Journal of Chromatography B, 1053 (2017) 72–80

Contents lists available at ScienceDirect

Journal of Chromatography B
journal homepage: www.elsevier.com/locate/chromb

Lipidomics by ultrahigh performance liquid chromatography-high

resolution mass spectrometry and its application to complex
biological samples
Alexander Triebl a , Martin Trötzmüller a,∗ , Jürgen Hartler b , Tatjana Stojakovic c ,
Harald C. Köfeler a,d
a
Core Facility for Mass Spectrometry, Center for Medical Research, Medical University of Graz, Stiftingtalstrasse 24, 8010 Graz, Austria
b
Institute of Computational Biotechnology, Graz University of Technology, Petersgasse 14, 8010 Graz, Austria
c
Clinical Institute of Medical and Chemical Laboratory Diagnostics, Medical University of Graz, Auenbruggerplatz 15, 8036 Graz, Austria
d
Omics Center Graz, Stiftingtalstrasse 24, 8010 Graz, Austria

a r t i c l e i n f o a b s t r a c t

Article history: An improved approach for selective and sensitive identification and quantitation of lipid molecular
Received 29 November 2016 species using reversed phase chromatography coupled to high resolution mass spectrometry was devel-
Received in revised form 8 March 2017 oped. The method is applicable to a wide variety of biological matrices using a simple liquid-liquid
Accepted 22 March 2017
extraction procedure.
Available online 24 March 2017
Together, this approach combines multiple selectivity criteria: Reversed phase chromatography sep-
arates lipids according to their acyl chain length and degree of unsaturation and is capable of resolving
Keywords:
positional isomers of lysophospholipids, as well as structural isomers of diacyl phospholipids and glyc-
High-resolution mass spectrometry
High performance liquid chromatography
erolipids. Orbitrap mass spectrometry delivers the elemental composition of both positive and negative
Quantitative lipidomics ions with high mass accuracy. Finally, automatically generated tandem mass spectra provide structural
Lipid structural isomers insight into numerous glycerolipids, phospholipids, and sphingolipids within a single run.
Calibration showed linearity ranges of more than four orders of magnitude, good values for accuracy
and precision at biologically relevant concentration levels, and limits of quantitation of a few femtomoles
on column.
Hundreds of lipid molecular species were detected and quantified in three different biological matrices,
which cover well the wide variety and complexity of various model organisms in lipidomic research.
Together with a software package, this method is a prime choice for global lipidomic analysis of even the
most complex biological samples.
© 2017 Elsevier B.V. All rights reserved.

1. Introduction

The general term “lipids” is used to describe a plethora of

Abbreviations: Cer, ceramide; CL, cardiolipin; DG, diacylglycerol; EIC,
extracted ion chromatogram; FTICR, fourier transform ion cyclotron reso-
nance; FWHM, full width half maximum; HexCer, hexosyl ceramide; HPLC,
high performance liquid chromatography; LC/MS, liquid chromatography mass
spectrometry; LLOQ, lower limit of quantitation; LPC, lysophosphatidylcholine;
LPE, lysophosphatidylethanolamine; MS/MS, tandem mass spectrometry; MTBE,
methyl tert butyl ether; PA, phosphatidic acid; PC, phosphatidylcholine; PE,
phosphatidylethanolamine; PG, phosphatidylglycerol; PI, phosphatidylinositol; PIP,
phosphatidylinositolphosphate; PS, phosphatidylserine; SFC, supercritical fluid
chromatography; SM, sphingomyelin; TG, triacylglycerol; UHPLC, ultrahigh perfor-
mance liquid chromatography; ULOQ, upper limit of quantitation.
∗ Corresponding author.
E-mail address: martin.troetzmueller@medunigraz.at (M. Trötzmüller).
biomolecules that differ in structure as well as in function, rang-
ing from simple fatty acids to complex saccharolipids and from
use in energy storage to signal transduction and as vitamins [1,2].
Lipidomics is the general study of lipids and their contributions to
both physiological processes and pathological conditions, such as
cancer and diabetes [3].
The two approaches in lipidomic analyses are shotgun tech-
niques and separation techniques [4]. Shotgun techniques rely on
stable infusion of sample into the ion source without prior sep-
aration and subsequent detection by either high-resolution mass
spectrometers [5–12] or a combination of tandem mass spectrom-
etry (MS/MS) scans [13–15] to identify lipids by their mass and/or
characteristic head group and chain fragments. Similar techniques
are intrasource separation [16], where addition of modifiers causes

