Trends in Analytical Chemistry 55 (2014) 113

Contents lists available at ScienceDirect

Trends in Analytical Chemistry

journal homepage: www.elsevier.com/locate/trac

Review

Dilute-and-shoot-liquid chromatography-mass spectrometry for urine analysis

in doping control and analytical toxicology
K. Deventer a,, O.J. Pozo b, A.G. Verstraete c, P. Van Eenoo a
a
DoCoLab (Doping Control Laboratory), Ghent University (UGent), Department of Clinical Chemistry, Microbiology and Immunology, Technologiepark 30, BE-9052
Zwijnaarde, Belgium
b
Bioanalysis research group, IMIM-Hospital del Mar, Doctor Aiguader 88, 08003 Barcelona, Spain
c
Ghent University (UGent), Department of Clinical Chemistry, Microbiology and Immunology, De Pintelaan 185, BE-9000 Gent, Belgium

a r t i c l e

i n f o

Keywords:
Dilute-and-shoot
Direct detection
Direct injection
Doping
Drugs-of-abuse
LC-MS
Liquid chromatography
Mass spectrometry
Toxicology
Urine

a b s t r a c t
In the past 20 years, liquid chromatography-mass spectrometry (LC-MS) has become a standard analytical technique in doping control and toxicology laboratories. Research groups have successfully applied it
to detect substances by direct injection, or dilute-and-shoot-LC-MS (DS-LC-MS).
However, some urinary components can precipitate into the vial, hampering the correct injection. Dissolved urinary matrix is responsible for shifted retention times and ion suppression or ion enhancement.
To compensate for the effect of the matrix, an isotope-labeled internal standard (IL-ISTD) is the best choice.
Dilution can also minimize the matrix effect, but can result in reduced analyte detectability. Hence,
DS-LC-MS methods are predominantly available for substances for which the required urinary detection
levels are high and that show good ionization efciency.
Taking into account the progressive increase in instrument sensitivity, we expect that the application of
DS-LC-MS will also come available for substances with low required detection levels or limited ionization
efciency.
2013 Elsevier Ltd. All rights reserved.

Contents
1.
2.

3.

4.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Applications and benefits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
2.1.
Common DS-LC-MS applications in doping control and analytical toxicology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
2.2.
Doping-specific applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
2.3.
Applications to analytical toxicology and workplace drug testing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Challenges. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
3.1.
Analyte detectability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
3.2.
Matrix effects. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
3.2.1.
Particulates and sediment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
3.2.2.
Retention-time shift . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
3.2.3.
Ion suppression (IS) or ion enhancement (IE). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
3.2.4.
Other interferences. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Perspectives and conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

1. Introduction
Increasing sample throughput is a challenge for every analytical
laboratory. An important factor that limits the throughput is
Corresponding author. Tel.: +32 (0)9/3313295; Fax: +32 (0)9/3313299.
E-mail address: Koen.Deventer@UGent.be (K. Deventer).
0165-9936/$ - see front matter 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.trac.2013.10.012

sample preparation and instrument-analysis time. Omission or

reduction of the sample clean-up and a faster analysis can result
in shorter reporting times in addition to cost savings for labor,
reagents and consumables. For anti-doping purposes, fast analysis
can be important during major sports events, where 24-h reporting
times are mandatory. From a toxicological point of view, fast

K. Deventer et al. / Trends in Analytical Chemistry 55 (2014) 113

analysis facilitates prompt diagnosis which is required in some

emergency cases.
Without doubt, the introduction of liquid chromatographymass spectrometry (LC-MS) in the early 1990s was a major step towards fullling this desire. Especially, the elimination of lengthy
derivatization procedures, needed prior to GC-MS analysis, and
MS detection showing greater selectivity than diode-array detection (DAD), allowed samples to be processed faster. Consequently,
LC-MS is now ubiquitous in doping control and toxicology laboratories and several recently published reviews described emerging

applications [18]. Most of these applications use sample-preparation steps normally based on liquid-liquid extraction (LLE) or
solid-phase extraction (SPE) in order to clean up and/or concentrate the analytes. Further increase in sample throughput can be
achieved by leaving out the chromatography or removing the sample preparation prior to the LC-MS analysis.
Omission of chromatography prior to MS analysis, also referred
to as ow-injection MS (FI-MS) was already introduced in the mid1990s [9]. Other techniques omitting chromatographic separation
are desorption electrospray ionization MS (DESI-MS) [10] and

Table 1
DS-LC-MS methods for urine in the eld of doping control
Compounds1

Ref.

Sample preparation

CU

DF

[26] diuretics, stimulants,

beta-blockers
[60] HIF-stabilizers

500 lL urine + 10 lL ISTD (H2O) + 500 lL H2O

190 lL urine + 10 lL ISTD

[45] THCA

200 lL urine + 50 lL ISTD (MeOH) + 50 lL NaOH(10 M)/7 min heating @

56C + 200 lL MeOH/HOAc(1/1)
20 lL urine + 180 lL ISTD (H2O)
60 lL urine + 1.14 mL ISTD (aqueous 1% HOAc)

no

2.5

no
no

10
20

10 lL urine + 240 lL ISTD (aqueous 0.001% HOAc,1 mM NH4OAc)

25

[77] steroids

10-fold DIL with H2O + 50 lL ISTD (H2O)

no

10

[76] steroid-GLUC

25-fold dilution

c+f

25

[19] ephedrines

45-fold DIL with H2O/Then 200 lL DU + 200 lL of ISTD (H2O)

90

[50] ETG

2-fold DIL with aqueous 2mM NH4OAc

[24] diuretics, stimulants,

narcotics
[53] HES, dextran
[23] diuretics stimulants

90 lL urine + 10 lL ISTD (MeOH)

no

90 lL urine + 10 lL ISTD (MeOH)

100 lL urine + 900 lL ISTD (aqueous 50 mM NH4OAc/0.001% HOAc)

no
c

1
10

[54] HES dextran

[21] ephedrines
[21] salbutamol, morphine

100 lL urine +100 lL H2O/ACN (5 mM NH4OAc/0.1% HCOOH)

