavonoids (2009–2015) summarising the different separation modes (a), hyphenation modes (b), column formats (c) and mass spectrometers (d) used in
the reports listed in Table 2.
Virtually all classes of flavonoids, including flavonols [344,367],
flavanols and procyanidins [81,147,344], chalcones [127], (iso)flavones
[357,368], flavanones [357,368] and anthocyanins
[248] have been separated successfully in LC × LC. Furthermore, the
suitability of LC × LC-UV for quantitative analysis of flavonoids has been
demonstrated [343,344]. The authors noted that LC × LC provided higher
limits of detection than 1-dimensional HPLC, a consequence of greater
dilution occurring in two dimensions (cf. Eqs. (6)–(8)), but that in some cases
improved chromatographic resolution provided more accurate results.
Hyphenation of LC × LC separation with MS is clearly beneficial from
the perspective of compound identification and selectivity. The improved
separation offered by LC × LC separation is also ben-eficial from the point of
view of MS, since reduced co-elution minimises the number of potential
interfering ions and thereby matrix effects [321]. However, especially on-line
LC × LC also places strict demands on the speed of MS detection due to the
2 Alternative separation modes have also found application in the 2-
very nar-row peak widths commonly obtained for fast D separations. This is
reflected in the fact that most (48%) of LC × LC–MS methods for flavonoid
analysis during the review period were performed using TOF (or Q-TOF)
instruments (Table 2). IT detectors have also been used [230,369], while
Zhang et al. [370] recently used an Orbitrap instrument operated at a
resolving power of 50,000 and scan speed of 2 Hz in combination with off-
line SEC × RP-LC analysis.
Qiao et al. [364] reported an interesting heart-cutting/RP-LC × RP-LC
approach for the analysis minor compounds in herbal medicines. Major
1 greatly improved as a consequence of less co-elution provided by the
compounds were removed following D separa-tion using a time-activated
heart-cutting method, whereas minor compounds were separated in
comprehensive mode using shifted gradients in the second dimension to
improve the orthogonality of the RP-LC × RP-LC system. The instrumental
configuration and an example contour plot obtained in this work are presented
in Fig. 11. Separated compounds were identified based on MS/MS spectra
obtained using IT-MS.
SEC and RP-LC have also been combined for flavonoid analysis, first in
heart-cutting mode by de Souza et al. [80] for the separation of flavonol
glycosides. In a follow-up report, the same group used an off-line SEC × RP-
LC method for the analysis of green and black tea flavonoids [81]. The SEC
mobile phase was collected automatically and evaporated between the two
dimensions. The authors reported
39
surprisingly good size-based separations in the first dimension, with tetra-,
tri-, di- and monoglycosylated flavonoids eluting in the order of decreasing
size. The separation mechanism is not clearly defined, however, with isomeric
flavonol glycosides also being separated to some extent in this dimension. In
◦
the sec-ond dimension, a conventional RP-LC column operated at 60 C
◦
[80] or a UHPLC column at 80 C [81] was used. The SEC × RP-LC
combination showed good orthogonality, and by using an off-line hyphenation
strategy involving fraction concentration, good sensi-tivity was obtained.
Thirty six flavonoid-glycosides were identified in the heart-cutting 2D LC
analysis of Maytenus ilicifolia, whereas 54 flavonoids were identified in green
and black teas using the off-line comprehensive approach with detection
performed by means of a triple quadrupole MS. An example of the contour
plot obtained for the off-line SEC × RP-LC analysis of a black tea is presented
in Fig. 12.
dimensional analysis of flavonoids, for example the combination of CPC
[371] or CCC [372] with RP-LC and RP-LC with micellar elec-trokinetic
chromatography (MEKC) [87], although these fall outside the scope of the
current report.
While many of the applications of LC × LC report similar num-bers of
compounds than are nowadays also identified using advanced LC–MS
methods (Section 4), the benefits of improved chromatographic resolution
cannot be overstated. Especially in qualitative analyses of flavonoids, the
main application area of LC × LC to date, the quality of mass spectral data is
combination of complementary separation modes [321].
A field where LC × LC demonstrates clear benefits is in the analysis of
proanthocyanidins. The complexity of these com-pounds, with the number of
isomeric structures increasing exponentially with DP, means that they cannot
be resolved using any available HPLC method [373]. Furthermore, because of
the large number of isomeric structures, high resolution MS and MS/MS data
are also of limited utility. LC × LC operation com-bining HILIC separation
according to MW and RP-LC separation according to isomeric composition
has been shown to offer a powerful approach for the elucidation of
procyanidin content in a variety of natural products, especially when
hyphenated to
40 A. de Villiers et al. / J. Chromatogr. A 1430 (2016) 16–78

Fig. 11. (A) Instrumental configuration used for the on-line heart-cutting/RP-LC × RP-LC analysis of Puerarialobata herbal medicine. Valve 1 was used to selectively cut the major constituents
(underlined peak numbers in (B)), and valve 2 for the comprehensive LC × LC analysis of the remainder of the constituents. (B) shows the UV contour plot obtained for this analysis, with a zoom of
the separation area in the red block on the right. Numbered compounds except for 12, 13 and 19 are isoflavones. Analysis conditions:
◦ 2 ◦
1D: 100 mm × 2.1 mm CSH C18 column, 50 C, 0.1 mL/min 0.1% formic acid/methanol gradient; D: 50 mm × 3.0 mm 2.7 m superficially porous C18 phase, shifted gradients, 50 C, 2.5 mL/min
(split 1:9 before ESI-IT-MS detection).
Source: Reproduced with permission from [364].

MS [126,147,148,230,340,342,363,369]. As an example, Fig. 13
demonstrates the information obtained for the on-line HILIC × RP-LC-FL-
ESI-TOFMS analysis of grape seed procyanidins [148]. Mass spectral data
allowed extraction of information for particular classes of compounds, as
illustrated in Fig. 13D for the mono-galloylated procyanidins. While this
methodology provides much more detailed molecular information than could
be obtained using 1-dimensional LC–MS, the complexity of the high MW
fraction (Fig. 13C) also serves to emphasise the challenges associated with
proanthocyanidin analysis. For such applications, LC × LC is expected to
play an increasingly important role in the future.
4. Advances in LC–MS analysis of flavonoids
instruments [383]. By the middle of the next decade [59,62] ESI and APCI
had become the norm in flavonoid analysis, while time-of-flight (TOF) and
quadrupole-time-of-flight (Q-TOF) instruments were only just finding
application in flavonoid analysis [384].
As will be shown in the following sections, the contemporary sit-uation is
significantly different. In addition to very significant gains in the sensitivity
and speed of MS systems which have been pro-gressively attained with newer
generation instruments, a range of new high resolution analysers and tandem
MS configurations have also found application in flavonoid analysis. We will
not attempt to address in any detail instrumental developments or the
operation of all instruments, but rather to highlight the potential benefits of the
different types of mass analysers and the types of analyses for which they are
suited on the hand of recent applications.

From the perspective of LC–MS, technological advances in MS
instrumentation over the last fifteen years have largely over-shadowed those
in HPLC. Since the beginning of the century, the commercial availability of
more robust and more advanced LC–MS instruments, coupled to the
reduction in the price of these instru-ments, has certainly contributed to the
much more widespread use and utility of the technique in flavonoid analysis.
The extent of these improvements, as well as the most important
developments, are clearly highlighted when comparing current practice with
the state of affairs at the time of several important reviews. By the end of the
previous century, the majority of LC–MS applications utilised fast atom
bombardment (FAB) and thermospray (TSP); ESI and APCI were recent
additions to the ion sources used for flavonoid analysis [55,380–382].
Furthermore, most analyses were performed using quadrupole (single stage or
triple quadrupole, QqQ) or ion trap
From the perspective of hyphenation of MS to high-performance
separations, several pertinent aspects should be considered. First, the fact that
optimal ESI performance is attained at flow rates of 0.2–0.4 mL/min is
entirely compatible with the trend in UHPLC of a reduction in column
diameter from conventional 4.6–2.1 mm i.d. columns dictated by frictional
heating considerations [199,213]. On the other hand, commercial monolithic
columns are mostly used in 4.6 mm i.d. formats, which, coupled to the higher
flow rates used for fast analysis on these columns, implies flow splitting prior
to MS detection. Superficially porous phases, due to their relatively high
permeability and favourable thermal conductivity properties
[249] are viable to use in both 4.6 and 2.1 mm formats, although clearly the
latter is preferred from the perspective of hyphenation to MS. Indeed, of the
applications listed in Tables 4–7, the majority employ columns of internal
diameter 2.1 mm.
 A. de Villiers et al. / J. Chromatogr. A 1430 (2016) 16–78
Fig. 12. Contour plot (325 nm UV detection) obtained for the off-line SEC × RP-LC separation of a black tea hydro-alcoholic extract (note that the first dimension SEC separation is on the y-axis).
Compounds 24–63 are flavonoids, as well as 5, 9–10, 12, 15–18 and 21 and 22. Analysis conditions:
◦
mm 1.7 m porous C18 phase, 80 C, 1 mL/min.
Source: Reproduced with permission from [81].
Lower column volumes however also place stricter demands on
instrumentation in terms of extra-column volume. For example, Wolfender
and co-workers [283] showed that peak capacities of UHPLC separations are
typically reduced by between 15% and 30% upon connection to MS
detection, even if care is taken to reduce the volume of the connection tubing.
Indeed, comparison of UV and MS chromatograms in many literature reports
visually reveals the effect of this phenomenon. Finally, the scan speed is of
obvious importance for hyphenation to very fast separations – this aspect will
be addressed in more detail for the different mass spectrome-ters below.
4.1. Ionisation modes
Early ionisation techniques applied for the direct analysis of flavonoid
analysis include chemical ionisation (CI) [385] and electron impact (EI) [386]
(these also in combination with GC sep-aration), field desorption [387],
plasma-desorption-MS (PD-MS) [388,389], FAB [169,390–392] and matrix-
assisted laser desorp-tion/ionisation (MALDI) [393,394].
However, it is only with the development of ionisation sources that
allowed coupling of HPLC with MS that the technique found the pre-
eminence it enjoys today in flavonoid analysis. Initial ionisation techniques
used for this purpose such as TSP [395,396], moving belt (MB) [397,398],
continuous-flow FAB [399] have now largely been replaced by API
techniques such as ESI [400–403], APCI [404–406] and to a lesser extent
APPI.
Of these, ESI is undoubtedly the most important. Indeed, in the advanced
LC–MS analysis of flavonoids during the period cov-ered by this review, only
three reports utilised APCI [163,368,407], mostly involving analysis of
isoflavones, and only one used APPI
41
1 ◦ 2
D: 300 mm × 7.8 mm SEC column, 60 C, 1 mL/min water/ACN gradient; D: 50 mm × 2.1
[293]; the rest without exception used ESI. Compared to the global picture for
LC–MS reported in 2012 [41] (82% ESI, 16% APCI, 2% APPI), the higher
prevalence of ESI is a consequence of the fact that most flavonoid classes are
more efficiently ionised using this mode. ESI was used in negative ionisation
mode in 48% of all applications, and both positive and negative ionisation
modes in 33% of applica-tion during this time (refer to Section 2.3 for a
general discussion of the MS behaviour of flavonoids).
APCI is generally considered an alternative to ESI for relatively non-polar
compounds which are less efficiently ionised in ESI. While APCI shows
comparable to better sensitivity compared to ESI for flavonoid aglycones
[161], and therefore is well suited to the analysis of hydrolysed samples, the
latter typically outperforms the former for natural glycosylated species
[165,407,408]. Two poten-tial advantages of APCI over ESI are better
compatibility with high flow rates, and lower susceptibility to matrix effects
during the ioni-sation process [383]. APCI continues to find application in
flavonoid analysis, and indeed has been used in recent years in combination
with UHPLC-(HR)–MS [163,407] and LC × LC [368], especially in the
analysis of more apolar isoflavonoids and isoflavones. Nevertheless, the better
performance of ESI for a wide range of flavonoid species is likely an
important contributing factor to the dominance of this form of ionisation in
contemporary flavonoid analysis.
APPI presents an alternative API source which is complementary to ESI or
APCI, especially for the analysis of non-polar compounds. Similar to APCI,
the liquid sample is vaporised by a modified heated nebuliser, but instead of
using a corona discharge to establish ioni-sation, the process relies on charge
transfer from dopant or solvent molecules ionised by 10 eV photons produced
by a discharge lamp [409]. Rauha et al. [165] compared ESI, APCI and APPI
with a vari-ety of mobile phases in positive and negative ionisation modes for
42 A. de Villiers et al. / J. Chromatogr. A 1430 (2016) 16–78

Fig. 13. Total ion chromatogram (TIC) (A) and fluorescence (B) contour plots for the HILIC × RP-LC–FL–ESI-MS separation of a grape seed extract. (C) A detailed section of the TIC and (D) the
1
extracted ion contour plot for monogalloylated procyanidins. Analysis conditions: D: 250 mm × 1 mm Diol column, 100 min gradient at 25 L/min; sampling time 2 min (split 1:24 between two
2 ◦
dimensions); D: 50 mm × 4.6 mm 1.8 m porous C18 phase, 50 C, 1.5 mL/min (split 1:1.2 before ESI-QTOF-MS detection). Peak labels: PC X GY refers to a procyanidin of degree of polymerisation
X and galloylation Y. F denotes the z-axis scale.
Source: Adapted from [148].

