Research articleDrug Testing

and Analysis

Received: 16 November 2015 Revised: 15 December 2015 Accepted: 16 December 2015 Published online in Wiley Online Library: 16 March 2016

(www.drugtestinganalysis.com) DOI 10.1002/dta.1951

Specific characterization of non-steroidalselective androgen peceptor modulators usingsupercritical fluid chromatography coupled toion-mobility mass spectrometry: application tothe detection of enobosarm in bovine urineLaure Beucher, Gaud Dervilly-Pinel,* Nora Cesbron, Mylène Penot,Audrey Gicquiau, Fabrice Monteau and Bruno Le Bizec

Currently under development for therapeutic purposes in humanmedicine, non-steroidal selective androgen receptormodulators(non-steroidal SARMs) are also known to impact growth associated pathways. As such, they present a potential for abuse in sportsand food-producing animals as interesting alternative anabolic substances. Forbidden since 2008 by the World Anti-DopingAgency (WADA) these compounds are however easily available and could be (mis)used in livestock production as growthpromoters. To prevent such practices, dedicated analytical strategies have to be developed for specific and sensitive detectionof these compounds in biological matrices. Using an innovative analytical platform constituted of supercritical fluid chromatog-raphy coupled to ionmobility-mass spectrometry, the present study enabled efficient separation and identification in urine of 4 ofthese drugs (andarine, bicalutamide, hydroxyflutamide, and enobosarm) in accordancewith EuropeanUnion criteria (CommissionDecision 2002/657/EC). Besides providing information about compounds structure and behaviour in gas phase, such a couplingenabled reaching low limits of detection (LOD< 0.05ng.mL�1 for andarine and limits of detection< 0.005ng.mL�1 for the threeothers) in urine with good repeatability (CV< 21%). The workflow has been applied to quantitative determination of enobosarmelimination in urine of treated bovine (200mg, oral). Copyright © 2016 John Wiley & Sons, Ltd.

Keywords: SARMs; Q-TWIM-TOF MS; supercritical fluid chromatography; anabolic practices; bovine; urine; trace concentrations

* Correspondence to: Gaud Dervilly-Pinel, LUNAM Université, Oniris, Laboratoired’Etude des Résidus et Contaminants dans les Aliments (LABERCA), Nantes,F-44307, France. E-mail: gaud.dervilly@oniris-nantes.fr

LUNAM Université, Oniris, Laboratoire d’Etude des Résidus et Contaminants dansles Aliments (LABERCA)Nantes F-44307, France

179

Introduction

Since the late nineteenth century, a new family of ligands of the an-drogen receptor (AR) has been synthesized.[1,2] They are known asnon steroidal selective androgen receptor modulators (SARMs)[3]

and comprise compounds such as andarine, bicalutamide,hydroxyflutamide, or enobosarm. These benzene-propanamideshave been developed during the past decade as tissue-selectivecompounds with potential therapeutic applications in male fertilityor hormone replacement therapy.[4–7] Presenting the same ana-bolic activity than steroids, these compounds however aretissue-specific in their action, targeting mainly bone and muscle,and do not present any androgenic activity and therefore noneof the associated side effects upon treatment. Although devel-oped for therapeutic purposes, these orally active compoundshave already been reported in human doping cases,[8,9] eventhough they have been forbidden by the World Anti-DopingAgency (WADA) since January 2008.[10,11] As formany illegal anabolicsubstances finding their way from sports doping to livestock produc-tion, their potential misuse in veterinary practice has to be consid-ered. These compounds have not yet been related to animalwelfare or consumers issues, although some risks associated to theiruse for therapeutic purposes have already been reported.[11,12] Since

Drug Test. Analysis 2017, 9, 179–187

no risk assessment has been conducted yet on these compoundsand because the use in stock farming of substances having a hor-monal action is prohibited in Europe [Dir 96/22/EC], analytical strate-gies have to be developed, as a precautionary principle, to preventtheir use and ensure consumers with chemically safe food from ani-mal origin.

Efficient analytical strategies to detect the use of SARMs havealready been proposed and main developments have been real-ized in the human anti-doping arena,[8,9,13,14] with one applica-tion available in food producing animals.[15] This particularstudy focused on development of a protocol to detect, in bo-vine urine, several non-steroidal SARMs and their metabolites,based on liquid or solid-phase extraction (SPE) followed by ultraperformance liquid chromatography- quadrupole -time of flightmass spectrometry (UPLC-Q-TOF MS) analysis using negativeelectrospray. Reported performances were described in

Copyright © 2016 John Wiley & Sons, Ltd.