10-fold DIL with H2O/Then 160 lL DU + 40 lL ACN
1 mL urine + 1 mL buffer pH 6.7 + 50 lL ES/1 h heating @ 55C + 0.5 mL K2CO3
solution Then 160 lL DU + 40 lL ACN
20 lL urine + 20 lL ISTD (ACN) + 760 ACN

c
c
c

2
10
2.5

40

[18] ephedrines
[42] Morphine-GLUC,
morphine
[25] diuretics, stimulants

[59] nicotine
[56]
[52]
[74]
[57]

salbutamol
diuretics
steroid-GLUC
formoterol

nr
20 lL urine + 960 lL H2O + 20 lL ISTD (aqueous 0.1% HCOOH)
200 lL urine + 50 ll ISTD (MeOH)
200 lL urine + 200 lL ISTD (10 lL ES, 10 lL ISTD, 180 lL buffer pH 7)/1.5 h
heating @ 56C
[20] efedrines
nr
[20] salbutamol, morphines nr
[51] ETG, ETS
2-fold DIL with aqueous 2 mM NH4OAc

no
c
c+f
c
c+f
c+f
c

100 lL urine + 100 lL ISTD (aqueous 20 mM NH4OAc)

no

100 lL urine + 5 lL ISTD + 95 lL PBS-solution

[78] hydrocortisone

1 mL urine + 1 mL H2O + 50 lL ISTD

c+f

[79] theobromine

0.5 mL urine + 1 mL aqueous 0.1% HOAc

c+f

[81] 3-methoxytyramine

2.5 mL urine + 1 mL buffer pH 5 +50 lL ISTD + 50 lL ES/2.5 h heating @ 50C/ c+f 20

Then 100 lL DU + 500 lL ACN
no 900
100 lL urine+9900 lL H2O/Then 1 mL DU + 8 mL mobile phase

[55] Salbutamol
[61] benuorex +
metabolites

0.5 mL urine + ISTD(MeOH) +0.5 mL H2O

0.1 mL urine + ISTD + 0.9 mL

c
c

multi-class
screening
research
quantitation/
conrmation
quantitation
quantitation
multi-class
screening
horse-doping
research
horse-doping
research
conrmation/
quantitation
quantitation/
research
multi-class
screening
research
multi-class
screening
screening
quantitation
quantitation

research/
quantitation
1
quantitation
50
screening
1.25 quantitation
2
quantitation/
conrmation
1
quantitation
1
quantitation
2
quantitation/
research
2
multi-class
screening
2
research/
quantitation

[22] diuretics, stimulants

narcotics
[58] AICAR

[80] Salicylic acid

Purpose

2
10

quantitation/
horse doping
quantitation/
horse doping
quantitation/
horse doping
quantitation/
horse doping
quantitation
research

Interface/
MS-type

Matrix
effect2

ESI/FSHRMS

7-127%

ESI/FSHRMS
ESI/MS/MS
ESI/SRM

nr
97%

ESI/SRM
ESI/SRM

85%
64-120%

ESI/SRM

15- 105%

APCI/ITMS/MS

nr

ESI/ITMS/MS

nr

APCI/SRM

87%

ESI/SRM

nr

ESI/SRM

negligible

ESI/SRM
ESI/FSHRMS

no ME
43-168%

ESI/FSHRMS
ESI/SRM
ESI/SRM

48-70%
nr
nr

ESI/SRM

70-98%

ESI/SRM
ESI/SRM
ESI/SRM
ESI/SRM

no ME
82-117%
81-97%
93-103%

ESI/ITMS/MS
ESI/ITMS/MS
ESI/SRM

nr
nr
nr

ESI/SRM

50%-130%

ESI/SRM

no
signicant
ME
49-75%

ESI/ ITMS/MS
ESI/FSHRMS
ESI/ITMS/MS
ESI/FSHRMS
ESI/FSHRMS

40-90%
64-88%

ESI/FSHRMS

84-86%

ESI/SRM
ESI/SRM ESI/
FSHRMS

negligible
nr

CU: Clean-up; c: centrifugation; f: Filtration; IV: Injection volume; FSHRMS: Full scan high resolution mass spectrometry; APCI: Atmospheric pressure chemical ionization;
ESI: Electrospray ionization; IT: Ion trap; DU: Diluted urine; ES: Enzyme solution; DIL: Diluted; nr: Not reported; DF: Dilution factor
1
For multi-class methods, only the three principal classes are mentioned. Abbreviated substances are explained in the main text.
2
Value = 100%, no ME; value < 100%, ion suppression; value >100%, ion enhancement.

K. Deventer et al. / Trends in Analytical Chemistry 55 (2014) 113

Table 2
DS-LC-MS methods for urine in analytical toxicology
Compounds1

Ref.

Sample preparation

CU

DF

Purpose

Matrix effect2

Interface/
MS-type

[32]

amphetamines

20 lL urine + 80 lL ISTD (H2O)

no

quantitation/
conrmation

ESI/SRM

[13]

methylphenidate, RA

2 lL urine + 200 lL ISTD (MeOH)

no

100

ESI/SRM

[71]

designer amphetamines

ESI/SRM

75%

[49]

ETG, ETS, ETP

1 mL urine + 50 lL ISTD + 4 mL H2O/ACN (98:2)

(20 mM NH4OAc-buffer pH 3)
50 lL urine + 50 lL ISTD + 900 lL ACN/H2O (50:50)

screening/
quantitation/
conrmation
screening

25-fold suppression at void

volume Negligible attR of
analytes
nr

no

20

ESI/SRM

[72]

psychoactives from
plants

no

ME observed at void volume

50%
signal reduction at void
volume

[31]
[36]

Opioids, cocaine and

metabolites
ETS

50 lL urine + 150 lL ISTD (H2O) Or 300 lL

urine + 50 lL ES/ Heating 1h @ 37C/ Then
50 lL + 150 lL ISTD (H2O)
100 lL urine + 10 lL ISTD (MeOH/ACN)

quantitation/
conrmation
multi-class
screening

no

[30]

700 drugs

100 lL urine + 20 lL ISTD (H2O) + 100 lL aqueous

0.1% HCOOH
100 ll urine + 100 lL ISTD (ACN) + 800 ll H2O

10

[30]

700 drugs

100 ll urine + 100 lL ISTD (ACN) + 100 ll H2O

[40]

opiates

1 mL urine + 10 lL ISTD (H2O/ACN) (95:5)

[44]

THCA, THCA-GLUC

500 lL urine + ISTD (MeOH) + 500 lL MeOH

[29]

200 lL urine + 50 lL ISTD + 1800 lL H2O

10

[29]

amphetamines,
benzoylecgonine
opiates

1.4

[39]

opiates

1 mL urine + 50 lL ISTD (MeOH) + 400 lL ES Heating

2 h @ 50C
20 lL urine + 80lL ISTD (H2O)

no

[48]
[73]

10 lL urine + 90 lL ISTD (H2O)

100 lL urine + 25 lL ISTD + 100 lL aqueous 20 mM
NH4OOCH buffer, pH 3
200 lL urine + 200 lL ACN

no
f

10
2

[69]

ETS
BUP, NORBUP, BUPGLUC, NORBUP-GLUC
MDPV

[34]

MDMA, MDA

10 fold DIL by ISTD (H2O)

[28]

