High Resolution Full Scan Liquid Chromatography Mass Spectrometry Comprehensive Screening in Sports Antidoping Urine Analysis

University of Groningen

High resolution full scan liquid chromatography mass spectrometry comprehensive screening in sports antidoping urine analysis Abushareeda, Wadha; Vonaparti, Ariadni; Saad, Khadija Al; Almansoori, Moneera; Meloug, Mbarka; Saleh, Amal; Aguilera, Rodrigo; Angelis, Yiannis; Horvatovich, Peter L.; Lommen, Arjen Published in: Journal of Pharmaceutical and Biomedical Analysis

DOI: 10.1016/j.jpba.2017.12.025

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Citation for published version (APA): Abushareeda, W., Vonaparti, A., Saad, K. A., Almansoori, M., Meloug, M., Saleh, A., Aguilera, R., Angelis, Y., Horvatovich, P. L., Lommen, A., Alsayrafi, M., & Georgakopoulos, C. (2018). High resolution full scan liquid chromatography mass spectrometry comprehensive screening in sports antidoping urine analysis. Journal of Pharmaceutical and Biomedical Analysis, 151, 10-24. https://doi.org/10.1016/j.jpba.2017.12.025

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Journal of Pharmaceutical and Biomedical Analysis 151 (2018) 10–24

Contents lists available at ScienceDirect

Journal of Pharmaceutical and Biomedical Analysis

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

High resolution full scan liquid chromatography mass spectrometry

comprehensive screening in sports antidoping urine analysis

a a a a

Wadha Abushareeda , Ariadni Vonaparti , Khadija Al Saad , Moneera Almansoori ,

a a a b c

Mbarka Meloug , Amal Saleh , Rodrigo Aguilera , Yiannis Angelis , Peter L. Horvatovich

d a a,∗

, Arjen Lommen , Mohammed Alsayraﬁ , Costas Georgakopoulos

Anti-Doping Lab Qatar, Sports City, P.O. Box. 27775 Doha, Qatar

Doping Control Laboratory of Athens, Olympic Athletic Center of Athens ‘Spiros Louis’, 37 Kiﬁssias Ave., 151 23 Maroussi, Greece

University of Groningen, P.O. Box. 196, 9700 AD Groningen, The Netherlands

RIKILT Wageningen University and Research, P.O. Box 230, 6700 AE Wageningen, The Netherlands

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

Article history: The aim of this paper is to present the development and validation of a high-resolution full scan

Received 13 November 2017

(HR-FS) electrospray ionization (ESI) liquid chromatography coupled to quadrupole Orbitrap mass

Received in revised form

spectrometer (LC/Q/Orbitrap MS) platform for the screening of prohibited substances in human urine

10 December 2017

according to World Antidoping Agency (WADA) requirements. The method was also validated for

Accepted 11 December 2017

quantitative analysis of six endogenous steroids (epitestosterone, testosterone, 5␣-dihydrotestosterone,

Available online 21 December 2017

dehydroepiandrosterone, androsterone and etiocholanolone) in their intact sulfates form. The sample

preparation comprised a combination of a hydrolyzed urine liquid–liquid extraction and the dilute &

Keywords:

shoot addition of original urine in the extracted aliquot. The HR-FS MS acquisition mode with Polarity

Liquid chromatography

Switching was applied in combination of the Quadrupole-Orbitrap mass ﬁlter. The HR-FS acquisition of

Full scan high-resolution

Mass spectrometry analytical signal, for known and unknown small molecules, allows the inclusion of all analytes detectable

Small molecule prohibited substances with LC–MS for antidoping investigations to identify the use of known or novel prohibited substances and

Sulfo-conjugate steroids metabolites after electronic data ﬁles’ reprocessing. The method has been validated to be ﬁt-for-purpose

Human urine

for the antidoping analysis.

1. Introduction ments for Minimum required performance levels (MRPL) [3] which

describes the main speciﬁcations for the analysis and detection

The World Antidoping Agency (WADA) is an international of exogenous substances in human urine. Another example is the

agency, which has the task to prevent and monitor any doping WADA Endogenous Anabolic Androgenic Steroids technical doc-

in sport. Sports movements and governments around the world ument TD2016EAAS [4] that provides the speciﬁcations for the

collaborate with WADA to prevent, deter and deﬁne the rules analysis of the endogenous proﬁle of small molecules present in

with which such doping practices and duplicitous behavior of ath- urine, such as testosterone and testosterone-likes substances used

letes using doping substances in sports can be challenged and for doping. The evaluation of the steroidal proﬁle is done by the

fought on a united and global basis. The WADA prohibited List [1] adoptive module of Urinary Athlete Biological Passport and a thor-

includes among other substances pharmacological classes of drugs, ough review of the steroidal ABP has been published [5].

either exogenous substances or endogenous substances adminis- The doping control laboratories implement different analytical

tered exogenously, which deﬁne doping. The WADA antidoping techniques in order to be able to detect a large variety of classes

control activity is based on the WADA accredited laboratories oper- of prohibited substances. For that purpose, a Mass spectrometer

ating under the speciﬁcations of the WADA International Standard (MS) coupled either with Gas Chromatography (GC/MS) or Liquid

of Laboratories (ISL) [2]. In addition to these criteria, WADA pub- Chromatography (LC/MS), is the most standard technology used

lishes technical documents which provide analytical requirements to ensure compliance with [1–4] in the analysis of urine samples.

to be fulﬁlled by laboratories. Examples are the Technical docu- The use of GC/MS and LC/MS is complementary for the speciﬁ-

cations [1–4]. The GC/MS is used as standard technique for small

molecules, in particular for anabolic steroids, because these com-

∗ pounds provide a weak ionization yield when the atmospheric

Corresponding author.

LC/MS interfaces like the electrospray ionization (ESI) is used [6].

E-mail address: [email protected] (C. Georgakopoulos).

https://doi.org/10.1016/j.jpba.2017.12.025

W. Abushareeda et al. / Journal of Pharmaceutical and Biomedical Analysis 151 (2018) 10–24 11

Several studies have been conducted by LC/MS concerning the and detection of intact sulfo-conjugated metabolites of Phase II

screening for small molecules following the WADA’s technical doc- metabolism.

uments. The urine sample preparation can either be applied as

direct urine LC/MS analysis using dilute &shoot (D&S) approach 2. Experimental

[7,8] or following urine extraction procedures such as [9] applied

during the Olympics games in 2012 in the London Olympic Labo- 2.1. Material and methods

ratory. The LC/MS systems are operated either in tandem MS/MS

mode [7] or HR-FS acquisition that either with Quadrupole/Time- 2.1.1. Reagents

␤

Of-Flight (LC/QTOF/MS) [10] or Orbitrap (LC/Q/Orbitrap/MS) [9] -Glucuronidase from Escherichia Coli (E.coli), for the enzy-

instruments. matic hydrolysis, was purchased from Roche Diagnostics GmbH

The WADA prohibited List [1] includes prohibited pharmaco- (Mannheim, Germany). Methanol (HPLC grade), di-potassium

logical classes and the therein particular molecules are referred as hydrogen phosphate trihydrate (K2HPO4·3H2O), potassium dihy-

examples, however it is open for other substances with a similar drogen phosphate (KH2PO4), sodium bicarbonate (NaHCO3),

chemical structure or similar biological effect. Consequently, the sodium carbonate (Na2CO3) and acetonitrile were supplied by

screening procedure should be generic and comprehensive for all Sigma Aldrich (Darmstadt, Germany). Ethylacetate was supplied

compounds detectable by the molecular proﬁling platform such by Merck (Darmstadt, Germany). Formic acid (HCOOH) and 5 M

as LC/MS; this should include the sample preparation and HR-FS ammonium formate (HCOONH4) were from Agilent Technology.