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accordance with EU analytical criteria [Decision 2002/657/EC],with decision limits (CC ) in the range 0.025 to 0.05 μg.L�1 anddetection capabilities (CC ) of 0.025 μg.L�1 (relative standard de-viation of intra-laboratory assays< 20 %). Using such a protocol,enobosarm could be detected in the urine of a treated calf(200mg once) until 6 days after administration. On the basis ofthis first proof of concept to the bovine, we propose in thisstudy to investigate alternative analytical strategies to furtherimprove the performances of detection of these substances,and therefore achieve better sensitivity and selectivity.Despite its predominant and efficient role in doping control,

liquid chromatography may generate insufficient sensitivity orspecificity of the detection because of co-elution or ion suppres-sion issues[14] when dealing with the analysis of complex bio-logical matrices. Supercritical fluid chromatography (SFC)[16]

represents an interesting separation alternative that has alreadyshown its potential in enhancing detection performances ofresidues of growth promoters.[17] Using mainly liquid carbon di-oxide as a mobile phase, this separation technique is based onsimilar retention mechanisms of both gas and liquid chromatogra-phy, depending on the mobile phase density, the flow rate, the col-umn temperature and the stationary phase’s properties[18] with alarger range of solvent selectivity choice. De facto, it enables anincreased separation of relatively similar and numerous chemicalcompounds as described in for peptides,,[19] drugs, chiral ornot,[20] or even lipids separation. As illustrated in a recent studyon pharmaceuticals,[21] SFC has further proven its efficiency inseparating diastereoisomers. Moreover, the relatively lowamount of solvents used with SFC could also allow reaching bet-ter sensitivity results by decreasing the chemical backgroundnoise and potential ion suppression.Until now, one of the very few brakes on the use of this sep-

arative technique was the control of the pressure which is nowovercome using new generation pumps that enable reproduc-ible results.[16]

In parallel of these chromatographic innovations, other novelseparation techniques such as ion mobility coupled to massspectrometry (IM-MS) have been reported to help face these is-sues (chemical noise, ion suppression, co-detection). Challengesdue to the complexity of biological matrices could therefore beovercome using promising technologies like the traveling-waveion mobility (TWIM) cell included in an orthogonal Q-TOF-MS.[22]

This device enables separation of ionic species based on theirsize, charge and shape. Traveling through neutral gas at re-duced pressure and driven by sequential high electrical field,ions express a specific time to drift along the TWIM part andpresent then a specific average collision cross section (CCS)leading to a strong signal clean-up. Although this technologyhas mainly been investigated for large molecules[23] for thepurpose of structural analyses, recent studies have presentedinteresting applications for small molecules detection in termsof separation efficiency[24] and specificity.[25–27] This approachhas also recently been assessed in the context of chemical foodsafety applied to the specific and sensitive detection of smallmolecules like β-adrenergic agonists in complex biologicalmatrices.[28]

The idea behind this study was first to compare LC and SFC sep-aration performances of non-steroidal SARMs and secondly, toassess the potential of IMS-MS in relation with the analysis of thesecompounds when present at trace levels in biological matrices, butalso to provide data related to enobosarm elimination and detect-ability in bovine urine.

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Experimental

Chemicals and reagents

Non-steroidal SARMs have been provided by Sigma-Aldrich (StLouis, MO, USA): enobosarm ((2S)-3-(4-Cyanophenoxy)-N-[4-cyano-3-(trifluoromethyl)phenyl]-2-hydroxy-2-methylpropanamide, SARMS22, GTx-024, MK-2866 or Enobosarm), andarine ((2S)-3-(4-Ace-tamidophenoxy)-2-hydroxy-2-methyl-N-[4-nitro-3-(trifluoromethyl)phenyl]propanamide or SARM S4), bicalutamide (N-[4-Cyano-3-(trifluoromethyl)phenyl]-3-[(4-fluorophenyl)sulfonyl]-2-hydroxy-2-methylpropanamide or Casodex) and hydroxyflutamide (2-Hy-droxy-2-methyl-N-[4-nitro-3-(trifluoromethyl)phenyl]propanamide).The bicalutamide-d4 has been purchased at Toronto ResearchChemical (North York, Canada).

High performance liquid chromatography (HPLC) grade ace-tonitrile and formic acid were respectively purchased fromSigma-Aldrich (St Louis, MO, USA) and from LGC StandardsGmbH (Wesel, Germany).

Standards solutions were prepared to obtain final concentra-tion of 10 ng.μL�1 of each non-steroidal SARMs (enobosarm,andarine, bicalutamide, hydroxyflutamide) diluted in water: ace-tonitrile: formic acid solution (v:v:v, 70:30:0.1). Bicalutamide-d4has been employed as internal standard and prepared in a wa-ter: acetonitrile: formic acid solution (v:v:v, 70:30:0.1) at a con-centration of 0.1 ng.μL�1.