[70]
[85]
[43]

phenylethylamines,
hypnotics, Nbenzylpiperazine
DMA+ metabolites
ETG
THCA

[47]

no
2.2

no

APCI/
SRM
ESI/SRM

no ME observed beyond
k0 = 1
nr

ESI/SRM

nr

ESI/SRM

nr

ESI/SRM

quantitation/
conrmation
quantitation/
conrmation
quantitation/
conrmation
quantitation/
conrmation
research
research

ESI/SRM

highest for hydromorphone,

norcodeine, codeine,
oxycodone
72117%

ESI/SRM

98-132%

ESI/SRM

11%85%

ESI/SRM

35-fold at void volume 37%

ESI/MS
ESI/SRM

nr
nr

ESI/
FSHRMS
APCI/
SRM
APCI/
SRM

nr

ESI/FSMS
ESI/FSMS
ESI/SRM
ESI/SIM

nr
nr
90% loss of signal at void
volume 36%
nr

ESI/SRM
ESI/SRM

nr
89%

ESI/SRM

90%

APCI/
SRM
ESI/SRM

9194%

ESI/SRM

negligible

ESI/
FSHRMS
ESI/SRM

28%

ESI/SRM

nr

ESI/
FSHRMS
ESI/SRM
ESI/FSMS

nr

Research

no

10

screening

10 lL urine + 90 lL ISTD (H2O)

no

10

multi-class
screening

c+f
no
no

1.3
10
10

ETG

700 lL urine + 300 lL ACN

10 lL urine + 90 lL ISTD (H2O)
120 lL urine + 60 lL ISTD (H2O) + 30 lL NH4OH
(10 M) Heating for 15 min @ 100C
100 lL urine + 10 lL ISTD (H2O) + 250 lL MeOH

3,5

[46]
[66]

ETS
GH, GBL, 1,4-BD

100 lL urine + 20 lL ISTD 100 lL ACN

1:20 dilution with ISTD (H2O)

no
no

2
20

[65]

GHB, BHB, 1,4-BD, GBL

20

[63]

unitrazepam

100 lL urine + 100 lL H2O + 50 lL ISTD

(MeOH) + 1.75 mL aqueous 0.2% HCOOH
non

research
quantitation
conrmation/
quantitation
conrmation/
quantitation
research
quantication/
conrmation
screening

[37]

opioids, amphetamines,
cocaine analogues
GHB, 1-4-BD, GVL

100 lL urine + 100 lL ISTD (MeOH) + 1800 lL MeOH/

H2O (1:1)
20 lL urine + 40 ll ISTD (aqueous 0.1%
HCOOH) + 140 lL MeOH/H2O (10/90/0.1% HCOOH)
25 lL urine + 225 lL ISTD in ES

20

10

no

10

500 lL urine + 50 lL ES/ Heating for 60 min @ 60C/

Then 20 lL + 180 lL ISTD (H2O/MeOH)
1 mL urine + 13 lL ISTD

no

10

[33]

no

[38]

narcotics, stimulants,
benzodiazepines
stimulants, narcotics,
benzodiazepines
cocaine, BE

[41]

stimulants, narcotics

50 lL urine + 200 lL ISTD (aqueous 0.1% HCOOH)

no

[68]
[64]

GHB, GHB-GLUC
benzodiazepines

100 lL urine + 900 lL ISTD (aqueous 0.4% HCOOH)

1 mL urine + 50 lL ISTD Then 500 lL + 1 mL H2O

no
c+f

10
3

[35]

ESI/SRM

quantitation,
clinical research
quantitation/
conrmation
multi-class
screening
multi-class
screening
bioanalytical
applications

[67]

toxicological
studies
conrmation/
quantication
conrmation/
quantication
multi-class
screening
multi-class
screening
conrmation/
quantitation
multi-class
screening
research
screening

nr
nr

nr

nr

815%
20%

CU: Clean-up; c: centrifugation; f: Filtration; FSHRMS: Full scan high resolution mass spectrometry; FSMS: Full scan mass spectrometry; SIM: Selected ion monitoring; APCI:
Atmospheric pressure chemical ionization; ESI: Electrospray ionization; IT: Ion trap; DU: Diluted urine; ES: Enzyme solution; DIL: Diluted; nr: Not reported; DF: Dilution
factor.
1
For multi-class methods, only the three principal classes are mentioned. Abbreviated substances are explained in the main text.
2
Value = 100%, no ME; value <100%, ion suppression; value >100%, ion enhancement.

K. Deventer et al. / Trends in Analytical Chemistry 55 (2014) 113

direct analysis in real time MS (DART-MS) [11]. Despite the short

analysis time, which can be obtained with these techniques, their
application in doping control analysis and analytical toxicology is
limited for the urinary matrix [12,13]. Indeed, isobaric substances
(either from exogenous or endogenous sources) are frequently
encountered, so retention time (tR) is an obligatory identication
criterion in conrmation analysis [14,15].
Instead of leaving out the chromatographic separation, a successful trend in doping control and analytical toxicology is removal
of the extraction step prior to the analysis of urine. As a result, a socalled dilute-and-shoot-LC-MS (DS-LC-MS) method is created.
The application of the DS-LC-MS strategy in analytical toxicology
was briey discussed in two recent reviews [4,16].
The aim of this review is to summarize the current applications
of DS-LC-MS in doping control and analytical toxicology (including
workplace drug testing) for the analysis of urine samples. Since the
major drawbacks are the limited analyte detectability (sensitivity)
and the presence of the unextracted matrix, we describe their effects on DS-LC-MS methods in a separate section. In a nal section,
perspectives and conclusions are presented. For clarity, DS-LC-MS
is used throughout the paper to describe the technology in general
and covers all types of mass spectrometers. Details on the applied
(coupled) MS are presented in Tables 1 and 2.

2. Applications and benets

The ability to-skip the preconcentration sample preparation
steps successfully in favor of DS-LC-MS mainly depends on two
prerequisites, as follows.

Table 3
Selected examples of urinary MRPLs and cut-offs, applied in doping control and
workplace drug testing. [14,17]
Class
Exogenous anabolic Steroids
(AAS)
Peptide-hormones
Beta-2-Agonists
salbutamol
formoterol
clenbuterol
Hormone Antagonists and
Modulators
Diuretics
Stimulants
ephedrine
pseudoephedrine
Narcotics
morphine
fentanyl and metabolites
buprenorphine (or
metabolites)
6-mono-acetyl morphine
codeine
Cannabinoids
tetrahydrocannabinol-acid
cannabimimetics
Glucocorticosteroids
Alcohol
Beta-Blockers
Barbiturates
Propoxyphene
LSD
Benzodiazepines
temazepam
oxazepam
desmethyldiazepam

Doping control
(ng/mL)
25

NS

NS
20
1,000*
40*
0.2
20

NS
NS

NS

200
100
10,000*
150,000*
50
1,000*
2
5

NS
150500
500
500
52,000
200300
NS
5

50
NP

10
3002,000

150*
1
30
NP
100
NP
NP
NP
NP

15
NS
NS
NS
NS
150
300
1

NS, Not specied; NP, Not prohibited.

threshold.