the analysis of five selected flavonoids, and concluded that nega-tive mode
ESI with ammonium acetate containing mobile phases provided the lowest
limits of detection (LODs). While this was espe-cially true for flavonoid-
glycosides, similar LODs were obtained between all three modes for
flavonoid aglycones in both positive and negative ionisation, although
different optimal mobile phase compositions were used for each ionisation
mode [165]. APPI has also more recently been used in flavonoid analysis.
Riffault et al. chose this ionisation method for the phytochemical characterisa-
tion of rose flowers, although the choice of ionisation mode was primarily
based on its performance for more apolar compounds such as fatty acids and
sesquiterpenoids [293].
The ability to perform swift polarity switching between posi-tive and
negative ionisation modes has proven beneficial in several research fields due
to the complementary information that may be gained for compound classes
preferentially ionised in either mode. In the case of flavonoid analysis, this is
however less important since most flavonoids are sufficiently ionised in either
mode (the exception being anthocyanins, where mobile phase considerations
for efficient separation in any case preclude the use of nega-tive ionisation).
Nevertheless, when flavonoids are determined together with other classes of
compounds, such as for example in metabolomic research or studies aimed at
comprehensive char-acterisation of natural product constituents, polarity
switching becomes a valuable tool. A common example is where complete
phenolic analysis is the goal and flavonoids are analysed together with
phenolic acids. However, Wolfender and co-workers showed that polarity-
switching, using a switch time of 0.3 s on a TOF
instrument, leads to an additional 30% loss in peak capacity for highly
efficient separations [283]. Therefore this option should be employed with
care, especially for ultra-fast separations.
It is relevant to note that in addition to the extensive use of MALDI, a
range of ionisation sources suitable for the direct analysis of flavonoids from
liquid or solid samples have been developed and found application in
flavonoid analysis. The most important of these are desorption electrospray
ionisation (DESI) [40] and direct anal-ysis in real time (DART) [410–412].
Since these ionisation sources are not applicable in LC–MS, they will not be
discussed here.
4.2. Mass analysers
Mass analysers can broadly be divided into two main classes, namely high
and low resolution (or nominal mass) instruments, based on their ability to
distinguish ions with small mass-to-charge (m/z) differences. The critical
parameter here is the resolving power, defined as the ratio of an ions’ mass to
the width of its peak at half height (full width at half maximum, FWHM). The
resolving power is normally specified with the mass at which it is determined,
as resolving power decreases with ion mass. High resolution mass
spectrometers (HR-MS) are generally considered to provide resolving power
greater than 10,000, while for ultrahigh resolving power instruments this value
is above 100,000 [41]. The former class includes TOF analysers, while the
Fourier transform (FT) instruments Orbitrap and ion cyclotron resonance
(ICR) fall in the latter class. Mass accuracy is the relative difference between
measured and theoretical m/z values, reported in parts per million
 A. de Villiers et al. / J. Chromatogr. A 1430 (2016) 16–78
Table 3
Overview of the specifications of various mass analysers used in the LC–MS analysis of flavonoids. The table is based on the work of Holcapekˇ et al. [41], updated using the latest specifications for
QqQ, Q-LIT and Orbitrap instruments.
a 3 Mass range m/z (upper limit)
Mass analyser Resolving power (FWHM) [×10 ]
QqQ – 2000–3000
a 6
IT – 2048–6000
a 4
TOF 10–60 100,000
ICR 750–2500 4000–10,000
a 100–500 2000–6000
Orbitrap
a Includes hybrid configurations.
b
Calculated for a mass range of m/z 1000.
(ppm) units. Sufficient mass accuracy provides the key benefit of allowing
determination of elemental composition [413]. The mass accuracy required
for this however depends on the mass of the ion, with mass accuracy of <5
ppm typically sufficient for compounds of m/z < 300; for higher MW ions,
this degree of mass accuracy is no longer sufficient because of the much
larger number of potential combinations of elements [41].
HR-MS instruments such as double-focusing sector and modi-fied
magnetic sector instruments used in the early LC–MS analysis of flavonoids
[108,171] have nowadays largely been replaced by TOF, Orbitrap and to a
lesser extent FT-ICR systems.
A second classification of MS instruments is based on whether they are
n efits of HR-MS data to increase selectivity, with the added benefit that full-
single or multistage systems. Multistage instruments allow MS (where n > 1)
operation either in time, as in ion trap (IT) instruments, or by combining
multiple mass analysers in space. Especially in combination with LC–MS
using soft ionisation tech-niques such as ESI, where limited fragmentation
n
information is obtained, tandem MS (MS/MS) or MS capabilities are
essential for structural elucidation purposes. An array of very powerful hybrid
instruments is nowadays commercially available, including QqQ, quadrupole-
linear ion trap (Q-LIT), Q-TOF [414,415], ion-trap-TOF (IT-TOF) [416–418],
quadrupole- and linear ion trap-Orbitrap [419] and quadrupole- and linear ion
trap-ICR instruments. With the exception of the ICR instruments, all of these
have found increasing application in flavonoid analysis during the last decade.
From the perspective of hyphenation to advanced LC separa-tions such as
UHPLC and LC × LC, a critical requirement of any mass analyser is a
sufficiently high acquisition speed [420]. Peak widths in the order of a few
seconds are often encountered, which implies that fast acquisition rates, in the
order of 5–10 Hz and higher, are required to obtain the requisite ∼15 data
points across each peak. The latest generation of quadrupole and ion trap
based analysers are mostly capable of meeting this requirement, while TOF
systems provide the best scanning speeds of all analysers.
Another important characteristic of mass analysers is the mass range they
are capable of covering. This value is relatively limited for quadrupole, ion
trap and Orbitrap instruments, with TOF sys-tems providing the highest mass
range of all systems.
An overview of the performance characteristics of mass anal-ysers
currently used in LC–MS analysis of flavonoids is presented in Table 3. It
should be noted that these are generalised specifica-tions, with some variation
occurring between instruments and the mode in which each instrument is
operated. For example, the scan speed is inversely related to the resolving
power in FT instruments (Orbitrap and ICR) and the mass range in scanning
instruments (Q and IT) as well as the sensitivity of all analysers to varying
degrees. For a relatively recent overview of the performance of the differ-ent
instruments, the reader is referred to the excellent reviews by Holcapekˇ et al.
[41] and Forcisi et al. [42], while more dedicated reviews may be consulted
for details on particular instruments such as Q-TOF [415], Orbitrap [421,422]
and FT-ICR [423] systems.
Flavonoid analyses may broadly be divided into targeted and untargeted
applications. In the case of the former, a finite number of known target
molecules are determined, mostly quantitatively,
43
Mass accuracy (ppm) b Dynamic range
Scanning speed (Hz)
– 10–30 6
10
– 10–60 10
5
<2–5 10–100 10 –10
4
<0.25–1 2 10
<1–3 1–20 4000–5000
whereas for untargeted or screening analyses the goal is to iden-tify as many
compounds as possible, often only qualitatively. The nature of the analysis
being performed will determine the best suited mass analyser for the given
application.
In the case of targeted analyses, selectivity and quantifica-tion
specifications (linearity, dynamic range, sensitivity) are of paramount
importance. Currently the most widely used approach for targeted analysis
relies on the high selectivity inherent to tan-dem MS operation, most
commonly exploited by utilising QqQ instruments operated in multiple
reaction monitoring (MRM) mode. While IT instruments are also capable of
this measurement mode, the quantitative performance of these detectors is
generally inferior to QqQ. An alternative strategy involves exploiting the ben-
scan data are also available. Especially Orbitrap instru-ments are increasingly
being used for this purpose, where the high resolving power of these
instruments is beneficial.
For untargeted analysis, resolving power is critical. In combi-nation with
high efficiency or fast separations, however, the duty cycle of the mass
spectrometer is equally important. Currently, TOF-MS instruments still
provide the best compromise between these parameters, although
developments in Orbitrap systems show promise to obtain higher resolving
power for similar scan times in the future. Secondly, tandem MS
measurements are highly beneficial, especially so in the case of ESI-MS
analysis of flavonoids due to the amount of information regarding substitution
patterns of isomeric compounds that may be obtained (cf. Section 2.3). The
combination of both MS/MS and high resolving power is ideal, and this
explains the contemporary popularity of Q-TOF systems for the non-targeted
analysis of flavonoids. On the other hand, the latest generation of Orbitrap
instruments are capable of providing the same information at high resolving
power (although lower scan speeds), and have gained more widespread
acceptance in the last few years. In the following sections, the applications of
advanced LC separation with each of the different types of mass
spectrometers for flavonoid analysis during the last seven years will be
outlined.
4.2.1. Nominal mass instruments: ion trap (IT) and triple
quadrupole (QqQ)
Ion trap instruments use oscillating electric fields to trap and store ions in
two or three dimensions, based on which 3D (QIT or IT, also called Paul
traps) and 2D (linear (quadrupole) ion trap (LQIT or LIT) systems can be
distinguished. The latter have higher trapping efficiencies and are less
susceptible to space-charge effects, pro-viding higher sensitivity and an
increased dynamic range [413]. Accordingly, most recent applications in
flavonoid analysis utilise LIT instruments (Table 4). The main advantage of
n
ion trap analysers is that they enable the true MS operation by allowing
selective trapping and fragmentation of parent and/or daughter ions as a
function of time [161]. The primary application of these instru-ments has
therefore been in qualitative analysis.
Ion trap instruments are extremely versatile and played a cru-cial role in
the early years of flavonoid analysis by LC–MS, primarily in terms of
n
structural elucidation by MS experiments. Nowadays,
44 A. de Villiers et al. / J. Chromatogr. A 1430 (2016) 16–78

Table 4
Applications of advanced LC hyphenated to ion trap MS detectors to flavonoid analysis (2009–2015).

Sample Flavonoids Ionisation Detector(s) Stationary phase (column Mobile phase(s) References
source dimensions), flow rate,
temperature, analysis time
Licorice (Glycyrrhiza Prenylated and ESI+/− LIT
a C18 (150 mm × 2.1 mm, c
A: 0.1% AA , B: 0.1% [428]
c
glabra) glycosylated flavanones, UV 1.7 m dp ), AA in ACN
chalcones, isoflav-3-enes, b
FR = 0.3 mL/min, 25 min
isoflavones and
isoflavanes (18
flavonoids) ESI− C18 (150 mm × 2.1 mm,
Peanut skins A- and B-type LIT A: 0.1% AA, B: 0.1% [286]
proanthocyanidins (83 UV 1.7 m dp ), FR = 0.6 mL/min AA in ACN
◦
flavonoids) (0.3 mL/min to MS), 50 C,
27 min
Berry skins (Vitis Anthocyanins (18 ESI+ a C18 (250 mm × 4.6 mm, c [284]
QIT A: 2% FA , B: 2% FA
vinifera L.) flavonoids) 5 m dp ), FR = 1.0 mL/min, in ACN
◦
ESI− 50 C, 45 min
Black currant juice Flavone, flavonol and a C18 (50 mm × 2.1 mm, A: 0.1% FA, B: 0.1% [430]
Q-LIT
flavanone aglycones and 1.8 m dp ), FA in ACN
glycosides (13 flavonoids) ESI− FR = 0.3 mL/min, 45 min
Rooibos (Aspalathus Flavonol and flavone QIT C18 (150 mm × 4.6 mm, A: 1% FA in [424]
linearis) aglycones and glycosides, a
TOF 1.8 m dp ), FR = 0.5 mL/min H2 O/ACN (90/10,
◦
flavanone and chalcone (0.2 mL/min to MS), 25 C, v/v), B: ACN
glycosides (26 flavonoids) ESI+/− 35 min
Asian plant dyes Flavone aglycones LIT C18 (150 mm × 2.1 mm, c [431]
A: 0.03% TFA , B:
(Arthraxon hispidus glycosides (9 flavonoids) UV 1.8 m dp ), 0.024% TFA in ACN
and Miscanthus FR = 0.1 mL/min, 40 min
tinctorius) ESI+/− C18 (50 mm × 2.0 mm,
Pomegranate (Punica Flavan-3-ols, flavonols, LIT A: 0.1% FA, B: 0.1% [429]
granatum L.) juice flavanones, 1.8 m dp ), FA in ACN
◦
dihydrochalcones, and FR = 0.2 mL/min, 30 C,
flavones (14 flavonoids) ESI− 20 min
Eucalyptus wood Flavan-3-ols, flavonol QIT C18 (50 mm × 2.1 mm, A: H2 O/ACN (99:1, [432]
aglycones and glycosides, UV 1.9 m dp ), v/v) containing
◦
flavanones and FR = 0.6 mL/min, 25 C, 0.1% FA, B: 0.1% FA
flavanonols (18 37 min in ACN
flavonoids) ESI+/− C18 (50 mm × 2.0 mm,
Pomegranate (Punica Flavonol glycosides, LIT A: 0.1% FA, B: 0.1% [433]
granatum L.) juice anthocyanins, taxifolin 1.8 m dp ), FA in ACN
◦
hexoside and naringenin FR = 0.2 mL/min, 30 C,
(11 flavonoids) 20 min
Rat serum Isoflavones (4 flavonoids) ESI+ Q-LIT C18 (50 mm × 4.6 mm, A: 0.1% FA, B: 0.1% [92]
b FA in ACN
2.6 m dp SP ),
ESI− FR = 0.5 mL/min, 9 min
Halimione portulacoides Flavonol and flavone QIT C18 (50 mm × 2.1 mm, A: H2 O:ACN (99:1) [434]
glycosides (13 flavonoids) UV 1.9 m dp ), containing 0.1% FA,
◦
FR = 0.6 mL/min, 25 C, B: 0.1% FA in ACN
ESI+/− 37 min
Eastern Teaberry Flavan-3-ols, QIT C18 (150 mm × 2.1 mm, A: H2 O/ACN/FA [435]
(Gaultheria procyanidins and flavonol UV 1.7 m dp ), (95:5:0.1, v/v), B:
◦
procumbens L.) aglycones and glycosides FR = 0.3 mL/min, 25 C, 0.1% FA in ACN
(22 flavonoids) ESI+/− 80 min
Viola yedoensis Makino Flavone glycosides (35 LIT C18 (50 mm × 2.1 mm, A: H2 O, B: ACN [154]
flavonoids) UV 1.9 m dp ),
◦
FR = 0.25 mL/min, 30 C,
ESI− 33 min
Green tea, oolong tea Flavonol glycosides (18 QIT C18 (100 mm × 2.1 mm, A: 2% AA, B: ACN [436]
and black tea flavonoids) UV 1.8 m dp ),
◦
FR = 0.4 mL/min, 20 C,
ESI− 20 min
Forsythia flowers Rutin QIT C18 (150 mm × 2.1 mm, A: H2 O/ACN/FA [313]
UV 1.9 m dp ), (95:5:0.1, v/v/v), B:
◦
FR = 0.2 mL/min, 25 C, 0.1% FA in ACN
68 min
Dietary supplements Flavonol and flavanones ESI+ QIT C18 (100 mm × 0.075 mm, Isocratic: [437]
and food matrices (5 flavonoids) UV c
<2 m dp ), FR = 450 nL/min, ACN/MeOH /H2 O/FA
10 min (32.5:10:57:0.5,
ESI+/− C18 (150 mm × 1.0 mm, v/v/v/v).
Rosé wines Flavonols, flavan-3-ols, QIT A: 1% FA, B: 1% FA [438]
dihydrochalcones and ESI+/− QqQ 1.8 m dp ), in MeOH
◦
anthocyanins (113 UV FR = 0.08 mL/min, 40 C,
flavonoids) 46 min
a b
LIT, linear ion trap; Q-LIT, Quadrupole-linear ion trap; QIT, quadrupole ion trap; TOF, time of flight; QqQ, triple quadrupole. FR, flow rate;
SP, superficially porous.
c
AA, acetic acid; ACN, acetonitrile; FA, formic acid; TFA, trifluoroacetic acid; MeOH, methanol.
 A. de Villiers et al. / J. Chromatogr. A 1430 (2016) 16–78
however, IT instruments are less commonly used on their own, but are often
combined with HR analysers in powerful hybrid configu-rations such as IT-
TOF and IT-Orbitrap instruments, which have largely superseded
conventional IT-MS in flavonoid analysis. In such instruments, the ion storage
and sequential fragmentation capabilities of IT instruments are highly
beneficial, particularly in structural elucidation studies. Alternatively, IT
multi-stage frag-mentation is often used in combination with separate LC–
HR-MS analysis for structural elucidation [424–426]. From the perspec-tive
of hyphenation to ultra-fast and high resolution separations, several latest
generation IT and especially LIT instruments provide sufficient acquisition
speeds of up to ∼60 Hz (Table 3).
Ion trap instruments are generally considered inferior to quadrupole or
triple quadrupole instruments for quantitation. This is because of the need to
account for variable trapping times and also the lower dynamic range of these
instruments (the latter limited by the capacity and need to avoid space-charge
effects). Nevertheless, Stanoeva et al. [427] have demonstrated the quanti-
2 structural studies, product ion scan (Q1 in SIM, Q3 in scan), neutral loss (Q1
tative analysis of flavonoids in full-scan MS and MS modes using an IT
instrument, providing similar results but better sensitivity than UV detection.
Gavina et al. [92] also utilised an IT instrument in MRM mode for the
quantitative analysis of isoflavones in rat serum, reporting a dynamic range of
3 orders of magnitude and LOD’s of 0.01–3 pg on-column. An overview of
the application of IT instruments in combination with advanced separation
methods is presented in Table 4; some noteworthy reports are discussed
briefly below.
A good example of the utility of IT instruments in the struc-tural
elucidation of flavonoids is the work of Cao et al. [154], who reported an
extensive study aimed at characterising flavone-di-C-glycosides using
n
UHPLC-ESI-MS spectra obtained on an IT instrument. Negative ionisation
was found to provide more diag-nostic information, and the authors were able
2 UHPLC separations [420,441]. Because of the operation principles of
to show that the first sugar cleavage in MS spectra occurs at the C-6
3 quadrupole instruments, however, a compromise between speed and
position, fol-lowed by the C-8 sugar in MS spectra. Based on this,
information about the nature of the sugars can be derived. Furthermore, the
0,2 0,2 −
X1 X 2 ion could be used to identify the aglycone species. The utility of
the approach was demonstrated for the analysis of a butanol extract of Viola
yedoensi, where 26 flavone-di-C-glycosides could be identified.
The group of Gruppen [286] used both QIT and LIT instruments to study
in detail the proanthocyanidins present in peanut skins. NP-LC fractionation
of the sample was followed by RP-HPLC and UHPLC to simplify the
analysis due to the extreme complexity of the sample. The authors found that
A-type oligomers were pre-dominant in this sample, and were able to assign
the position of the A-linkages in 43 trimeric and tetrameric procyanidins
based on MS/MS fragments resulting from QM fission (Fig. 4) [182,184].
The same group reported a UHPLC-LIT method for the rapid screening of
prenylated flavonoids [428]. The methodology is based on the detection of
neutral losses of 56 and 42 amu associated with prenyl and pyran ring
moieties. The ratio of these two neutral losses could then be used to
distinguish between these two classes of com-pounds.
He et al. [284] used a conventional HPLC column operated at
◦ flavonoids, where the latter is used for compound identification and the
50 C in combination IT detection to confirm for the first time the presence of
pelargonidin-3-O-glucoside in Vitis vinifera L. grape skins. Mena et al. [429]
proposed an UHPLC-IT method-ology using three different sets of MS
conditions (scan times 0.19–0.38 s) for the screening of phenolics in
pomegranate juice. Flavonoids and hydolyzable tannins were identified in
nega-tive ionisation mode, and anthocyanins in positive ionisation mode.
Rak and co-workers used a hybrid triple quadrupole-LIT instru-ment to
devise a method for untargeted screening of flavonoids [430]. Two separate
experiments were used: in the first instance,
45
MRM scanning was used to detect the presence of selected agly-cones
following in-source fragmentation of glycosylated flavonoids using a high
declustering potential. This was followed by a second full scan detection
mode in the same analysis to allow tenta-tive assignment of the molecular
weight of the suspected target analytes. Finally, a second LC analysis was
performed with the detector used in product ion scan mode (with the third
quadrupole used as a linear ion trap) for the identification of flavonoid
derivatives.
Within the scope of this review, we will limit our atten-tion to triple
quadrupole (QqQ) instruments. These combine two quadrupoles with a RF
collision chamber in-between to allow tandem MS operation in space. Since
both quadrupoles may be operated in selected ion monitoring (SIM) or scan
modes, a range of different modes can be used on QqQ instruments. Scan
mode, where either of the quadrupoles is scanned, is less commonly used in
flavonoid analysis, in part because better performance in terms of sensitivity
and accurate mass information can be obtained on other instruments. For
and Q3 in SIM mode at a fixed off-set) and precursor ion scan (Q1 scan, Q3
SIM) modes may be used [439]. QqQ instruments are however mostly utilised
as selective and sensitive detectors in selected or multiple reaction monitoring
mode (SRM or MRM), where Q1 is used in SIM mode to isolate par-ent ions,
and Q3 in SIM to monitor selected daughter ions. For such targeted analysis,
QqQ instruments provide very high sensitivity and selectivity as a
consequence of the combination of two mass filters in SIM mode.
The latest generation quadrupole systems can achieve scan rates as high as
10–15,000 amu/s [321,420], and using this technology in QqQ format, dwell
times as low as 5 ms can be achieved in MRM mode [420,440], which are
mostly sufficient for quantification of narrow peaks commonly obtained in
sensitivity has to be made; this is especially relevant when relatively large
numbers of MRM transitions are to be monitored.
Indeed, in the majority (87%) of the studies listed in Table 5, QqQ
instruments are used in MRM mode for targeted analysis. Two examples of
the power of this approach for flavonoid anal-ysis will be highlighted.
Vrhovsek et al. [442] reported a targeted metabolomic approach based on
UHPLC-QqQ for the quantification of 135 phenolics, including 66 flavonoids,
in fruits and beverages within 15 min. Quantification limits varied between
0.5 and >50 pg, while linear responses spanning more than four orders of
mag-nitude were reported. Lambert et al. [438] used a 1 mm UHPLC column
and QqQ detection in MRM mode to quantify 152 pheno-lics in Rosé wines
(Fig. 14). In this work, dwell times were varied for each transition to obtain
10–15 points across each peak (most values were in the order of 5 ms).
QqQ instruments are nowadays less often used in the other operational
modes for structural elucidation or untargeted analy-sis, with more advanced
n
HR-MS instrumentation having surpassed QqQ systems for such
applications. In fact, triple quadrupole instru-ments are often used together
with HR-MS systems such as TOF or Orbitrap instruments in the study of
former for quantification [425,443,444]. Product ion scans are also used to
assist in tentative compound identification [96,180,445] and to determine
suitable CID conditions and transitions for MRM operation [446,447]. Some-
what surprisingly, Spácilˇ et al. [447] reported a 10-fold higher sensitivity in
positive ionisation mode, although negative ionisa-tion provided better
selectivity. A similar observation (2–3 times higher sensitivity in positive
ionisation) was made by Nagy et al. [408], and might be due to reduced
background noise in QqQ instru-ments operated in MRM mode.
46 A. de Villiers et al. / J. Chromatogr. A 1430 (2016) 16–78

Table 5
Applications of advanced LC hyphenated to triple quadrupole MS detectors to flavonoid analysis (2009–2015).

