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a r t i c l e
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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
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
[45] THCA
no
2.5
no
no
10
20
25
[77] steroids
no
10
[76] steroid-GLUC
25-fold dilution
c+f
25
[19] ephedrines
90
[50] ETG
no
no
c
1
10
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
no
[78] hydrocortisone
c+f
[79] theobromine
c+f
[81] 3-methoxytyramine
[55] Salbutamol
[61] benuorex +
metabolites
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
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.
Ref.
Sample preparation
CU
DF
Purpose
Matrix effect2
Interface/
MS-type
[32]
amphetamines
no
quantitation/
conrmation
ESI/SRM
[13]
methylphenidate, RA
no
100
ESI/SRM
[71]
designer amphetamines
ESI/SRM
75%
[49]
screening/
quantitation/
conrmation
screening
no
20
ESI/SRM
[72]
psychoactives from
plants
no
[31]
[36]
quantitation/
conrmation
multi-class
screening
no
[30]
700 drugs
10
[30]
700 drugs
[40]
opiates
[44]
THCA, THCA-GLUC
[29]
10
[29]
amphetamines,
benzoylecgonine
opiates
1.4
[39]
opiates
no
[48]
[73]
no
f
10
2
[69]
ETS
BUP, NORBUP, BUPGLUC, NORBUP-GLUC
MDPV
[34]
MDMA, MDA
[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
ESI/SRM
98-132%
ESI/SRM
11%85%
ESI/SRM
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
no
10
multi-class
screening
c+f
no
no
1.3
10
10
ETG
3,5
[46]
[66]
ETS
GH, GBL, 1,4-BD
no
no
2
20
[65]
20
[63]
unitrazepam
research
quantitation
conrmation/
quantitation
conrmation/
quantitation
research
quantication/
conrmation
screening
[37]
opioids, amphetamines,
cocaine analogues
GHB, 1-4-BD, GVL
20
10
no
10
no
10
[33]
no
[38]
narcotics, stimulants,
benzodiazepines
stimulants, narcotics,
benzodiazepines
cocaine, BE
[41]
stimulants, narcotics
no
[68]
[64]
GHB, GHB-GLUC
benzodiazepines
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.
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
100200
100200
100200
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
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
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
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
40
20
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
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
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).
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).
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)
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)
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
11
Morphinemetabolite
M3G
M6G
Morphine
ND 16.8
D
16.2
ND 29.0
D
14.7
ND 9.2
D
24.5
100
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
12
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13