Workplace drug testing

(ng/mL)

100200
100200
100200

 The rst is ionization behavior. Without doubt, compounds that

show efcient ionization are more likely to be analyzed by a DSLC-MS approach. For example, opiates and stimulants contain
an easily-ionizable basic nitrogen atom. Diuretics can include
sulfonamide groups, carboxylic acids or basic amines that can
be easily protonated or deprotonated.
 The second is the detection level required. In doping, these
are called the minimum required performance limits
(MRPLs), which depend on the class of compounds [17]. In
analytical toxicology, detection levels are called cut-offs
[14]. Selected examples of MRPLs and cut-offs are presented
in Table 3.
Each anti-doping laboratory should develop its methods so that
it can detect all substances at the MRPL routinely in every sample.
The MRPL is not a reporting level, and positive ndings may also
originate from concentrations below it. A qualitative determination is sufcient for most doping agents. For some substances, a
threshold is in place, and only concentrations above this (taking
into account measurement uncertainty) are considered as a doping
violation, so quantitative analysis is compulsory for threshold
substances.
In contrast to the MRPLs, urinary concentrations below the cutoff should not be reported. Both in workplace drug testing and
doping control, similar detection levels are required for stimulants
and opiates (Table 3). Groups, for which low detection levels are
required include anabolic steroids in doping control and LSD in
analytical toxicology.
2.1. Common DS-LC-MS applications in doping control and analytical
toxicology
Taking into account the two prerequisites above, the majority of
DS-LC-MS applications can be found for stimulants and opiates
(Tables 1 and 2).
Ephedrines are an important group of stimulants in anti-doping
analysis. The required detection levels for these substances are in
the range 5150 lg/mL. Their quantication by DS-LC-MS has been
investigated by several research groups [1821]. The differences in
dilution factors are noteworthy and range from no dilution [20],
over 10-fold dilution [18,21] to 90-fold dilution [19]. In addition
to these dedicated detection methods for ephedrines, other
stimulants, related to doping-control analysis, can also be analyzed
by DS-LC-MS [2226]. DS-LC-MS methods for stimulants have also
been described in analytical toxicology for the detection of (synthetic) phenylethylamines [2735] and cocaine [29,31,33,3538].
One of the benets of detecting stimulants by DS-LC-MS is the
improved detection capability of benzoylecgonine (BE) and ritalinic acid (RA), which are metabolites of cocaine and methylphenidate, respectively. Both metabolites have a zwitter structure and
are in a permanent charged state. Hence, they are difcult to extract using LLE. A comparative study between LLE and DS for these
substances showed that a 25-fold and 10-fold better detectability
was obtained by DS-LC-MS, due to the low recovery of LLE [25]
(Fig. 1).
Routine detection of narcotics by DS-LC-MS has also been described in both doping control [22,24,26] and in analytical toxicology [2931,33,35,37,3941]. In general, excellent ionization is also
observed for this class of substances, due to the presence of a basic
nitrogen in most of them. As a result, the low detection levels for
fentanyl (2 ng/mL) could also be reached in several DS-LC-MS
methods [24,26].
An important application of DS-LC-MS can be found in the
quantication of morphine. Since morphine is excreted both glucuronidated and free, DS-LC-MS allows quantication of the morphine-glucuronides (morphine-GLUC) directly together with the

K. Deventer et al. / Trends in Analytical Chemistry 55 (2014) 113

Cocaine

Benzoylecgonine

60
40
5.89

6.97

20
4.35
4

60
40
20

6.68

4.61

5
6
Time (min)

Relative Abundance

6.58

80
60
40
20
4.48

5.07

5.95
6

NL: 8.36E6
m/z= 167.50-168.50 F: + c
ESI SRM ms2
290.000@cid31.00
[82.001-82.011,
167.983-167.993] MS
Genesis T320D

80
60
40
20
0

5
6
Time (min)

4.555.17
4.5

6.22
5.0

5.5
Time (min)

6.0

RT: 4.18 - 6.68 SM: 5G

NL: 9.24E2
m/z= 149.50-150.50 F: + c
ESI SRM ms2
304.000@cid30.00
[149.995-150.005,
181.995-182.005] MS ICIS
T320D

100
RT: 5.27
6.09
4.06

20
0

80
60
40
20
0

5
6
Time (min)

NL: 7.26E5
m/z= 81.50-82.50 F: + c
ESI SRM ms2
290.000@cid31.00
[82.001-82.011,
167.983-167.993] MS
Genesis T320D

RT: 5.43

100
Relative Abundance

RT: 3.27 - 7.27 SM: 5G

NL: 7.04E5
m/z= 81.50-82.50 F: + c
ESI SRM ms2
290.000@cid31.00
[82.001-82.011,
167.983-167.993] MS
Genesis T320

RT: 4.40 - 6.40 SM: 1G

RT: 5.40
100

Relative Abundance

Direct-injection

3.99 4.58
20

60

6.0

RT: 5.43

NL: 4.70E3
m/z= 181.50-182.50 F: + c
ESI SRM ms2
304.000@cid30.00
[149.995-150.005,
181.995-182.005] MS ICIS
T320D

5.92

80

5.5
Time (min)

Time (min)

60

5.0

80

40

6.02

4.64 4.94

100

RT: 3.43 - 7.43 SM: 5G

RT: 5.43
100

40

20

RT: 4.18 - 6.68 SM: 5G

80

40

4.5

NL: 2.31E4
m/z= 149.50-150.50 F: + c
ESI SRM ms2
304.000@cid30.00
[149.995-150.005,
181.995-182.005] MS ICIS
T320

RT: 5.30

3.96

60

RT: 3.30 - 7.30 SM: 5G

100

NL: 8.95E6
m/z= 167.50-168.50 F: + c
ESI SRM ms2
290.000@cid31.00
[82.001-82.011,
167.983-167.993] MS
Genesis T320

80

5
6
Time (min)

Relative Abundance

Relative Abundance

Relative Abundance

80

3.66

Relative Abundance

Liquid/liquid-extraction

NL: 6.46E4
m/z= 181.50-182.50 F: + c
ESI SRM ms2
304.000@cid30.00
[149.995-150.005,
181.995-182.005] MS ICIS
T320