Sample Flavonoids Ionisation Detector(s) Sensitivity Stationary phase Mobile References

source (injection (column dimensions), phase(s)
volume) flow rate, temperature,
analysis time

Vitaflavan
®
and cocoa Flavanol ESI− b
QqQ (MRM )
b 1.7–692 g C18 (100 mm × 2.1 mm, d
A: 2% AA , B: [452]
d
nibs monomers and (not specified) 1.8 m dp ), ACN
procyanidins (4 c
FR = 0.4 mL/min, room
flavonoids) temp, 13 min
Milk-based food Anthocyanins and ESI+ QqQ (MRM) 3–3000 pg C18 (50 mm × 2.1 mm, d [408]
A: 10% FA , B:
products flavonols (26 on-column 1.7 m dp ), ACN
flavonoids) (10 L) FR = 0.4 mL/min, room
ESI− temp, 13 min
Cocoa Procyanidins (10 QqQ (MRM) 1–30 pg/ L C18 (100 mm × 2.1 mm, A: 0.2% AA, B: [453]
flavonoids) 1.8 m dp ), ACN
◦
FR = 0.4 mL/min, 30 C,
12.5 min
GegenQinlian Puerarin, ESI+ b 12.7–51 pg C18 (100 mm × 2.1 mm, A: 0.1% FA and [454]
QqQ (SIM )
a
decoction (TCM ) daidzein, baicalin, on-column 1.7 m dp ), 5 mM AA, B:
wogonoside and (5 L) ◦ d
FR = 0.2 mL/min, 30 C, MeOH
liquiritin ESI− 6 min
Black tea Proanthocyanins QqQ (MRM) LOD’s not C18 (100 mm × 2.1 mm, A: 0.1% FA, B: [88]
(8 flavonoids) UV specified 1.7 m dp ), 0.1% FA in ACN
◦
FR = 0.5 mL/min, 30 C,
ESI+/− 7 min
Rat plasma Procyanidins and QqQ (MRM) 1.5–606 g C18 (100 mm × 2.1 mm, A: 0.2% AA [455]
anthocyanins (21 (not specified) 1.8 m dp ), (procyanidins),
flavonoids) FR = 0.4 mL/min, 10% AA
12.5 min (anthocyanins),
ESI+/− C18 (100 mm × 2.1 mm, B: ACN
Tea Catechins (8 QqQ (MRM) LOD’s A: 0.05% AA [447]
compounds) 4.5–7.2 pg 1.7 m dp ), (+mode) or
◦
(+mode) and FR = 0.45 mL/min, 35 C, 0.01% FA
45–72 pg 5.5 min (−mode), B:
(−mode) MeOH
on-column
ESI+/− (1.5 L) C18 (100 mm × 2.1 mm,
Chamomile flowers Flavones and QqQ (MRM) LOD’s A: 0.1% FA, B: [456]
and Chamomile tea flavonols (9 2.6–12.9 pg 1.7 m dp ), MeOH
◦
extracts flavonoids) (−mode), FR = 0.45 mL/min, 30 C
4.9–408 pg
(+mode)
on-column
ESI− (3 L) C18 (50 mm × 2.1 mm,
Radix puerariae Isoflavones QqQ (MRM) A: 0.2% FA, B: [457]
aglycones and c MeOH
2.6 m dp SP ),
◦
glycosides (13 FR = 0.5 mL/min, 35 C,
flavonoids) 12 min
Red wine Anthocyanins ESI+ QqQ (Neutral Qualitative C18 (100 mm × 2.1 mm, A: 7.5% FA, B: [180]
(121 flavonoids) loss, survey 1.7 m dp ), ACN
◦
scan) FR = 0.1 mL/min, 50 C,
30 min
Mung bean (Vigna Flavanones, ESI+ QqQ (MRM) LOD’s C8 (150 mm × 2.1 mm, A: 10 mM FA, [83]
radiate) flavones and UV 0.04–3173 pg 1.7 m dp ), B: MeOH
◦
isoflavones (24 on-column FR = 0.2 mL/min, 40 C,
flavonoids) ESI+/− (2 L) 17 min
Apple, berries, green Dihydrochalcones, QqQ (MRM) LOD’s C18 (100 mm × 2.1 mm, A: 0.1% FA, B: [442]
tea and red wine isoflavones, 0.5–50 pg 1.8 m dp ), 0.1% FA in ACN
◦
flavones, on-column FR = 0.4 mL/min, 40 C,
flavanones, (2 L) 15 min
flavan-3-ols and
flavonols (63
flavonoids) C18 (150 mm × 2.1 mm,
Sangiovese wines Pyranoanthocyanins ESI QqQ (MRM) LOD’s 2–860 pg A: 5% FA, B: 5% [458]
and anthocyanin UV on-column 1.7 m dp ), FA in ACN
◦
flavanol adducts (2 L) FR = 0.4 mL/min, 40 C,
(92 flavonoids) ESI− 18 min
Cherry tomatoes, Flavonols and QqQ (MRM, LOD’s C18 (50 mm × 2.1 mm, A: 0.1% FA, B: [445]
tomato sauce and flavanones (3 product ion 100–1540 pg 1.7 m dp ), 0.1% FA in ACN
◦
juice flavonoids) and neutral on-column FR = 0.4 mL/min, 30 C,
ESI− loss scan) (10 L) 3 min
Cocoa beans Procyanidins (3 QqQ (MRM, Semi- C18 (150 mm × 2.1 mm, A: 0.1% FA, B: [446]
flavonoids) product ion quantitative 1.7 m dp ), 0.1% FA in ACN
◦
scan) FR = 0.3 mL/min, 30 C,
UV 35 min
 A. de Villiers et al. / J. Chromatogr. A 1430 (2016) 16–78 47
Table 5 (Continued )

Sample Flavonoids Ionisation Detector(s) Sensitivity Stationary phase Mobile References

source (injection (column dimensions), phase(s)
volume) flow rate, temperature,
analysis time
Tomato, broccoli, Isoflavones, ESI+/− QqQ (MRM) 5–100 g/kg C18 (100 mm × 2.1 mm, A: 30 mM AA [440]
carrot, eggplant and flavones and 1.8 m dp ), adjusted to pH
◦
grape flavonols (22 FR = 0.2 mL/min, 30 C, 5 with FA, B:
flavonoids) ESI− 11 min MeOH
Fermented orange juice Flavanones (9 QqQ (MRM) 6.25–44.08 pg C18 (150 mm × 2.1 mm, A: 0.1% FA, B: [459]
compounds) on-column 1.7 m dp ), 0.1% FA in ACN
(10 L) FR = 0.32 mL/min,
ESI− 12 min
Huang-Lian-Jie-Du Flavones and QqQ (MRM) 0.02–1.0 pg/ L C18 (50 mm × 2.1 mm, A: 0.1% FA, B: [444]
decoction (TCM) flavanones (24 (injection 1.7 m dp ), 0.1% FA in ACN
◦
flavonoids) volume not FR = 0.45 mL/min, 35 C,
ESI− specified) 12 min
Apple peel Flavonol QqQ LOD’s not C18 (100 mm × 2.1 mm, A: 0.1% FA, B: [460]
glycosides, ESI− TOF specified 1.8 m dp ), 0.1% FA in ACN
◦
flavan-3-ols and FR = 0.3 mL/min, 30 C,
chalcone (8 14 min
compounds) and
procyanidins
(DP1–10) c
PFP (50 mm × 2.1 mm,
Citrus juices and Flavanones (13 ESI+ QqQ (MRM) 500–2900 pg A: 0.1% FA, B: [461]
beverages flavonoids) on-column 2.6 m dp SP), ACN
(10 L) FR = 0.3 mL/min, 16 min
Standards and urine Flavone ESI+ QqQ (MRM) LOD’s not C18 (50 mm × 0.3 mm, A: 2% AA, B: 2% [462]
b
metabolites ESI+ IM -LIT specified 2.7 m dp SP), AA in MeOH
(diosmetin-3-O- b ◦
(FAIMS ) FR = 35 L/min, 50 C,
glucoronide, 6 min
-7-O-glucoronide
and -3,7-O-
diglucoronide)
Grape berries Flavonols, ESI+ QqQ (MRM) LOD’s not Non-anthocyanins: Non- [463]
flavan-3-ols, specified C18 (100 mm × 2.1 mm, anthocyanins:
dihydrochalcones 1.8 m dp ), A: 0.1% FA, B:
◦
and anthocyanins FR = 0.4 mL/min, 40 C, 0.1% FA in ACN
(37 flavonoids) 15 min Anthocyanins:
Anthocyanins: C18 A: 5% FA, B: 5%
(150 mm × 2.1 mm, FA in MeOH
1.7 m dp ),
◦
FR = 0.4 mL/min, 40 C,
ESI+/− 14 min
Tomato, carrot, grape, Flavonols, QqQ (MRM) LOD’s not C18 (100 mm × 2.1 mm, A: 30 mM [464]
eggplant and brocolli isoflavones and specified 1.8 m dp ), ammonium
◦
flavones (16 FR = 0.2 mL/min, 30 C, acetate, pH 5,
flavonoids) ESI− 12 min B: MeOH
Olive oil Flavonol and QqQ (MRM) LOD’s 5–137 pg C18 (50 mm × 2.1 mm, A: 0.2% AA, B: [465]
flavones (3 on-column 1.8 m dp ), ACN
◦
flavonoids) (10 L) FR = 0.4 mL/min, 30 C,
ESI− 5 min
Gingko biloba tablets Flavonol QqQ (MRM) LOD’s C18 (50 mm × 4.6 mm, A: 1% FA, B: [450]
aglycones and 0.035–5.1 pg 1.8 m dp ), MeOH
◦
glycosides (13 on-column FR = 0.5 mL/min, 30 C,
flavonoids) ESI+/− (5 L) 28 min
Gingko biloba seeds Flavan-3-ols, QqQ (MRM) LOD’s C18 (100 mm × 2.1 mm, A: 0.1% FA, B: [466]
flavonol aglycones 0.1–5.7 pg 1.7 m dp ), 0.1% FA in ACN
◦
and glycosides on-column FR = 0.4 mL/min, 35 C,
and flavones (19 (2 L) 11 min
flavonoids) ESI− C18 (150 mm × 1.0 mm,
Sausages with added Flavonol- QqQ (MRM) 15–510 g/kg A: ACN/0.1% FA [467]
cocoa and grape seed glycosides and 1.7 m dp ), (5/100, v/v), B:
◦
extracts proanthocyani- FR = 0.13 mL/min, 35 C, ACN/0.1% FA
dins (10 15 min (60/100, v/v)
flavonoids) ESI+/− C18 (100 mm × 2.1 mm,
Artichoke, garlic and Flavonols, QqQ (MRM) 1–50 g/kg A: 30 mM [451]
spinach isoflavonols and 1.8 m dp ), ammonium
◦
flavones (22 FR = 0.2 mL/min, 30 C, acetate, pH 5,
flavonoids) ESI+/− 11 min B: MeOH
Sweet cherry (Prunus Anthocyanins, QqQ (MRM, full LOD’s 3–76 pg C18 (50 mm × 2.1 mm, A: 1% FA, B: 1% [294]
avium L.) flavonols and scan and on-column 1.8 m dp ), FA in ACN
◦
flavan-3-ols (18 product ion (1.1 L) FR = 0.5 mL/min, 60 C,
flavonoids) scan) 15 min
UV
48 A. de Villiers et al. / J. Chromatogr. A 1430 (2016) 16–78
Table 5 (Continued )

Sample Flavonoids Ionisation Detector(s) Sensitivity Stationary phase Mobile References

source (injection (column dimensions), phase(s)
volume) flow rate, temperature,
analysis time
Lentil seed coats Anthocyanins, ESI+ QqQ (MRM) Qualitative PFP (100 mm × 2.1 mm, A: 1% FA, B: [91]
flavan-3-ols, UV 2.6 m dp SP), H2 O/ACN/FA
proanthocyani- FR = 0.4 mL/min, 50 min (5:90:5, v/v/v)
dins, flavanones,
flavones and
flavonols (21
flavonoids) ESI− C18 (150 mm × 4.6 mm,
Wild Berberis species Flavonol QqQ (product LOD’s not A: 0.1% FA, B: [260]
glycosides (16 ion) specified 2.6 m dp SP), ACN
◦
flavonoids) UV FR = 1.0 mL/min, 30 C
15.1 min
Belamcanda chinensis Isoflavone ESI+ QqQ (product 0.14–1.02 pg C18 (100 mm × 2.1 mm, A: 0.1% FA, B: [443]
(TCM) aglycones and ESI+ ion) on-column 1.7 m dp ), 0.1% FA in ACN
◦
glycosides (26 Q-TOF (2 L) FR = 0.4 mL/min, 40 C,
flavonoids) ESI− 17 min
Brazilian pepper Anthocyanins and QqQ (product Qualitative C18 (150 mm × 2.1 mm, A: 1% FA [96]
(Schinus biflavones (7 ion scan, 1.8 and 1.7 m dp ), (anthocyanins),
◦
terebinthifolius Raddi) flavonoids) neutral loss, FR = 0.4 mL/min, 40 C, 0.1% FA
exocarp CID) 18 and 23.5 min (nonantho-
ESI+ Q-TOF cyanins), B:
ESI− UV C18 (100 mm × 2.1 mm, 0.1% FA in ACN
Grape (V. vinifera, V. Flavonol QqQ (MRM, Semi- A: 5% FA, B: [449]
riparia, V. labrusca, glycosides (24 precursor-ion quantitative 1.7 m dp ), ACN
◦
Vitis lincecumii and V. compounds) and FR = 0.45 mL/min, 40 C,
rupestri) species neutral-loss 10 min
ESI− scan)
Mulberry (Morus alba Flavan-3-ols, Q-TOF C18 (100 mm × 2.1 mm, A: 0.2% FA, B:
ESI− QqQ (MRM) 0.14–77.28 mg/kg [425]
L.) anthocyanins and ESI+/− LIT-Orbitrap 1.7 m dp ), ACN
◦
flavonol UV FR = 0.4 mL/min, 40 C,
glycosides (34 15 min
flavonoids) ESI+/− C18 (100 mm × 1.0 mm,
Rosé wines Flavonols, QqQ (MRM) 10–50,000 pg A: 1% FA, B: 1% [438]
flavan-3-ols, ESI+/− QIT on-column 1.8 m dp ), FA in MeOH
◦
dihydrochalcones UV (2 L) FR = 0.17 mL/min, 40 C,
and anthocyanins 30 min
(113 flavonoids) ESI− C18 (100 mm × 2.1 mm,
Green, white and black Flavan-3-ol QqQ (MRM) 7.5–37.5 pg A: 0.1% FA, B: [468]
teas derivatives (8 on-column 1.7 m dp ), 0.1% FA in
◦
compounds) (5 L) FR = 0.3 mL/min, 40 C, MeOH
4 min
Gentiana cruciata L. Flavone ESI+ QqQ (MRM) LOD’s not C18 (50 mm × 2.1 mm, A: 0.1% FA, B: [469]
roots glycosides (4 UV specified 1.9 m dp ), 0.1% FA in ACN
compounds) ◦
FR = 0.4 mL/min, 30 C
a
TCM, traditional Chinese medicine.
c
b QqQ, triple quadrupole; MRM, multiple reaction monitoring; SIM, selected ion monitoring; IM, ion mobility; FAIMS, field-asymmetric waveform ion mobility spectrometry. FR, flow rate; PFP,
pentafluorophenylpropyl; SP, superficially porous.
d
AA, acetic acid; ACN, acetonitrile; FA, formic acid; MeOH, methanol.