http://dx.doi.org/10.1016/j.jchromb.2017.03.027
1570-0232/© 2017 Elsevier B.V. All rights reserved.
 A. Triebl et al. / J. Chromatogr. B 1053 (2017) 72–80
selective ionization of lipid classes, and ion-mobility mass spec-
trometry, where the physicochemical properties of ions can be used
to separate lipid classes and structural or even positional isomers
[17–23].
While shotgun methods are an excellent tool for high-
throughput profiling, ion suppression effects may limit their ability
to detect low-abundance components, and the lack of separation
prior to mass analysis may diminish the informative value of MS/MS
spectra. Adducts of lipids belonging to different classes, or their iso-
topes, may have the same nominal mass which cannot be resolved
during MS/MS isolation, leading to an MS/MS spectrum with sig-
nals from multiple precursor lipids [24]. Also, some labile lipids,
such as phosphatidylserine [25–27] or cerebrosides (unpublished
observation), may undergo spontaneous in-source fragmentation,
artificially forming ions of phosphatidic acid (PA) or ceramides,
respectively. Chromatography causes these lipids to elute at dif-
ferent retention times so that the artificially generated ions can be
clearly discriminated from the authentic sample components.
Furthermore, separation of lipids has the advantage of minimiz-
ing the negative influence of interfering compounds in terms of ion
suppression and mixed MS/MS spectra. Ultrahigh performance liq-
uid chromatography (UHPLC), the evolution of conventional high
performance liquid chromatography (HPLC) brought by the advent
of sub-2 m particle columns, is the most widespread separation
method. Additionally, core-shell columns are capable of delivering
performance comparable to UHPLC at HPLC pressures [28]. Super-
critical fluid chromatography (SFC) is a method with promising
potential for lipidomic analyses providing shorter analysis times
and highest chromatographic resolution [29,30].
The method previously developed in our lab [31], while at the
time able to generate unprecedented data quality, still suffered
from some drawbacks, which collectively made it unsuitable for
analysis of highly complex lipidomes. Firstly, use of different HPLC
mobile phases for positive and negative polarity caused lipids to
elute at different retention times in positive and negative mode,
complicating data analysis. Secondly, the chromatographic reso-
lution and separation power proved to be insufficient for more
sophisticated lipidomic samples. Additionally, the hybrid ion trap-
Fourier transform ion cyclotron resonance (FTICR) instrument used
had limited scan speed, causing long duty cycles. Furthermore, the
sensitivity of both the ion trap and the FT analyzer (both are tech-
nologies from more than a decade ago) are quite limited when
compared to more timely mass analyzers.
While most lipidomic research is focused on study of mam-
malian organisms containing almost exclusively fatty acyls with an
even number of carbon atoms, lower organisms such as Caenorhab-
ditis elegans have a high amount of odd-carbon fatty acyls in their
lipids [6,32–34]. This drastically increases the complexity of the
lipidome as well as the number of potentially interfering com-
pounds for each analyte and requires an appropriately powerful
method for correct analysis.
Here we present a massively improved approach for lipidomic
profiling of even the most complex biological samples with the
potential to structurally analyze and quantify hundreds of glycero-
, phospho- and sphingolipids in a single run. Highest selectivity
is ensured by combining information from chromatography, high-
resolution full scans and tandem mass spectra in conjunction with
automated data analysis.
2. Material and methods
2.1. Chemicals
Acetonitrile, isopropanol, methanol, tert-methyl-butyl ether
(MTBE) (all Chromasolv grade) and ammonium formate (LC/MS
73
grade) were purchased from Sigma-Aldrich (St. Louis, MO, USA);
chloroform and formic acid were purchased from Merck (Darm-
stadt, Germany). Deionized water was obtained from an in-house
MilliQ Gradient A10 system (Millipore, Billerica, MA, USA).
2.2. Lipid standards
Lipid standards used for quantification and validation (listed
in Supplementary Tables 2 and 3, respectively) were purchased
from Avanti Polar Lipids (Alabaster, AL, USA) and from Larodan Fine
Chemicals AB (Malmö, Sweden).
2.3. Lipid extraction
The MTBE based lipid extraction protocol employed is a mod-
ified version of the original extraction protocol published by
Matyash et al. [32]. Methanol (1.5 mL) and MTBE (5 mL) were added
to the samples (50 mg murine liver, 100 mg of C. elegans embryos,
or 106 A549 adenocarcinomic human alveolar basal epithelial cells,
respectively) in 12 mL glass tubes with teflon lined caps and the
mixture was incubated for 10 min in an overhead shaker at room
temperature. After addition of 1.25 mL deionized water and 10 min
of additional shaking, the mixture was centrifuged for 5 min at 1
350 × g and the upper phase was transferred to a new glass tube.
The lower phase was re-extracted with 2 mL of the upper phase
of MTBE/methanol/deionized water (10:3:2.5, v/v/v) and again the
upper phase was collected, combined with the upper phase from
the first extraction, evaporated in a vacuum centrifuge (Thermo
Fisher Scientific, Waltham, MA, USA) and dissolved in chloro-
form/methanol (1:1, v/v) for storage at −20 ◦ C. The liver extract was
dissolved in a volume of 30 mL, the C. elegans extract in 500 ␮L and
the A549 cell extract in 1 mL to account for the varying lipid content.
Prior to analysis, the internal standards were added (see Section
2.6), the storage solvent was evaporated under a gentle stream
of nitrogen and the sample reconstituted in the same volume of
injection solvent isopropanol/chloroform/methanol (90:5:5, v/v/v).
2.4. LC method
Chromatographic separation was performed on a Waters
(Waters, Milford, MA, USA) BEH C8 column (100 × 1 mm, 1.7 ␮m),
thermostatted to 50 ◦ C in a Dionex Ultimate 3000 RS UHPLC sys-
tem. Mobile phase A was deionized water containing 1 vol% of 1 M
aqueous ammonium formate (final concentration 10 mmol/L) and
0.1 vol% of formic acid as additives. Mobile Phase B was a mixture of
acetonitrile/isopropanol 5:2 (v/v) with the same additives. Gradient
elution started at 50% mobile phase B, rising to 100% B over 40 min;
100% B were held for 10 min and the column was re-equilibrated
with 50% B for 8 min before the next injection. The flow rate was
150 ␮L/min, the samples were kept at 8 ◦ C and the injection volume
was 2 ␮L.
2.5. MS method
The Orbitrap Velos Pro hybrid mass spectrometer (Thermo
Fisher Scientific Inc., Waltham, MA, USA) was operated in Data
Dependent Acquisition mode using a HESI II ion source. Prior to
experiments, lens settings were tuned and source parameters were
optimized to the signal of PE 16:0/18:1 using the protonated and the
deprotonated molecule. Every sample was measured once in pos-
itive polarity and once in negative polarity. Ion source parameters
for positive polarity were as follows: Source Voltage: 4.5 kV; Source
Temperature: 275 ◦ C; Sheath Gas: 25 arbitrary units; Aux Gas: 9
arbitrary units; Sweep Gas: 0 arbitrary units; Capillary Tempera-
ture: 300 ◦ C. Ion source parameters for negative ion mode were:
Source Voltage: 3.8 kV; Source Temperature: 325 ◦ C; Sheath Gas:
74 A. Triebl et al. / J. Chromatogr. B 1053 (2017) 72–80