Animal experiment

Six Prim’Holstein male calves were enrolled in the experiment.All were aged three months (56 ± 5 days) (solid feed and hay)and weighed approximately 100 ± 5 kg at the beginning of theexperiment. One animal was orally administered a unique doseof enobosarm (GTx-024, GTx Inc., Memphis, TN, USA) (200mg) inethanolic solution diluted in apple juice, adapted from a proto-col described by De Rijke et al.[15] The five others formed thecontrol group and were orally administrated the sameethanol/apple juice mix without enobosarm. Urine sampleswere collected before (J-3, J-2, and J-1), and after treatment(J0 + 3 h and J0 + 5 h), then at regular intervals within 21 daysfollowing the drug administration.

Extraction and purification from urine

Aliquots of 3mL urine were supplemented with 20μL of internalstandard (bicalutamide-d4; C = 0.1 ng.μL�1) and 3mL of acetatebuffer 0.25M pH4.8. The pH was then controlled and, adjusted to5 if necessary with the acetate buffer. Phase II metabolites weredeconjugated through enzymatic hydrolysis using 20μL of β-glucu-ronidase/aryl sulfatase enzyme (EMDMillipore Corporation, Billerica,Massachusetts, USA) over 1 h at 50 °C. Then, a solid phase extractionphasewas undertaken using Oasis HLB 3 cc 60mg column (Waters,Milford, Massachusetts, USA). Conditioning was realized with 6mLof methanol and 3mL of water, previously to sample dropping.Columns were then washed with 3mL of two successive differentmethanol:water solutions (S1= v:v, 5:95; S2= v:v, 20:80). Targetedcompounds were then eluted using 4mL of methanol. Sampleswere evaporated under nitrogen gas flow at 45 °C (VAC ELUT,VWR BDH Prolabo, Pessac, France). Finally, extracts were re-suspended in 250μL of water: acetonitrile: formic acid solution (v:v:v, 70:30:0.1) for injection on the chromatographic system.

hn Wiley & Sons, Ltd. Drug Test. Analysis 2017, 9, 179–187

sarm

andhydroxyflu

tamide)iden

tificationusingLC

-QqQMSan

dSFC-Q

-TWIM-

USF

C-Q-TWIM

-TOFMS

USFC:

Retentio

ntim

e(m

in)

Precursorion

Targeted

frag

men

ts

m/z

CCS(A2)

Frag

men

t1:

m/z

Frag

men

t2:

m/z

97.63

440.1064

195.76

150.055

261.0487

25.00

429.0527

182.53

255.0376

185.0321

45.00

433.0783

182.63

255.0376

185.0321

35.72

388.0904

182.62

118.0287

269.046

24.35

291.0593

155.03

205.1042

-

Hyphenation of SFC and IM-MS for growth promoters’ detection

Drug Testing

and Analysis

Chromatographic conditions

The liquid chromatography workflow was based on an AcquityUHPLC TM System (Waters, Manchester, UK) using an Acquity BEHC18 column (1.7μm, 2.1 × 100mm) at 50 °C. Mobile phase A wascomposed of water with 20mM of ammonium acetate whereasmobile phase B was 100% acetonitrile. Using a flow rate of 0.6mL.min�1, the gradient started with 70/30 (phase A/B) for 1min to upto 70/30 (B/A) at 9min and 100/0 at 9.5min. After 1min at 100/0,the proportion was decreased to 30/70 (B/A) at 11min and stayeduntil the end of the run at 15min.

For supercritical fluid chromatographic separation, an AcquityUPC2 TM System (Waters, Manchester, UK) has been used, fittedwithan Acquity UPC2 TM BEH column (1.7μm, 3.0 × 100mm) at 55 °C.Mobile phase was constituted of liquid carbon dioxide as phase Aand methanol with 20mM of ammonium bicarbonate and 0.1%formic acid as phase B. The flow rate was set to 2mL.min�1 for15min. Initially, the proportion of phase A/phase B was 99/1 for2min then up to 80/20 from 10 to 12min. From 12 to 15min, theflow was set up as 99/1. The cross contamination between experi-ments has been carefully checked and solved with appropriatescleanings. Considering the interface between SFC and IM-MS, itshould be noted that nomake-up solvent has been employed sinceassociated with a high chemical background noise was tested.