Nevertheless, neutral loss and precursor ion scans are use-ful in group-
type identification of flavonoids, for example by studying glycosylated and/or
n rutinosides, di-hexosides and hexosyl-glucoronides, respectively) was used to
acylated flavonoid derivatives, prior to HR-MS identification of compounds
[180,448]. As an exam-ple, Alberts et al. [180] applied UHPLC-QqQ in
neutral loss and ‘survey scan’ (neutral loss-triggered product ion scan mode)
for the analysis of red wine anthocyanins (Fig. 15). In neu-tral loss mode,
class-type detection of anthocyanidin-glycosides, -acetylglycosides,
-coumaroylglycosides, -caffeoyglycosides and -di-glycosides was performed.
Compound identity was then con-firmed based on characteristic high-energy
CID spectra obtained for the anthocyanidin aglycones in survey scan mode.
Significantly, the characteristic product ion spectra showed consistent
behaviour also for the derived anthocyanins encountered in wine, which
allowed the authors to identify 121 compounds in red wine using this
approach.
De Rosso et al. [449] used UHPLC-QqQ in various modes for the detailed
analysis of red wine flavonols. First of all, precursor ion analysis of the six
known wine flavonol aglycones was used
to identify compounds of this class. Neutral loss mode (monitor-ing losses of
146, 162, 176, 308 and 324 for pentosides, hexosides, glucoronides,
confirm putative compounds of interest. The authors reported that the latter
operation mode was less sen-sitive and relied on slower scan times compared
to precursor ion scan mode, such that closely eluting isobaric compounds were
not resolved. Further confirmation of the tentatively identified com-pounds
was obtained using high resolution MS and MS/MS data acquired on a Q-TOF
instrument [449].
Obtaining a general sense of the sensitivity of QqQ detection in MRM
mode is complicated by the fact that sensitivity varies between instruments
and as a function of the number and nature of target analytes, ionisation mode
and chromatographic conditions. Nevertheless, reported limits of detection
(LODs) fall in the range of 0.02 pg–3 ng on-column (Table 5), with higher
values reported for aglycones [83,408]. Usual LOD values for glycosylated
species are in the region of 0.1–10 pg on-column, and typical linear ranges are
in the order of 2–5 orders of magnitude [408,442,450], which
 A. de Villiers et al. / J. Chromatogr. A 1430 (2016) 16–78 49

◦
Fig. 14. Overlay of MRM traces acquired for the UHPLC–ESI-QqQ-MS analysis of 152 phenolics in rosé wine. Analysis conditions: 100 mm × 1 mm 1.7 m porous C18 phase, 40 C, 0.17 mL/min.

Source: Reproduced from [438].

Fig. 15. (A) Chromatogram obtained for the UHPLC–MS/MS analysis of red wine anthocyanins on a QqQ instrument in neutral loss mode for the detection of anthocyanin-glycosides (neutral loss of
m/z 162). The insert shows the fragmentation pattern at low collision energies (25 V). (B) The characteristic product ion spectrum of malvidin-3-O-glucoside obtained in ‘survey scan’ mode (neutral
loss-triggered product ion scan) at 50 V collision energy. The insert shows the fragmentation scheme for the aglycone following the loss of the neutral sugar moiety. Analysis conditions: 100 mm × 2.1
◦
mm 1.7 m porous C18 phase, 50 C, 0.1 mL/min.
Source: Adapted from [180].
50 A. de Villiers et al. / J. Chromatogr. A 1430 (2016) 16–78
explains the widespread use of these detectors for targeted quan-titative
analysis. It is relevant to note that matrix effects remain a concern for such
applications using ESI. This can be minimised by using standard addition
quantification – the increased number of injections is then offset by the
shorter analysis times offered by UHPLC separation [440,451].
4.2.2. Time-of-flight (TOF) instruments
Time-of-flight mass spectrometers [470] work on the principle that lighter
ions travel faster than heavier ions following an initial acceleration by an
electric field. All ions acquire the same kinetic energy during this initial
acceleration period, and are separated in the field-free flight tube according to
their different velocities. The physical property that is measured is flight time,
which is directly related to the mass-to-charge ratio of the ion. Due to this
mode of operation, TOF instruments offer very high mass ranges (without
compromising sensitivity, as is the case in scanning mass spectro-meters),
very high acquisition rates and for the latest generation of instruments,
relatively high resolving power and good sensitivity (although the latter
decreases with high acquisition rates) (Table 3).
Several important developments have improved the per-formance of TOF
instruments [471]; from the perspective of hyphenation to LC with ionisation
sources such as ESI or APCI the introduction of orthogonal acceleration to
transform continuous ion beams into a pulsed source is especially relevant
[472,473]. Fur-thermore, reflectron instruments improve the resolving power
and mass accuracy of TOF instruments through the use of an electro-static
reflector (ion mirror) to correct for kinetic energy dispersion of ions leaving
the ion source [474], although this comes at the cost of somewhat reduced
sensitivity and dynamic range [42].
In addition, several hybrid TOF instruments are nowadays com-mercially
available, which provides the option of tandem MS operation with high
resolving power acquisition. The most com-mon of these, the quadrupole-
TOF (Q-TOF) [414,415] has found extensive use in flavonoid analysis (Table
6). The Q-TOF configu-ration allows collimation of the ion beam as well as
the option of mass selection in Q1 and fragmentation in a RF only
quadrupole. A further variant of the Q-TOF system equipped with an ion
mobility cell between the quadrupole and the TOF analyser is available (see
below for a discussion on ion mobility) [475]. In another hybrid instrument,
the ion trap TOF (IT-TOF) [416], the IT is used to store ions from a
continuous source such as ESI, from where they are ejected via a dc pulse to
the TOF, delivering enhanced sensitivity through the storage of ions in the IT.
n
This configuration can also be used to perform MS experiments prior to high
resolution TOF detection.
The high acquisition speed of TOF instruments (already high-lighted for
GC in the 1980s [476]) makes this an ideal MS detector for non-targeted
analysis in combination with very fast UHPLC sep-arations [42,420] and
therefore also on-line LC × LC systems [321]. Combined with the relatively
high resolving power and excellent mass range of TOF-MS, this characteristic
is responsible for the extensive application of the detector in contemporary
LC–MS anal-ysis of flavonoids. QqQ instruments operated in MRM mode
still provide lower limits of detection than TOF systems for targeted analyses,
although at the cost of loss of information about the com-plete ion population.
Similar quantitative performance (although not sensitivity) has been reported
for TOF and QqQ instruments by exploiting the selectivity of high resolution
mass data [477].
An illustrative example of the advantage of single-stage TOF analysis
may be found in the work of Abrankó et al. [193], who reported a systematic
‘bottom-up’ approach for the screening analysis of flavonoid derivatives. The
methodology is based on the use of two slightly different LC–MS analyses
with in-source CID used to produce characteristic fragments (aglycones for
+ +
flavonoid-O-glycosides and [Y0+C2H2O+H] and [Y1+C2H2O+H]
ions for flavonoid-C-glycosides). Further putative identification of the parent
ions was then obtained based on extracted ion chro-matograms (using a 10
mDa window) of the characteristic ion and an automated screening approach
where known ‘adducts’ such as hexosides, pentosides, etc. are searched for in
the full scan spectral data acquired. Sodium adducts were used as added
confirmation of the proposed structure (based on the observation that fragment
ions do not form sodium adducts).
A trend in the last few years has been increasing use of hybrid TOF
instruments, with single stage TOF analysers increasingly being replaced by
especially Q-TOF systems in flavonoid analyses. While in-source CID can be
used to obtain fragmentation information needed to identify compounds
[164,193,297,478], hybrid systems are much more flexible in allowing dual
3
scan modes, true MS/MS and also pseudo MS operation by utilising in-
source CID [479,480]. The most used configuration for flavonoid analysis is
the Q-TOF (two-thirds of the papers listed in Table 6 used this instrument). In
addition to the tandem MS capabilities of Q-TOF systems, alterna-tive
acquisition modes are also possible, such as recording spectra at both high and
E
low collision energy settings within a single analy-sis (referred to as MS by
one vendor). An example of the application of this approach for the
identification of phenolic compounds in apple products by UHPLC–Q-TOF-
MS analysis is presented in Fig. 16 [481]. Fig. 16B shows the extracted ion
chromatograms for two partially co-eluting flavonoids, with the corresponding
E
MS spec-tra obtained using a collision energy ramp of 10–40 eV presented in
Fig. 16C. While this approach is useful for the fast screening of unknown
flavonoids, providing as it does useful information for tentative compound
identification based on the molecular ion and major fragments in a single
analysis, it also lacks the selectivity offered by the MS/MS mode. This is
E
evident from Fig. 16C where MS spectra contain fragment ions from co-
eluting compounds, which may hamper structural elucidation of co-eluting
flavonoids of the same class.
UHPLC-Q-TOF has also been used in the detailed analysis of the MS/MS
behaviour of flavonoids. For example, Guo et al. [177] reported a systematic
approach to differentiate between isomeric 6-C and 8-C flavone glycosides
based on negative and positive ionisation MS/MS spectra of the deprotonated
and protonated molecular ions, with the latter proving to be more
discriminative. Ma et al. [482] reported a MS/MS method on a Q-TOF
instrument to allow differentiation of regioisomers of methylated flavonols
+
based on the position-specific [M+H−CH4] ion and methylated RDA frag-
ments, as well as complementary information obtained from the RP-LC
elution order.
On the other hand, the utility of Q-TOF-MS for targeted analysis of
selected flavonoids is illustrated by the work of Delcambre and Saucier [483],
who were able to demonstrate the presence of 14 flavanol-glucosides in
Merlot grapes and wine for the first time. To do this, the authors exploited the
MS/MS capabilities of the Q-TOF to confirm the identity of the parent ion
based on the fragments observed and their chemical formula obtained from
accurate mass data.
Ion trap time-of-flight (IT-TOF) instruments have found rela-tively limited
application in flavonoid analysis compared to Q-TOF systems [484–486],
especially in combination with advanced LC separation methods.
Nevertheless, the work of Dugo et al. and Som-mella and co-workers
illustrates the utility of such an approach, where superficially porous phases
were combined with the MS/MS and high resolution capabilities of the IT-
TOF for the characteri-sation of flavonoids in mulberry leave extracts [258],
Mate [367], citrus juices [487] and apples [124].
Li et al. [488] reported an interesting approach based on
UHPLC separation and Q-TOF detection for the identification
of antioxidants in licorice. To do this, the authors compared
chro-matographic profiles before and after spiking the sample
with the
 A. de Villiers et al. / J. Chromatogr. A 1430 (2016) 16–78 51

Table 6
Applications of advanced LC hyphenated to time-of-flight MS detectors to flavonoid analysis (2009–2015).

Sample Flavonoids Ionisation Detector(s) Stationary phase Mobile References

source (column dimensions), phase(s)
flow rate, temperature,
analysis time
Rose species (Rosa Flavonol ESI+/− b
Q -TOF
b C18 (100 mm × 2.1 mm, A: 0.05% TFA ,
d [489]
d
damascene, Rosa aglycones and UV 1.7 m dp ), B: ACN
bourboniana and glycosides (14 c ◦
FR = 0.3 mL/min, 30 C,
Rosa brunonii) flavonoids) ESI+/− 4.5 min
Plant extracts Kaempferol, TOF C18 2× d [283]
A: 0.1% FA , B:
(Arabidopsis thaliana quercetin, rutin (150 mm × 2.1 mm, 0.1% FA in ACN
and Ginkgo biloba) and silibinin 1.7 m dp ),
◦
FR = 0.35 mL/min, 90 C,
ESI+/− 120 min
Buyang Huanwu Flavan-3-ols, TOF C18 (50 mm × 4.6 mm, A: 0.3% FA, B: [490]
a chalcones, UV ACN
decoction (TCM ) 1.8 m dp ),
◦
flavonols FR = 0.8 mL/min, 25 C,
glycosides and 30 min
isoflavones (24
flavonoids) ESI+/− C18 (50 mm × 4.6 mm,
Flos Lonicerae Japonicae Flavone and TOF A: 0.1% FA, B: [297]
(TCM) flavonol UV 1.8 m dp ), FR = 0.7 and ACN
glycosides (10 0.8 mL/min (0.35 and
flavonoids) 0.4 mL/min to MS),
temperature gradient
ESI− ◦
22–35 C, 25 min
b
Morus alba leaves Flavonol IT -TOF C18 (150 mm × 4.6 mm, A: H2 O (pH 3, [258]
glycosides (13 UV c FA), B: ACN (pH
2.7 m dp SP ),
flavonoids) FR = 1.0 mL/min 3, FA)
(0.2 mL/min to MS),
◦
25 C, 50 min
Hops (Humulus lupulus Flavan-3-ols, ESI+ Q-TOF C18 (50 mm × 2.1 mm, A: 0.1% FA and [491]
L.) flavonol UV 1.8 m dp ), 50 M
◦
aglycones and FR = 0.25 mL/min, 45 C, ammonium
glycosides 36 min formate, B: 95%
ACN containing
0.1% FA and
50 M
ammonium
ESI− C18 (50 mm × 4.6 mm, formate
Radix paeoniae rubra Catechin TOF A: 0.3% FA, B: [492]
(TCM) UV 1.8 m dp ), 0.3% FA in ACN
◦
FR = 0.8 mL/min, 25 C,
ESI− 24 min
Durum wheat Anthocyanidins, TOF C18 (150 mm × 4.6 mm, d [493]
A: 0.5% AA , B:
flavone UV 1.8 m dp ), ACN
aglycones and FR = 0.5 mL/min
glycosides, (0.125 mL/min to MS),
isoflavones and ◦
40 C, 62 min
proanthocyani-
dins (42
flavonoids) ESI− C18 (50 mm × 4.6 mm,
Radix Puerariae (TCM) Isoflavones (13 TOF A: 0.1% FA, B: [494]
compounds) UV d
1.8 m dp ), MeOH
FR = 2.0 mL/min
(0.5 mL/min to MS),
ESI+/− ◦
46 C, 8 min
Wild Mexican Lupine Flavone and TOF C18 (100 mm × 2.1 mm, A: 0.5% FA, B: [495]
(Lupinus reflexus) isoflavone ESI+/− IT 1.8 m dp ), 0.5% FA in ACN
conjugates (62 FR = 0.5 mL/min
flavonoids) (0.2 mL/min to MS),
ESI+/− 60 min
Granule decoctions of Flavone Q-TOF C18 (100 mm × 2.1 mm, A: 50 mM [496]
TCM aglycones and UV 1.7 m dp ), ammonium
◦
glycosides, FR = 0.5 mL/min, 35 C, acetate, B: ACN
flavonols and 12 min
flavanones (13
flavonoids) ESI− C18 (150 mm × 4.6 mm,
Buckwheat Flavonols, TOF A: 1% AA, B: [497]
flavan-3-ols d
1.8 m dp ), MP A/ACN
and FR = 0.5 mL/min (60/40, v/v)
procyanidins (0.125 mL/min to MS),
(28 flavonoids) ESI− ◦
25 C, 70 min
Rock rose (Cistus Flavan-3-ols, TOF C18 (150 mm × 4.6 mm, A: 0.5% AA, B: [498]
ldanifer) flavonols and ESI− IT 1.8 m dp ), ACN
flavones (13 UV FR = 0.8 mL/min
flavonoids) (0.25 mL/min to MS),
70 min
52 A. de Villiers et al. / J. Chromatogr. A 1430 (2016) 16–78
Table 6 (Continued )

Sample Flavonoids Ionisation Detector(s) Stationary phase Mobile References

source (column dimensions), phase(s)
flow rate, temperature,
analysis time
Erigeron injection Flavonol, ESI− Q-TOF C18 (50 mm × 4.6 mm, A: 0.2% FA, B: [499]
flavone and ESI− QIT 1.8 m dp ), FR not ACN
◦
flavanone specified, 40 C, 30 min
glycosides (11
flavonoids) ESI+/− C18 (100 mm × 2.1 mm,
Tempranillo red wine Proanthocyanidins TOF A: 5% FA, B: [500]
(9 compounds) UV 1.7 m dp ), ACN
◦
FR = 0.2 mL/min, 40 C,
ESI+/− 13.6 min
Tomato fruit Aglycones and TOF C18 (150 mm × 4.6 mm, A: 0.5% AA, B: [501]
glycosides of ESI+/− IT 1.8 m dp ), ACN
flavonols, UV FR = 1.8 mL/min
flavanones and (0.2 mL/min to MS),
flavones (20 ◦
37 C, 46 min
flavonoids) C18 (100 mm × 2.1 mm,
Fructus Aurantii, TCM Flavanone ESI+ Q-TOF A: 0.1% FA, B: [502]
extracts and rat glycosides and UV 1.7 m dp ), ACN
◦
plasma methoxylated FR = 0.4 mL/min, 25 C,
flavones (21 15 min
flavonoids) and
4 metabolites ESI+/− C18 (100 mm × 4.6 mm,
Standards Flavonols, TOF A: 0.5% FA, B: [164]
flavones, 1.8 m dp ), 0.5% FA in ACN
◦
anthocyanins FR = 0.5 mL/min, 40 C,
and flavanone 45 min
aglycones and
glycosides (19
flavonoids) C18 (50 mm × 4.6 mm,
Yinhuang granules Flavone ESI+ TOF A: 0.2% FA, B: [478]
(TCM) aglycones and UV 1.8 m dp ), ACN
◦
glycosides (7 FR = 0.6 mL/min, 25 C,
flavonoids) 17 min
Rooibos tea (Aspalathus Flavones, ESI+ Q-TOF C18 (200 mm × 2.1 mm, A: 2% AA, B: [216]
linearis) flavanone dihy- UV 1.7 m dp ), ACN
◦
drochalcones FR = 0.3 mL/min, 30 C,
and flavonols 31 min
(28 flavonoids) ESI+/− C18 (100 mm × 2.1 mm,
Avocado fruit Flavan-3-ols, TOF A: 0.1% FA, B: [285]
flavanones and UV 1.8 m dp ), 0.1% FA in ACN
◦
flavonols (6 FR = 0.6 mL/min, 50 C,
flavonoids) ESI− 45 min
Wheat varieties Flavone-O,C- TOF C18 (150 mm × 4.6 mm, A: 0.5% AA, B: [503]
diglycosides, 1.8 m dp ), ACN
flavones, FR = 0.5 mL/min
isoflavones, (0.125 mL/min to MS),
anthocyanins ◦
40 C, 72 min
and proantho-
cyanidins (67
flavonoids) ESI+/− C18 (150 mm × 4.6 mm,
Rosemary (Rosmarinus Flavone TOF A: 0.1% FA, B: [504]
officinalis) aglycones and UV 1.8 m dp ), ACN
glycosides, FR = 0.8 mL/min
flavonol (0.2 mL/min to MS),
glycosides, 55 min
flavanones and
flavonols (24
flavonoids) ESI− C18 (50 mm × 4.6 mm,
Licorice (Glycyrrhiza) Flavones, Q-TOF A: 0.2% FA, B: [488]
isoflavones, UV 1.8 m dp ), ACN
◦
isoflavane, FR = 0.7 mL/min, 30 C,
chalcones and 36 min
flavanones (21
flavonoids) C18 (100 mm × 2.1 mm,
Medicago truncatula Flavones, ESI+ Q-TOF A: 0.5% FA, B: [479]
root samples isoflavones and 1.8 m dp ), 0.5% FA in ACN
flavanones (13 FR = 0.5 mL/min
flavonoids) (0.2 mL/min to MS),
19 min
Red wine Anthocyanins ESI+ Q-TOF C18 A: 7.5% FA, B: [218]
and derived UV (2 × 100 mm × 2.1 mm, 7.5% FA in ACN
pigments and 1.7 m dp ),
◦
proantho- FR = 0.06 mL/min, 50 C,
cyanins (137 98 min
flavonoids)
 A. de Villiers et al. / J. Chromatogr. A 1430 (2016) 16–78 53
Table 6 (Continued )