30 arbitrary units; Aux Gas: 10 arbitrary units; Sweep Gas: 0 arbi-
trary units; Capillary Temperature: 300 ◦ C. Automatic gain control
target value was set to 106 ions to enter the mass analyzer, with a
maximum ion accumulation time of 500 ms. Full scan profile spec-
tra from m/z 400–1200 for positive ion mode and from 400 to 1600
in negative ion mode were acquired in the Orbitrap mass analyzer
at a resolution setting of 100 000 at m/z 400. For MS/MS experi-
ments, the 10 most abundant ions of the full scan spectrum were
sequentially fragmented in the ion trap using He as collision gas
(CID, Normalized Collision Energy: 50; Isolation Width: 1.5; Acti-
vation Q: 0.2; Activation Time: 10) and centroided product spectra
at normal scan rate (33 kDa/s) were collected. The exclusion time
was set to 10 s.
2.6. Data processing and quantitation
LC/MS data were processed using Lipid Data Analyzer (LDA), as
previously described [35]. Briefly, the algorithm identifies lipids
with a 3D algorithm, using the three dimensions m/z, retention
time, and intensity to correctly integrate peaks, while also taking
into account the isotopic distribution. MS/MS spectra (displayed,
but not automatically interpreted by the program) were manually
inspected to obtain information about characteristic head group
fragments and fatty acyl composition and, if possible, positions, as
described previously [36–38]. Lipid species contributing less than
1% to the total amount of the respective lipid class were omitted
from the analysis.
For quantitation purposes, phosphatidylcholine (PC), phos-
phatidylethanolamine (PE), phosphatidylserine (PS), lysophos-
phatidylcholine (LPC), lysophosphatidylethanolamine (LPE) and
sphingomyelin (SM) were analyzed as [M+H]+ ions, ceramides (Cer)
and hexosylceramides (HexCer) as [M+H−H2 O]+ , and diacylglyc-
erols (DG) and triacylglycerols (TG) as [M+NH4 ]+ ions in positive
polarity, while cardiolipin (CL), phosphatidylinositol (PI) and phos-
phatidylglycerol (PG) were quantified in negative ion mode as
[M−H]− ions.
Lipids were quantified using LIPID MAPS internal standards con-
taining odd-chain fatty acyls for phospholipids or sphingolipids, or
deuterated glycerolipids. Quantitation of naturally occurring lipids
was performed using a one-point calibration. To 60 ␮L of the liver
extract and the A549 cell extract, all LIPID MAPS internal standards
listed in chapter 2.2 were added to a concentration of 1.5 ␮M prior
to measurement. To 60 ␮L of the C. elegans sample, only PC 25:0,
PE 25:0, PS 25:0, PG 25:0, PI 25:0, the deuterated TG and DG mix,
and ceramide/sphingoid internal standard mixture were added at
a concentration of 5 ␮M. Lysophospholipids, for which there is no
quantitative LIPID MAPS standard available, were quantified using
a 25:0 diacyl species.
Lipid shorthand nomenclature is used according to Liebisch et al.
[39]. Furthermore, we adhere to the recommendations by D.A.
Volmer concerning nomenclature in mass spectrometry publica-
tions [40].
2.7. Method validation
Method validation was performed similar to [31]: A liver
lipid extract was spiked with synthetic lipid standards (PC
12:0/12:0, PE 12:0/12:0, PS 12:0/12:0, PG 12:0/12:0, LPC 15:0/0:0,
Cer d18:1/17:0, SM d18:1/17:0, TG 17:0/17:0/17:0 and DG
12:0/12:0/0:0) to 13 concentration levels ranging from 1 nM to
100 ␮M to assess linearity (0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 5, 7.5
10, 25, 50, and 100 ␮mol/L). Each concentration level, along with
an unspiked lipid extract, was analyzed in triplicate as described in
Sections 2.4–2.6. Lipids used for validation purposes were analyzed
in both polarities (see also Table 1). Internal standards listed in Sup-
plementary Table 2 were added to concentrations of 2 ␮mol/L for