MS acquisition of known and unknown molecules. Moreover, HR-

FS MS acquisition and polarity switching for positive and negative 2.1.2. Reference materials

ionized molecules are important parameters to be considered [11] The Reference Materials used in the current study were from

to obtain comprehensive molecular proﬁle detectable by an LC/MS Sigma Aldrich (Darmstadt, Germany), Steraloids (Newport, USA),

system. LGC (Wesel, Germany), Toronto Research Chemicals TRC (Toronto,

The limitations of the steroid proﬁles in anti-doping applica- Canada), Cerilliant (Round Rock, USA), Lipomed (Arlesheim,

tions have been addressed for several years [5] and studies have Switzerland), Cayman (USA), NMI Australian Government National

been conducted to improve its effectiveness as a forensic biomarker Measurement Institute (Pymble, Australia). Reference compounds

tool to monitor and identify testosterone and analogs abuse [12]. In that are not commercially available were provided by WADA

1996, a study conducted by Dehennin et al. [13] proposed the intro- Accredited Laboratories of Cologne (Germany), Ghent (Belgium),

duction of epitestosterone sulfate in the athlete’s steroid proﬁle Rome (Italy), Athens (Greece), Barcelona (Spain) and Sydney

as a ratio of testosterone glucuronide to the total epitestosterone (Australia), and the World Association of Antidoping Scientists

fractions. In another study, several additional endogenous steroid (WAADS).

forms from those referred in [4] have been identiﬁed [14,15]. Untar- The deuterated internal standards of morphine 3-␤-d-

geted metabolomics studies for the detection of steroid biomarkers glycuronide-d3 and mefruside-d3 used for qualitative screen-

have been performed as well [16–18]. Complementary to these ing were obtained from TRC (Toronto, Canada). The deuter-

studies [13], the sulfate fraction of endogenous steroid proﬁle, in ated internal standards used for quantitative screening were:

addition to the free and glucuronide fractions of [4], has been stud- 5␣-Dihydrotestosterone Sulfate-d3 (5␣-DHT-d3), Testosterone

ied by several research groups in relation to the genotype for the sulfate-d3 (TS-d3) and Androsterone sulfate-d4 (AS-d4) were all

UGT2B17 enzyme., where it is proposed that in case of exogenous purchased from NMI (Pymble, Australia).

testosterone intake, the ratio between androsterone glucuronide Stock standard solutions of the standard analytes were indi-

and epitestosterone glucuronide as a complement to testosterone vidually prepared in methanol. For validation purposes, working

and epitestosterone glucoronide ratio can be used especially in standard solution containing the standard analytes was prepared in

UGT2B17 del/del individuals. In addition, etiocholanolone sulfate methanol by subsequent dilutions of the stock solutions. The sulfo-

was excreted at signiﬁcantly higher levels in those individuals conjugated steroids analytes were included in a different working

◦

[19–22]. solution. All solutions were stored at −20 C in amber vials until

The detection and quantiﬁcation of the intact conjugated use.

endogenous and exogenous steroids by LC/MS has been studied and

reviewed by several groups since ‘90s [23] and [24]. The quantiﬁca- 2.1.3. Sample preparation

tion of glucuronidated and sulfated endogenous steroids by LC/MS To 5 mL urine, 1 mL phosphate buffer at pH 7 (Na2HPO4 0.8 M

has been reported earlier [25]. A similar approach has been fol- and NaH2PO4 0.4 M), 100 ␮L of ␤ −Glucuronidase from E. coli

−1

lowed in other studies for the analysis of intact conjugated steroids and 50 ␮L of a methanolic solution of mefruside-d3 (10 ␮g mL )

−1

of endogenous and exogenous origin [26–28]. morphine-3␤-d-glucuronide-d3 (5 ␮g mL ) were used as internal

This article focuses on the application and validation of an LC/MS standards (ISTDs). After addition of the internal standards to the

◦

screening for small molecules using HR-FS acquisition with polarity sample, the mixture was incubated for 1.5 h at 50 C. After hydroly-

switching. The method has been developed using an LC/Q/Orbitrap sis, the pH was adjusted to 9–10 with a mixture of sodium hydrogen

MS with a combination of D&S and a solvent extraction proce- carbonate and sodium carbonate (10:1) (w/w). A liquid–liquid

dure for urine sample preparation. The method is intended to be extraction with 5 mL of ethylacetate was performed using anhy-

generic in order to allow the detection of known designer drug drous sodium sulfate as salting-out agent. After centrifugation, the

urine metabolites [29] together with quantiﬁcation of six intact organic layer was separated from the aqueous phase by freezing

◦

endogenous sulfate steroids: epitestosterone (ES), testosterone the sample at −80 C. The organic phase was then acidiﬁed with

␣ ␣

(TS), 5 -dihydrotestosterone (5 DHTS), dehydroepiandrosterone 200 ␮L of 3 M acetic acid in ethylacetate and evaporated under

◦

(DHEAS), androsterone (AS) and etiocholanolone (ETIOS) using nitrogen stream at 50 C. The remaining residue was reconstituted

negative ionization mode, with possibility to detect unknown with 200 ␮L of 80:20 LC mobile phase A/B (v/v) (see description

exogenous substances. This method is developed as a complemen- of eluent at Section 2.1.4.1). 20 ␮L of the original urine were mixed

tary for a previously published GC/QTOF/MS screening method [30]. with the reconstituted extract and 5 ␮L of the mixture were injected

Accordingly, the antidoping laboratories will have a completed to the LC/MS system for analysis.

GC and LC HR FS screening method with simultaneous measure-

ment of steroid proﬁle according to WADA Code speciﬁcations [4] 2.1.4. Instrumentation

12 W. Abushareeda et al. / Journal of Pharmaceutical and Biomedical Analysis 151 (2018) 10–24

2.1.4.1. Chromatographic conditions. A Dionex UHPLC system were determined for the same analytes by comparing the MS sig-

(Thermo Scientiﬁc, Bremen, Germany) was used for the chromato- nal at the 50% MRPL concentration urine sample spiked after the

graphic separation. The system consisted of a vacuum degasser, a extraction to the MS signal of the analytes spiked in the reconstitu-

high-pressure binary pump, an autosampler with a temperature tion solvent at equal concentrations. Matrix effects and extraction

◦ ◦

controlled sample tray set at 7 C and a column oven set at 30 C. recovery were evaluated in 5 different urine matrices. The mass

◦

Chromatographic separation was performed at 30 C using a Zor- accuracy has been calculated in one urine sample used for identiﬁ-

bax Eclipse Plus C18 column (100 × 2.1 mm i.d., 1.8 ␮m particle cation capability experiment to assess eventual mass bias in the

size; Agilent Technologies). The mobile phase consisted of 5 mM entire range of the full scan MS acquisition, for both ionization

ammonium formate in 0.02% formic acid (solvent A) and a mixture polarities.

of acetonitrile/water (90:10 v/v) containing 5 mM ammonium for-

mate and 0.01% formic acid (solvent B). A gradient elution program

2.1.5.2. Quantitative validation. For the endogenous steroids quan-

−1

was employed at a constant ﬂow rate of 0.2 mL min with solvent

titative validation, the following validation parameters have been

B starting at 5% for 1 min, increasing to 32% in 2.5 mins and stay-

determined, in ﬁve experimental days. The linearity range of the

ing constant at 32% for 13 mins and then, set back to 100% within

calibration curves for each sulfate steroid in various concentrations

8 mins, where the eluent composition was held for 2.5 min before

has been evaluated. Seven points’ calibration curves for quantiﬁca-

returning to 5% within 1 min. The analysis run time was 28 min and

tion purposes were generated from spiking standards in steroid

the post-run equilibrium time was 4 min. The injection volume was

depleted urine samples. The depleted urine sample was prepared

5 uL.

as follow: blank urine was collected from female children and

was depleted from endogenous steroids using C18 SPE extrac-

2.1.4.2. Mass spectrometric conditions. The mass spectrometer was

tion. The linearity of the calibration curves were assessed in the

−

a QExactive benchtop Orbitrap-based mass spectrometer (Thermo 1

concentration range of 1–200 ng mL for TS, ES and 5␣DHTS;

−

Scientiﬁc, Bremen, Germany) operated in the positive–negative 1

10–2000 ng mL for AS and EtioS and DHEAS. The calibration

polarity switching modes and equipped with a heated electro-

curves were built from the peak area ratio of sulfo-conjugates

spray ionization (HESI) source. Source parameters were: sheath

steroids and TS-d3 for TS and ES, AS-d4 for AS EtioS and DHEAS and

gas (nitrogen) ﬂow rate, auxiliary gas (nitrogen) ﬂow rate and

TS-d3 for 5␣DHTS. The intermediate precision and the accuracy (%

sweep gas ﬂow rate: 40, 10 and 1 arbitrary units respectively,

bias) for each sulfate steroid has been determined by the analysis of

◦ ◦

capillary temperature: 300 C, heater temperature: 30 C, spray

the spiked quality control (QC) samples at two concentration levels

voltage: +4.0 kV (positive polarity) and −3.8 kV (negative polar-

(low and high), which were prepared during 5 different experimen-

ity). The instrument operated in FS mode from m/z 100–1000 at

tal days and injected twice for each calibration curves. Both the

17,500 resolving power and duty cycle of 100 ms for both polari-

intermediate precision and bias from the QC samples were used

ties and in MS/MS mode from m/z 100–1000 at 17,500 resolving

to estimate the combined Measurement Uncertainty (MU) for each

power and duty cycle of 62 ms (product ion mode) for the analytes

steroid according to the following equation:

that showed matrix interfering peaks in the FS mode. The auto-

6 2 2

matic gain control (AGC) was to 10 . The mass calibration of the Combined MU = intermediate precision + Bias