RT: 5.30

100

RT: 4.43 - 6.43 SM: 1G

RT: 5.43
100
Relative Abundance

RT: 3.30 - 7.30 SM: 5G

4.68 5.04
5

5.96

6.41

6
Time (min)

Fig. 1. Extracted ion chromatograms for cocaine (ions at m/z 182 (top) and m/z 150 (bottom)) and benzoylecgonine (BE) (ions at m/z 168 (top) and m/z 82 (bottom)) after
analysis of a routine sample. Upper chromatograms present the result after liquid-liquid extraction (LLE) (including a 10-fold concentration step) and after 25-fold dilution
(lower chromatograms). Zwitter compound BE shows equal signal intensities after dilute-and-shoot-liquid chromatography-mass spectrometry (DS-LC-MS) due to its poor
extraction recovery (<1%) in the LLE. However, after LLE, parent drug cocaine could be detected while, after DS-LC-MS analysis, cocaine was not detectable. (Reprinted from
[25] with permission).

aglycone. Applications can be found in doping control [20,21,2] and

analytical toxicology [30,37,40,41].
Tetrahydrocannabinol (THC) is the psychoactive substance
present in Cannabis sativa. It is generally screened for by immunoassay (IA) in toxicology and by GC-MS in doping control. However,
in conrmation analysis, quantitation should be performed.
Dedicated DS-LC-MS methods have been described to quantify
the principal metabolite tetrahydrocannabinol acid (THCA)
[4345]. Felli et al. [44] used the DS-LC-MS approach to determine
simultaneously free THCA and THCA-glucuronide (THCA-GLUC).
Their ratios allow discriminating between frequent and infrequent
users. Chebbah et al. [45] investigated the detection of THCA in
urine related to doping control analysis. Because THCA can form

positively and negatively charged ions, its quantication in both

ionization modes was investigated and validated.
In toxicology, the phase II metabolites of ethanol, ethyl sulfate
(ETS) and ethyl glucuronide (ETG) are analyzed by DS-LC-MS within the framework of alcohol-withdrawal programs or longer-term
detection of alcohol use [36,4649]. One paper also describes the
detection of ethyl phosphate (ETP) as an alternative marker to
monitor the abuse of ethanol [49]. The successful detection of
these substances by DS-LC-MS lies in the permanent ionized state
of the phosphate and sulfate groups and the easily-ionizable glucuronide moiety.
Currently, the effect of ethanol consumption on the endogenous
steroid prole is under investigation in doping control.

K. Deventer et al. / Trends in Analytical Chemistry 55 (2014) 113

ISTd (Mefrusid)
9.24e+005

acetazolamide
7.16e+004

althiazide
4.10e+004

amiloride
1.78e+005

5.43 min

4.22 min

5.52 min

4.12 min

azosemide
9.78e+004

bemethiazide
1.64e+005

bendroflumethiazide
3.32e+005

benzoylecgonine
3.98e+004

5.76 min

5.70 min

5.74 min

4.75 min

benzthiazide
9.44e+005

bumetanide
2.35e+005

buthiazide
4.74e+004

chlorazanil
4.80e+004

5.62 min

5.79 min

5.56 min

5.41 min

chlorothiazide
6.88e+004

chlorthalidone
3.83e+004

clopamide
1.09e+005

clorexolone
1.30e+005

4.31 min

5.12 min

5.28 min

5.58 min

cyclopenthiazide
1.43e+005

cyclothiazide
6.03e+004

diclofenamide
9.17e+004

epithiazide
2.41e+005

5.76 min

5.67 min

5.12 min

5.58 min

ethacrynic acid
3.67e+005

ethiazide
5.34e+004

furosemide
1.21e+005

hydrochlorothiazide
6.71e+004

5.86 min

5.13 min

5.50 min

4.50 min

Fig. 2. Extracted ion chromatograms of analytes from different doping classes in a quality control sample analyzed with dilute-and-shoot-liquid chromatography-mass
spectrometry (DS-LC-MS): diuretics (125 ng/mL) (acetazolamide, althiazide, amiloride, azosemide, bemethiazide, bendroumethiazide, benzthiazide, bumetanide, buthiazide,
chlorazanil, chlorothiazide, chlorthalidone, clopamide, clorexolone, cyclopenthiazide, cyclothiazide, diclofenamide, epithiazide, ethacrynic acid, ethiazide, furosemide,
hydrochlorothiazide, hydroumethiazide, indapamide, mefruside metabolite, methyclothiazide, meticrane, metolazone, muzolimine, piretanide, polythiazide, torasemide,
triclomethiazide, xipamide), stimulants and phase I metabolites (500 ng/mL) (benzoylecgonine, ritalinic acid, ephedrine, pseudoephedrine, norephedrine, norpseudoephedrine), stimulant phase II metabolites (500 ng/mL) (p-OHAM-sulfate, ethamivan-sulfate), uricosuric agent (125 ng/mL) (probenecid), allosteric modier of hemoglobin
(100 ng/mL) (RSR 13), nucleoside (1500 ng/mL) (AICAR), peptide-fragment (10 ng/mL) (GHRP-2 metabolite), selected androgen receptor modulator (100 ng/mL) (Andarine),
plasma volume expanders (500 lg/mL) (HES, dextran), beta-2-agonists (100 ng/mL) (formoterol, salbutamol, salmeterol), phase II metabolites of plasticizers (550 ng/mL)
(5oxo-MEHP glucuronide, 5OH-MEHP-glucuronide), and narcotics (200 ng/mL) (methadone, pethidine, fentanyl, oxycodone). (Reprinted from [24] with permission).

K. Deventer et al. / Trends in Analytical Chemistry 55 (2014) 113

hydroflumethiazide
1.42e+005

indapamide
2.58e+005

mefruside metabolite
4.45e+005

methyclothiazide
4.38e+004

4.94 min

5.59 min

5.24 min

5.42 min

meticrane
1.89e+004

metolazone
5.76e+004

modafinilic acid
1.75e+005

muzolimine
1.88e+005

4.94 min

5.48 min

5.39 min

5.50 min

piretanide
2.89e+005

polythiazide
1.52e+005

probenecid
6.41e+005

ritalinic acid
2.76e+004

5.68 min

5.73 min

5.81 min

4.48 min

RSR 13
3.30e+006

torasemide
5.62e+005

triclomethiazide
1.31e+005

xipamide
1.49e+005

5.80 min

5.25 min

5.36 min

5.76 min

formoterol 1
9.83e+004

salbutamol
1.61e+005

salmeterol
2.39e+004

methadone
1.56e+006

4.63 min

3.93 min

5.15 min

5.12 min

pethidine
2.89e+006

fentanyl
1.39e+006

oxycodone
8.42e+004

ephedrine
6.16e+004

4.72 min

4.92 min

4.28 min

3.84 min

Fig. 2 (continued)