normalization.
The matrix effect was evaluated by comparing the responses of
the LIPID MAPS internal standards (listed in Supplementary Table
2) analyzed both alone and spiked into a liver lipid extract to con-
centrations of 2 ␮mol/L.
3. Results and discussion
3.1. Chromatography
We evaluated multiple columns in terms of chromatographic
resolution, peak shape, reproducibility and practicability and found
the BEH C8 column to suit our needs. Supplementary Figs. 1 and 2
show example chromatograms, and Supplementary Table 1 shows
performance characteristics for five different RP-HPLC columns
evaluated. The BEH C8 column provides the necessary chromato-
graphic performance to separate lipids spanning a broad range of
polarity: The very polar species LPC 13:0 and LPC 14:0 are chro-
matographically separated and both elute well after the solvent
peak; and there is over 3 min of elution time left after the retention
time of TG 60:1 to allow for elution of even longer triacylglycerols.
Using the same acidic mobile phase for positive and negative
ionization we did not observe drastically reduced ionization in neg-
ative ion mode but significantly gained selectivity, compared to a
formerly used mobile phase without formic acid for analysis in neg-
ative ion mode. Every lipid elutes at the same retention time both
in negative and in positive ion mode, allowing for alignment of
chromatograms to additionally confirm correct species identifica-
tion (Supplementary Figs. 3 and 4). Almost all lipids form sodium
adducts in positive ion mode and for some lipids (CL and PS), sodium
adducts even occur in negative ion mode as [M−2H+Na]− [41].
Sodium adducts can be particularly misleading, since they have
only a marginal m/z difference from the protonated form of another
lipid of the same class. For example, [PC 38:4+Na]+ and [PC 40:7+H]+
have a difference in molecular mass of only 2.4 mDa which cannot
be baseline separated at the mass resolution used (Supplementary
Fig. 4).
Retention time is an essential parameter in lipidomic LC/MS
data analysis. As previously shown and well-described by the
equivalent carbon number model [31,42–44], retention of lipids
in reversed phase chromatography increases with total number of
carbon atoms and decreases with the number of double bonds. The
construction of homologous series for each lipid class can be used
to discard false positive results from structural isomers belonging
to different lipid classes, or nominally isobaric interferences such
as the aforementioned sodium adducts (Fig. 1). Odd-carbon PE and
PC species, in use as quantitative internal standards, are structural
isomers of even-carbon PC and PE, respectively (e.g. internal stan-
dard PC 31:1 and analyte PE 34:1), but are clearly distinguishable
by retention time.
The method presented delivers good chromatographic per-
formance for more than 10 phospho-, sphingo- and glycerolipid
classes, but analysis of classes such as phosphatidic acid (PA)
and phosphatidylinositolphosphate (PIP) is hindered by poor chro-
matographic peak shapes. The strong peak tailing of PA and PIP
is attributed to interactions of the terminal phosphate group with
stainless steel surfaces [45,46], an effect avoided by using phos-
phate buffers as HPLC solvent [42], chemical derivatization [47] or
hydrophilic interaction liquid chromatography [27,48].
The duration of a chromatographic run is an important parame-
ter influencing total analysis time, and often shorter analysis times
are preferred. However, when analyzing larger sample batches, the
contribution of chromatographic run time to total analysis time is of
somewhat lower impact, since sample analysis is completely auto-
 A. Triebl et al. / J. Chromatogr. B 1053 (2017) 72–80 75