Orbitrap instrument was evaluated in both positive and negative

modes daily and external calibration was performed prior to use

following the manufacturer’s calibration protocol. 3. Result and discussion

2.1.5. Method validation 3.1. Method development and optimization

2.1.5.1. Qualitative method validation. The following validation

parameters have been included in the experimental protocol and The method development was oriented to create a compre-

have been determined for all substances of interest. The identiﬁca- hensive analytical procedure to cover as many different small

tion capability is the ability of the method to detect the substance molecules as possible. Liquid-liquid extraction by ethylacetate as

at the 50% of the Minimum Required Performance Levels (MRPL) organic solvent was selected in order to achieve recovery of the

concentration [3] in ten (10) different urine routine antidoping sulfates phase II metabolites that remain unaffected after the enzy-

␤

negative samples. In this study, 10 different urine matrices were matic hydrolysis by the -Glucuronidase from E.Coli. The extracted

spiked with the standard multicomponent solutions at concen- and reconstituted urine aliquot was enriched by 10% (v/v) original

trations equal to the 50% of MRPL or below (See Table 1). The urine sample as part of D&S approach. The combined sample was

speciﬁcity of the method was determined using the same exper- analyzed by LC/MS. The D&S part of the analyzed sample is impor-

imental data obtained to assess the identiﬁcation capability after tant for small molecules that are not extracted in the extraction

checking the detection of substance of interest in the selected ion process, such as melidonium, ethylglycuronide, FG4592, AICAR,

chromatographic time window obtained at speciﬁc m/z value. The ﬁnasteride carboxylic acid metabolite and ritalinic acid. The use of

absence of matrix interferences was evaluated by the analysis of the ethylacetate as extraction solvent and the D&S contribution of

the urine samples (blank sample) used also for the identiﬁcation the analyzed sample do not contaminate the LC/MS system and do

capability without being spiked. The Limit of detection (LOD) was not change the maintenance schedule of the LC/MS system com-

determined as the lowest detectable concentration per substance pared with an LC/MS system used to analyze extracted aliquots

with subsequent dilution cycles up to 1% of the concentration of only. Moreover, the liquid chromatographic column was replaced

the respective MRPL concentration. Sample carry over was evalu- after approximately 1200 urine sample injections.

ated by the detection of substances in blank urine samples injected One of the main aspects during method development was to

after the injection of a urine sample spiked with the analytes at a optimize the liquid chromatographic separation of AS to ETIOS.

concentration 20 times higher than the respective MRPL level. The To achieve this goal in the current instrumental conﬁguration and

target analyte recovery was determined for some selected analytes settings, several isocratic sections as part of the overall eluent gra-

listed in Table 1 by comparing of the MS signal of the standards dient program, were tested to achieve the liquid chromatographic

at 50% MRPL concentration spiked in urine sample prior sample method described in the experimental part 2.1.4.1.

preparation to the MS signal in urine sample spiked after sample The MS acquisition was performed using an Orbitrap mass

preparation and prior evaporation step. Similarly, the matrix effects analyzer in order to maximize the advantages from simultaneous

W. Abushareeda et al. / Journal of Pharmaceutical and Biomedical Analysis 151 (2018) 10–24 13 (SD) b , a (4.8) (6.3) (6.5) (9.3) (9.4) (8.8) (7.4) (5.6) (5.1) (4.6) (6.3) (6.8) (12)

(14) (10)

Extraction

% 82.5 93.3 91.6 28.3 26.8 84 35.1 101 42.2 92.8 95.2 96.1 94 78.1 86.7 Recovery b , a

Effect

(7.2) (8.3) (4.8) (3.8) (6.8) (9.9) (6.4) (7.2) (7.1)

(7.6) (12) (12) (21)

(18) (15)

Matrix 19.3 29.5 20 25.1 21.5 48 2.5 12 44.2 45.8 63.2

(SD) − − − − − − − +5 +5 − +23.2 +21.5 − − − % ) 1 − a mL

LOD (ng 2 5 2 1 2 2 2.5 2 2 2 2 2 2 1 2 10 2 2 2 1 2 2 2 1 5 5 1 2 2 2 1 2 2 1 1 1 2 2 2 2 2 1 1 1.25 0.6 0.6 0.25 0.25 0.6 0.25 0.25 0.25

) Mass z

/ m ( Ion 222.9954 401.0168 228.0406 439.0711 431.9908 363.1020 371.0604 339.0201 346.0987 323.0199 303.0185 314.9978 376.9901 364.0528 364.0528 361.0864 457.0042 447.1481 347.1183 379.892 355.0510

Ion

− − − − − − − − H] H] H] H] H] H] H] H] − − − − − − − − Secondary [M+H]+ [M+NH4]+ [M+H]+ [M+NH4]+ [M+H]+ [M [M+NH4]+ [M+H]+ [M+H]+ [M [M+H]+ [M+NH4]+ [M+NH4]+ [M [M [M [M+NH4]+ [M [M [M [M+H]+

) z / m (

>121.0650 >135.0806

Mass

Ion 220.9809 381.9762 230.0552 369.0001 420.0305 429.9762 384.0716 365.1166 352.0198 341.2111 293.9415 337.0055 390.0896 499.2129 378.0355 388.0198 302.9073 325.0345 423.9480 415.2115 301.0040 329.0004 295.9570 329.9836 472.1234 380.0511 237.0111 357.9495 366.0674 366.0674 428.2333 363.1006 437.9636 284.0962 290.0361 637.1285 283.9572 317.9835 449.1626 349.1330 254.1147 377.8949 353.0368 329.2224 329.2224 329.2224 397.2032 287.2 287.2 285.1849 317.2475 317.2475

−

(+) (+) − − − − − − − − − − − − − − − − − − − − − − − − − −

Ion

H] H] H] H] H] H] H] H] H] H] H]- H] H] H] H] H] H] H] H] H] H] H] H] H] H] H] H] − − − − − − − − − − − − − − − − − − − − − − − − − − − Main [M [M [M [M [M [M [M+H]+ [M+H]+ [M [M+H]+ [M [M [M+HCOO] [M+H]+ [M [M [M [M+H]+ [M [M+H]+ [M [M [M [M [M [M [M+H]+ [M [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M [M [M+H]+ [M+H]+ [M [M [M+H]+ [M+H]+ [M+H]+ [M [M [M+H]+ [M+H]+ [M+H]+ [M MSMS MSMS [M+H]+ [M+H]+ [M+H]+

(min)

g analytes.

RT 5.1 13.9 5.0 15.3 21.4 19.4 6.45 21.9 15.1 22.8 5.7 7.6 9.0 19.2 21.5 20.5 7.8 5.3 16.5 14.5 20.9 11.8 6.0 7.0 24.2 22.0 6.2 11.0 12.8 16.4 9.6 21.0 21.4 21.7 6.3 22.1 5.4 6.7 23.0 8.65 6.0 9.9 21.6 11.5 15.2 16.9 7.6 19.7 20.5 21.2 22.7 23.0 304

) 1 Conc. −

validation

(ng Spiked 100 25 100 50 100 100 50 100 100 100 100 100 100 50 100 100 100 100 100 50 100 100 100 100 50 50 50 25 100 100 50 100 100 25 50 50 100 100 100 100 100 25 20 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5

qualitative

for

f Agents

used

ACB ATFB

data

Masking

acid

and Artifact Artifact

Agents

-Hydroxyprostanazol -Hydroxyfluoxymesterone ␤ ␤ experimental Compound Diuretics Acetazolamide Althiazide Amiloride Asozemide Bendroflumethiazide Benzthiazide Brinzolamide Bumetanide Buthiazide Canrenone Chlorothiazide Chlorthalidone Clopamide Conivaptan Cyclopenthiazide Cyclothiazide Dichlorphenamide Dorzolamide Epitizide Eplerenone Ethacrynic Furosemide Hydrochlorothiazide Hydroflumethiazide Lixivaptan Mebutizide Methazolamide Methylchlorthiazide Metolazone Indapamide Mozavaptan Piretanide Polythiazide Probenecid Quinethazone Relcovaptan Thiazide Thiazide Tolvaptan Torasemide Triamterene Trichlormethiazide Xipamide 16 3-Hydroxyprostanazol 4-Hydroxyprostanazol 6 Boldenone Methyldienolone Boldione Bolasterone Calusterone Anabolic

and

No. 1 44 21 2 3 4 6 7 9 10 11 12 13 14 18 22 23 24 25 26 27 29 30 31 33 34 35 38 39 42 43 45 46 47 48 49 51 52 5 8 15 16 17 20 28 32 36 37 41 50 19 40 Theoretical Table

14 W. Abushareeda et al. / Journal of Pharmaceutical and Biomedical Analysis 151 (2018) 10–24 (SD) b , a (5.4) (9.1) (7.1) (4.1) (6.3) (3.8) (5.2) (4.8)