Table

1.Synthesisof

crite

riadefined

fornon

-steroidalselectiveandrogen

receptormod

ulators(andarine,bicalutam

ide,en

obo

TOFMS

Com

pou

nds

Mon

oisotopic

mass(Da)

UPLC-QqQMS

UPLC:

Retentio

ntim

e(m

in)

Transitio

ns

Nam

eChe

micalform

ula

12

3

Andarine

C19H18F3N3O

6441.114777

3.31

440,16

>150,05

440,16

>261,02

440,16

>204,9

Bicalutamide

C18H14F4N2O

4S430.061035

3.77

429,10

>255,10

429,10

>185,04

429,10

>173,0

Bicalutamide-d4

C18H10D4F4N

2O4S

434.0861

3.77

433,16

>255,10

433,16

>184,97

433,16

>177,0

Enobosarm

C19H14F3N3O

3389.098724

4.30

388,16

>118,01

388,16

>269,02

388,16

>185,0

Hyd

roxy

flutamide

C11H11F3N2O

4292.067078

3.08

291,10

>205,02

291,10

>175,04

291,10

>155,0

181

MS analysis

• Coupledwith liquid chromatography, a QqQMS fittedwith anESI source operating in negative mode (2 kV). Source temper-ature was 150 °C and offset of 30 V. Desolvation temperaturewas set at 450 °C. Parameters were specifically optimized forSARMs detection as follows: tensions of cone (Volts) werefixed as following: 6, 24, 30, and 42 for andarine, bicalutamide,enobosarm, and hydroxyflutamide, respectively. Collision en-ergies (eV) applied on precursor to monitor ion reactions(MRM) were adapted to 3 transitions (Table 1) for each com-pound. The acquisitions respected time window between2.5min and 5min.

• IM-MS experiments were performed in negative electrospraymode on a hybrid quadrupole/ traveling-wave ion mobility/orthogonal acceleration time-of-flight geometry instrument(Synapt G2-S HDMS, Waters, Manchester, UK). Optimizedparameters were fixed as follows: capillary voltage of 2.5 kVand cone voltage of 40 kV. The source and desolvation tem-peratures were set at 80 °C and 350 °C, resp., with cone anddesolvation gas flows of respectively 50 and 500mL.min�1.Sampling cone and Source offset were both set to 10. Radio-frequency stepwave voltagewas set to 100 and the ion guideto 300. The resolution of the quadrupole was fixed for the lowmass at 12.5 and highmass at 15. The nitrogen gas flow in theIMS cell was 90mL.min�1 (3.2mbar). The IMS wave velocityand height were 650m.s�1 and 30V, respectively. The Trans-fer wave velocity and height were 450m.s�1 and 4V. Collisionenergy from 10 to 60 eV were applied after the ion mobilitycell, so in the transfer cell. Full scan mass spectra (MSE) analy-ses were acquired in sensitivity mode for a mass range from50 to 600 at a mass spectra resolving power of 20 000 FullWidth Half Maximum (FWHM) (m/z 526) at 3GHz in contin-uum mode. External calibration of the instrument was per-formed with solution of sodium formate in accordance withthe manufacturer’s instructions. Lockspray solution of Leucineenkephalin (200pg.mL�1; water:acetonitrile:formic acid, v:v:v,

Drug Test. Analysis 2017, 9, 179–187 Copyright © 2016 John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/dta

Fig

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182

50:50:0.2) was infused along the acquisitions for post-run ex-act mass corrections according to a flow rate of 10μL.min�1

(inter-scan delay: 0.1ms).

Data processing and analysis

Compounds were visualized with Advanced Chemistry Develop-ment software V12.01 (Ontario, Canada). Mass spectra andmobilograms were analyzed using MassLynx 4.2. Precise mobil-ity data were extracted with DriftScope 2.7 and UNIFI 2.2.

CCS measurements

Collision cross sections (CCSs) were fixed at the apex of the ionmobility peak. Calculations have been based on correlation be-tween drift times and known CCS values for singly protonatedoligomeric ions from Poly[D,L]Alanine in nitrogen (acetonitrile:water: formic acid, 50:50:0.2, v:v:v; n = [3;11]) acquired underthe same instrument parameters by direct injection and basedon the work of Bush et al.[29,30]