Table 1
Validation data (unweighted) for lipids in positive and negative mode, determined for one molecular species per lipid class. Values for lower limit of quantitation (LLOQ)
were conservatively determined because of the minimal but detectable natural abundance of the measured analytes. n = 5; n.a.: not applicable (not within linear range).

Accuracy Precision
(%) (RSD%)

Lipid molecular Type of ion LLOQ Linear range R2 Low Medium High Low Medium High
species (fmol on column) (␮M) (0.05 ␮M) (1 ␮M) (50 ␮M) (0.05 ␮M) (1 ␮M) (50 ␮M)

TG 51:0 [M+NH4 ]+ 100 0.05–7.5 0.9874 176 95 n.a. 7.1 1.9 1.6
DG 24:0 [M+NH4 ]+ 100 0.05–10 0.9918 0 1 n.a. 27.1 5.2 4.9
PC 24:0 [M+H]+ 10 0.005–25 0.9981 144 115 n.a. 2.0 1.7 1.9
[M+HCOO]− 10 0.005–25 0.9886 91 113 n.a. 5.4 0.9 2.1
PE 24:0 [M+H]+ 10 0.005–100 0.9957 97 91 59 1.5 2.1 3.2
[M−H]− 10 0.005–10 0.9955 109 121 n.a. 4.1 3.6 2.9
PS 24:0 [M+H]+ 10 0.005–100 0.9981 54 120 82 5.8 1.7 2.1
[M−H]− 10 0.005–100 0.9976 57 126 83 6.8 1.6 1.1
PG 24:0 [M+H]+ 100 0.05–100 0.9982 28 90 102 6.3 1.6 2.5
[M−H]− 10 0.005–100 0.9945 117 122 67 2.6 0.8 1.8
LPC 15:0 [M+H]+ 10 0.005–25 0.9954 124 119 n.a. 2.3 1.6 2.4
[M+HCOO]− 20 0.01–100 0.9939 51 102 71 3.1 1.0 2.0
Cer 17:0 [M+H−H2 O]+ 10 0.005–10 0.9867 78 112 n.a. 4.5 2.8 3.2
[M−H]- 100 0.05–10 0.9940 100 80 n.a. 3.0 2.1 2.1
SM 17:0 [M+H]+ 20 0.01–50 0.9968 135 121 95 1.4 2.4 3.6
[M+HCOO]- 100 0.05–10 0.9940 76 90 n.a. 3.7 1.8 2.6

mated, and dwarfed by the time required for sample preparation
particularly data processing.
3.2. Lipid detection and identification
The Orbitrap instrument used is able to provide exceptional
spectral quality, and routinely reaches values for resolution
(FWHM) of over 110 000 for lysophospholipids, over 90 000 for
phospholipids, over 80 000 for triacylglycerols and over 70 000 for
cardiolipins. Mass accuracy is usually well below 3 ppm, and below
10 ppm even for low-abundance analytes (see also Supplementary
Tables 7–9). The ability to obtain MS/MS spectra in the ion trap
in parallel to full scanning in the Orbitrap is an enormous bene-
fit. Using this technique, we achieve cycle times of 1.7–2 s (for one
Orbitrap full scan at full resolution and 10 ion trap MS/MS scans),
which is sufficient to describe almost all analytes with more than
10 data points per mass chromatographic peak. Even without an
inclusion list, for one representative liver sample, tandem mass
spectra were available for 127 of the 195 quantified lipid molec-
ular species, and additionally for all internal standards, resulting
in MS/MS coverage of 65%. This completely untargeted approach
is suitable for identification of unexpected or novel lipids, which
requires no additional chromatographic runs, but only re-analysis
of existing data.
In contrast to techniques without precursor ion selection such
as MSE , All Ion Fragmentation, All Ions MS/MS or MS/MS ALL, our
approach relies on generation of fragment spectra of only one m/z
value. Assignment of fatty acyl composition and position is facil-
itated when all fragments displayed ideally belong to only one
precursor ion. As is the case with all ion trap scans, our MS/MS
spectra suffer from low mass cutoff. While the major diagnos-
tic head group fragment (protonated phosphocholine, m/z 184) of
long-chain protonated PC is lost by low mass cutoff, MS/MS spec-
tra of the corresponding sodium adducts may be interpreted. These
show characteristic head group neutral losses of 59 Da (trimethy-
lamine) and 183 Da (phosphocholine) which are not affected by low
mass cutoff. For almost all lipid classes investigated, interpretation
of both positive and negative MS/MS spectra delivers specific head
group fragments as well as fragments indicative of both identities
and positions of the fatty acyls. Exceptions are glycerolipids (TGs
and DGs) of which the positions of the fatty acyls cannot easily be