Sample Flavonoids Ionisation Detector(s) Stationary phase Mobile References

source (column dimensions), phase(s)
flow rate, temperature,
analysis time
Rooibos tea (Aspalathus Flavonol and ESI− TOF C18 (150 mm × 4.6 mm, A: 1% FA in [424]
linearis) flavone IT 1.8 m dp ), H2 O/ACN
aglycones and FR = 0.5 mL/min (90/10, v/v), B:
glycosides, (0.2 mL/min to MS), ACN
flavanone and ◦
25 C, 35 min
chalcone
glycosides (26
flavonoids) ESI+/− C18 (150 mm × 2.0 mm,
Hop cones (Humulus Flavonol TOF A: 0.1% FA, B: [259]
lupulus L.) aglycones and 2.6 m dp SP), 0.1% FA in ACN
◦
glycosides, FR = 0.2 mL/min, 45 C,
flavan-3-ols, 78 min
chalcones and
naringenin (13
flavonoids) ESI− C18 (50 mm × 4.6 mm,
Polygonaceae herbs Epicatechin Q-TOF A: 0.1% FA, B: [505]
(TCM) UV 1.8 m dp ), ACN
FR = 0.8 mL/min
(0.4 mL/min to MS),
◦
25 C, 32 min
Plant material Flavonol and ESI+ TOF C18 (100 mm × 4.6 mm, A: 0.5% FA, B: [193]
flavone 1.8 m dp ), 0.5% FA in ACN
glycosides, FR = 0.5 mL/min, 45 min
anthocyanins
and flavanones
(17 flavonoids) ESI− C18 (50 mm × 2.1 mm,
Merlot (Vitis vinifera L.) Flavan-3-ol Q-TOF A: 0.1% FA, B: [483]
seeds and wine monoglyco- UV 1.8 m dp ), 0.1% FA in ACN
◦
sides (14 FR = 0.4 mL/min, 30 C,
compounds) 27 min
Fructus aurantii (TCM) Flavanone ESI+ Q-TOF C18 (50 mm × 2.1 mm, A: 1% AA, B: [506]
glycosides and UV 1.7 m dp ), ACN
◦
methylated FR = 0.2 mL/min, 40 C,
flavones (6 12 min
flavonoids) ESI+/− C18 (100 mm × 4.6 mm,
Rooibos tea (Aspalathus Flavones, Q-TOF A: 2% AA, B: [210]
linearis) flavanones UV 1.8 m dp ), ACN
◦
dihydrochal- FR = 1.0 mL/min, 38 C,
cones and 45 min
flavonols (26
flavonoids) ESI+/− C18 (100 mm × 1.0 mm,
Hops (Humulus lupulus Chalcones, Q-TOF A: 0.1% FA, B: [507]
L.) flavanones and 1.8 m dp ), 0.1% FA in ACN
flavonols (12 FR = 0.15 mL/min,
flavonoids) ESI+/− 20 min
a Isoorientin, TOF C18 (100 mm × 2.1 mm, A: 0.1% FA, B: [508]
Galium spurium (TTM )
rutin, 1.7 m dp ), 0.1% FA in ACN
◦
isoquercitrin FR = 0.25 mL/min, 40 C,
and quercetin ESI+/− 26 min
Wheat (Triticum Flavone Q-TOF C18 (100 mm × 2.1 mm. A: 0.5% FA, B: [480]
aestivum L.) glycosides (41 c 0.5% FA in ACN
2.7 m dp SP ),
compounds) FR = 0.6 mL/min
(0.2 mL/min to MS),
15 min
Blueberries, red Anthocyanins ESI+ Q-TOF Amide A: 0.4% TFA in [128]
cabbage, red radish, (64 UV (150 mm × 1.0 mm, ACN, B: 0.4%
grape skins and black compounds) 1.7 m dp ), TFA
beans FR = 0.025 mL/min,
ESI+/− ◦
50 C, 27–40 min
Potato extracts Anthocyanins Q-TOF C18 (100 mm × 2.1 mm, A: 0.5% FA, B: [292]
and flavonol 1.8 m dp ), 90% MeOH
◦
derivatives (11 FR = 0.4 mL/min, 50 C,
flavonoids) ESI− 14 min
Hops (Humulus lupulus Proanthocyanidins TOF C18 (50 mm × 2.0 mm, A: 0.1% FA, B: [509]
L.) (40 1.7 m dp ), 0.1% FA in ACN
◦
compounds) FR = 0.4 mL/min, 35 C,
ESI− 9 min
Artichoke leaf Flavones Q-TOF C18 (100 mm × 1.0 mm, A: 0.1% FA, B: [510]
aglycones and ESI− IT 1.8 m dp ), 0.1% FA in ACN
glycosides and UV FR = 0.15 mL/min,
hesperetin 16 min
glycoside (12
flavonoids)
54 A. de Villiers et al. / J. Chromatogr. A 1430 (2016) 16–78
Table 6 (Continued )

Sample Flavonoids Ionisation Detector(s) Stationary phase Mobile References

source (column dimensions), phase(s)
flow rate, temperature,
analysis time
Lettuce (Lactuca sativa) Flavonol, ESI− Q-TOF C18 (150 mm × 4.6 mm, A: 0.5% AA, B: [511]
flavone and 1.8 m dp ), ACN
flavanone FR = 0.8 mL/min, 40 min
glycosides (23
flavonoids) ESI−
Trollius species (TCM) Flavone Q-TOF Phenyl A: 0.05% FA, B: [89]
glycosides (34 (100 mm × 2.1 mm, ACN
flavonoids) 1.7 m dp ),
◦
FR = 0.4 mL/min, 25 C,
30 min
Tea plant (Camellia Methylated ESI+ Q-TOF C18 (100 mm × 2.1 mm, A: 0.5% FA [482]
sinensis) kaempferol and 1.8 m dp ), 2 mM
◦
quercetin FR = 0.2 mL/min, 30 C, ammonium
regioisomers 30 min formate, B:
(10 flavonoids) ESI− C18 (150 mm × 4.6 mm, ACN
Peppers (Capsicum Flavonol, TOF A: 0.5% AA, B: [191]
annuum L.) flavone and 1.8 m dp ), ACN
flavanone FR = 0.8 mL/min, 62 min
glycosides and
phloretin
dihexoside (23
flavonoids) ESI+/− C18 (100 mm × 4.6 mm,
Dan-Lou tablets (TCM) Isoflavone and Q-TOF A: 0.1% FA, B: [288]
flavanone UV 1.8 m dp ), ACN
◦
glycosides (28 FR = 0.4 mL/min, 50 C,
flavonoids) ESI+/− 40 min
Canola (Brassica napus Flavonol Q-TOF C18 (100 mm × 1.0 mm, A: 0.1% FA, B: [512]
L.) organs aglycones and UV 1.8 m dp ), 0.1% FA in ACN
glycosides and FR = 0.15 mL/min,
naringenin (26 20 min
flavonoids) ESI− C18 (150 mm × 4.6 mm,
Whole lemon powder Flavanone, TOF A: 0.5% AA, B: [513]
flavonol and UV 1.8 m dp ), ACN
flavone FR = 0.8 mL/min
aglycones and (0.2 mL/min to MS),
glycosides (16 ◦
25 C, 35 min
flavonoids) ESI− C18 (50 mm × 2.0 mm,
Halophyte Zygophyllum Isorhamnetin- TOF A: 0.1% FA, B: [514]
album Desf. 3-O-rutinoside, UV 1.8 m dp ), ACN
◦
malvidin FR = 0.4 mL/min, 23 C,
3-rhamnoside 70 min
and quercetin
3-sulphate ESI− C18 (50 mm × 2.1 mm,
Cranberrybush Procyanidins (6 TOF A: 0.1% FA, B: [515]
(Viburnum Opulus L.) compounds) UV 1.7 m dp ), MeOH
◦
FR = 0.4 mL/min, 25 C,
ESI− 8 min
Citrus bergamia juice Flavanone IT-TOF C18 (100 mm × 2.1 mm, A: 0.1% FA, B: [487]
glycosides (17 UV 1.7 m dp SP), 0.1% FA in ACN
compounds) FR = 0.7 mL/min
(0.35 mL/min to MS),
◦
45 C, 6.1 min
Licorice (Raddix Liquiritin, ESI+ Q-TOF C18 (50 mm × 2.1 mm, A: 0.5% AA, B: [516]
Glycyrrhizae) liquiritin UV 1.8 m dp ), ACN
◦
apioside and FR = 0.2 mL/min, 30 C,
liquiritigenin ESI+/− 10 min
Apple, apple juice and Flavanols, Q-TOF C18 (100 mm × 2.1 mm, A: 0.1% AA, B: [481]
apple cider flavonols, dihy- 1.7 m dp ), 0.1% AA in
◦
drochalcones FR = 0.35 mL/min, 40 C, MeOH
and 22 min
anthocyanins
(42 flavonoids) ESI− C18 (100 mm × 2.1 mm,
Licorice (Glycyrrhiza Flavanones, Q-TOF A: 0.1% FA, B: [143]
species) flavones, UV 1.8 m dp ), ACN
◦
isoflavones and FR = 0.6 mL/min, 50 C,
chalcones (45 9 min
flavonoids) ESI− C18 (150 mm × 4.6 mm,
Melon (Cucumis melo Eriodictoyl and Q-TOF A: 0.5% AA, B: [517]
L.) diosmetin 1.8 m dp ), ACN
rutinosides and FR = 0.8 mL/min
hesperidin (0.2 mL/min to MS),
24 min
 A. de Villiers et al. / J. Chromatogr. A 1430 (2016) 16–78 55
Table 6 (Continued )

Sample Flavonoids Ionisation Detector(s) Stationary phase Mobile References

source (column dimensions), phase(s)
flow rate, temperature,
analysis time
Green beans (Phaseolus Flavonols, ESI− Q-TOF C18 (150 mm × 4.6 mm, A: 0.5% AA, B: [518]
vulgaris L.) flavones, 1.8 m dp ), 0.5% AA in ACN
flavanones, FR = 0.8 mL/min
flavan-3-ols (0.2 mL/min to MS),
and isoflavones ◦
25 C, 35 min
(60 flavonoids) ESI+/− C18 (100 mm × 2.1 mm,
Standards Flavone Q-TOF A: 0.1% FA [177]
glycosides (4 1.8 m dp ), (−mode) or
compounds) FR = 0.2 mL/min, 0.1%
analysis time not ammonium
specified formate
(+mode), B:
0.1% FA
(−mode) or
0.1%
ammonium
formate
(+mode) in
ESI− C18 (150 mm × 2.1 mm, MeOH
Knotgrass (Polygonum Flavonol Q-TOF A: H2 O/ACN/FA [407]
aviculare L.) glycosides (24 ESI/APCI+/− IT 1.9 m dp ), (95:5:0.1,
◦
compounds) UV FR = 0.2 mL/min, 25 C, v/v/v), B: 0.1%
ESI+/− 100 min FA in ACN
Shenqi Fuzheng Flavone and Q-TOF C18 (100 mm × 2.1 mm, A: 0.1% FA, B: [519]
Injection (TCM) isoflavone 1.8 m dp ), MeOH
◦
glycosides (12 FR = 0.3 mL/min, 25 C,
flavonoids) ESI+/− 40 min
Siwu decoction (TCM) Flavonols and Q-TOF C18 (100 mm × 2.1 mm, A: 0.1% FA, B: [520]
flavone UV 1.7 m dp ), ACN
◦
glycosides (18 FR = 0.4 mL/min, 35 C,
flavonoids) ESI− 25 min
Waterlemon (Citrullus Flavonol and Q-TOF C18 (150 mm × 4.6 mm, A: 0.5% AA, B: [521]
lanatus) extract flavone 1.8 m dp ), ACN
glycosides (23 FR = 0.8 mL/min
flavonoids) (0.2 mL/min to MS),
ESI− ◦
25 C, 35 min
‘Chemlali’ Olive Flavone, TOF C18 (150 mm × 4.6 mm, A: 0.5% AA, B: [426]
cultivar flavonol ESI− IT 1.8 m dp ), ACN
◦
aglycones and UV FR = 0.8 mL/min, 25 C,
glycosides and 35 min
taxifolin (10
flavonoids) ESI− C18 (50 mm × 2.1 mm,
Sprouts (Lepidium Flavonol Q-TOF A: 4.5% FA, B: [522]
sativum L.) glycosides (4 UV 1.7 m dp ), ACN
◦
compounds) FR = 0.45 mL/min, 30 C,
ESI− 15 min
Wine Flavan-3-ols, TOF Serially coupled C18 C18: A: 10 mM [132]
flavonols and (50 mm × 3.0 mm, ammonium
eriodictyol-7- 2.7 m dp SP), acetate/ACN
glucoside (5 FR = 0.05–0.1 mL/min (90:10, v/v), B:
flavonoids) and HILIC A: 10 mM
(150 mm × 2.1 mm, ammonium
5 m dp ), acetate/ACN
FR = 0.4 mL/min, 27 min (10:90, v/v)
HILIC: A: ACN,
ESI− C18 (150 mm × 4.6 mm, B: H2 O
Spanish Cistus species Flavan-3-ols, TOF A: 0.5% AA, B: [523]
flavonol 1.8 m dp ), ACN
aglycones and FR = 0.8 mL/min, 60 min
glycosides,
flavanone and
isoflavone
glycosides (29
flavonoids) ESI− C18 (100 mm × 3.0 mm,
Apple peel Flavonol TOF A: 0.1% FA, B: [460]
glycosides, ESI− b 0.1% FA in ACN
QqQ 2.7 m dp SP),
◦
flavan-3-ols FR = 0.3 mL/min, 50 C,
and 40 min
procyanidins ESI− C18 (100 mm × 2.1 mm,
Gegen-Qinlian-Wan Flavones, Q-TOF A: 0.1% FA, B: [524]
(TCM) isoflavones, UV 1.8 m dp ), ACN
◦
chalcones and FR = 0.3 mL/min, 40 C,
flavanones (42 30 min
flavonoids)
56 A. de Villiers et al. / J. Chromatogr. A 1430 (2016) 16–78
Table 6 (Continued )