Performances

Performances of the detection strategy were assessed based on thefollowing parameters: specificity, accuracy, linearity, and limit of de-tection (LOD). The robustness and the reliability of both the super-critical fluid chromatography and the ion mobility separation werechecked for concentrations of 0.25, 0.5, 1.25, 2.5, 5 and 25ng.mL�1

of 4 non-steroidal SARMs and 10ng.mL�1 of internal standard(bicalutamide-d4) in urine (N: 70 experiments). A calibration curvewas built based on related results. The linearity and LOD (S/Nratio> 3) were evaluated in the concentration range 0–5ng.mL�1.The noise has been fixed based on detected ion with close mass-to-charge ratios ( : 5 ppm) and retention times (Rt ± 0.1min) of eachnon-steroidal SARM. Precision and accuracy (of both mass and CCS)were defined on the standards solutions for all detected concentra-tions (N: 70 experiments) repeated over five months. 37 urine sam-ples of untreated calves were analyzed for specificity as well as 70blank mobile phases for potential cross contamination andinterferents identifications between injections. The same protocolwas followed for UPLC – QqQ MS experiments (N: 36).

ure 1. Drift times of bicalutamide-d4 and andarine in both positive (above

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Results and discussion

IMS characterization

The study started with the IMS characterization of the non-steroidalSARMs of interest to assess the separation efficiency of thetraveling- wave ion mobility cell. The approximated structural infor-mation thus obtained is also called collision cross sections (CCSs). Itrefers to the conformation in space of the ionized species and wasassessed based on corresponding observed drift times as explainedin the Experimental section.

Impact of protonation/deprotonation on structures

Pure compounds have been directly injected in the spectrometer inboth positive (ESI+) and negative (ESI-) electrospray to characterize(Q-TOF) the behaviour of the molecular ions in gas phase.

Protonated (M+H)+ and deprotonated (M-H)� species could beobserved in positive and negative ionization modes, respectively.Corresponding drift times could be determined as follows: andarine(DtESI+: 3.50± 0.03ms; DtESI-: 4.23± 0.02ms) (Figure 1), enobosarm(DtESI+: 3.08± 0.02ms; DtESI-: 3.85± 0.01ms), and hydroxyflutamide(DtESI+: Not detected; DtESI-: 2.55 ± 0.01ms). For these compounds,smaller drift times were thus observed for protonated species,considering both molecular and sodium adduct ions, comparedto deprotonated ions. On the opposite, bicalutamide (DtESI+:4.02 ± 0.04ms; DtESI-: 3.66 ± 0.03ms) and bicalutamide-d4 (DtESI+: 4.07 ± 0.04ms; DtESI-: 3.68 ± 0.03ms) presented higher drifttimes for deprotonated ions than protonated species (Figure 1).It means that, in the case of deprotonation, bicalutamide andbicalutamide-d4 present a larger conformation in space thanin their protonated forms, while the opposite is observed forandarine and enobosarm. Such a phenomenon reveals the im-pact of protonation/deprotonation on the structure in space ofthe ions. The main difference which could explain the different be-haviour between bicalutamide/bicalutamide-d4 and others is thesulfur atom located between the two benzene cycle of bothbicalutamide and bicalutamide-d4 (Figure 3) while enobosarmand andarine own a carbon atom instead. The resulting orbitalsof these two groups of molecules differ then, which may explainthe observations. Indeed, and according to Thevis et al.,[31] the de-protonation site is expected to be the hydroxyl function between

) and negative (below) electrospray ionization using the Q-TWIM-TOF MS.

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Hyphenation of SFC and IM-MS for growth promoters’ detection

Drug Testing

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this two benzene cycle. The observed difference could therefore re-sults from an elbow formed upon protonation of enobosarm andandarine between cycles, leading to a more compact structurethen, when, on the other hand, deprotonation would lead to astructure elongation for bicalutamide and bicalutamide-d4resulting in a larger space occupation compared to the protonatedforms.

Regarding the efficiency of the separation between targetanalytes in the ion mobility cell, slightly better performances wereobserved in positive compared to negative ionization modes as fol-lows: ESI+ (Rs: [0.2; 0.5]) and ESI- (Rs: [0.6; 1.1]). Such results highlightthe impact of the ionizationmode on the 3D structures of the targetanalytes under their ionized forms and subsequently the conse-quences in terms of species separation, based on their drift times.Although the compounds of interest in the present study may alsobe separated according to their retention times and masses, suchresult propose an alternative physical parameters of interest to sep-arate ionized species.

However and since higher ionization efficiency has been ob-served for the four SARMs of interest in the present study usingnegative electrospray, which was in agreement with previousfindings,[31] Subsequent experiments have therefore beenconducted only using ESI- and based on the deprotonatedforms.