Fig. 1. Construction of homologous series to aid in identification of lipid molecular species. Reversed phase retention behavior of phospholipids is based on number of carbon
atoms and degree of unsaturation. Extracted ion chromatograms of protonated PE 37:4 shows numerous interferences from (a) isobaric PC 34:4, (b) the second isotope of
PE 37:5, and (c) sodiated PE 35:1. Correct peaks identified by Lipid Data Analyzer are filled red, green background denotes that tandem mass spectra of the corresponding
analytes are available. Note that most of the lipid molecular species depicted show multiple chromatographic peaks belonging to structural isomers differing in fatty acid
composition. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
76 A. Triebl et al. / J. Chromatogr. B 1053 (2017) 72–80
Fig. 2. Number of glycerolipids (GL), glycerophospholipids (GP), and sphingolipids
(SP) identified in murine liver, A549 cells and C. elegans with a 1% cutoff for each
lipid class. Glycerolipids were not analysed in the A549 sample.
deduced, since the fatty acyls at the sn-1 and sn-3 position behave
identically upon collisional activation.
Using this approach, hundreds of lipids can be identified in a
single lipidomic sample. It is necessary to limit oneself concerning
the number of lipid species for several reasons: Firstly, the pareto
principle also applies to lipidomics, and a small number of lipid
species usually constitutes the bulk of the lipid biomass. An in-
depth analysis of TGs in murine liver shows that out of a total of
375 identified molecular species, 18 species already represent over
90%, and 128 species represent over 99.9% of the total class amount
(Supplementary Fig. 5). This effect is even more pronounced for the
class of PI, where only 5 molecular species represent over 95% of the
total class amount. Thus it is necessary to introduce a cut-off simply
to reduce the number of molecular species which contribute only
negligibly to the whole lipid class. Another reason why we do not
advocate analyzing extremely low-abundance species with global
methods is that these species tend to have high values of standard
deviation, increasing the chances of finding no difference where in
fact there is one. We instead argue that extremely low-abundance
compounds should be quantified with specialized methods and not
with global approaches. For these reasons, a class-specific cut-off
value of 1% was chosen, which means that for each lipid class, all
lipid species which together contribute less than 1% to the total
class amount were omitted from the analysis. We argue that this is
a good compromise between comprehensiveness and data analysis
effort for a global untargeted approach.
In total, 195 lipid species were identified in murine liver (58
glycerophospholipids, 106 glycerolipids, and 31 sphingolipids),
while a total of around 350 lipid species were identified in C. elegans
(see Fig. 2 for overview and Supplementary Tables 2–4 for complete
results). The difference in the number of species found is simply due
to the fact that while liver contains in its lipids almost exclusively
fatty acyls with an even number of carbon atoms, lower organisms
such as C. elegans contain a large amount of fatty acyls with an odd
number of carbon atoms, thereby drastically increasing the num-
ber of lipid species. These numbers of lipid species identified are
in accordance with existing lipidomic methods employing a sin-
gle LC/MS method [49–51], while analysis of a substantially larger
number of lipids often requires use of multiple analytical methods
[52], specialized chromatographic methods, such as supercritical
fluid chromatography [30], or hyphenated chromatographic meth-
ods, such as on-line or off-line 2D-LC [49,53].
Most importantly however, the presented method includes all
necessary steps for large-scale lipidomic measurements for highly
complex organisms, such as C. elegans; a sample preparation that
is applicable to a wide range of biological matrices, a powerful
chromatographic approach including separation of many fatty acyl
structural isomers, and a software for data processing, lipid identi-
fication and quantitation.
3.3. Separation of structural and positional isomers
The chromatographic approach used is able to separate a wide
variety of structural isomers, which can then be identified based
on their retention behavior or tandem mass spectra. The simplest
cases are PC or PE odd-carbon internal standards, which are struc-
tural isomers of even-carbon PE or PC analytes, but can easily be
differentiated. However, many other diacyl phospholipids or tria-
cylglycerols show more than one peak on the lipid species/bond
type level (Fig. 3). Inspection of the tandem mass spectra reveals
that these peaks almost always correspond to structural isomers
with different fatty acyl compositions which are completely or at
least partially chromatographically separated. This is a substantial
improvement over the method formerly employed in our lab [31].
Furthermore, it is an advantage over using HILIC, where individ-
ual species within lipid classes are not separated, thus structural
isomers can also not be chromatographically separated.
Many lysophospholipids also show double peaks, which are
most likely positional isomers (Supplementary Fig. 6). Our obser-
vations are consistent with published data [54–57] stating that the
regioisomers with the acyl group at the sn-2 position elute ear-
lier in reversed phase HPLC and upon collisional activation of the
protonated molecule show a higher relative intensity of the head
group-derived fragment (m/z 184 and neutral loss of 141 for LPC
and LPE, respectively) as compared to the neutral loss of water.
However, it is possible that intra-molecular acyl migration, at least
to some extent, may occur artificially, during sample preparation
or storage, depending on the exact circumstances [58].
3.4. Quantitation
One advantage of using reversed phase chromatography is that
lipid species differing in one double bond are baseline separated by
chromatography, so there is no interference in m/z from isotopo-
logues containing two 13 C atoms. This is an advantage over shotgun
or HILIC approaches, where either all analytes, or all lipid species
of one class, respectively, enter the mass analyzer at the same time,
thereby requiring complicated isotope correction or de-isotoping
algorithms [4,30,59–61], except when using mass analyzers with
ultra-high resolving powers [62].
Classical quantitative analysis, as is customary in LC analysis,
relies on calibration curves for each analyte, ideally in an analyte-
free matrix. In LC/MS lipidomics however, this is not possible due
to the lack of analyte-free matrices and the sheer number of ana-
lytes investigated. Stable isotope labeled internal standards, the
gold standard of absolute quantitation, are hardly available and
their use is rarely economical. While absolute amounts are often
required, they should be treated with care and knowledge of the
limitations of their accuracy. For these reasons, absolute quanti-
tation at the highest level of quality is very difficult to achieve.
Nevertheless, even if absolute values may be subject to error, up-
or downregulations of analytes between sample groups are clearly
detectable. Therefore we believe that absolute quantification in the
field of LC/MS lipidomics is best considered semi-quantitative and
its value and significance lies in comparing multiple groups (phys-
iological/developmental states, wild-type vs. knockout, treatment
vs. control etc.) and focusing on fold changes rather than on abso-
lute amounts, similar to pure profiling methods.
The selection of suitable internal standards for lipidomic
analysis is challenging. While quantitative standards containing
odd-chain fatty acyls are used for lipidomic analysis of higher
organisms, they are unsuitable when the organism of interest –
such as C. elegans – contains high amounts of odd chain fatty acyls
 A. Triebl et al. / J. Chromatogr. B 1053 (2017) 72–80 77

