R. Ventura et al., Eur. J. Mass Spectrom. 14,191-200 l20081 Received: 6 June 2008 m Revised: 27 June 2008 m Accepted: 30 June 2008 IR Publication: 1 August 2008
/ EJMS 1 TfN SPECTROMETRY Special Issue: Sports Drug Testing by Mass Spectrometry
High-throughput and sensitive screening by ultra-performance liquid chromatography tandem mass spectrometry of diuretics and other doping agents
-. ~ -~ ...... , ...... ~... - ~...... ~ ...... ~.-.... ~ ...... ~ . . Rosa Ventura,aobo'Meritxell Roig,=~~Núria M~nfort,~~~Pilar Sáe~,~ Rosa Bergesa and Jordi Seguraamb aGrup de Recerca en Bioanalisis, IMIM-Hospital del Mar, Barcelona, Spain. E-mail: [email protected] 'Departament de Ciencies Experimentals i de la Salut, Universitat Pompeu Fabra. UPF, Barcelona. Spain
The reliability of ultra-performance liquid chrornatography coupled to tandern rnass spectrornetry IUPLC-MS/MSl for high throughput screening in anti-doping control has been tested. A rnethod to screen for the presence of diuretics and other doping agents in urine has been optirnised and validated. The extraction procedure consisted of an alkaline extraction IpH9.51 with ethyl acetate and salting-out effect lsodiurn chloride]. The extracts were analysed by UPLC-MSIMS. Analysis of 31 forbidden drugs and metabolites was achieved in a total run time of 5rnin, using a C,, colurnn 1100 rnmx2.1 mrn ¡.d., 1.7pm particle sizel and a rnobile phase containing deionised water and acetonitrile with forrnic acid, with gradient elution ata flow-rate of 0.6rnLrnin-l. ldentification of the cornpounds was perforrned by rnultiple reaction monitoring, using electrospray ionisation in positive- or negative-ion rnode. Precursor and product ions were studied for each cornpound and cone voltage and collision energy were optirnised. Due to the different chernical structure of the cornpounds under study, extraction recoveries varied from less than 10% to 100% depending on the analyte. The lirnits of detection ranged frorn 50ngrnL-' to 200ngrnL'1, and al1 the cornpounds cornply with the requirernents of quality established by the World Anti-doping Agency. Intra-assay precision was evaluated at two concentrations for each cornpound and, in most cases. a relative standard deviation of the signal ratio lower than 20% was obtained. The rnethod has demonstrated to be reliable when analysing routine samples and the short analysis time resulting frorn a simple sample preparation and a rapid instrumental analysis allow a fast turn-around time and rnakes it of great interest for routine anti-doping control purposes.
Keywords: UPLC-MS/MS, doping control, diuretics. stimulants. probenecid, finasteride, efaproxiral. esmolol
lntroduction The current list of prohibited substances in sports includes nine beta-blockersl.' The list is updated each year with new groups of prohibited substances Ianabolic agents. hormones substances by the World Anti-doping Agency [WADA]. More and related substances, beta-2-agonists, hormone antago- than two hundred pharmacologically and chemically different nists and modulators. diuretics and other masking agents, substances are included in the present List of forbidden stimulants. narcotics, cannabinoids, glucocorticosteroids], substances. The task of the doping control laboratories is three prohibited methods [enhancement of oxygen transfer, to screen for the detection of the administration of all these chemical and physical manipulation and gene doping1 and two banned substances by the analysis of the unchanged drugs groups of substances prohibited in particular sports [alcohol, andlor their metabolites in urine and. in case of suspicious
ISSN: 1469-0667 O IM Publications LLP 2008 dni. ln 13C;Sloirnc 97n AlI riohts reserved 192 Screening of Diuretics and Other Doping Agents using UPLC-MS/MS