Sample Flavonoids Ionisation Detector(s) Stationary phase Mobile References

source (column dimensions), phase(s)
flow rate, temperature,
analysis time
Citrus grandis Flavanones, ESI+/− Q-TOF C18 (100 mm × 2.1 mm, A: 0.1% FA, B: [525]
Tomentosa (TCM) flavones and 2.6 m dp SP), MeOH
◦
flavonols (19 FR = 0.3 mL/min, 40 C,
flavonoids) ESI− 16 min
Horehound Flavone Q-TOF C18 (150 mm × 4.6 mm, A: 0.5% AA, B: [526]
(Marrubium vulgare) aglycones and 1.8 m dp ), ACN
leaves glycosides (9 FR = 0.5 mL/min
flavonoids) (0.167 mL/min to MS),
ESI+/− ◦
25 C, 45 min
Brazilian pepper Anthocyanins Q-TOF C18 (150 mm × 2.1 mm, Anthocyanins: [96]
exocarp (Schinus and biflavones QqQ 2.6 m dp SP), A: 1% FA, B:
◦
terebinthifolius Raddi) (7 flavonoids) UV FR = 0.4 mL/min, 40 C, 0.1% FA in ACN;
28 min non-
anthocyanins:
A: 0.1% FA, B:
ESI+/− C18 (100 mm × 2.1 mm, 0.1% FA in ACN
Orris root (Iris Flavonol TOF A: 5 mM [527]
rhizomes) aglycones and UV 1.8 m dp ), ammonium
◦
glycosides and FR = 0.4 mL/min, 45 C, formate, pH
isoflavones (18 12 min 3.8, B:
flavonoids) MeOH/ACN
(50:50, v/v)
with 5 mM
ammonium
formate and
C18 (100 mm × 2.1 mm, 0.1% FA
Rhizome of Belamcanda Isoflavone ESI+ Q-TOF A: 0.1% FA, B: [443]
chinensis (TCM) aglycones and ESI+ QqQ 1.7 m dp ), 0.1% FA in ACN
◦
glycosides (26 FR = 0.4 mL/min, 20 C,
flavonoids) ESI− 25 min
Mugwort (Artemisia Flavonol Q-TOF C18 (150 mm × 4.6 mm, A: H2 O/ACN [528]
vulgaris) leaves glycosides and 1.8 m dp ), (90:10, v/v)
luteolin FR = 0.5 mL/min, room with 0.1% FA, B:
rutinoside (5 temp, 50 min ACN
flavonoids) ESI+/− C18 (50 mm × 2.1 mm,
Raw and Isoflavones and Q-TOF A: 0.1% FA, B: [109]
honey-processed isoflavanes (12 1.8 m dp ), ACN
◦
astragalus (TCM) flavonoids) FR = 0.3 mL/min, 25 C,
ESI+/− 23 min
Wu-tou decoction Isoflavone, Q-TOF C18 (50 mm × 2.1 mm, A: 0.1% FA, B: [529]
(TCM) chalcones and 1.7 m dp ), ACN
◦
flavanones (9 FR = 0.3 mL/min, 35 C,
flavonoids) ESI− 25 min
Leaves of Myrtus Flavan-3-ol Q-TOF C18 (150 mm × 4.6 mm, A: 0.5% AA, B: [530]
communis L. derivatives and 1.8 m dp ), ACN
◦
flavonol FR = 0.8 mL/min, 25 C,
glycosides (10 32 min
flavonoids) ESI− C18 (150 mm × 4.6 mm,
Taselgha (Globularia Flavone, Q-TOF A: 0.5% AA, B: [531]
alypum L.) flavanone and 1.8 m dp ), ACN
flavonol FR = 0.5 mL/min
glycosides, (0.167 mL/min to MS),
flavan-3-ol ◦
25 C, 45 min
derivatives,
flavonols and
flavanonols (11
flavonoids) ESI− C18 (100 mm × 2.1 mm,
Goji berry (Lycium Flavonol Q-TOF A: 0.1% FA, B: [289]
barbarum) extracts glycosides (8 1.8 m dp ), 0.1% FA in
◦
flavonoids) FR = 0.3 mL/min, 50 C, MeOH
ESI− 19.5 min
Commercial extra Apigenin and TOF C18 (100 mm × 4.6 mm, A: 0.1% FA, B: [532]
virgin olive oil luteolin 1.8 m dp ), ACN
ESI+/− FR = 0.5 mL/min, 30 min
Da-Huang-Gan-Cao- Flavan-3-ols, Q-TOF C18 (50 mm × 4.6 mm, A: 0.1% FA, B: [533]
Tang chalcones, ESI+/− IT 1.8 m dp ), ACN
◦
(TCM) isoflavones and UV FR = 0.6 mL/min, 30 C,
flavones (25 50 min
flavonoids) ESI−
Hedyotis diffusa Willd. Flavonol and Q-TOF c A: 0.1% FA, B: [97]
C18 PE
(TCM) flavone UV (100 mm × 2.1 mm, ACN
glycosides (7 1.7 m dp ),
flavonoids) ◦
FR = 0.25 mL/min, 25 C,
65–77 min
 A. de Villiers et al. / J. Chromatogr. A 1430 (2016) 16–78 57
Table 6 (Continued )

Sample Flavonoids Ionisation Detector(s) Stationary phase Mobile References

source (column dimensions), phase(s)
flow rate, temperature,
analysis time
Danggui San (TCM) Flavone ESI+/− Q-TOF C18 (100 mm × 2.1 mm, A: 0.1% FA, B: [534]
aglycones and 1.7 m dp ), 0.1% FA in ACN
◦
glycosides (18 FR = 0.3 mL/min, 35 C,
flavonoids) ESI− 18 min
Fabaceae (Vicia faba L.) Flavan-3-ols, Q-TOF C18 (150 mm × 4.6 mm, A: 0.5% AA, B: [535]
proanthocyani- 1.8 m dp ), ACN
dins, flavonols, FR = 0.8 mL/min
flavanonols, (0.2 mL/min to MS),
flavones and ◦
25 C, 40 min
flavanones (86
flavonoids) ESI− C18 (100 mm × 1.0 mm,
Black cumin seeds Flavonol Q-TOF A: 0.1% FA, B: [536]
(Nigella sativa) glycosides (10 1.8 m dp ), 0.1% FA in ACN
flavonoids) FR = 0.15 mL/min,
ESI− 20 min
Hawthorn fruits Epicatechin, Q-TOF C18 (50 mm × 2.1 mm, A: 0.1% FA, B: [537]
hyperoside, 1.8 m dp ), ACN
◦
isoquercitrin FR = 0.4 mL/min, 35 C,
and quercetin ESI− 12 min
Annurca apple (Malus Dihydrochalcones, IT-TOF C18 (150 mm × 2.1 mm, A: 0.1% AA, B: [124]
pumila Miller cv anthocyanins, 2.6 m dp SP), 0.1% AA in ACN
◦
Annurca) flavonols, FR = 0.5 mL/min, 40 C,
flavanones, 27 min
flavan-3-ols
and
procyanidins
(57 flavonoids) ESI− C18 (150 mm × 4.6 mm,
Leaves of Adhatoda Flavone and Q-TOF A: 0.1% FA, B: [261]
vasica flavonol UV 2.7 m dp SP), MeOH
◦
aglycones and FR = 0.6 mL/min, 20 C,
glycosides (29 30 min
flavonoids) ESI+/− C18 (150 mm × 4.6 mm,
‘Temri’ and ‘Tounsi’ Flavonols, Q-TOF A: 0.5% AA, B: [538]
(Ficus carica L.) fruits flavones, UV 1.8 m dp ), ACN
flavanones, FR = 0.5 mL/min, room
flavanonols, temp, 40 min
flavanols,
isoflavones and
anthocyanins
(39 flavonoids) ESI+/− C18 (100 mm × 2.1 mm,
Scutellariae species Flavone Q-TOF A: 0.1% FA, B: [539]
(TCM) aglycones and QqQ 1.7 m dp ), ACN
glycosides, UV FR = 0.4 mL/min, 20 min
flavanonol
glycosides,
isoflavones,
flavanones and
flavonols (28
flavonoids) ESI+/− C18 (150 mm × 4.6 mm,
Araceae (Arum Flavone, Q-TOF A: 0.5% AA, B: [540]
palaestinum) flavonol, UV 1.8 m dp ), ACN
◦
flavanone and FR = 0.8 mL/min, 25 C,
flavanonol 70 min
glycosides,
anthocyanins,
isoflavones,
flavonol-
phenolic acid
derivatives (57
flavonoids) ESI+/− C18 (100 mm × 2.1 mm,
Euphorbiaceae Flavanone, Q-TOF A: 0.1% FA, B: [541]
(Phyllanthus amarus) flavonol, ESI+/− IT 1.7 m dp ), ACN
◦
flavone UV FR = 0.3 mL/min, 30 C,
aglycones and 57 min
glycosides (18
flavonoids) ESI− C18 (150 mm × 4.6 mm,
Egyptian chickpea Flavonols, Q-TOF A: 0.5% AA, B: [262]
cultivars (Cicer isoflavones, UV 2.7 m dp SP), ACN
arientinum L.) isoflavanones FR = 0.5 mL/min, 35 min
and
flavan-3-ols
(47 flavonoids)
58 A. de Villiers et al. / J. Chromatogr. A 1430 (2016) 16–78
Table 6 (Continued )

Sample Flavonoids Ionisation Detector(s) Stationary phase Mobile References

source (column dimensions), phase(s)
flow rate, temperature,
analysis time
Berries (Rhodomyrtus Flavones and ESI+ Q-TOF C18 (100 mm × 2.1 mm, A: H2 O, B: ACN [542]
tomentosa (Ait.) flavonols (6 1.7 m dp ),
◦
Hassk) flavonoids) FR = 0.3 mL/min, 30 C,
ESI+/− 30 min
Jinqi Jiangtang tablet Rutin, Q-TOF C18 (50 mm × 2.1 mm, A: 0.2% [543]
(TCM) luteoloside and 1.7 m dp ), phosphoric
◦
isoquercitrin FR = 0.3 mL/min, 35 C, acid, B: MeOH
ESI− 21 min
Rhubarb species Flavan-3-ol Q-TOF C18 (150 mm × 2.1 mm, A: 0.1% FA, B: [265]
derivatives, 1.6 m dp SP), ACN
◦
procyanidins FR = 0.3 mL/min, 40 C,
and flavanone 29 min
glycosides (15
flavonoids) C18 (150 mm × 2.1 mm,
Ginkgo biloba Isoflavone and ESI+ Q-TOF A: 0.1% FA, B: [544]
flavonol UV 2.7 m dp SP), 0.1% FA in ACN
aglycones and FR = 0.2 mL/min, 21 min
glycosides (22
flavonoids) ESI− C18 (150 mm × 4.6 mm,
Mentha pulegium and Flavanols, Q-TOF A: 0.5% AA, B: [545]
Origanum Majorana flavones, 1.8 m dp ), ACN
◦
species flavanonols, FR = 0.8 mL/min, 25 C,
flavanone and 35 min
flavonols (33
flavonoids) ESI− C18 (100 mm × 2.1 mm,
Shuang-Huang-Lian Flavone Q-TOF A: 0.1% FA, B: [546]
oral liquid (TCM) aglycone and b 0.1% FA in ACN
ELSD 1.7 m dp ),
◦
glycosides (4 FR = 0.4 mL/min, 40 C,
flavonoids) 24 min
a
TCM, traditional chinese medicine, TTM, traditional Turkish medicine.
c
b Q, quadrupole; TOF, time-of-flight mass spectrometer; IT, ion trap; QqQ, triple quadrupole mass spectrometer; ELSD, evaporative light scattering detector. FR, flow rate; SP,
superficially porous; PE, polar embedded.
d
TFA, trifluoroacetic acid; ACN, acetonitrile; FA, formic acid; AA, acetic acid; MeOH, methanol; MP, mobile phase.

Fig. 16. (A) UV chromatogram obtained at 280 nm for the UHPLC–Q-TOF-MS analysis of an apple extract. (B) The extracted ion chromatograms for phloretin-2 -O-xylosyl-glucoside (m/z 569) and
E
quercetin-3-O-xyloside (m/z 435) and (C) the corresponding MS spectra for the compounds obtained using the simultaneous (non-selective) acquisition of spectral data using a collision energy ramp
◦
of 10–40 eV. Analysis conditions: 100 mm × 2.1 mm 1.7 m porous C18 phase, 40 C, 0.35 mL/min; ESI+ detection at 10 Hz.

Source: Reproduced with permission from [481].

 A. de Villiers et al. / J. Chromatogr. A 1430 (2016) 16–78
1,1-diphenyl-2-picrylhydrazyl (DPPH) radical to determine the most active
antioxidants. This methodology proved to be more sensitive than the
conventional on-line DPPH assay.
Q-TOF-MS has also been hyphenated with HILIC [128], with an amide
◦ high as 500,000, depending on the particular instrument, scan mode and scan
column operated at 50 C being used for the analysis of diverse anthocyanins
in blueberries, grape skins, black beans, red cabbage and red radish.
The widespread use of TOF, and especially Q-TOF detectors in
contemporary LC–MS analysis of flavonoids makes sense in light of the high
resolution mass data and high acquisition rates offered by these detectors and
their relative cost compared to higher resolv-ing power instruments. The mass
accuracy of the latest generation TOF detectors are sufficient to allow
determination of molecular formulae for flavonoids of MW up to ∼1200
[128] and to distinguish between isobaric compounds based on this data.
Furthermore, the MS/MS capabilities of Q-TOF systems are critical in
tentative struc-tural elucidation of flavonoids. Wojakowska et al. [480] found
a resolving power of higher than 15,000 sufficient for molecular for-mula
determined for flavonoids of MW up to 700 in first order spectra, but
estimated that a resolving power of 40,000 would be needed to attain the
same level of mass accuracy of MS/MS spec-tra. This is within the reach of
several commercial instruments. While the mass accuracy of Orbitrap and
especially FT-ICR sys-tems are certainly much better than TOF systems, and
therefore hold clear benefits for the separation of high MW compounds such
as proanthocyanins, the performance of TOF systems for ultra-fast separations
currently remains unmatched between the high reso-lution instruments.
4.2.3. Fourier transform ion cyclotron resonance (FT-ICR) MS FT-ICR
[547,548] is the mass spectrometric technique with the
6
highest mass resolution (up to greater than 10 ) and sub-femtomol sensitivity.
The technique works on the principle of exciting all ions in the cyclotron
simultaneously and measuring the ion image resulting from their combined
trajectories, which is amplified and translated to the frequency domain using
FT [423]. The res-olution depends on the observation time, and as a
consequence FT-ICR instruments are too slow to allow on-line hyphenation to
UHPLC separation, and are generally used only in infusion mode for
flavonoid analysis [507]. An alternative is to use at-line or off-line coupling to
HPLC separations, which essentially involves collection of fractions from the
chromatographic system and subsequent infu-sion into the FT-ICR. While
time consuming and labour intensive, this is currently the most practical way
to exploit the high resolving power of FT-ICR in combination with HPLC
separation, and shows promise for example in metabolomic analysis [42]. By
2006 FT-ICR had not been used in the LC–MS analysis of flavonoids [145],
and to the best of our knowledge the only application of LC–FTICR-MS to
flavonoid analysis since then was reported by Suzuki et al. [549]. These
authors used conventional HPLC (5 m ODS phase) with posi-tive mode ESI
to characterise flavonoids in Lotus japonicas. The mass spectrometer was
operated with a resolving power of 100,000 (at m/z 400), although no
information was provided on the scan time and the TIC was not presented.
On-going developments aimed at extending the resolving power and reducing
the time domain tran-sient length of FT-ICR instruments may in future allow
hyphenation of the detector to fast HPLC separations [42].
4.2.4. Orbitrap instruments
The Orbitrap mass analyser, first described by Makarov [550,551], was
introduced on the market in 2005. This system relies on the orbital trapping of
ions around a spindle-like central elec-trode and an outer flared barrel-like
electrode using electrostatic fields. The frequency of the harmonic ion
oscillations, which are inversely proportional to the square root of the m/z
ratios, is mea-sured as an image current transient which is converted to
frequency
59
spectrum using FT [421]. In the first commercial configuration (LTQ-
Orbitrap), ions are initially stored in a linear ion trap, from where they are co-
axially ejected into a bent quadrupole or ‘C-trap’ to focus and quickly inject
them into the Orbitrap. The resolving power of these instruments can be as
time.
Various forms of bench-top Orbitrap mass spectrometers have been
TM
utilised in the analysis of flavonoids. In the LTQ-Orbitrap XL a collision
cell is added after the C-Trap to perform CID for MS/MS analyses; the LTQ-
Orbitrap Velos shows increased MS/MS sensi-tivity due a more effective ion
TM
guide. The Orbitrap Elite uses a smaller Orbitrap with an increased electric
field, increasing the resolving power of the instrument to 240,000 at m/z =
400. A mod-ified version of this instrument utilising a compact analyser has
been shown to attain a resolving power of 960,000 at m/z 400 for a 3 s
TM
detection time [552]. In the Orbitrap Exactive , the ion source is directly
connected to the ‘C Trap’ followed by a collision cell before the Orbitrap
analyser. Ions that underwent fragmentation in the collision cell are
transferred back into the ‘C-Trap’ before being injected into the Orbitrap for
TM
mass analysis. The latest edi-tion is the non-hybrid Q-Exactive , which has
a lower resolving power (140,000 at m/z 200) but provides higher scan speeds
than previous versions (12 Hz at a resolving power of 17,500), making it
compatible with high speed separations.
The utility of high resolution MS and MS/MS data obtained through
utilisation of Orbitrap instruments in infusion mode has been demonstrated for
the identification of polyphenols [553,554], while a number of reports
utilising conventional HPLC with Orbit-rap detectors have also been reported
[150,192,555–560]. For example, Lamuela-Raventós and co-workers used
conventional HPLC methods in combination with an LTQ-Orbitrap Velos to
study the phenolic composition of tomatoes [448], beer [561], walnut
[562] and sofrito [563], while Cahill et al. [564] developed and validated an
HPLC-Orbitrap method for quantifying isoflavones in waste water following
APCI ionisation. In the following discussion pertaining to Table 7, the focus
will however be on selected appli-cations of advanced LC methods
hyphenated to Orbitrap detection for flavonoid analysis.
The majority of these have to date been performed using the LTQ-Orbitrap
TM
XL instrument. Li et al. [565] used this instru-ment coupled to a UHPLC
separation to identify 15 flavonoids in the traditional Chinese medicine
(TCM) Sarcandra glabra, including glycosylated flavanones, flavanonols and
n
a flavanol, based on neg-ative ESI MS spectra and accurate mass
information. Analysis of Serbian honeys [566–568] was achieved on a 1.9 m
dp column, while Orbitrap MS/MS was used to identify up to 31 flavonols,
flavanonols, flavanones and flavones. An example of the benefit of high
resolution MS data is demonstrated for these samples in Fig. 17, where four
peaks are observed in the EIC obtained at 269.06 (Fig. 17(b), 200 ppm mass
precision). Extraction of masses 269.0459 and 269.0822 at 1 ppm mass
precision (Fig. 17(c) and (d), respec-tively) shows the peak pairs observed for
closely eluting apigenin and galangin (m/z 269.0459) and alpinetin and
pinostrobin (m/z 269.0822), respectively, as confirmed by data-dependent
MS/MS operation [566].
TM
Lebel et al. [290] used a LTQ-Orbitrap XL instrument for the
quantitative analysis of erectile dysfunction ingredients, including 9 natural
flavonoids. The authors used full scan mode for deter-mination of accurate
mass and data-dependent MS/MS of targeted precursors. Limits of detection
for the flavonoids varied between 3 and 23 pg on-column.
Tchoumtchoua et al. [163] reported a comprehensive structure-oriented
approach based on UHPLC–ESI-MS/MS using an LTQ-Orbitrap
TM
Discovery instrument for the characterisation of isoflavonoids (isoflavones,
dihydroisoflavones and their cor-responding glycosides) in natural products.
The proposed
60 A. de Villiers et al. / J. Chromatogr. A 1430 (2016) 16–78

Table 7
Applications of advanced LC hyphenated to Orbitrap and ion mobility mass spectrometry to flavonoid analysis (2009–2015).