Fig. 3. Separation of PE structural isomers from a C. elegans lipid extract. The chromatogram shows extracted ion chromatograms of deprotonated PE 34:2. Manual inspection
of tandem mass spectra reveals three structural isomers, differing in fatty acyl composition separated by reversed phase chromatography. The positions of the fatty acyls can
be determined by comparing the relative intensities of the carboxylate anions. Tandem mass spectrum of the internal standard PC 12:0/13:0 shows the carboxylates of the
sn-2 fatty acyl of higher relative intensity than of the sn-1 fatty acyl, in accordance with literature data [72].

[63]. A compromise would be the use of internal standards which
merely correspond to low-abundance endogenous lipid species
and spiking them at relatively high concentrations (e.g. 25:0 phos-
pholipid species, as performed here), thereby superimposing the
endogenous concentrations. The C. elegans lipid extract was mea-
sured with and without addition of the internal standards and the
baseline abundance of the analytes corresponding to the internal
standards was found to be negligible – either not detected or at least
90-fold lower than when internal standards were added. 1-(1Z-
alkenyl)-2-acyl-PC and -PE, also termed plasmalogens, for which
there are no LIPID MAPS quantitative internal standards available,
may be quantified with diacyl–PC and −PE internal standards, as
the responses of PC P-36:1 and PE P-36:1 are within 20% of the
respective diacyl species, investigated in triplicate measurements
at concentrations of 0.5 and 5 ␮M for both positive and negative
polarities. We also argue that lysophospholipids may be quantified
using a short-chain diacyl species if no other suitable standard is
available.
Another point to be considered is that the LIPID MAPS diacyl
phospholipids internal standards may contain small amounts of
lysophospholipids, probably derived from chemical degradation
during storage etc. This results in the corresponding lysophospho-
lipid molecular species being present in an injection containing
only LIPID MAPS diacyl phospholipid internal standards. Although
their relative abundance may seem negligible (in the case of LPE,
0.5–2% of the area of the respective diacyl PE species), it should
be noted that lysophospholipids usually represent only a fraction
of the respective diacyl phospholipid class and therefore, degrada-
tion of only a minor amount of diacyl phospholipids could already
result in an overestimation of the corresponding lysophospho-
lipid species. However, this problem concerns only few molecular
species, but their results should be critically evaluated. Source
fragmentation in contrast, which would also lead to an artificial
increase of lysophospholipids, plays no role here, as the authentic
lysophospholipids are easily distinguished by retention time from
those generated in the ion source.
Although almost all lipid classes are detectable in both polarities,
quantitation was only performed for one adduct ion per lipid class.
To increase selectivity, tandem mass spectra of another adduct in
the same polarity, or in the other polarity, can be inspected for
further confirmation. E.g., while PE was quantified in positive polar-
ity (due to the selectivity offered by the neutral loss of the head
group), negative polarity is far better suited for identification of
the identities and positions of the fatty acyls.
3.5. Method validation and evaluation of matrix effect
To preface this chapter, any validation in large-scale lipidomics
is a compromise: Ideally, and classically, method validation is
performed with one validation standard per analyte in an analyte-
free matrix. Using hundreds of validation standards is simply not
feasible, and analyte-free matrices for lipidomics are simply non-
existent.
The chromatographic setup used is capable of reproducible per-
formance. Retention times of naturally occurring lipids from 13
lipid classes (TG 52:2, Cer 42:2, HexCer 42:1, CL 72:8, DG 34:1,
LPC 18:0, LPE 18:0, PC 34:1, PE 34:2, PG 34:1, PI 38:4, PE P-38:4,
PS 38:4, and SM 42:2) remained constant over the course of 48
consecutive injections of a liver extract (average relative standard
deviation 0.09%). Autosampler stability over 48 h was assessed by
calculating RSD for the concentrations of these 13 lipid species,
which were on average 8.6%, indicating no changes in concentra-
tion with prolonged analysis time and storage in the autosampler
(Supplementary Table 5). Relative standard deviation of the peak
area of naturally occuring PC 34:1 in five replicate injections of
liver, C. elegans and A549 cell lipid extracts was 6.8%, 5.3% and 2.2%,
respectively, and retention time was unaffected by the different
sample matrices (overall relative standard deviation 0.4%).
78 A. Triebl et al. / J. Chromatogr. B 1053 (2017) 72–80
It is well established that the electrospray ionization process has
a narrow linear dynamic range and the upper concentration limit is
frequently cited as 10−5 M [64–66], although this value is strongly
expected to depend on the analytes and exact environment. The
linearity of the presented method was evaluated for 9 different
lipid classes by spiking a liver extract with a non-endogenous or
at least low-abundance lipid species in the range of 1 nM–100 ␮M.
While some lipids were found to exhibit almost perfect linear-
ity over the whole concentration range investigated (e.g. PG 24:0
[M+H]+ , R2 = 0.9989 and [M−H]− R2 = 0.9945), others show a dis-
tinct saturation-like response at higher concentrations (e.g. PC 24:0