(15) (13) (11)

Extraction

% 98 89.6 92.9 88 88.7 85.4 86.2 87.1 77 87.8 Recovery b , a

Effect

(9.4) (7.9) 90.3 (4.9) (5.4)

(11) (13) (17) (16) (19) (11) (13)

Matrix 71.2 67.1 28.4 13.1 14 15 23 25 35 21

(SD) − − − − − − +13 − − − − % ) 1 −

a mL

LOD 0.25 0.1 0.1 (ng 0.5 0.5 0.05 1 0.5 1 1 0.25 0.05 0.05 0.5 0.1 0.1 0.05 0.025 0.1 0.05 0.025 0.25 0.2 0.1 0.1 0.1 0.1 0.4 0.5 0.5 0.5 0.1

) Mass z

/ m ( Ion 363.1977 381.2083 300.1605 442.1219 407.1320 202.1338 343.1663

Ion − −

O]+ 2

H − − − H] H] − − Secondary [M+HCOO] [M+HCOO] [M [M+H]+ [M+NH4]+ [M+H [M

) z / m (

281.1898 269.1894 159.0804 257.1900

>147.0804 > >147.0804 > >227.1430 > >275.2006 >

Mass

305.2 347.2 299.2 285.2 Ion 319.2068 337.2173 319.2268 309.1849 345.2537 345.2537 345.2537 299.2 305.2 329.2584 313.2162 271.1693 271.1693 399.1847 302.1751 383.0836 401.0766 440.1075 307.0547 417.0473 388.0915 287.0647 217.0972 368.2182 366.9838 220.1444 234.1601 291.1025 263.0712 304.1543 316.1543 345.1809 272.1272 286.1438 393.2173 311.1133 383.1898

−

(+) 347.2 (+) (+)(+) 285.2 − − − − − − − −

Ion

H] H] H] H] H] H] H] H] − − − − − − − − Main [M+H]+ [M+H]+ [M+H]+ MSMS [M+H]+ [M+H]+ [M+H]+ [M+H]+ MSMS MSMS MSMS [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M [M [M+H]+ [M+HCOO] [M [M [M [M [M [M [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+

(min)

g 23.8 15.4 11.1 8.3 22.3 16.0 15.1 14.2 21.9 20.4 22.1/22.7 22.3 23.3 17.2 19.8 16.9 8.5 6.2 23.7 23.9 21.9 14.9 24.4 23.0 11.5 4.7 7.1 7.2 4.8 5.4 7.5 6.3 5.5 5.7 6.6 5.3 5.8 12.2 7.7 RT ) 1 Conc. −

2.5 2.5 (ng 2.5 Spiked 2.5 2.5 1 2.5 * 1 2.5 2.5 2.5 * 10 1 2.5 2.5 2.5 2.5 2.5 2.5 10 5 5 5 5 5 10 20 10 10 5 5 2.5 1

␣ MT)

MTs

- ␤

,17 term-MT) sulfate

(Andarine ␣

(Ostarine MT * watch”

MT)

long (Formebolone

-ol-3-one -methylandrost- MT)

␤

␣ “night

sulfate hydroxy

-hydroxymethyl,17

␤ Artifact

Agonists

(Ostarine) -Hydroxymethyltestosterone -Hydroxystanozolol 1

(Andarine) -ﬂuoro-18-nor-17,17-dimethyl- -Hydroxystanozolol ␣ ␤ )

-Hydroxystanozolol 1 ␣ ␤ methylandrost-1,4,13-trien-3-one (Methandienone Compound 9 4,13-diene-11 (Fluoxymesterone Fluoxymesterone 11 (Formebolone 2-hydroxymethyl-11 Gestrinone 16 4 3 18-nor-17 Methyltrienolone Oxandrolone Stanozolol THG Trenbolone 17-Epitrenbolone Mesterolone Mesterolone Ractopamine LGD4033 S1 S4 O-dephenylandarine S9 S22 O-dephenylostarine Zilpaterol Bambuterol Brombuterol Cimaterol Cimbuterol Clenpenetrol Clenproperol Fenoterol Fenoterol Formoterol Higenamine Coclaurine Indacaterol Mabuterol MT) MT) dihydroxy-17 1,4-diene-3-one BETA-2 Continued (

No. 53 79 70 83 86 87 64 57 69 54 63 72 74 81 82 84 85 55 56 58 59 60 61 65 66 67 68 71 73 75 76 78 80 88 89 90 62 77 91 Table

W. Abushareeda et al. / Journal of Pharmaceutical and Biomedical Analysis 151 (2018) 10–24 15 (SD) b , a (8.1) (5.8) (4.3) (4.2) (6.0) (5.6)

(13) (11)

Extraction

% 85.5 88.3 86.1 82 88.7 91.5 91.9 68 Recovery b , a

Effect

(17) (14) (16) (7.4) (18) (16) (12)

Matrix 30 24 24 31 50 62 43

(SD) − − − − − − − % ) 1 − a mL

0.2 12.5 LOD (ng 1.0 0.1 2.0 0.5 0.5 0.5 1.0 0.2 0.2 0.1 0.1 0.5 0.2 0.2 0.2 1 1 1 1 0.2 0.4 1

) Mass z

/ m ( Ion 238.1449 414.2650 224.1292 279.0775 257.0891

Ion

− − − >135.0804

H] H] H] H]- − − − − Secondary [M [M [M MSMS 299.2 [M+HCOO]- [M

) z /

m (

Mass

Ion 325.1289 387.1914 241.1547 291.1703 402.1772 288.1594 240.1594 416.2795 226.1438 228.1147 486.1809 305.2111 294.1713 283.16930 285.1849 422.1881 452.1987 394.1568 297.1849 299.2006 452.0607 468.0557 233.0720 474.1734 418.2377 301.1798 305.2111 406.1932 402.2064 267.1703 259.1037 177.1386

58.0659

> − − −

Ion

H] H] H] − − − Main [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M [M [M MSMS 147.11 [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+

(min)

g RT 9.0 7.0 4.6 5.1 5.3 5.7 4.7 20.8 4.7 6.6 18.6 8.2 16.0 21.0 18.3 21.7 21.8/22.1 21.3 22.5 21.9 24.5 21.8 17.9 1.15 11.5 22.2 12.6 21.4 23.1 13.8 5.6 1.5 5.1 ) 1 Conc. −

10 10 8 * * 10 (ng Spiked 5 5 5 5 5 10 100 10 10 5 5 10 * * 10 10 50 10 10 10 * 10 10 4700 50 10

MODULATORS

METABOLIC

MT)

4-hydroxy 3-hydroxy-4-methoxy

Artifact

AND

MT)

sulfoxide

MT)

(ATD

-Hydroxyandrost-1,4,6-triene- - - ␤ ␤ ) ␣ MT) Compound Mapenterol Olodaterol Pirbuterol Procaterol Reproterol Ritodrine Salbutamol Salmeterol Terbutaline Tulobuterol Vilanterol 6 Hydroxytestosterone (6-OXO Anastrozole Androsta-1,4,6-triene-3,17-dione (ATD) 17 3-one Clomiphene Clomiphene-hydroxy-methoxy Clomiphene-N-desethyl-hydroxy MT Exemestane 17 Hydroxyexemestane (Exemestane GW1516 GW1516 Bis(4- cyanophenyl)methanol (Letrozole Melidonium Raloxifene Tamoxifene Testolactone Tetrahydrotestolactone Toremifene Toremifene- carboxy metabolite Tamoxifene- carboxy metabolite Trimetazidine AICAR 1-Benzylpiperazine STIMULANTS HORMONE Continued (

No. 92 103 108 109 110 113 114 120 121 122 116 124 95 96 97 98 99 100 101 105 106 111 115 117 118 119 93 94 102 104 107 112 123 Table

16 W. Abushareeda et al. / Journal of Pharmaceutical and Biomedical Analysis 151 (2018) 10–24 (SD) b , a (5.0) (5.5) (4.5) (9.0) (8.1)

(15) (12) (16) (10)

Extraction

% 87 88.8 65 79.6 42 55 71.7 78.9 Recovery b , a

Effect

(7.7) (9.5) (8.0) (9.2)

(12) 91.2 (12) (15) (12) (26)

Matrix 61.9 57 38.6 42.0 38 35 18 42.4

% (SD) − − − +11 − − − − − ) 1 −

a mL

LOD (ng 1.25 1.25 2.5 1 1 1 1 1 5 1 1 1.25 1 20 1 1 1 6.25 1 1 1 1 1 1 1 5 1 1 1 1 20 1 1 1 1 1 1 1 1 1 10 5 1 1 1 1 0.2 1 1.25 1

) Mass z

/ m 288.0700 ( Ion 176.1434 324.0781 119.0855 119.0855 174.0913 152.1070 241.1911 227.1754 217.0972 300.1605 148.1121 262.0744 755.5007 383.2201 165.0910 165.0910 128.1434 135.0440 163.0754 160.1121 149.0961