K. Deventer et al. / Trends in Analytical Chemistry 55 (2014) 113

pseudoepehdrine
1.49e+005

norephedrine
5.86e+004

norpseudoephedrine
6.24e+004

AICAR
6.63e+004

3.96 min

2.68 min

3.34 min

1.62 min

GHRP-2 metabolite
1.16e+006

andarine
1.19e+006

andarine metabolite
1.17e+003

dextran
2.45e+004

4.74 min

5.56 min

5.32 min

3.76 min

HES
3.42e+004

p-OHAM-sulf
1.04e+004

p-OHMA-sulf
1.77e+004

ethamivan-sulf
2.05e+003

4.02 min

3.85 min

3.87 min

5.02 min

5oxo-MEHP gluc
2.90e+005

5OH-MEHP gluc
2.63e+005

5.18 min

5.15 min
Fig. 2 (continued)

Consequently, DS-LC-MS methods for the detection of ETS and ETG

were described [50,51].
Besides these classes, which can be found in both elds, several
other groups of substances are relevant to only either doping control or analytical toxicology.
2.2. Doping-specic applications
Diuretics are used to mask the abuse of other doping agents or
to lose weight in sports where weight classes are in force. Several
DS-LC-MS protocols have been successfully developed for the comprehensive detection of these substances [2226,52]. A driving
force for the analysis of diuretics by DS-LC-MS is the occurrence
of large differences in physico-chemical properties for these compounds, complicating extraction procedures.
DS-LC-MS in doping-control analysis also allows detection of
the highly polar plasma-volume-expanders hydroxyethyl starch
(HES) and dextran [53,54]. These substances are used as masking
agents or to prevent dehydration by athletes. The analytical challenge for this class of substances is the high-molecular-weight
fragments present in urine (150 kDa) and their high solubility in
water (polysaccharide structure).

For salbutamol, a beta-2-agonist considered to have anabolic

properties at elevated doses, a threshold of 1 lg/mL is in force. Taking into account this high concentration and the excellent ionization behavior of beta-2-agonists, several DS-LC-MS methods have
been described for the quantitation of salbutamol [20,21,55,56].
For another beta-2-agonist, formoterol, a threshold of 40 ng/mL
is in force and a DS-LC-MS quantitative method has also been described [57].
DS-LC-MS was also successfully applied for the detection of
highly-polar 5-amino-4-imidazolecarboxyamide (AICAR) that is
claimed to result in enhanced performance without the need for
training [58]. A DS-LC-MS method was developed for nicotine, an
alkaloid drug for which performance-enhancing effects are currently under investigation [59]. The authors state that the 40-fold
dilution of the samples with ACN and subsequent ultra-centrifugation allows precipitation of urinary protein fragments. DS-LC-MS
was also presented in a research method to investigate the routine
detection of hypoxia-inducible factor (HIF)-stabilizers [60] and to
investigate the metabolites of benuorex [61].
Many of these examples highlight one of the main advantages
of DS-LC-MS (i.e. the lack of a sample preparation step) (and the
inherent loss of any analyte) and the universal tness of the

K. Deventer et al. / Trends in Analytical Chemistry 55 (2014) 113

methodology regardless of the chemical structure, unlike methods

involving extraction steps.
For screening purposes, the trend is therefore to combine different classes of substances in a single DS-LC-MS method. Hence,
doping-control DS-LC-MS methods for diuretics can also include
stimulants [2226], narcotics [22,24,26] beta-blocking agents
[26] or miscellaneous substances, such as AICAR, HES and dextran,
and intact phase-II metabolites [24] (Fig. 2).
2.3. Applications to analytical toxicology and workplace drug testing
Benzodiazepines are therapeutically used for the treatment of
insomnia, anxiety and muscle relaxation. Because they are addictive and can be used in sexual assaults, these substances are of
toxicological relevance. Moreover, they are involved in >50% of
overdoses in several developed countries [62]. Several DS-LC-MS
methods have been described for their detection [30,33,63,64].
Also, for hypnotic Z-drugs zolpidem, zopiclone and zaleplon, that
have similar pharmacological effects to benzodiazepines, DS-LC-MS
methods were described [28,30].
Successful detection of polar, thermally-unstable gammahydroxybutyric acid (GHB) and analogues by DS-LC-MS has also
been described [6567]. Wood et al. compared a 20-fold dilution
of the samples with a purication by SPE [66]. They concluded that
the gain in sensitivity with SPE was negligible compared to the
simpler procedure using DS-LC-MS. Petersen et al. evaluated
GHB-GLUC as a biomarker to detect GHB intoxication [68].

DS-LC-MS has also found its way into research in analytical

toxicology and was shown to be helpful in the identication of
phase-II metabolites of methylenedioxy-pyrovalerone (MDPV)
[69]. Another research application was a simple method for
quantitative detection of DMA and its metabolites [70]. The
authors stated that LC-MS analysis was benecial because DMANO
cannot be analyzed by GC-MS, due to its thermolability.
Multi-class DS-LC-MS methods can be found in toxicology for
the detection of (designer) stimulants [2731,33,35,37,71] together
with narcotics [2931,33,35,37], benzodiazepines [30,33,5], other
hypnotics [27,28,30,33] and cannabinoid metabolites [30,35]. A
multi-class DS-LC-MS method was developed to detect plantderived psychoactive substances [72]. Fig. 3 shows a relevant
example of a toxicologically-positive urine containing different
types of substances from different classes and with different
physico-chemical properties.

3. Challenges
3.1. Analyte detectability
The main drawback of DS-LC-MS is its limited sensitivity due to
the absence of a preconcentration step.
To be able to reach the low cut-offs for LSD, Eichhorst et al. had
to include an extraction step, whereas the other compounds in the
method were detected by DS-LC-MS [35]. Kronstrand et al. applied

Fig. 3. Chromatogram displaying 10 labeled standards from 10 selected reaction monitoring (SRM) function windows, and nine drugs/metabolites detected in a urine
specimen from a drug-dependent patient: stimulants (methylphenidate, ritalinic acid), narcotics (methadone, EDDP), benzodiazepines (oxazepam, lorazepam, nordiazepam,
temazepam), and cannabis metabolite (THCA). (Reprinted from [35] with permission).