CCSs determination

Based on 70 experiments repeated over five months, CCSs havebeen calculated for andarine, bicalutamide, bicalutamide-d4,hydroxyflutamide and enobosarm, as follows and respectively:195.76± 0.36Å2, 182.53± 0.14Å2, 182.63± 0.15Å2, 155.03± 0.20Å2

and 188.62± 0.21Å2 (Figure 2). The relative standard deviations ofmeasured CCSs ranged from 0.14 to 0.28 (RSDmean: 0.21 or 0.11%)for each targeted molecular ion attesting for the good specificityand robustness of this parameter in mobile phase. As observed inFigure 2, a slight difference of CCS is observed betweenbicalutamide and bicalutamide-d4; this difference was found stableand repeatable all over the experiments ( bicalutamide-d4/bicalutamide: 0.1± 0.08Å2) attesting for the efficiency of the sepa-ration and its ability to resolve deuterated forms.

Figure 2. Ion mobility separation of 5 non-steroidal selectiveandrogenic receptor modulators (andarine, bicalutamide, bicalutamide-d4,hydroxyflutamide and enobosarm) observed on the Q-TWIM-TOF MSusing negative electrospray ionization.

Drug Test. Analysis 2017, 9, 179–187 Copyright © 2016 John W

Q-TWIM-TOF MS/MS: fragmentation patterns

First, standards have been characterized by both Q- TOF MS/MSand Q-TWIM-TOF MS/MS, i.e., with and without the activation ofthe ion mobility cell. Collision energies applied in the transfer cell(CE) for all compounds have been optimized. The required CE to ob-tain similar fragmentation patterns were lower for MS/MS experi-ments (CE: [10; 18]) than for IM-MS/MS experiments (CE [18; 30]).When ion mobility separation is activated, transmitted energy toions is supposed of higher level than without which could explainthis observation. SFC-IM-MS/MS experiments were then acquiredin MSE mode with respect to CE defined in IM-MS/MS mode. Thistype of experiment (MSE) allows monitoring all ionic signals andfragmentation reactions occurring during the same mass analysis.Both precursor and fragment ions are visualized. The loss in termsof transmission and then sensitivity is expected to be reduced com-pared to classical simultaneous andmultiple MS/MS acquisitions. Inthat case, collision energies are applied as a ramp.

Patterns of fragmentation and fragments identification have al-ready been proposed by Thevis et al.[31] and De Rijke et al.[15] forandarine and enobosarm. They have therefore been consideredas a basis to explain observed fragmentation using MS/MS, IM-MS/MS or IM-MSE (Figure 3).

One of the andarine (m/z[M-H]-: 440.1064) fragments wasobserved in accordance with those previously described[31,32]

(m/z[M-H]-: 261.0487, chemical formula (CF): C10H8N2F3O3;

m/z[M-H]-: 150.0555, chemical formula CF: C8H8NO2) corre-sponding to inductive effect coming from the nitrogen atomincluded between the two benzene cycles. Bicalutamide(m/z[M-H]-: 429.0527) and bicalutamide-d4 (m/z[M-H]-: 433.0783)exhibited the same two major fragment ions: m/z 255.0376(CF: C11H6F3N2O2) and 185.0321 (CF: C8H4F3N2). This observa-tion confirms the position of deuteriums as indicated by thesupplier which means they are located on the benzene cyclepresenting the fluoric atom (see bicalutamide structure inFigure 3). Hydroxyflutamide (m/z[M-H]-: 291.0593) presented amain fragment of m/z: 205.0225 (CF: C7H4F3N2O2) whileenobosarm (m/z[M-H]-: 388.0904), fragmented into m/z: 118.0287(CF: C7H4NO) and 269.0460 (CF: C12H8F3N2O2).

In the following experiments, two major fragments were consid-ered for each molecular ion as synthesized in Table 1, except forhydroxyflutamide for which a second fragment could not easilybe observed based on SFC - IM-MSE experiments.

183

Chromatographic separation

To evaluate the separation efficiency of the developed protocolusing supercritical fluid chromatography, LC experiments havebeen reproduced based on the protocol developed by De Rijkeet al. [15] for enobosarm’s detection with in-lab optimizations. Thesame length has been chosen for the two methods to guaranteeappropriate cleaning of stationary phases and equal basis for com-parison. Retention times (Rt) as well as peak resolution have beencompared. Results are presented in Figure 4.

With LC method (Figure 4, Chromatogram A), andarine,hydroxyflutamide, bicalutamide, bicalutamide-d4 and enobosarmwere separated (Rtandarine: 3.31 ± 0.15min; Rthydroxyflutamide: 3.08± 0.2min; Rtbicalutamide: 3.77 ± 0.2min; Rtbicalutamide-d4: 3.77± 0.2min; Rtenobosarm: 4.30± 0.18min) with peak resolutions in therange [0 – 6.6] using bicalutamide-d4 as reference (R: 2x[(Rtbicalutamide-d4-Rtx)/(W bicalutamide-d4 +Wx)] where W is the basepeak width and x, the analyte of interest).