Sample Flavonoids Ionisation Detector(s) Stationary phase Mobile References

source (column dimensions), phase(s)
flow rate, temperature,
analysis time
Sarcandra glabra Flavanonols, ESI− b
LIT -Orbitrap C18 (100 mm × 2.1 mm, d
A: 0.1% FA , B: [565]
a d
(TCM ) flavanones, flavonones UV 1.9 m dp ), ACN
and flavonols (15 c
FR = 0.6 mL/min,
flavonoids) ESI+/− 20 min
Red mustard greens Anthocyanins and LIT-Orbitrap C18 (200 mm × 2.1 mm, A: 0.1% FA, B: [189]
flavonol-glycosides UV 1.9 m dp ), 0.1% FA in ACN
(169 flavonoids) ESI− FR = 0.3 mL/min, 65 min
Blueberry, Hongcaitai Anthocyanins and LIT-Orbitrap C18 (200 mm × 2.1 mm, A: 0.1% FA, B: [287]
and red radish flavonol aglycones and 1.9 m dp ), 0.1% FA in ACN
◦
glycosides (64 FR = 0.3 mL/min, 50 C,
flavonoids) ESI+/− 65 min
Huang-Lian-Jie-Du Flavones and b C18 (100 mm × 2.1 mm, A: 0.05% FA, B: [444]
Q -Orbitrap
b
decoction (TCM) flavanones (24 QqQ 1.7 m dp ), 0.05% FA in
flavonoids) ◦ d
FR = 0.45 mL/min, 35 C, MeOH
ESI+/− 12 min
Vitus vinifera L. juice Flavonoids Q-Orbitrap C18 (100 mm × 2.1 mm, A: 0.1% FA, B: [572]
(unspecified) c 0.1% FA in
2.6 m dp SP ),
◦
FR = 0.3 mL/min, 40 C, MeOH
ESI− 20 min
Apple Flavonol, flavone, Orbitrap C18 (150 mm × 3.0 mm, A: 0.1% FA, B: [112]
flavanone aglycones (single-stage) 1.7 m dp ), 0.1% FA in ACN
◦
and glycosides, UV FR = 0.5 mL/min, 40 C,
chalcones, flavanonols, 23 min
flavanols and
anthocyanins (27
flavonoids) ESI− C18 (150 mm × 3.0 mm,
Whole coffee fruit Procyanidins, Orbitrap A: 0.1% FA, B: [263]
flavan-3-ols and (single-stage) 2.6 m dp SP), ACN
flavonol glycosides (12 UV FR = 0.7 mL/min
flavonoids) (0.2 mL/min to MS),
ESI− ◦
40 C, 20 min
Euterpe oleracea palm Flavonol aglycones and LIT-Orbitrap C18 (100 mm × 2.1 mm, A: 0.1% FA, B: [575]
juice glycosides, flavone 1.8 m dp ), 0.1% in MeOH
◦
aglycones and FR = 0.4 mL/min, 30 C,
glycosides, 35 min
anthocyanins,
flavanonols and
flavan-3-ols (29
flavonoids) ESI− C18 (50 mm × 2.1 mm,
Berry extracts Flavonols, flavanones LIT-Orbitrap A: 0.1% FA, B: [576]
and flavones (18 1.9 m dp ), 0.1% FA in ACN
flavonoids) ESI− FR = 0.4 mL/min, 9 min
Olive oil Flavone aglycones and LIT-Orbitrap C18 (100 mm × 2.1 mm, A: 0.1% AA, B: [577]
glycosides and 2.7 m dp SP), ACN
quercetin-3-O- FR = 0.4 mL/min, 33 min
rutinoside (7
flavonoids) ESI− C18 (50 mm × 2.1 mm,
Serbian unifloral Flavonols, flavanonols, LIT-Orbitrap A: 0.1% FA, B: [566]
honeys flavanones and 1.9 m dp ), 0.1% FA in ACN
flavones (31 FR = 0.4 mL/min, 9 min
flavonoids) ESI+/− C18 (50 mm × 2.1 mm,
Stem bark of Amphimas Isoflavones, LIT-Orbitrap A: 0.1% FA, B: [163]
pterocarpoides isoflavanones and APCI+/− UV 1.9 m dp ), MeOH
flavonols (19 FR = 0.2 mL/min, 12 min
flavonoids) ESI+/− C18 (200 mm × 2.1 mm,
Brassica species Anthocyanins and LIT-Orbitrap A: 0.1% FA, B: [569]
microgreens flavonol glycosides UV 1.9 m dp ), 0.1% FA in ACN
(135 flavonoids) ESI− FR = 0.3 mL/min, 65 min
Rorippa indica (Linn.) Flavonol glycosides LIT-Orbitrap C18 (200 mm × 2.1 mm, A: 0.1% FA, B: [570]
Hiern. (Cruciferae) and acylated glycosides UV 1.9 m dp ), 0.1% FA in ACN
(46 flavonoids) ESI+/− FR = 0.3 mL/min, 65 min
Strawberry (Fragaria Dihydroflavonols, LIT-Orbitrap C18 (200 mm × 2.1 mm, A: 0.1% FA, B: [217]
vesca) flavonol aglycones and UV 1.9 m dp ), 0.1% FA in ACN
◦
glycosides, FR = 0.3 mL/min, 60 C,
proanthocyanidins and 65 min
anthocyanins (19
flavonoids) ESI+/− C18 (100 mm × 4.6 mm,
Lithocarpus Dihydrochalcones, LIT-Orbitrap A: 0.1% FA, B: [578]
polystachyus (TCM) flavan-3-ols, 1.8 m dp ), MeOH
flavanones and FR = 0.6 mL/min, 50 min
flavanonols (46
flavonoids)
 A. de Villiers et al. / J. Chromatogr. A 1430 (2016) 16–78 61
Table 7 (Continued )

Sample Flavonoids Ionisation Detector(s) Stationary phase Mobile References

source (column dimensions), phase(s)
flow rate, temperature,
analysis time
Lime honey Flavonol glycosides (15 ESI− LIT-Orbitrap C18 (100 mm × 2.1 mm, A: 0.1% FA, B: [568]
flavonoids) 1.9 m dp ), 0.1% FA in ACN
ESI− FR = 0.3 mL/min, 13 min
Rhizoma Smilacis Flavanonols, LIT-Orbitrap C18 (100 mm × 3.0 mm, A: 0.1% FA, B: [579]
glabrae flavan-3-ols and UV 1.7 m dp ), 0.1% FA in ACN
◦
procyanidins (23 FR = 0.3 mL/min, 30 C,
flavonoids) ESI− 10 min
Serbian lime honey Flavones, flavonols and LIT-Orbitrap C18 (50 mm × 2.1 mm, A: 1% FA, B: [567]
flavanones (24 1.9 m dp ), ACN
flavonoids) ESI+/− FR = 0.3 mL/min, 15 min
Soy based nutraceutical Isoflavones, flavones, Orbitrap C18 (100 mm × 2.1 mm, A: 30 mM [574]
products flavonols, flavanones (single-stage) 1.7 m dp ), ammonium
◦
and chalcones (49 FR = 0.2 mL/min, 30 C, acetate, B:
flavonoids) 47.5 min MeOH
Adulterated or Flavone aglycones and ESI+ LIT-Orbitrap C18 (100 mm × 2.1 mm, A: 0.1% FA, B: [290]
counterfeit erectile glycosides, isoflavones 2.6 m dp SP), 0.1% FA in ACN
◦
dysfunction products and prenylated FR = 0.35 mL/min, 50 C,
flavonols (9 flavonoids) ESI− 10 min
Jujubu, fuji apple, Proanthocyanidins (76 LIT-Orbitrap C18 (200 mm × 2.1 mm, A: 0.1% FA, B: [571]
litchi, mangosteen, flavonoids) UV 1.9 m dp ), 0.1% FA in ACN
chocolate, grape and FR = 0.3 mL/min, 65 min
cranberry extracts ESI− C18 (50 mm × 2.1 mm,
Serbian poplar type Flavonols, flavanonols, LIT-Orbitrap A: 1% FA, B: [580]
propolis flavones and 1.9 m dp ), ACN
flavanones (56 FR = 0.3 mL/min, 15 min
flavonoids) ESI+/− C18 (50 mm × 2.1 mm,
Fufang decoction Flavanones aglycones LIT-Orbitrap (+mode) A: [581]
(TCM) and glycosides and 1.9 m dp ), 0.1% FA, B:
◦
flavones (10 FR = 0.3 mL/min, 30 C, MeOH;
flavonoids) 31 min (−mode) A:
ESI+/− C18 (100 mm × 2.1 mm, H2 O, B: MeOH
Mulberry (Morus alba Anthocyanins, flavonol LIT-Orbitrap (−mode) A: [425]
L.) aglycones and ESI− QqQ 1.9 m dp ), 0.1% FA, B: 0.1%
◦
glycosides, flavan-3-ol UV FR = 0.3 mL/min, 40 C, FA in ACN;
derivatives and 12 min (+mode) A: 1%
flavanone glycosides FA, B: ACN
(35 flavonoids) ESI− C18 (100 mm × 2.1 mm,
Olive tree (Olea Flavone aglycones and LIT-Orbitrap A: 0.1% FA, B: [291]
europaea) organs glycosides, flavonol UV 2.7 m dp SP), ACN
aglycones and FR = 0.8 mL/min, 35 min
glycosides, flavanols
and flavanonols (20
flavonoids) ESI− C18 (50 mm × 2.3 mm,
Ragweed (Ambrosia Flavonol aglycones and LIT-Orbitrap A: 0.1% FA, B: [582]
artemisiifolia L.) glycosides (16 1.8 m dp ), 98% ACN
pollen flavonoids) ESI− FR = 0.3 mL/min, 9 min
Nepeta species Flavonols, flavones and LIT-Orbitrap C18 (100 mm × 2.1 mm, A: 1% FA, B: [583]
flavanones (18 QqQ 1.7 m dp ), ACN
◦
flavonoids) UV FR = 0.3 mL/min, 30 C,
ESI+/− 15 min
Green tea nutraceutical Flavan-3-ol derivatives, Orbitrap C18 (100 mm × 2.1 mm, A: 30 mM [573]
products flavonol aglycones and (single-stage) 1.7 m dp ), Ammonium
◦
glycosides, flavone FR = 0.2 mL/min, 30 C, acetate
aglycones and 16 min acidified to pH
glycosides (33 5 with FA, B:
flavonoids) MeOH
Standards and urine Flavone metabolites ESI+ b c d [462]
IM -LIT PFP A: 2% AA , B:
(diosmetin-3-O- b (100 mm × 2.1 mm, 2% AA in MeOH
(FAIMS )
glucoronide, ESI+ QqQ (MRM) 5 m dp ),
-7-O-glucoronide and FR = 0.4 mL/min, room
-3,7-O-diglucoronide) ESI− temp
Cauliflower waste Flavonol-glycosides Q-IM-TOF C18 (150 mm × 2.1 mm, A: 0.1% FA, B: [584]
and acylated b 0.1% FA in
(TWIMS ) 1.7 m dp ),
◦
flavonol-glycosides (24 FR = 0.25 mL/min, 40 C, MeOH
flavonoids) ESI− 45 min
Black tea thearubigins Theasinensins, B-type Q-IM-TOF C18 (100 mm × 2.1 mm, A: 0.1% AA, B: [585]
proanthocyanidin (TWIMS) 1.7 m dp ), 0.1% AA in ACN
◦
dimers and rutin (5 FR = 0.3 mL/min, 35 C,
flavonoids) 35 min
a
TCM, traditional Chinese medicine.
b
LIT, linear ion trap; Q, single quadrupole; QqQ, triple quadrupole; IM, ion mobility; FAIMS, field-asymmetric waveform ion mobility spectrometry; TWIMS, travelling wave ion mobility
spectrometry.
c
FR, flow rate; SP, superficially porous; Pentafluorophenylpropyl.
d
FA, formic acid; ACN, acetonitrile; MeOH, methanol; AA, acetic acid.
62 A. de Villiers et al. / J. Chromatogr. A 1430 (2016) 16–78
Fig. 17. UHPLC–ESI-Orbitrap analysis of an ethyl acetate extract of Serbian honey. (a) depicts the TIC obtained in negative ionisation mode, (b) the extracted ion chromatogram for m/z 269.09 at 200
ppm mass precision, and (c) m/z 269.0459 and (d) 269.0822 at 1 ppm mass precision, respectively. Apigenin and galangin (c) and pinetin and pinostrobin (d) were identified based on MS/MS spectra.
Source: Reproduced with permission from [566].
methodology was illustrated for the determination of isoflavones in an
Amphimas pterocarpoides crude extract. Chromatographic, UV and MS
information was combined to prioritise potential com-pounds of the target
class, which were further characterised based on accurate mass fragmentation
information. The structures of selected tentatively identified compounds were
further confirmed by NMR, and investigated using APCI and ESI MS/MS
following infusion of the isolated compounds. For methylated isoflavones,
positive mode APCI was found to provide useful information for structural
elucidation purposes.
n
The utility of UHPLC–DAD-ESI-HR-MS in the profiling of flavonoids
in foods is amply illustrated by a series of papers from the group at the US
TM
Department of Agriculture. These authors used an LTQ-Orbitrap XL
(operated at a resolving power of 15,000) with positive and negative ESI and
a 1.9 m RP-LC column to screen a range of phenolics in various sam-ples.
For example, 30 anthocyanins and 105 flavonol glycosides were identified in
microgreens [569], 67 anthocyanins and 102 flavonol glycosides in red
mustard greens [189], 19 flavonoids in strawberries [217], 46 flavonol
glycosides and acylated gly-cosides in Rorippa indica [570] and 247
proanthocyanidins in seven different food materials [571]. In the latter report,
the authors derived relative molar response factors for ion counts in SIM
operation from UV data, and used these to quantify (sepa-rated)
proanthocyanidins in grape seeds. Despite the assumptions inherent to this
approach, the methodology represents a signif-icant contribution in the field
of proanthocyanin analysis, since
quantification of these compounds is impossible using UV detection only due
to extensive co-elution and the lack of suitable commercial standards.
In another interesting paper, the group investigated the use of negative ESI
behaviour of anthocyanins [287]. These compounds are normally detected in
positive ionisation mode due to the preva-lence of the flavylium cationic
species in solution, but may be hard to distinguish from flavonol-O-glycosides
due to identical nominal masses and (low-energy) fragmentation patterns. The
motivation here was to use negative ionisation to discriminate between these
flavonoid classes. Indeed, unusual [M−2H] − and [M−2H+H2O]− ions were
observed for the anthocyanins, for which the authors pro-posed a possible
mechanism of formation. However, a more likely explanation is that these
ions are due to the deprotonated quinoidal and carbinol pseudobasic forms of
anthocyanins, which are present in the column due to the low acidity of the
mobile phase (0.1% formic acid) – these species are not separated
chromatographically due to their relatively fast inter-conversion [105]. In our
opinion negative ionisation of anthocyanins makes little sense in light of the
fact that these compounds can normally be distinguished from other classes
based on UV characteristics, and failing that based on distinct high collision
energy CID spectra of anthocyanidins [180]. Furthermore, analysis of
containing only 0.1% formic acid
anthocyanins using mobile phases
leads to significant and evident band-broadening for these
compounds, thereby exacerbating co-elution and providing an
additional criterion to distinguish them from flavonols, if
needed.
 A. de Villiers et al. / J. Chromatogr. A 1430 (2016) 16–78
The Q-Exactive
TM
Orbitrap has also found application in flavonoid
analysis. Palermo et al. [557] used this instrument in combination with HPLC
separation to identify 40 flavonoids in arti-chokes. The resolving power of the
instrument was set to 25,000 FWHM for a scan time of 1 s. Yang et al. used
this instrument in combination with an HPLC method for the qualitative
analysis of the TCM Huang-Lian-Jie-Du decoction (HLJDD). 24 flavonoids
were identified, some of which were then used as markers for quality control
and quantified in selected samples using a UHPLC–QqQ-MS method utilising
polarity switching [444]. Tarr et al. [572] used a superficially porous phase
TM
and Q-Exactive Orbitrap MS in a metabolomics study to investigate the
effect of terroir and variety on Vitis vinifera L. grape juices. The metabolic
signatures in negative and positive ionisation showed clustering according to
varietal and terroir properties, respectively, while flavonoid profiles obtained
from identification of 466 compounds using a flavonoid database were found
to be good indicators of varietal character indepen-dent of terroir. Garrido
Frenich and co-workers reported UHPLC–MS methods based on the Q-
TM
Exactive Orbitrap for the qualitative and quantitative analysis of
flavonoids in green tea [573] and soy based nutraceutical products [574]. MS
data were acquired in full MS (no fragmentation, resolving power 25,000
FWHM; scan time = 0.25 s) and all-ion fragment (collision energy 30 eV,
10,000 FWHM; scan time = 0.10 s) modes in both positive and negative
ionisation. The overall scan rate for the four acquisition functions was 0.56
Hz. LOD values varying between 1 and 50 g/L were reported for a range of
different flavonoids. Mullen et al. [263] described an HPLC method
TM
employing a superficially porous phase and an Exactive Orbitrap mass
spectrometer for the (semi)-quantitative analysis of procyani-dins, flavonols
and hydroxycinnamic acids in whole coffee fruits, beans and husks from
China, India, and Mexico. The instrument resolving power was set to 60,000,
although the scan speed was not specified.
It should be noted that although the Orbitrap is a true high resolution
instrument (providing for example resolving powers of 60,000–100,000
FWHM for flow injection analysis [190,553,554]), this performance is highly
dependent on the scan time and mode. In LC–MS applications where fast or
high-resolution separation is performed, the instrument is commonly used at a
resolution set-ting of between 7500 and 30,000 to acquire a sufficient number
of spectra across each peak, and lower values for MS/MS acquisition [422].
(In fact, depending on the number of scan modes utilised, scan speeds are
often insufficient for high-speed separations on older Orbitrap instruments).
In practice therefore Orbitrap instru-ments provide little gain in resolving
power compared to the latest generation TOF systems for such applications.
On the other hand, the compromise between sensitivity and scan time is lower
than for other HR mass analysers [422].
The compromises involved in parameter selection for quanti-tative
UHPLC-Orbitrap analysis have been elegantly addressed by De Paepe et al.
[112]. These authors found a scan speed of 2 Hz, obtained at a resolving
power of 50,000 (WFHM at m/z 200) in MS mode, to be optimal using an
TM
Exactive instrument (for an anal-ysis time of 23 min; for fast gradients
resolution would need to be adapted to increase the scan speed). Furthermore,
mass accuracy performance is also related to the dynamic range, which in turn
is related to the scan range. It is therefore clear that careful opti-misation of
MS (and MS/MS) parameters is required for optimal performance of Orbitrap
instruments.
Current generation Orbitrap instruments do not compete with TOF
systems in terms of scan speed, which explains the more widespread use of
the latter in very fast separations and LC × LC, for example. However,
considering the extensive developments in Orbitrap technology which have in
a relatively short time resulted in improved mass resolution on the one hand
and scan speeds on the other, it is possible that this will become
63
a real alternative to TOF analysers for such analyses in the future.
4.2.5. Ion mobility spectrometry
Ion mobility spectrometry (IMS) measures the drift-time of ions through a
buffer gas in the presence of an electric field. The drift-time of an ion depends
on its size/charge ratio – ions can therefore be separated based on differences
in their net charge or collision cross-sections, or by interactions with the gas.
The technique has found extensive application in the detection of explo-sives,
drugs and chemical warfare agents. IMS is ideally compatible with mass
spectrometry and provides complementary informa-tion, which explains the
growth in popularity of ion mobility-mass spectrometry (IM-MS) [586]. IM-
MS is especially used in the sep-aration of isobaric compounds and isomers,
conformers and in the measurement of ion size. Interestingly, because of the
rela-tive timeframes of chromatographic separations (s), IMS (ms) and MS
( s), the technique may be considered a form of comprehen-sive 3D
separation, since the separation obtained in each previous ‘dimension’ is
essentially maintained. A range of different IMS methods may be used in IM-
MS, including traditional drift-time ion mobility (DTIMS), aspiration ion
mobility (AIMS), field-asymmetric waveform ion mobility (FAIMS) and
travelling wave ion mobility (TWIMS). A diverse range of IM-MS
instrumentation is furthermore available, with IMS having been hyphenated to
magnetic sector, TOF, quadrupole, IT and FT-ICR instruments. For further
details on the various instrumental configurations available, the reader is
referred to [587].
IM-MS has found relatively sparse application in flavonoid anal-ysis to
date (Table 7), with most work done in infusion mode (i.e. without
chromatographic pre-separations). For example, Clowers et al. [588]
described a method using a dual gate IM-LIT mass spectrometer to separate
cation adducts of flavonoid-diglycosides. This type of mass spectrometer can
operate either in a dual gate scanning (DGS) or single mobility monitoring
(SMM) mode. In DGS mode a complete mobility spectrum was obtained.
Once the drift-time(s) were obtained using DGS, ions can be selectively
transmitted to accumulate a specific ion population to improve efficiency.
Troc´ et al. [589] demonstrated the (travelling wave) IM-MS separation of the
epimers (+)-catechin and (−)-epicatechin by complexation with chiral
modifiers and transition metals.
In contrast, flavone-glycosides were separated under ambi-ent pressures
using a differential ion-mobility spectrometer [462]. This method included a
chromatographic separation using a PFP column. For the identification of
diosmetin-7-O-glycoside and diosmetin-3-O-glycoside an IMS coupled to a
Q-LIT analyser was used.
More recently, IM-MS has also been used in combination with UHPLC
separation for flavonoid applications. Yassin et al. [585] described a method
employing UHPLC coupled to a Q-IM-TOF mass spectrometer to separate
isomeric flavanols unresolved by chromatographic separation. The authors
showed that isomeric structures of B-type prodelphinidins and theasinensins
as well as the isobaric compound rutin could be resolved based on their
mobility drift times in the negative ion mode. Gonzales et al. [584] used the
additional selectivity offered by ion mobility separation to obtain cleaner mass
spectra to assist in the identification of acyl-ated and non-acylated flavonol-
glycosides. In both of these works, a commercial instrument equipped with a
TWIMS cell [475] was used.
In TWIMS, the continuous low electric field of traditional IMS is replaced
by sweeping a high field sequentially through the cell [587]; this approach
provides low IMS resolution, but high sensitiv-ity and is ideally suited for
IMS-MS. This system is equipped with trap and transfer cells before and after
the IMS cell, and manip-ulation of collision energy in these can be used for
example to
64 A. de Villiers et al. / J. Chromatogr. A 1430 (2016) 16–78