10

K. Deventer et al. / Trends in Analytical Chemistry 55 (2014) 113

additional SPE when concentrations of BUP or BUP-GLUC were

below 20 lg/L [73].
Analyte detectability is a key factor for those doping substances
forbidden at any time. So the better the sensitivity, the longer the
period in which the misuse can be detected. As an example, Guddat
et al. illustrated the possibility of urinary detection of a GHRP2
metabolite by DS-LC-MS (Fig. 2). However, they concluded that,
for doping control purposes, the detection window was too short
using the DS-LC-MS approach and they suggest a preconcentration
step to increase the detectability [24].
Similarly, no paper has so far been described the routine detection of anabolic steroid aglycones with DS-LC-MS due to the low
detection levels that they require (Table 1) and their limited
ionization potential. For endogenous steroids, the maximum
sensitivity is not required since they are always present in urine.
Analytical approaches should be able to quantify them at physiological levels. This goal could be achieved by DS-LC-MS by direct
quantication of the steroid glucuronides [74]. The required detection levels could be reached only due to the specic transitions of
the steroid glucuronides.
An important step to reduce the matrix effect (ME) is dilution
(sub-section 3.2.). Unfortunately, dilution can also hamper analyte
detectability. This is illustrated in Fig. 1 for cocaine. A 25-fold dilution of the sample did not allow cocaine to be detected in the
sample. With a sample-preparation step, including a 10-fold concentration, detection could be achieved. Also several research
groups described that further dilution would hamper the required
analyte detectability in their assays [26,59,71]. Other research
groups described different dilution steps or injection volumes,
depending on the required detection levels of the target analyte(s)
[20,21,30,41].
3.2. Matrix effects
3.2.1. Particulates and sediment
There can be several sources of sediment, including crystallization of inorganic salts, such as calcium phosphate and calcium
oxalate in the hours after collecting the samples, and proteinuria,
which can be up to 10 times higher in urine from endurance sport
athletes compared to a control group [75]. A direct threat from the
particulates is clogging of the chromatographic system.
Fortunately, this type of interferent can be easily removed by centrifugation or ltration (Tables 1 and 2). For economic and practical
reasons, we prefer centrifugation in our laboratory. After centrifugation, the devices are placed directly into the autosampler and
injection from the clean supernatant is performed. Using a lter
device involves additional costs and manipulations. Approximately
half of the papers consulted do not describe any method to remove
sediment or particulates.
3.2.2. Retention-time shift
A problem rarely described is shifted retention time (tR). These
shifts can depend on the nature of the compounds and on the
urinary matrix. The shifts were observed as systematic: neutral
compounds did not shift; basic compounds showed increased
retention; and, acidic ones shifted to the front [18,25,42,45]. The
origin of these shifts is still not clear, although two authors attributed them to the high concentration of the analytes of interest
[18,41]. It was also observed that shifted tR occurred more in urine
samples with a gravity greater than 1.030 g/mL (non-published
results).
An isotope-labeled internal standard (IL-ISTD) is best suited to
identify these shifts due to its resemblance to the analyte of interest; it prevents reporting of a false negative result, when the compound shifts outside the detection window. If no IL-ISTD is used, it
is very important that the magnitude of the shifts is determined

during method validation and is reduced to an acceptable level.

and that, particularly for multi-target screening methods with only
a few IL-ISTDs, sufciently-wide detection windows are applied.
Dilution can have a tremendous effect on tR stability. Crews
et al. evaluated stability of the tR of 10 IL-ISTDs in 80 authentic urine samples after a 10-fold dilution of the samples, of which 90%
had stability of 0.05 min compared with a neat standard, 8% 0.1
min and 2% 0.15 min. Nevertheless, in one sample, a shift of
0.19 min was still present [33] and might have been urine with
an exceptionally high value for specic gravity.
The effect of dilution was also observed in a screening method
for diuretics where a dilution of factor of 25 reduced the shift of
amiloride from 0.8 min to 0.17 min compared to a neat standard.
Stabilization of tR was also observed for ephedrines after a fourfold dilution [18] and for morphine-GLUC after a 20-fold dilution
[42]. Also, deformed peaks were avoided.
3.2.3. Ion suppression (IS) or ion enhancement (IE)
As already mentioned, urine is a complex matrix, including, but
not limited to, phospholipids, protein fragments, salts, urea, and
creatinine and a wide range of organic acids and other inorganic
compounds. These compounds are permanently eluting from the
column throughout the run. To visualize the continuous eluting
matrix, Badoud et al. plotted a 2D graph of interferences observed
during the analysis [26]. Consequently, it is difcult to assign
which urinary compound interferes with the analytes of interest.
This is particularly true for multi-target screening methods where
analytes are eluting throughout the run.
Since salts are not removed in DS-LC-MS methods, IS is
generally highest at the void volume of the column and salts are
probably more prone to affect early-eluting analytes. Signicant
IS has been described by several authors for opiates, a group of
compounds that elute close to the void column of reversed-phase
columns [29,39,40,42].
Few DS-LC-MS methods are described in horse doping probably
because of IS caused by the high protein concentration and the
complexity of the equine-urine matrix. The methods are limited
to research in which the equine urine is diluted [76,77] or to
methods for the detection of a single compound for which high
detection levels are required and consequently a multifold dilution
can be applied [7881].
IS/IE should not be underestimated in LC-MS analysis, particularly for DS-LC-MS methods where the whole urinary matrix is still
present. Indeed, while tR instability can be directly observed in the
chromatogram, signs of pronounced reduction or enhancement of
the signal are often not present in the chromatogram.
An IL-ISTD compensates best for IS. The World Anti-Doping
Agency (WADA) even states explicitly in its identication criteria
that, for DS-LC-MS methods, the use of an IL-ISTD is strongly advised, due to the potential for matrix IS or IE [15]. The adequate
compensation of IS/IE by an IL-ISTD in quantitative DS-LC-MS
methods has been described by several authors despite an IS being
higher than 20%. Gustavson observed 33% of IS for M3G [39]. After
correcting with IL-ISTD, the quantitation was acceptable. Stephanson et al. described an IS of 36% for THCA-D9 [43] and also a good
corrective effect of the IL-ISTD. Deventer et al. described IS values
up to 61.6% for morphine and its metabolites [42]. However, the
IL-ISTDs were able to compensate appropriately (Table 4).
Despite this proof of the compensation effects of the IL-ISTD,
IS/IE in a DS-LC-MS method should still be determined. A recent
review on MEs in the eld of toxicology correctly claimed that
no LC-MS method should be published without investigating
IS/IE [82]. Tables 1 and 2 present values for IS/IE from the different
published DS-LC-MS methods.
The effect of IS/IE in qualitative analysis is less critical
mainly for those analytes showing high ionization efciency. For

11

K. Deventer et al. / Trends in Analytical Chemistry 55 (2014) 113

Morphinemetabolite

Ion suppression/ ion

enhancement (%)
Calculated from
absolute peak areas

M3G
M6G
Morphine

ND 16.8
D
16.2
ND 29.0
D
14.7
ND 9.2
D
24.5

Calculated from area ratio

correction with IL-ISTD
2.7
4.9
0.3
5.3
1.3
3.7

100

Accuracy compared with the SPE

LC-MS/MS reference method (%)