results during the screening step, to perform a second anal- the method allows the detection of diuretics and other ysis for confirmation of the screening result. These analyses masking agents [such as probenecid or 5cr-reductase inhib- have to be performed in a short period of time according to itor metabolites], some stimulants, efaproxiral [enhancer of the lnternational Standard for ~aboratories.~For this reason, oxygen transfer] and metabolite of esmolol [beta-blocker laboratories are forced to develop high throughput screening drug]. and confirmation methods. The conventional analytical strategy of doping control labo- ratories has been to use multi-analyte screens for groups of prohibited sub~tances.~For long time, screening and confir- Experimental mation methods have been based mainly on gas chroma- Reagents and solvents tography coupled to mass spectrometry [GC-MS]. The polar Standards used were: acetazolamide, adrafinil, chlortalidone, nature of the metabolites of most of the banned substances ethacrynic acid, furosemide. hydrochlorothiazide, inda- and the low concentrations detected in urine lead to complex pamide, metolazone, ritalinic acid and trichlormethiazide sample preparation procedures. including specific derivatisa- [Sigma-Aldrich, Madrid, Spain]; bendroflumethiazide degra- tions to form suitable derivatives for GC-MS analysis of the dation product ~4-amino-6-~trifluoromethyllbenzene-1.3-di- different compounds. In recent years, the availability of robust sulfonamide], canrenone and piretanide impurity [European and reliable mass spectrometric detectors for liquid chro- Pharmacopoeia,Strasbourg. France1;amilorideand probenecid matography [LCl based on electrospray ionisation [ESII has [Merck, Darmstadt. Germany]; bendroflumethiazide [Davur SA. promoted the use of LC-MS or LC-tandem mass spectrometry San Sebastián de los Reyes, Madrid, Spainl; benzoylecgonine [MS/MSl systems for screening and confirmation of most of and methylphenidate [Cerilliant. Austin, TX, USA]; benzthiazide the banned cornp~unds."~~LC-MS/MS is currently the tech- [A.H. Robbins Company, Richmond. Virginia, USA]; bumeta- nique of choice for the screening and confirmation of most of nide [Boehringer Ingelheim, Ingelheim, Germanyl; buthiazide the groups of substances of doping interest. [Boehringer Mannheim. Mannheim, Germanyl; chlorothiazide Diiferent strategies have been described,in order to [Acofarma. Terrassa, Barcelona, Spain]; clopamide [Sandoz, reduce the complexity of the doping control analysis and the Barcelona. Spain), diclofenamide [Farmaquímica Española total analysis time. Fast GC-MS methods based on the use SA, Barcelona. Spain]: efaproxiral !Doping Control Laboratory, of shorter columns and higher carrier gas velocities with Cologne, Germany]; esmolol metabolite, 4-[2-hydroxy-3- respect to conventional GC conditions have been described (11 -methylethyllaminolpropoxylbenzene propanoic acid to reduce the chromatographic analysis time.24,25The use of [SynFine Research, Ontario. Canada); finasteride metabolite, LC-MS/MS or LC-time-of-flight [ToFI/MS allowed the devel- u-carboxyfinasteride [NMl Australian Government. Sydney, opment of comprehensive screening procedures including Australia]; modafinil [Cephalon. Maisons-Alfort. France]; pire- different groups of substances with a single sample prepara- tanide [Hoechst Ibérica SA, Barcelona, Spain]; polythiazide 'tion without the need of specific derivatisations as was needed [Service Central d'Analyse, Vernaisson, Fancel; spironolac- for GC-MS-based method~.'~'~,~~The direct analysis of conju- tone [Searle Ibérica. Alcobendas, Madrid, Spainl; torasemide gated metabolites by LC-MS/MS has also been described to INational Doping Control Centre, Bangkok, Thailand]; triam- avoid the hydrolysis step during sample ~re~aration.'~ terene [Laboratorios Almirall, Barcelona. Spain]; and xipa- In recent years, new LC strategies have been developed with mide [Laboratorios Lácer SA, Barcelona. Spainl. 