Fig. 18. Methodology for assignment of daughter ions based on two separate ion mobility-MS experiments and classical least squares data modelling. Data shown for isovitexin (apigenin-6-C-
glucoside).
Source: Reproduced with permission from [590].

allow parent ions to enter the IMS cell (low collision energy in trap cell) and
fragment the separated ions before transfer to the TOF (high transfer cell
collision energy) [585]. Garmón-Lobato et al.
[590] reported an interesting chemometric method for using IMS-MS
measurements to assign daughter ions formed in the transfer cell (i.e. after
IMS separation) to specific pre-cursor ions. Since CID fragmentation is also
performed in the trapping cell, this approach provides equivalent information
3
to MS experiments, but for all precursor ions simultaneously. The procedure
is illustrated schematically in Fig. 18.
4.2.6. Overview of mass spectrometers used in flavonoid analysis Fig. 19
summarises graphically the relative proportion of appli-
which utilised different MS
cations listed in Tables 1, 2 and 4–7
detectors (single quadrupole applications are not included). It
should be pointed out that this is not an accurate reflection of
the global picture in flavonoid analysis because of the focus in
the cur-rent work on advanced LC separations (and inevitable
bias based on the incomplete data set). Nevertheless, for
applications falling within the scope of this work, the relative
popularity of TOF sys-tems is clear (50% of all applications)
and not unexpected based on
 A. de Villiers et al. / J. Chromatogr. A 1430 (2016) 16–78 65

trace-level targeted analysis of flavonoids in complex mixtures in combination

with ultra-fast and high resolution separations. For structural elucidation and
non-targeted analyses, however, high resolution and hybrid HR-MS systems
have proven indispensable. The popularity of TOF, and especially Q-TOF
instruments in com-bination with advanced LC separation is due to the
performance of these systems in terms of acquisition speed, sensitivity, mass
range and tandem MS capabilities. In recent years, Orbitrap instruments have
gained increasing popularity in flavonoid analysis, although the resolving
power-scan speed compromise currently still favours TOF systems for
hyphenation to advanced separations.

In terms of chromatographic separation, the widespread contemporary

Fig. 19. Overview of the application of different MS instruments in the advanced LC–MS
analysis of flavonoids (data based on references listed in Tables 1, 2, 4–7, 2009–2015).
performance characteristics such as speed, resolving power and cost.
Furthermore, Q-TOF instruments in particular have found extensive
application due to the combination of tandem MS and high resolution data
provided by these systems, which is especially useful for structural
elucidation of flavonoids. Orbitrap instruments currently represent the only
alternative high resolution systems for use in combination with advanced LC
separation, but are still rel-atively new additions to the market; it is likely that
these systems will find increasing application in flavonoid analysis in the near
future due to similar advantages as TOF instruments (although cur-rently still
lower scan speeds). Triple quadrupole instruments are the second most
utilised, and will likely find continued applica-tion in the field due to their
suitability for trace-level and targeted analyses.
5. Conclusions and perspectives
availability of UHPLC instrumentation and the per-formance characteristics of
the latest generation sub-2 m porous and superficially porous phases have
made faster and more efficient separation of large numbers of flavonoids
possible without sac-rificing robustness. The recent availability of
superficially porous phases of particle sizes of 1.3 m promises to further
extend perfor-mance gains. The complementary benefits of elevated
temperature operation is increasingly being exploited in the modest range of
◦
50–80 C; higher temperatures are unlikely to become com-mon due to
column/instrument limitations and concerns regarding the thermal stability of
flavonoids. LC × LC has been shown to deliver high-resolution separation of
highly complex mixtures of flavonoids. The relative complexity of the
technique has limited its use to date to that of primarily being a research tool,
but the recent availability of commercial instruments will entice further
applica-tions to flavonoid analysis. One of the primary application areas of LC
× LC has been in the analysis of complex proanthocyanidin mixtures, where
the demonstrable benefits of the technique will ensure its continued
application.
All of these developments have ensured that many more flavonoids can
now be identified in a single analysis through judicious selection of separation
conditions and suitable MS instru-mentation, although it is worth noting that
these assignments remain tentative in the absence of reference standards;
NMR there-fore remains an essential tool in flavonoid characterisation.

Advances in LC–MS analysis of flavonoids largely mirror, and to a degree
build on those in fields such as proteomics and metabolomics, which are
underpinned by significant developments in both HPLC and MS
instrumentation. Most influential have been technological advances in MS,
and translation of these to commer-cial instruments which are becoming more
pervasive in routine laboratories. Notable are the performance of the latest
genera-tion of QqQ instruments, which makes these systems ideal for the
The extent of the advances in LC–MS and their implications in terms of
the quality of analytical data obtainable are often not fully appreciated. Fig. 20
presents an attempt to highlight the evolution of flavonoid analysis, using
anthocyanins as an example. Compar-ison of the contemporary situation with
the first HPLC separation of wine anthocyanins in 1978 shows a clear increase
in separation performance coinciding with improvements in column
technology and LC × LC operation. Concurrent progress in hyphenation to
MS

Fig. 20. Evolution of the HPLC analysis of anthocyanins. (A) One of the first applications of RP-LC-UV to the analysis of grape anthocyanins. Conditions: C18 column (250 mm × 4.6 mm, 5 m dp ),
10% formic acid/methanol gradient at 2.5 mL/min, UV detection at 520 nm. Reproduced with permission from [29]© 1978 American Society for Enology and Viticulture. AJEV 29:42–49. (B)
UHPLC–ESI-QqQ-MS analysis of rosé wine showing the MRM transitions for 152 phenolic compounds. Conditions: C18 column (150 mm × 1 mm, 1.8 m d p ), 1% formic acid/methanol gradient at
0.17 mL/min, 40 ◦ C. Reproduced from [438]. (C) depicts the high efficiency UHPLC–ESI-Q-TOF-MS analysis of red wine anthocyanins. Conditions: C18 column (200 mm × 2.1 mm, 1.7 m d p ),
7.5% formic acid/acetonitrile gradient at 0.06 mL/min, 50 ◦ C. Reproduced with permission from [218]. (D) presents a UV contour plot obtained for the off-line HILIC × RP-LC analysis of grape
anthocyanins. Conditions: 1 D: amide column (150 mm × 4.6 mm, 2.5 m d p ), 0.4% TFA/acetonitrile gradient at 0.2 mL/min, 50 ◦ C, 1 ts = 0.5 min; 2 D, superficially porous C18 column (50 mm × 4.6
mm, 2.6 m dp ), 7.5% formic acid/acetonitrile gradient at 0.5 mL/min, 50 ◦ C, ESI-Q-TOF-MS and UV detection at 500 nm. Reproduced with permission from [248].
66 A. de Villiers et al. / J. Chromatogr. A 1430 (2016) 16–78
detection now allows selective analysis of large numbers of target DESI desorption electrospray ionisation

compounds by tandem MS, whereas HR-MS hyphenated to high- DGS dual gate scanning
resolution separations enables identification of large numbers of DP degree of polymerisation
unknown compounds. In fact, comparison of the reports cited in DPPH 1,1-diphenyl-2-picrylhydrazyl radical
this work with literature from only ten years ago clearly highlights DTIMS drift-time ion mobility spectrometry
the performance gains in terms of both analysis time reduction and ED electrochemical detection
the number of compounds that can be separated and tentatively EI electron (impact) ionisation
identified allowed by the hyphenation of advanced separation and ELSD evaporative light scattering detector
MS technologies. Considering the pace of developments in HPLC ESI electrospray ionisation
and MS, any prediction of future progress is necessarily fraught FAB fast atom bombardment
with uncertainty; it is however clear that the growing application FAIMS field-asymmetric waveform ion mobility spectrometry
of advanced LC–MS methods to flavonoid analysis will increasingly FL fluorescence
play an important role in support of flavonoid research. FR flow rate
FT Fourier transform
Symbols and abbreviations GC gas chromatography
HILIC hydrophilic interaction chromatography
HPLC high performance liquid chromatography
Symbols HRF heterolytic ring fission
ˇ under-sampling correction factor HR-MS high resolution mass spectrometry
xD xth dimension HTLC high temperature liquid chromatography
Dm diffusion coefficient of the analyte in the mobile phase i.d. internal diameter
x dilution factor in the xth dimension ICR ion cyclotron resonance
DF
P pressure drop IEC ion exchange chromatography
Pmax maximum pressure IM-MS ion mobility-mass spectrometry
dp particle size IMS ion mobility spectrometry
ε external porosity IT ion trap
εT total porosity LC liquid chromatography
fc fractional surface coverage LC–MS liquid chromatography–mass spectrometry
xF volumetric flow rate in the xth dimension LC × LC comprehensive two-dimensional liquid
H height equivalent of a theoretical plate (typically in m) chromatography
H
min minimum height equivalent of a theoretical plate LOD limit of detection
Kvo 2 LIT linear ion trap
column permeability (typically in m )
L column length MALDI matrix-assisted laser desorption ionisation
max wavelength of maximum absorbance MB moving belt
N number of theoretical plates (dimensionless) MD-LC multidimensional liquid chromatography
mobile phase viscosity MRM multiple reaction monitoring
n c,2D (practical) peak capacity of a two-dimensional separation MS mass spectrometry
x
nc peak capacity of separation in the xth dimension MS/MS tandem mass spectrometry
x n
standard deviation in dimension x MS multistage mass spectrometry (where n ≥ 2)
1
ts first dimension sampling time MW molecular weight
x gradient time in the xth dimension NMR nuclear magnetic resonance
tg
uopt optimal mobile phase linear velocity (typically in mm/s) NP-LC normal phase liquid chromatography
u0 mobile phase linear velocity (determined using unre- ODS octadecyl-silica
tained marker) OEG oligo-ethylene glycol
V
frac fraction volume PD plasma-desorption
x injection volume in the xth dimension PEG polyethylene glycol
Vinj
wb peak width at baseline PFP pentafluorophenyl
ppm parts per million
Abbreviations Q quadrupole
1-D one-dimensional QM quinone methide fragmentation pathway
2-D two-dimensional QqQ triple quadrupole
ABTS 2,2 -azino-bis(3-ethylbenzothiazoline)-6 sulfonic acid RDA retro-Diels–Alder
AIMS aspiration ion mobility spectrometry RP-LC reversed phase liquid chromatography
amu atomic mass units SEC size exclusion chromatography
APCI atmospheric pressure chemical ionization SIM selected ion monitoring
API atmospheric pressure ionization SMM single mobility monitoring
APPI atmospheric pressure photoionisation SR split ratio
BFF benzofuran-forming fission SRM selected reaction monitoring
CCC countercurrent chromatography TCM traditional Chinese medicine
CD cyclodextrin TFA trifluoroacetic acid
CE capillary electrophoresis TLC thin layer chromatography
CI chemical ionisation TOF time-of-flight
CID collision induced dissociation TSP thermospray
CPC centrifugal partition chromatography TWIMS travelling wave ion mobility spectrometry
DAD diode array detection UHPLC ultra high pressure liquid chromatography (pressures
DART direct analysis in real time >400 bar)
 A. de Villiers et al. / J. Chromatogr. A 1430 (2016) 16–78
Acknowledgements
The authors gratefully acknowledge Stellenbosch University, Sasol, the
National Research Foundation (NRF, South Africa, grant 81830 to AdV),
Restek (Academic Support Program Grant to AdV) and the International
Foundation of Science (IFS, Sweden, Grant F/4904-1 to AdV) for financial
support.
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