]+ ]+ ]+ Ion 3 3 3

O]+ O]+ O]+ O]+ 2 2 2 2

NH]+ NH]+ NH]+ ]+ d 3 3 3 2 H NH NH H NH H H − − − O − − − − − − − 6 H] H] H] CH CH CH H − − − − − − 8 Secondary [M+H [M+H]+ [M [M+H [M+H [M+H]+ [M+H]+ [M+H]+ [M [M+H [M+H]+ [2M+H]+ [M [M+H [M [M+H [C [M [M+H [M

) z / m (

Mass

Ion 197.0840 178.1590 194.1539 116.1434 116.1434 116.1434 102.1277 322.0801 167.0855 192.0590 206.1539 136.1121 136.1121 352.1519 290.1387 240.1747 219.1128 150.0913 260.1201 196.1332 182.1176 215.0826 164.1434 164.1434 164.1434 302.1751 200.1281 166.1226 194.1539 224.1281 182.1176 260.0598 378.2540 368.2220 216.1747 385.2346 342.1925 232.1308 189.1386 182.0976 230.1539 182.1176 196.1332 146.1539 142.1590 163.0754 194.1176 212.1201 178.1226 194.1176 180.1383

]+ ]+ 2 2

NH NH 5 5 O]+ 134.0964 H H

2 2 2

H C C NH3]+ − Ion − − − −

10 H] H − 13 Main [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ C [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H [M+H]+ [M+H]+ [M+H [M+H [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+

(min)

g 6.8 6.7 6.8 5.9 6.2 6.0 5.1 23.9 9.0 5.1 6.0 5.6 5.7 20.8 6.0 9.4 6.8 5.0 5.1 10.8 10.4 7.9 8.2 6.0 6.2 6.2 6.2 1.3 5.3 5.8 8.1 3.1 3.0 23.5 23.3 8.1 6.3 6.6 8.6 6.1 5.6 7.6 4.8 5.0 4.1 6.6 5.8 6.0 7.4 6.1 6.0 6.3 RT ) 1 Conc. −

(ng Spiked 25 25 50 50 50 50 50 10 50 50 50 50 25 50 50 50 50 50 25 50 50 50 50 50 50 50 50 10 50 50 50 50 * 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 10 25 50

methylester

) -Methylphenethylamine Compound 1-(3-Chloro)phenyl-Piperazine 2Amino-NEthyl-Phenylbutane 4-Ethylephedrine 4-Methylhexanamine Tuaminoheptane 1.4-Dimethylpentylamine 1.3-Dimethylbutylamine 6-Hydroxybromantane Adrafinil Amiphenazole Amphepramone Amphetamine ␤ Benfluorex Benzoylecgonine Benzphetamine Carphedone Cathine Cathinone Clobenzorex Cropropamide Crotethamide Cyclazodone DiMethylamphetamine Ethylamphatemine Mephentermine Dobutamine Ecgonine Ephedrine/Pseudoephedrine Etafedrine Ethamivan Etilefrine Etilefrine-Sulfate Famprofazone Fenbutrazate Fencamfamine Fencamine Fenethylline Fenfluramine Fenproporex Flephedrone Furfenorex HMA HMMA Heptaminol Isometheptene MDA MDMA Mefenorex Mephedrone Methedrone Methoxyphenamine Continued (

No. 125 131 158 127 128 138 169 176 129 130 133 134 135 136 137 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 159 160 161 162 163 164 165 167 170 171 172 173 174 175 126 132 166 168 Table

W. Abushareeda et al. / Journal of Pharmaceutical and Biomedical Analysis 151 (2018) 10–24 17 (SD) b , a (4.0) (6.4) (9.6) (5.5) (8.7)

(0.8) (0.3) (13) (10)

Extraction

% 3.1 77.6 77 62.2 94 2.1 89.9 93.3 85.5 Recovery b , a

Effect

(5.4) (8.8)

(9.9) (6.9) (8.8) (10) (13) (13)

(10)

Matrix 46 11 44 43.3

(SD) − − − 79.4 60 96.6 40.9 +34.0 − % ) 1 −

a mL

LOD (ng 1 1 1 5 1 1.25 1 0.25 50 5 2.5 1 1 0.2 1 1 1 1 1 12.5 1 1 1 1 1 1 1 2.5 1 1 0.5 5 0.1 0.5 0.05 0.25 0.1 0.5 0.1 0.1 1 5 0.5 0.5 0.5 0.5 0.5

) Mass z

/ m ( Ion 162.1277 234.0431 234.0431 164.1070 260.0598 135.0804 135.0802 324.1206

]+ Ion 3

O]+ 2

NH]+

3 H NH − − − H] CH − − Secondary [M+H [M+H]+ [M+H]+ [M [M [M+H [M+Na]+

) z /

m (

Mass

Ion 180.1383 234.1489 167.0855 167.0855 378.2176 150.1277 179.1179 232.0285 204.0995 136.0757 232.0285 182.1176 262.0757 122.0969 232.1696 177.0659 139.0978 192.1383 178.1226 166.1226 330.2216 218.1903 156.1747 220.1332 150.1277 150.1277 150.1277 150.1277 150.1277 188.1434 174.1280 280.1827 266.1670 335.1754 323.1502 339.14520 152.1077 351.2431 328.1543 468.3108 414.2638 310.2165 278.1903 337.2274 233.1648 286.1438 286.1438 316.1543 302.1378 286.2165 248.1645 393.2537

O]+

H − − Ion −

10 10 H] H] H H − − 13 13 Main [M+H]+ [M+H]+ C C [M+H]+ [M+H]+ [M+H]+ [M [M+H]+ [M+H [M [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+

(min)

g RT 5.5 6.7 9.7 8.0 7.4 5.5 6.4 1.5 7.4 1.3 1.3 2.0 2.0 5.0 7.0 6.1 6.0 5.8 5.8 4.3 22.0 8.4 7.3 5.9 5.7 5.8 6.2 6.0 6.4 6.5 6.6 20.9 20.4 6.0 22.6 16.4 3.9 12.3 5.7 12.1 7.6 19.8 13.2 9.9 6.2 4.8 3.4 5.7 4.3 8.2 7.3 19.8 ) 1 Conc. −

(ng Spiked 50 50 50 50 50 25 50 * 10 500 * 50 * 500 50 50 50 10 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 10 50 1 25 2.5 2.5 5 25 1 1 25 25 25 25 25 25 25

MT)

sulfate

desmethyl

sulfate

desmethyl carboxylate

acid

(Methadone

) Compound Methylephedrine Methylphenitate Modafinil Modafinil Morazone N,N-dimethylphenethylamine Nikethamide Norfenefrine-Sulfate Norfenfluramine Octopamine Octopamine Oxilofrine Oxilofrine PEA PVP Pemoline Pentetrazol Phendimetrazine Phenmetrazine Pholedrine Prenylamine Prolintane Propylhexedrine Ritalinic D-methamphetamine Phenpromethamine Ortetamine Phentermine p-Methylamphetamine Selegiline Selegiline Sibutramine Sibutramine Strychnine Sydnocarb p-Hydroxysydnocarb p-Hydroxyamphetamine 3-Methylfentanyl 6-Acetylmorphine Buprenorphine Norbuprenorphine Methadone EDDP Fentanyl Norfentanyl Hydromorphone Morphine Oxycodone Oxymorphone Pentazocine Pethidine Racemoramide NARCOTICS Continued (

No. 177 208 185 188 190 181 201 214 178 179 180 184 186 187 189 193 194 195 196 197 198 199 200 202 203 204 205 206 207 209 211 212 215 216 217 218 219 221 222 223 224 225 226 227 228 182 191 210 213 220 183 192 Table

18 W. Abushareeda et al. / Journal of Pharmaceutical and Biomedical Analysis 151 (2018) 10–24 (SD) b , a (5.1) (7.1) (4.0) (3.9) (8.1) (3.4) (5.4) (5.1)

(11) (11)

Extraction

% 87.8 90 88.5 90.9 55.0 91.1 86.9 86.0 89.4 Recovery b , a

Effect

(9.0) (5.0) (7.0) 80 (9.4)

(8.6) (12) (11) (11) (12) (10)

Matrix 44.6 45 65.5 65.1 27 60.1 3.2 14 13 40

(SD) − − − − − − − − − − % ) 1 −

a mL

LOD (ng 0.02 0.1 0.1 0.05 0.2 0.05 0.05 0.2 0.05 1.5 0.3 0.3 0.3 0.3 4 1 1.5 0.3 0.3 0.3 0.3 1.5 0.75 0.3 1.5 0.3 1.5 1.5 1.5 0.3 0.3 0.3 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 1.25 0.5 0.5 1 0.5