7-propyltheo- the aim of reducing the analysis time and increasing separa- phylline, synthesised in our laboratory, was used as interna\ tion efficiency, sensitivity and resolution. Ultraperformance standard. liquid chromatography IUPLCI enables the use of columns Ethyl acetate [HPLC gradel, acetonitrile and methanol packed with small particles [<2pm] coupled to chromato- [LC gradient gradel, formic acid ILC/MS grade] and sodium graphic systems specially designed to run at the optimum chloride, 25% ammonia, ammonium chloride [all analytical linear velocities Ihigh pressures and minimal system volumesl. gradel were purchased from Merck [Darmstadt. Germanyl. UPLC allows the same resolution by using high Linear veloci- Milli Q water was obtained by a Milli-Q purification system ties, which results in a reduction of the analysis time. or allows [Millipore Ibérica, Barcelona, Spain]. an improvement in chromatographic resolution using conven- Organic layers were evaporated to dryness under nitrogen tional linear velocities. Fast data acquisition MS analysers stream with a Turbo-Vap LV evaporator from Zymark are needed to obtain enough data points to define the narrow Corporation [Hopkinto, MA. USA]. peaks obtained with the UPLC systems when running at high linear velocities. UPLC-MSIMS methods have been described Standards solutions for some doping agentsI9 and in other fields of analytical Stock standard solutions of each analyte (1 mgmL-', in free ~hemistry.~~,~~ base form] were prepared by dissolving 10 mg of the free base In this paper, the reliability of UPLC-MS/MS for high- form in 10mL of methanol. Working solutions of 100pgmL-' - . throughput screening methods in anti-doping control has 10pgmL-' and 1 pgmL-' were prepared by 1 : 10. 1 : 100 and been tested. A fast and reliable screening method based on 1 : 1000 dilutions of the 1 mg mL-' stock solutions with meth- UPLC-MS/MS has been developed and validated. At present, anol. Al1 solutions were stored at -20°C. R.Ventura etal., Eur. J. Mass Spectrom. 14,191-200 12008)
Sample preparation procedure delays of 5ms and inter-scan times of 20ms. All data w A modification of a sample preparation procedure previously acquired and processed using MassLynx 4.1 software. described was used 30 Aliquots of urine samples (2 5 mL] were added with a concentration of 100 ng mL-' of 7-propyl- Validation study theophylline, used as internal standard and adjusted to The following parameters were evaluated: selectivity, limits pH9 5 with ammonium chloride buffer 1100pLl. Then, sodium of detection [LODI. extraction recovery and intra-assay and chloride (1 gl was added to promote salting-out effect and intermediate precisions, according to a protoco[ previously samples were extracted with 8mL of ethyl acetate by shaking de~cribed.~'Selectivity and specificity were studied by the at 40 movements per rnin for 20rnin. After centrifugation analysis of 10 different blank urine samples obtained from 11500g For 5rn,n), organlc layers were evaporated to d&ness different healthy volunteers. The presence of any interfering under nitrogen stream in a water bafh at 10°C The extracts substance at the refention time of the compounds of interest were reconstituted with 100pL of a mixture of deionised and the internal standard5 was verified. water:acetonitrile (90: 10, vlvl. and aliquots of 5 pL were The extraction recovery of each analyte was calculated by analysed by UPLC-MSIMS. analysis of four replicates of a uririe sample spiked with the compound. The ratio of the peak areas of the analyte and the Ultraperformance liquid chromatography internal standard obtained from the extracted spiked samples tandem mass spectrometry conditions were compared with the mean of the peak areas obtained when j Chromatographic separations were carried out on a Waters the analytes were added to extracted blank urine samples Acquity UPLC system, equipped with a quaternary pump (representing 100% of extraction recovery]. system using an Acquity BEH C,, column (100 rnrn x 2.1 mm The LOD was estimated by analysis of four replicates of
' ¡.d.. 1.7pm particle sizel IWaters Corporation. Milford. MA, blank urine samples spiked with the analyte at a concentration USA]. The column temperature was set to 45OC. Separation corresponding to the rninirnum required performance limits was performed with a binary mobile phase at a flow rate of [MRPL] defined by WADA or l~wer.