Table 4
Average ion suppression/ ion enhancement calculated for morphine and metabolites
from 6 different urine samples. Calculations were performed on non-diluted (ND) and
20-fold diluted (D) samples. Additionally, for both the non-diluted and diluted
samples, calculations were also performed using area ratios of analytes vs. IL-ISTD.
(Adapted from [42] with permission)

95

90
Direct-injection LC-MS/MS
SPE LC-MS

85

Direct-injection LC-MS
Direct-injection LC-MS + EtS

80
0.10

0.30

0.50

0.75

1.00

Reporting limit for urinary EtG (mg/L)

qualitative purposes, IS/IE should be evaluated only because IS can

make an analyte undetectable at the required concentration level
in a specic urine, so qualitative methods can be considered validated with large values of IS/IE and the compensation of an IL-ISTD
is not required.
Dilution of the samples can be applied to minimize IS/IE, in
addition to the compensation made by an IL-ISTD. The effect of this
strategy can be explained by the ionization process (ESI-based MS),
for which efciency is proportional to the ease of evaporation of
the droplets. IS/IE occurs when there is competition between the
different species in the ESI source or when matrix increases the
surface tension of the droplets hampering efcient evaporation
[83]. Hence, when innite dilution of urine would be applied,
IS/IE should become negligible compared to that of a neat standard.
Hence, several research groups describe dilutions to reduce or to
eliminate IS/IE completely. Politi et al. described minimizing the
IS after a 50-fold dilution of the samples [52]. Vonaparti et al.
described a 100-fold dilution for the analysis of salicylic acid in
the heavy matrix of equine urine. They observed a nearly complete
removal of the ME [80] and were also able to omit ultracentrifugation and the ltration step. Eichorst and co-workers describe a
dilution of a factor of 100 to overcome IS/IE [13].
A comparison between LODs and IS/IE obtained with diluted
and non-diluted samples was performed within the framework
of developing a multi-component screening method [25]. Injection
of 25-fold diluted samples decreased sensitivity for several compounds compared to non-diluted samples. But the 25-fold dilution
did not lead to a 25-fold higher LOD. This was explained by the
lower IS/IE observed with the diluted samples.
Dilution does not necessarily mean that IS/IE is completely
eliminated, as illustrated in Table 4. The injection of non-diluted
samples showed average IS in the range 16.829%. After 20-fold
dilution of the urine samples, IS/IE was still invariably worse with
values of +16.2% to 24.5%. Acceptable results could only be
achieved after correcting with IL-ISTDs, irrespective of the dilution
step. The authors concluded that dilution was only useful to reduce
tR shifts and to avoid peak deformation.
3.2.4. Other interferences
Since no dedicated sample preparation is applied, the strength
of a DS-LC-MS method relies on the separation power of the
chromatographic system and the selectivity of the MS to avoid
interfering peaks.
Besides the effect on signal intensities, the matrix can also be a
problem when it is present as an interfering peak. Interfering peaks
in DS-LC-MS screening methods were described for 1-phenyl2-butylamine [27]. This interference was removed during
conrmation by applying a dedicated SPE. Guddat et al. attributed
interferences in the detection of dextran to the presence of

Fig. 4. Overall accuracy of the dilute-and-shoot-liquid chromatography-tandem

mass spectrometry (DS-LC-MS/MS) procedure for urinary ETG compared with the
solid-phase exteraction (SPE)-LC-MS/MS reference method. The frequency of truepositive plus true-negative results out of all results is shown for each of the
analytical procedures at the ve selected thresholds. (Reprinted from [84] with
permission).

polymeric glucose and also suggested using another method to

conrm a positive nding [53].
Generally, the ME on product-ion ratios is not discussed separately because it is an inherent part of validating a method.
However, Helander et al. presented an interesting comparison of
SPE-LC-MS(/MS) and DS-LC-MS(/MS) for the determination of
ETG [84]. Too many false-negative samples for the DS-LC-MS(/MS)
approach were encountered due to product-ion ratios being outside
the acceptance range, although the concentrations in the samples
were well above the limit of quantitation (LOQ) of the method. Only
when the reporting limit was increased the DS-LC-MS(/MS) protocols
have more than 95% accuracy with the SPE-LC-MS(/MS) method
(Fig. 4). Hence, we can conclude that the remaining matrix can cause
not only IS/IE but also invisible interferences resulting in unacceptable product-ion ratios.
4. Perspectives and conclusions
Without doubt, DS-LC-MS for the analysis of urine samples has
become a trend in the past 10 years in both analytical toxicology
and doping-control analysis. In particular, the economic benets
(easy sample preparation and omission of time-consuming extractions) are the driving forces behind this trend. IL-ISTDs are the best
choice to compensate for MEs. A multifold dilution can also reduce
MEs. Because the dilution step can decrease analyte detectability,
the majority of DS-LC-MS methods still focus on highly-ionizable
substances, for which the required detection levels in urine are
high. However, we can expect that, with increasing instrument
sensitivity, other classes, including anabolic androgenic steroids
or peptide-based drugs, will also be analyzed by DS-LC-MS methods.
Quantitation by DS-LC-MS can be advantageous within the
framework of quality assurance: DS-LC-MS allows sample manipulations to be minimized, thereby lowering the total uncertainty
budget. This was described by Eichhorst et al. [13], who reported
improved precision due to absence of both extraction and derivatization. Also, Lee et al. described that DS-LC-MS methods can
reduce quantication errors [21]. However, no quantitative data
support these ndings and, to prove these statements, further
research should be performed.
From a theoretical point of view, an unlimited number of
substances, having a wide variety of physicochemical properties,
can be analyzed simultaneously by applying DS-LC-MS. The main
limiting factor in these multi-class-screening methods is the
performance of the instruments.

12

K. Deventer et al. / Trends in Analytical Chemistry 55 (2014) 113

In particular, for triple-quadrupole-based selected reaction

monitoring (SRM) methods, cycle times limit the number of substances that can be screened for. To circumvent this disadvantage,
a high-resolution mass spectrometer operated in full-scan mode
(FS-HRMS) can be used. Also, an FS-HRMS method allows a truly
open screening method, because previously obtained MS data
can be reevaluated at any later stage for the presence of emerging
drugs. Consequently, several DS-LC-FS-HRMS methods have been
described in doping control and analytical toxicology in the past
four years [23,26,33,41]. Taking into account these benets and
that the price of HRMS instruments is coming into the range of
conventional triple-quadrupole instruments, we can expect that
the application of DS-LC-FSHRMS will increase further.
Acknowledgements
Financial support by the Instituto de Salud Carlos III (OJP) and
WADA (Project 11A17PV) is gratefully acknowledged.
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