) Mass z

/ m 460.1970 ( Ion 389.1970 356.1292 453.1686 437.1981 437.1981 447.2377 475.2337 444.2028 417.2272 425.1981 411.1978 421.2032 421.2032 423.1825 395.1676 451.1938 419.2075 405.1919 403.1762 479.2087 479.2087 439.1774 335.19760 405.1821 378.2399 327.1715

Ion − − − − − − − − − − − − − − − − −

− − − − − − − H] H] H] H] H] H] H] − − − − − − − Secondary [M+HCOO] [M [M+HCOO] [M+HCOO] [M+HCOO] [M+H]+ [M+HCOO] [M+HCOO] [M+HCOO] [M+H]+ [M+HCOO] [M+H]+ [M+HCOO] [M+HCOO] [M+HCOO] [M [M [M+HCOO] [M+HCOO] [M+HCOO] [M+HCOO] [M+HCOO] [M+HCOO] [M [M [M [M

) z / m (

Mass

491.2287 Ion 387.2101 343.1915 336.1958 352.1902 385.1918 356.2009 344.1645 358.1438 358.1802 372.1594 409.1776 393.2072 393.2072 431.2428 541.3160 467.1995 416.2068 400.2118 461.2184 381.2072 455.1887 377.2123 377.2123 379.1915 397.1821 453.2083 375.2166 361.2009 359.1853 435.2177 435.2177 395.1864 337.2122 250.1802 267.1703 292.1543 308.2220 326.2326 262.1802 272.1412 293.1860 407.1965 380.2544 296.1856 329.1860 292.1907 310.2013 268.1907

− − −

− Ion

H] − Main [M+H]+ [M [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+HCOO] [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+HCOO] [M+H]+ [M+HCOO] [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+

(min)

g 15.7 25.0 25.8 22.7 22.4 27.5 23.0 22.9 23.5 23.2 16.9 14.4 14.9 12.5 22.2 27.7 24.0 6.3 10.4 17.8 10.5 15.3 20.5 20.8 10.0 15.8 21.9 13.3 9.8 10.1 18.2 18.9 6.8 6.4 9.3 4.9 6.6 9.6 7.9 10.5 9.5 5.6 15.5 7.0 7.0 7.9 6.7 8.7 6.6 RT ) 1 Conc. −

(ng Spiked 1 75 0.5 0.5 0.5 0.5 0.5 0.5 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 50 50 50 25 50 50 25 25 50 50 50 25 50 50 25 50 15

MT 15

MT 0.5

MT 0.5 MT

MT desacetyl

MT -carboxy

acid

␤

acid

-carboxy

acetonide hydroxy

␤

6 desacetyl 17 prop.

9-tetrahydrocannabinol

propionate

5-hydroxypentyl

N-(4-hydroxbutyl) N-butanoic N-(4-hydroxypentyl) pentanoic

-Hydroxybudesonide 15 ) ␤ Compound Sufentanyl Carboxy- JWH-250 JHW-250 JWH-200 JWH-122 JWH-73 JWH-73 JWH-18 JWH-18 Beclomethasone Betamethasone Dexamethasone 6 Budesonide Ciclesonide Clobetasol Deﬂazacort Deﬂazacort Desonide Fludrocortisone Flumethasone Fluocortolone Fluorometholone Fluprednisolone Fluticasone Fluticasone Methylprednisolone Prednisolone Prednisone Triamcinolone Flunisolide Triamcinolone Acebutolol Alprenolol Atenolol Befunolol Betaxolol Bisoprolol Bufarolol Bupranolol Carteolol Carvedilol Celiprolol Esmolol Labetalol Levobunolol Metipranolol Metoprolol MT CANNABINOIDS GLUCOCORTICOIDS BETA-BLOCKERS Continued (

No. 229 231 253 256 270 233 246 266 269 274 230 239 262 232 234 235 236 237 238 240 241 242 244 245 248 249 250 251 252 254 255 257 258 259 260 261 263 264 265 267 268 271 272 273 275 276 277 243 247 Table

W. Abushareeda et al. / Journal of Pharmaceutical and Biomedical Analysis 151 (2018) 10–24 19 (SD) b , a (8.8) (5.1) (8.3) (9.1) (8.3) (3.2) (6.2)

(13)

Extraction

% 77.8 80.1 94.4 82.1 54.2 86 79.5 89.1 Recovery b , a

Effect

(7.7) (7.5)

(15) (14) (10) (10) (10) (12)

Matrix 35 25.5 20 63 17 54 16 17.3

(SD) − − − − − − − − % ) 1 −

a mL

LOD (ng 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 1.0 1.0 2.5 0.25 0.2 0.2 0.1 1 0.5 0.5 1 0.5 0.5 0.2

) Mass z

/ m ( Ion 354.1921 271.1122 221.0660 401.2446 342.1700 250.1590 152.1070

Ion −

O]+ O]+ 2 2

H H − − − − − H] H] H] − − − [M Secondary [M+HCOO] [M [M [M+H]+ [M+H [M+H

) z / m (

Mass

Ion 310.2013 406.1824 266.1751 292.2271 249.1598 260.1645 273.1267 317.1642 403.2591 307.1110 414.9924 531.1560 353.1132 529.2479 340.1553 291.0588 300.1594 300.1594 268.1696 515.2442 264.1958 240.1150 195.0877 399.2278 222.1852 134.0964

analytes.

>75.0070 − −

intensities.

Ion

H] H] − − ion

Main [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ MSMS 221.07 [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M [M [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ selective

main

the

(min)

g number

RT 5.7 20.2 7.5 20.5 6.0 9.0 4.9 6.3 1.1 10.3 6.7 23.8 20.3 23.1 16.3 22.0 19.6 5.9 5.4 7.7 22.7 6.7 7.8 5.7 13.4 6.8 4.8 according limited

) 1 Conc. −

for

estimated

(ng Spiked 50 25 50 25 50 50 50 50 5000 * 50 50 50 1 1 10 * 5 5 50 25 25 25 25 25 10 material.

evaluated were

were

reference

Recovery,

urine

Recovery,

benzene-1,3-disulfonamide.

FACTORS

time. Extraction

(FG4592)

carboxy-MT

excretion

and

SUBSTANCES

Extraction

from glucuronide

) and retention

Effects Cl. Br.

Compound Nadolol Nebivolol Oxprenolol Penbutolol Pindolol Propranolol Sotalol Timolol Ethyl Finasteride Fluconazole Miconazole Ketoconazole Roxadustat Ibutamoren Efaproxiral 2-Hydroxyﬂutamide Hydrocodone Codeine Pipradrol Telmisartan Tramadol Bupropion Caffeine Mitraginine Tapentadol Norephedrine CONFOUNDING OTHER to

37 81 available

of of

Effects

Matrix

Continued (

referred

LOD, Matrix Isotope Isotope 4-Amino-6-chloro-1,3-benzenedisulfonamide. 4-Amino-6(triﬂuoromethyl) RT f c a e No. 278 291 286 295 296 302 287 288 279 281 283 284 285 290 293 294 297 298 300 301 280 289 292 303 304 282 299 g b d Table *Compound

20 W. Abushareeda et al. / Journal of Pharmaceutical and Biomedical Analysis 151 (2018) 10–24

Fig. 1. Calibration curves of ES, TS, 5A-DHTS, DHEAS, AS and ETIOS.

Table 2

Summary of the data used for quantitative validation of endogenous sulfate steroids.

Compound name RT (min) Ion (m/z) QC level (ng/ml) bias% Intermediate precision (%) MU%

QC1 QC2 QC1 QC2 QC1 QC2 QC1 QC2

TS 10.1 367.1585 2.5 100 17.5 14.0 3.8 4.1 17.9 14.5

ES 10.9 367.1585 2.5 100 14.1 5.7 3.2 3.4 14.4 6.6

5˛DHTS 13.2 369.1741 2.5 100 2.8 7.5 2.3 0.3 3.6 7.5

DHEAS 12.7 367.1585 25 1000 9.9 2.4 2.4 2.2 10.1 3.2

ANDROS 16.3 369.1741 25 1000 17.9 15.7 1.6 1.7 18 15.8

ETIOS 17.2 369.1741 25 1000 10.3 4.8 3.0 1.2 10.7 4.9

TS-d3 10.1 370.1773 Internal

AS-d4 16.2 373.1992 stan-

5˛DHTS −d3 13.0 372.1929 dards

RT referred to retention time.

conditions of polarity switching, full scan in high-resolution mode resolution of the acquisition method deﬁned as full width at half

and tandem MS acquisition, which provide the best performance maximum (FWHM) at 200 m/z was of 70,000 and resulted in

for the selected substance classes that should be analyzed in chromatographic peaks with 4 acquisition points for each polarity

complex sample with important matrix interferences. The MS mode. Under those conditions, the introduction of simultaneous