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Figure 3. Fragments observed for 4 non-steroidal selective androgenic receptor modulators (andarine, bicalutamide, hydroxyflutamide and enobosarm;C: 10 ng.mL�1) using Q-(TWIM)-TOF MS in MS/MS or MSE modes.

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For SFC experiments (Figure 4, Chromatogram B), andarine,hydroxyflutamide and enobosarm presented respective retentiontimes (Rt) of 7.63± 0.02min, 4.35±0.01min and 5.72± 0.04min,while bicalutamide and its deuterated form presented intermediatesimilar retention times (Rtbicalutamide =Rtbicalutamide-d4: 5.00± 0.02min). Higher peak resolutions could be observed [0 – 28.5]upon SFC than in LC separations. A very stable separation was fur-ther more observed (mean of Rt variation coefficients of 0.0038±0.0018) for the 5 observed compounds, attesting for the repro-ducibility of the implemented separation approach.Comparing the two chromatographic techniques, the elution

order changed. Andarine and enobosarm were reversed whichresult from different binding mechanisms. LC separation is ex-plained by the polarity of the molecules when SFC showed herea size-related separation.

Figure 4. Chromatograms of 4 non-steroidal selective androgenic receptormoC: 10 ng.mL�1) using liquid chromatography (A) and supercritical fluid chromat

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SFC exhibiting better separation and resolution results than LCwith a reliable reproducibility can therefore be considered as effi-cient alternative to LC separation.

Consequently, further experiments were performed based onSFC – Q-TWIM-TOF MS coupling.

Application to the detection of SARMs in urine

Blank urine samples have been spiked with various concentrations(0.25, 0.5, 1.25, 2.5, 5, and 25ng.mL�1) of non-steroidal SARMs to as-sess matrix effect on both retention time and CCSs calculation andsensitivity performances of the SFC - Q-TWIM-TOFMSworkflow. Re-sults of this section are reported in Table 1.

All the experiments have been conducted in light of the analyti-cal requirements set within the Commission Decision 2002/657/EC,

dulators (1: hydroxyflutamide, 2: andarine, 3: bicalutamide, and 4: enobosarm;ography (B).

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and in particular with regard to those related to HR-MSN experi-ments. The following parts assessed if the proposed strategy fulfillssuch requirements.

Specificity of the detection

Relative retention times observed upon the analysis of the variousspiked urine extracts were found to differ from 0.2 to 0.46 %, so lessthan 0.5 %, when the requirements for such analytical methods de-velopment (Dec 2002/657/EC) are maximum of 2.5% for LC experi-ments and 0.5 % for GC analyses. SFC in particular was notconsidered as potential separation techniques in this Decision2002/657/EC, therefore, no specific criteria is defined, however, as-suming that SFC is somewhere between these two analytical tech-niques, it could be considered that this criterion is fully respected.

Regarding full scan characterization using high resolution massspectrometry, the European legislation (Decision 2002/657/EC) re-quires for identification purposes monitoring at least two signals pre-senting appropriate intensities, which enable ‘earning’ the fourminimum required identification points (IPs). Such a requirementwas fully answered for the SARMs studied in the present work. SinceHigh Resolution mass spectrometry was used, even hydroxyflutamide

Figure 5. Drift cleaning of matrix interferents for andarine, bicalutamideand hydroxyflutamide using SFC – Q-TWIM-TOF MS. Interferents arepresented as dashes with retention time close to targeted compounds.

Figure 6. Signal-to-noise ratios vs concentrations for 4 non-steroidal selectiveand enobosarm; C: 0.25, 0.5, 1.25, 2.5 and 5 ng.mL�1).

Drug Test. Analysis 2017, 9, 179–187 Copyright © 2016 John W

that could only be monitored through two ions (1 precursor+1 frag-ment) could reach the four necessary IPs.

Besides, interferents presenting relatively close m/z (mass accu-racy of 20 ppm in sensitivity mode and 5ppm in high resolutionmode) and retention times were identified. However, theyexpressed clearly different CCS values as illustrated in Figure 5 forandarine (CCSandarine interferents: 127.58Å

2; CCSandarine: 195.75Å2).

In addition, CCSs measured in matrix were found to differ onlyfrom 0.14 to 0.28 % when compared to results in standard solu-tions. Based on these observations, CCS could be considered as acomplementary parameter providing additional confidence in sig-nals assignments.

Since nomatrix effect on either the retention times or on the CCSvalues could be observed, together with relative standards devia-tions for both retention times and CCSs remaining below 0.5 %,SFC – Q-TWIM-TOF MS may be considered as an efficient workflowfor the specific detection of SARMs in complex biological matrix.