[M+H]+ ) (Supplementary Fig. 7). It is noteworthy that some lipid
classes may have a broader linear range in one polarity than the
other, and also adducts of the same lipid class deviate in their linear
range (Table 1).
While arguably a better choice as validation standards, the LIPID
MAPS internal standards used for quantitation are supplied only at
relatively low concentrations (425 ␮mol/L); thus the lipid species
listed in Chapter 2.7 were chosen as validation standards. The non-
spiked liver matrix already contains detectable although minute
amounts of the lipid standards added for validation purposes (e.g.
naturally occurring 12:0/12:0 phospholipid species). These were
nevertheless chosen for validation because their natural abundance
is insignificant compared to the vast excess of other sample com-
ponents and linearity is not negatively affected. While carryover
of the injection system can be ruled out, lower limits of quanti-
tation cannot be classically determined. The limit of quantitation
was therefore conservatively determined as the lowest concentra-
tion point which differed substantially from the non-spiked sample
(for an example, see Supplementary Fig. 8). LLOQs vary for different
lipid classes, but are generally in the range of a few fmol on column
(Table 1).
While using multiple internal standards for one lipid class is
beneficial, they may differ in their response (i.e. peak area), and
additionally suffer from matrix effects, a well-known phenomenon
in LC/MS [67–69]. For example, the four PS internal standards differ
in their responses by as much as 44% when analyzed at a concentra-
tion of 1.5 ␮M with and without sample matrix, whereas most other
lipid classes analyzed do not exhibit grave matrix effects (Supple-
mentary Table 1). Additionally, instrument response is known to
depend on factors such as lipid class, acyl chain length and degree
of unsaturation, even within the same lipid class [70,71]. Thus,
internal standards of differing molecular species and eluting at
different retention times may experience different degrees of ion
suppression, or even ion enhancement, caused by coeluting sample
components. Also, ionization efficiency (i.e. number of ions formed
in the electrospray process) may be different, as solvent composi-
tion is not constant during gradient elution. This effect is observable
to at least some degree in most lipid classes for which multiple
internal standards are available (Supplementary Fig. 9). It is par-
ticularly grave for the class of DG, where the internal standards
differ enormously with chain length and number of double bonds.
For example, under the conditions employed, the signal of IS d5DG
28:0 is only 2% of the signal of IS d5DG 40:8 even within the same
sample. On the other hand, most other lipid classes do not show
grave differences in instrument response between species differing
in chain length and degree of unsaturation.
To minimize these problems, the quantitation software
employed uses a sophisticated algorithm, explained in greater
detail in the original publication [35], which selects the best inter-
nal standard, if multiple internal standards are available – as is the
case for most glycerophospholipids. Despite the previously men-
tioned differences in response, this approach results in very good
values for accuracy for almost all lipid classes investigated while
precision is excellent throughout (Table 1), resulting in a mean
relative standard deviation of 7.1% for all analytes quantified.
4. Conclusion
A sensitive and highly reliable method for detection and
quantitation of molecular species of multiple glycerolipid, glyc-
erophospholipid and sphingolipid classes was developed. The
method is applicable to a wide variety of biological matrices with a
versatile but simple MTBE extraction procedure. Intact lipid species
are detected by a high mass resolution Orbitrap Velos Pro hybrid
mass spectrometer in both positive and negative polarity, while
tandem mass spectra acquired in the linear ion trap provide deep
insight into the molecular structure. The reversed phase chro-
matography approach used is capable of robust performance, offers
important additional selectivity, and is able to resolve structural
and positional isomers. Finally, lipids are identified and quanti-
fied by ample use of quantitative internal standards together with
an automated software package, thereby offering a flexible plat-
form for untargeted lipidomic analysis of even the most complex
biological samples.
Method validation indicates a linear range of four orders of mag-
nitude for most lipid classes investigated, while limits of detection
are in the low femtomolar range. The method was applied to murine
liver, adenocarcinomic cells, and C. elegans, resulting in quantita-
tive and structural information about hundreds of lipid species in
a manner applicable to large-scale lipidomic studies.
Conflict of interest
All authors declare no conflict of interest.
Acknowledgements
This work was supported by the Austrian Science Fund (FWF) [P
26148].
The authors would like to thank Professor Frank Döring
(Christian-Albrechts-Universität zu Kiel, Germany), Dr. Katharina
Leithner (Medical University of Graz, Austria), and Dr. Kathrin
Zierler (Karl-Franzens-Universität Graz, Austria) for providing C.
elegans, A549 cells, and mouse liver samples.
Appendix A. Supplementary data
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.jchromb.2017.
03.027.
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