W. Abushareeda et al. / Journal of Pharmaceutical and Biomedical Analysis 151 (2018) 10–24 21

Fig. 2. Extracted ion chromatograms at ±5 ppm mass window of two mesterolone sulfate metabolites in a urine sample suspected for illegal use of mesterolone anabolic

steroid and in a negative urine sample.

tandem MS acquisition would create long duty cycle leading to matograms for all analytes of interest, except for stimulants and

low sensitivity due to low number of sampling point of a chro- narcotics that are eluted at an early retention time where window

matographic peak. In the current application, however the FWHM of the extracted ion chromatogram window was set at ±10 ppm.

was set to 17,500, which resulted in 8–9 acquisition points for an

average chromatographic peaks in polarity switching mode, while 3.2. Validation results

addition of the acquisition up to three MS/MS resulted in 5–6

3.2.1. Qualitative validation results

acquisition points per chromatographic peak. Respectively, a mass

The identiﬁcation capability, speciﬁcity, matrix interferences,

window of ±5 ppm was applied for extraction of the ion chro-

LOD, recovery and matrix effects results for the validated target

22 W. Abushareeda et al. / Journal of Pharmaceutical and Biomedical Analysis 151 (2018) 10–24

substances are shown in Table 1. Substances ionizable by ESI with 3.3. Application to real samples

difﬁculties to meet the validation speciﬁcations were analysed

by the GC/MS screening, like aminoglutethimide, 4-androstene- The current method had been applied as screening method to

3,6,17-trione, HU-210 and norfenephrine. Table 1 summarizes the ofﬁcial antidoping samples, among others, reported as Adverse

results of qualitative validation and represents all classes of 304 Analytical Findings (AAF, positive case) based on the GC/MS tech-

prohibited substances that were detected with score 10/10 in the nology detection of AAS abuse. The examples presented herein

different urine matrices tested at concentrations equal to or below do not represent an exhaustive study of the sulfate metabolites

50% of the MRPL. Our approach was able to detect even substances of the respective AAS, but the detection of sulfate metabolites

showing ion suppression more than 70% or extraction recovery less based on matching the m/z of the sulfate conjugate metabo-

than 20%. For most of the substances the LOD are in the level of 10% lites published in literature. The ﬁrst example concerns with

MRPL or lower. Carry over between injections was not observed at a sample contained a metabolite (1␣-methyl-5␣-androstane-

injected concentration of 20 times higher than the MRPL levels [3]. 3␣-ol-17-one) of the anabolic steroid of mesterolone at the

−1

concentration of 3 ng mL , without the presence of mesterolone

parent compound. Our LC/MS method was applied to this sam-

3.2.2. Quantitative validation results

ple in order to test and corroborate the results obtained using

The validation data related to the sulfate endogenous steroids

standard GC/MS approach. Two mesterolone sulfate metabolites

are summarized as follow: the representative calibration curves

with parent ion m/z of 383.1898 and 399.1847 Da, and the non-

of the 6 steroids are presented in Fig. 1; precision, accuracy

sulfate metabolite detected by GC/MS with m/z of 488 > 433 were

expressed as% of bias and combined measurement uncertain-

detected in full scan LC/MS data. Fig. 2 shows the extracted

ties of the steroids are shown in Table 2; calibration curves

ion chromatogram for mesterolone sulfate metabolites in the

obtained following the protocol described in section 2.1.5.2 were

sample presented together with blank urine sample. These two

calculated from the peak area ratio of sulfo-conjugates steroids

mesterolone sulfate metabolites are considered to be mark-

and the deuterated standards except for AS and EtioS, calibra-

ers of mesterolone abuse [31]. The second example is related

tion curves for AS and EtioS were calculated from the height

to a positive case reported for the AAS nandrolone based on

ratio of the analytes due to the low chromatographic resolu-

the GC/MS detection of the metabolites 19-Norandrosterone

tion and considerable overlap of the chromatographic peaks of

and 19-Noretiocholanolone from the free and glucuronide urine

AS and EtioS at high concentration level. The validation results −1

fraction at a concentration of approximately 50 ng mL for 19-

demonstrated high analytical performance with respect of lin-

Norandrosterone. The current LC/MS method analysis revealed

earity, accuracy (2.4–17%), intermediate precision (1.2–4.1%), and

two peaks of m/z 355.1585 attributed to 19-Norandrosterone

combined measurement uncertainties (3.2–18%) over the range

−1 sulfate and 19-Noretiocholanolone sulfate not present in the

of 1–200 ng mL for TS, ES and 5␣DHTS and over the range

−1 LC/MS of negative samples [27] (Fig. 3). In a third exam-

10–2000 ng mL for AS, EtioS and DHEAS. Weighted linear regres-

ple, a sample reported for boldenone detected by GC/MS at a

sion was applied to calculate the calibration curves due the −1

concentration of the level of 500 ng mL analyzed in parallel

wide concentration range of the analytes, which resulted in

by the LC/MS method in MS/MS mode showed the peak m/z

larger biases at low concentration levels when non-weighted

365.1428 > 350.1188 for boldenone sulfate and epiboldenone sul-

linear regression was used. The analytical results described in

fate [26] (Fig. 4).

this study can be extended to other exogenous and endogenous

steroids.

Fig. 3. Extracted ion chromatograms at ±5 ppm mass window of 19-nornadrosterone sulfate and 19-noretiocholanolone sulfate in a urine sample suspected for use of

nandrolone anabolic steroid and a negative sample.

W. Abushareeda et al. / Journal of Pharmaceutical and Biomedical Analysis 151 (2018) 10–24 23

Fig. 4. Extracted ion chromatograms at ±5 ppm mass window of 19-nornadrosterone sulfate and 19-noretiocholanolone sulfate in a urine sample suspected for use of

nandrolone anabolic steroid and a negative sample.

4. Conclusion Acknowledgment

The presented LC/MS screening method was developed and val- The authors acknowledge the Qatar National Research Fund

−

idated for the analysis of endogenous and exogenous substances, (QNRF) for funding the current project under the NPRP 6 334

−

with aim to expands the analytical repertoire of small molecules to 3–087 contract. Mrs. Noor Al-Motawa, ADLQ Education and

the detection of sulfate Phase II metabolites and allow the quanti- Research Ofﬁce Director, is sincerely acknowledged for the constant

tative determination of endogenous sulfate steroids in their intact support on the research projects.

form. This becomes very important, because the sulfoconjugated

steroid proﬁle in this project can be analyzed in all samples without References

any further analysis and without creating any interferences to glu-

[1] World Anti-doping Agency, World Anti-doping Agency (WADA) Prohibited

curoconjugated steroid proﬁle required by WADA [4]. The use of the

List, 2017,

combined ethylacetate liquid–liquid extraction of hydrolyzed urine https://www.wada-ama.org/sites/default/ﬁles/resources/ﬁles/2016–09-29

and the D&S urine addition allow to extract both sulfate derivatives wada prohibited list 2017 eng ﬁnal.pdf (Accessed 12 August 2017).

[2] World Anti-Doping Agency, World Anti-Doping Agency (WADA) International

and intact steroids and drugs. In future study, we aim to assess

Standard for Laboratories (ISL 2016), 2016,

the method for analysis of large number of steroid sulfate metabo-

https.//www.wada-ama.org/sites/default/ﬁles/resources/ﬁles/isl june 2016.

lites. This expansion is important due to fact that several studies pdf (Accessed 6 August 2017).

[20,22,27] show that sulfate metabolites can be detected much later [3] World Anti-Doping Agency, World Anti-doping Agency (WADA)

TD2017MRPL, 2017, https.//www.wada-

after steroid abuse due to the longer half-life of sulfate metabo-

ama.org/sites/default/ﬁles/resources/ﬁles/wada-td2017mrpl-en 0. pdf

lites compared to non-conjugated forms. Besides the advantage of

(Accessed 31 August 2017).

the detection time of sulfoconjugated metabolites of exogenous [4] World Anti-Doping Agency, Endogenous Anabolic Androgenic Steroids:

Measurement and Reporting. TDAAS, 2016, ver. 1.0.

steroids, they are eliminated in greater quantities in comparison

https://www.wada-ama.org/en/resources/science-medicine/td2016-eaas

to their glucuronide analogues, resulting in better retrospective

(Accessed 15 August 2017).

detection capabilities [32]. [5] T. Kuuranne, M. Saugy, N. Baume, Confounding factors and genetic

polymorphism in the evaluation of individual steroid proﬁling, Br. J. Sports

The high-resolution full scan LC/MS data with the polarity

Med. 48 (2014) 848–855.

switching MS acquisition mode provides the ﬁngerprint of all com-

[6] O.J. Pozo, P. Van Eenoo, K. Deventer, F.T. Delbeke, Ionization of anabolic

pounds detected by an LC/MS(/MS) system with several advantages steroids by adduct formation in liquid chromatograph electrospray mass

spectrometry, J. Mass Spectrom. 42 (2007) 497–516.

over targeted analysis of compounds. These include the high MS

[7] K. Deventer, O.J. Pozo, A.G. Verstraete, P. Van Eenoo, Dilute-and-shoot-liquid

speciﬁcity without reduction in the sensitivity of detection, the

chromatography-mass spectrometry for urine analysis in doping control and

ability to identify all compounds, which can be detected by an analytical toxicology, Trends Anal. Chem. 55 (2014) 1–13.