Sensitivity of the detection

Usingworkflow based on LC-QqQMS, limits of detection have beendefined as 0.0675, 0.0771, 0.1415 and 0.01491ng.mL�1 forbicalutamide, enobosarm, andarine, and hydroxyflutamide, respec-tively. The repeatability of the detection for each concentrationlevel tested was estimated below 15%.

On the other side, matrix-matched calibration curves havebeen established to assess the sensitivity and linearity of the de-tection using SFC – Q-TWIM-TOF MS. As illustrated in Figure 6,correlation coefficients between 0.8428 and 0.9533 were obtainedfor the detection of andarine, bicalutamide, hydroxyflutamide andenobosarm. Limits of detection were fixed for a signal-to-noise ratioequal to 3 according to Decision 2002/657/EC. Considering the low-est point of the concentration range (C: 0.25ng.mL�1), S/N from 23to 304 have been observed (S/Nandarine: 23.73; S/Nbicalutamide: 67.44;S/Nhydroxyflutamide: 304.05; S/Nenobosarm: 38.65). Limits of detectionhave thus been estimated in urine as 0.0406, 0.0058, 0.0018 and0.0054ng.mL�1 for andarine, bicalutamide, hydroxyflutamide andenobosarm, respectively. The repeatability was calculated as below21%.

In view of these results, the coupling of SFC and IM-MS has beenpresented enabling highly sensitive detection compared to LC-QqQ MS association.

androgenic receptor modulators (andarine, bicalutamide, hydroxyflutamide

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Figure 7. Enobosarm excretion curve in urine after treatment of a male calf with extracted ion chromatograms corresponding to the lowest detectedconcentration with respect for European Commission Decision 2002/657/EC (m/z Enobosarm precursor [M-H]-: 388.0904; m/z Enobosarm fragment 1 [M-H]-: 118.0287;Rt: 5.72min).

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Application to samples collected on treated animals

Finally, based on the reliable performances of the detection strat-egy developed, the whole workflow has been applied to urine sam-ples collected on a calf treated with enobosarm. The urine sampleswere subjected to sample clean-up as described in the Experimen-tal Section.As expected, enobosarm signal was not detected in samples

collected before the administration of the drug (Figure 7), orin those arising from control animals. From the very first day af-ter treatment enobosarm signals were observed and corre-sponding concentration (CDay1) measured at: 77.78 ng.mL�1.Enobosarm could be identified in the urine samples duringseven days based on the monitoring of its two specific frag-ments and according to Dec 2002/657 criteria. Considering onlyone fragment but including CCS as an additional criterion,enobosarm could be detected over a longer period (14 days) af-ter administration with an associated detectable concentrationof 1.22 ng.mL�1. Monitoring two transitions, the excretion pro-file was very similar to that already reported by De Rijke et al.,[15]

who also described higher concentration at day 1 and the pos-sible identification of the substance in urine over 6 days.

Conclusion

Non-steroidal SARMS have been developed for replacement ofsteroid-based therapies, due to their higher tissue selectivity lead-ing to fewer side effects. In parallel and because of their provedanabolic properties, they may be used to enhance sportperformances[8] or to increase animal products yields. As relativelynew chemicals on market, they raise concerns about food safetyand regulation related to growth promoting practices in food pro-duction. Detection methods have then to be developed to ensurechemical safety.In this work, coupling of SFC with IM-MS has been developed

and compared to optimized LC-MS based analytical method.

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This workflow has shown to be efficient for the specific analysisof four different non-steroidal SARMs (andarine, bicalutamide,enobosarm, and hydroxyflutamide). With promising intra-laboratory repeatability and sensitivity results, the combinationof mass-to-charge ratio, retention time and cross collision sec-tion appears to provide a high level of confidence in the deter-mination of these compounds at trace concentrations in urinefrom bovine (LOD< 0.05 ng.mL�1; CV< 21 %). Moreover, analy-sis of kinetic elimination of enobosarm on male calf after singledose treatment (200mg) allowed comfortable detection of itsadministration over 14 days.

As a result, SFC coupled with IM-MS demonstrated to specificallyseparate targeted non-steroidal androgen receptor modulatorsions from matrix background noise, enabling efficient signalcleaning and lowering the sensitivity of their detection in urine.

Acknowledgements

The authors want to address their sincere thanks to Sara Stead,Mike McCullagh, and Kevin Giles (Waters, Manchester, UK) and LoicHerpin, Karinne Pouponneau, Cédric Vernet (LABERCA) for precioushelp as well as support.

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