[8] C. Görgens, S. Guddat, A. Thomas, P. Wachsmuth, A. Orlovius, G. Sigmund, M.

LC/MS system in positive and negative ionization modes, the ability

Thevis, W. Schänzer, Simplifying and expanding analytical capabilities for

to reprocess the collected data retrospectively, to search for new

various classes of doping agents by means of direct urine injection high

metabolites, unknown prohibited designer drugs, and to obtain performance liquid chromatography high resolution/high accuracy mass

information on the overall metabolomic state of a person, which spectrometry, J. Pharm. Biomed. Anal. 131 (2016) 482–496.

may reﬂect illegal use of performance enhancer compounds.

24 W. Abushareeda et al. / Journal of Pharmaceutical and Biomedical Analysis 151 (2018) 10–24

[9] A. Musenga, D. A.Cowan, Use of ultra-high pressure liquid chromatography [21] T. Piper, W. Schänzer, M. Thevis, Genotype-dependent metabolism of

coupled to high resolution mass spectrometry for fast screening in high exogenous testosterone –new biomarkers result in prolonged detectability,

throughput doping control, J. Chromatogr. A 1288 (2013) 82–95. Drug Test. Anal. 8 (2016) 1163–1173.

[10] A. Vonaparti, E. Lyris, Y.S. Angelis, I. Panderi, M. Koupparis, A. [22] J. Schulze, J. Tho¨ rngren, M. Garle, L. Ekström, A. Rane, Androgen sulfation in

Tsantili-KakouLidou, R.J.B. Peters, M.W.F. Nielen, C. GeorgakopouLos, healthy UDP-Glucuronosyl transferase 2B17 enzyme-deﬁcient men, J. Clin.

Preventive doping control screening analysis of prohibited substances in Endocrinol. Metab. 96 (11) (2011) 3440–3447.

human urine using rapid-resolution liquid chromatography/high-resolution [23] D. Borts, L. Bowers, Direct measurement of urinary testosterone and

time-of-ﬂight mass spectrometry, Rapid Comm. Mass Spectrom. 24 (2010) epitestosterone conjugates using high-performance liquid

595–1609. chromatography/tandem mass spectrometry, J. Mass Spectrom. 35 (2000)

[11] T. Friedmann, U. Flenker, C. Georgakopoulos, M. Alsayraﬁ, P. Sottas, S. 50–61.

Williams, R. Gill, Evolving concepts and techniques for anti-doping, [24] C. Gomez, A. Fabregat, Ó. Pozo, J. Marcos, J. Segura, R. Ventura, Analytical

Bioanalysis 4 (13) (2012) 1667–1680. strategies based on mass spectrometric techniques for the study of steroid

[12] D.W. Andersen, K. Linnet, Screening for anabolic steroids in urine of forensic metabolism, Trends Anal. Chem. 53 (2014) 106–116.

cases using fully automated solid phase extraction and LC–MS-MS, J. Anal. [25] F. Badoud, E. Grata, J. Boccard, D. Guillarme, J. Veuthey, S. Rudaz, M. Saugy,

Toxicol. 38 (2014) 637–644. Quantiﬁcation of glucuronidated and sulfated steroids in human urine by

[13] L. Dehennin, P. Lafarge, Ph. Dailly, D. Bailloux, J.P. Lafarge, Combined proﬁle of ultra-high pressure liquid chromatography quadrupole time-of-ﬂight mass

androgen glucuro- and sulfoconjugates in postcompetition urine of spectrometry, Anal. Bioanal. Chem. 400 (2011) 503–516.

sportsmen: a simple screening procedure using gas chromatography-mass [26] E. Tudela, K. Deventer, L. Geldof, P. Van Eenoo, Urinary detection of

spectrometry, J. Chromatogr. B 687 (1996) 85–91. conjugated and unconjugated anabolic steroids by dilute-and-shoot liquid

[14] P.Van Renterghem, P. Van Eenoo, P. Sottas, M. Saugy, F. Delbeke, Delbeke pilot chromatography-high resolution mass spectrometry, Drug Test. Anal. 7

study on subject-based comprehensive steroid proﬁling:novel biomarkers to (2015) 95–108.

detect testosterone misuse in sports, Clin. Endocrinol. (Oxf.) 75 (2011) [27] S. Rzeppa, G. Heinrich, P. Hemmersbach, Analysis of anabolic androgenic

134–140. steroids as sulfate conjugates using high performance liquid chromatography

[15] O.J. Pozo, J. Marcos, R. Ventura, A. Fabregat, J. Segura, Testosterone coupled to tandem mass spectrometry, Drug Test. Anal. 7 (2015) 1030–1039.

metabolism revisited: discovery of new metabolites, Anal. Bioanal. Chem. 398 [28] G. Balcells, O. Pozo, A. Esquivel, A. Kotronoulas, J. Joglar, J. Segura, R. Ventura,

(2010) 1759–1770. Screening for anabolic steroids in sports: analytical strategy based on the

[16] J. Boccard, F. Badoud, E. Grata, S. Ouertani, M. Hanaﬁ, G. Mazerolles, P. Lanteı´ detection of phase I and phase II intact urinary metabolites by liquid

ri, J. Veuthey, M. Saugy, S. Rudaz, A steroidomic approach for biomarkers chromatography tandem mass spectrometry, J. Chromatogr. A 1389 (2015)

discovery in doping control, Forensic Sci. Int. 213 (2011) 85–94. 65–75.

[17] M. Raro, M. Ibaın˜ez,´ R. Gil, A. Fabregat, E. Tudela, K. Deventer, R. Ventura, J. [29] W. Abushareeda, A. Fragkaki, A. Vonaparti, Y. Angelis, M. Tsivou, K. Saad, S.

Segura, J. Marcos, A. Kotronoulas, J. Joglar, M. Farreı,´ S. Yang, Y. Xing, P. Van Kraiem, E. Lyris, M. Alsayraﬁ, C. GeorgakopouLos, Advances in the detection of

Eenoo, E. Pitarch, F. Hernaındez,´ J. Sancho, Oı.J.´ Pozo, Untargeted designer steroids in anti-doping, Bioanalysis 6 (6) (2014) 881–896.

metabolomics in doping control: detection of new markers of testosterone [30] W. Abushareeda, E. Lyris, S. Kraiem, A.A. Wahaibi, S. Alyazidi, N. Dbes, A.

misuse by ultrahigh performance liquid chromatography coupled to Lommen, M. Nielen, P.L. Horvatovich, M. Alsayraﬁ, C. Georgakopoulos, Gas

high-resolution mass spectrometry, Anal. Chem. 87 (2015) 8373–8380. chromatographic quadrupole time-of-ﬂight full scan high resolution mass

[18] A. Palermo, F. Botrè, X. de la Torre, N. Zamboni, Non-targeted LC-MS based spectrometric screening of human urine in antidoping analysis, J.

metabolomics analysis of the urinary steroidal proﬁle, Anal. Chim. Acta 964 Chromatogr. B 1063 (2017) 74–83.

(2017) 112–122. [31] P. Kiousi, Y.S. Angelis, A.G. Fragkaki, W. Abushareeda, M. Alsayraﬁ, C.

[19] F. Badoud, J. Boccard, C. Schweizer, F. Pralong, M. Saugy, N. Baume, Proﬁling of Georgakopoulos, E. Lyris, Markers of mesterolone abuse in sulfate fraction for

steroid metabolites after transdermal and oral administration of testosterone doping control in human urine, J. Mass Spectrom. 50 (2015) 1409–1419.

by ultra-high pressure liquid chromatography coupled to quadrupole [32] A.T. Cawley, R. Kazlauskas, G.J. Trout, A.V. George, Determination of urinary

time-of-ﬂight massspectrometry, J. Steroid Biochem. Mol. Biol. 138 (2013) steroid sulfate metabolites using ion paired extraction, J. Chromatogr. B 825

222–235. (2005) 1–10.

[20] J. Schulze1, M. Johansson, J. Thörngren, M. Garle, A. Rane, L. Ekström, SULT2A1

gene copy number variation is associated with urinary excretion rate of

steroid sulfates, Front. Endocrinol. (Lausanne) 4 (2013), 88.