~' 0.6mLmin-'. The optimised separation conditions were as Intra-assay precision was assessed by analysis of four repli- follows: solvent A. deionised waterwith 0.01 % formic acid; and cates of samples spiked at two different concentrations on the solvent B. acetonitrile with 0.01% formic acid. The gradient same day. lntermediate precision was estimated by analysis elution was as follows: from Omin to 0.6min, 5% B; from of one replicate of a quality control sample spiked with some 0.6min to 3.8min, to 90% B; during 0.2min, 90% 6; frorn 4min of the compounds, on six different days and by two different to Ll min. to 5% 6; from 4.1 min to 5min. 5% B. The mobile analysts. The precisions were measured using the relative phases were filtered daily using filters of 0.22pm pore size. standard deviation [RSD] between the ratio of the areas of the The sample volurne injected was 5 pL. compound and the ISTD in the different analyses. The UPLC instrument was coupled to a Quattro Premier XE triple quadrupole mass spectrometer [Micromass, Waters Actual urine samples Corporation. Milford. MA. USA1 with an electrospray [Z-spray) The screening method was applied to urine samples obtained ionisation source working in positive or negative ionisation in excretion studies involving the administration of therapeutic mode. Acquisition was perforrned in rnultiple reaction rnoni- doses of the compounds to healthy volunteers and urine toring [MRMI mode. The protonated or deprotonated molec- collection. The clinical protocol was approved by the local ular ion of each compound was selected as the precursor Ethical Committee [CEIC-IMAS, lnstitut Minicipal dlssistencia ion, depending on the ionisation mode. Source conditions Sanitaria, Barcelona. Spainl. For all the compounds, single in positive-ion mode were fixed as follows: capillary voltage, doses were administered by oral route. Urine samples were 3 kv; Lens voltage, 0.2V; source temperature 120°C; desol- collected before administration and up to 24h or 48h after vation temperature. 450°C; cone gas flow rate, 50 L h.'; administration at different collection periods. The method desolvation gas flow-rate, 1200 L h.'. In negative mode. the was also applied to routine anti-doping control samples for a conditions were the same. except that the capillary voltage period of one year. was set at 2.5kV. High-purity nitrogen was used as desolva- tion gas and argon was used as collision gas. Electrospray ionisation working parameters (cone voltage and collision energies] were optimised for each compound using direct Results and discussion infusion of individual standard solutions of the compounds The compounds studied are listed in Tabie 1. Due to the differ- (10 pg m~-']at 10p~min-'with mobile phase [50:50, A: Bl ences in the mechanism of diuretic a~tivity,~~the diuretics at 200pLmin-l. group includes compounds with large differences in molecular Cone energy voltages, transitions monitored and collision structure and, thus. in physico-chemical properties. They energy voltages were established for each analyte. and the may be classified according to their acid-base behaviour in optimised values are listed in Table 1. Data acquisition was different sub-groups: basic compounds, such as amiloride performed in two acquisition groups [for positive and nega- and triamterene [potassium sparing diuretics]; neutral tive ions, respectively] with dwell times of 5rns. inter-channel diuretics, such as canrenone and spironolactone [aldosterone 194 Screening of Diuretics and Other Doping Agents using UPLC-MS/MS
Table 1. Cornpounds included in the screening procedure: rnonoisotopic rnass, ionization rnode [pos, positive; neg. negaüve], retention times (RTI, relative retention times [RRT), precursor [PI) and product (DI) ion, cone voltages [CV] and collision energies ICE].
? v.7: ~~~~;-.l~i:~i!-~.~~~~~-y~~.t~~~~.i~yi'~:~<~py,~q.~,y~a!~y;?n~~:i.q!~,~+~::.+y~~~r, ~xf,qaV;,y"<~;$~ .',
, , Amiloride 229.05 POS 1.22 0.56 230 25 171 Acetazolamide 221.99 neg 1.48 0.69 Acetazolamide 221.99 POS 1.48 0.69 Chlorothiazide 294.95 neg 1.49 0.69 Chlorothiazide 294.95 POS 1.53 0.71 Hydrochlorothiazide 296.97 neg 1.74 0.81 Triamterene 253.11 POS 1.81 0.84 Bt 2.03 0.94 Di 2.05 0.95
CtII"! IOLIUUI 2.16 1.00 CI 1.01 Tc 1 .O3 Tricn~ormethiazide 1.O3 Ft~rosemidf POS L.J~ 1.1 o Al.thiazide POS 2.38 1.10 M etolazone 365 POS 2.40 1.11 lndapamide 365.06 POS 2.49 1.15 Benzthiazide 430.98 POS 2.49 1.15 Buthiazide 353.03 POS 2.54 1.18 Polythiazide 438.97 POS 2.58 1.19 Piretanide 362.09 POS 2.64 1.22 Xipamide 354.04 POS 2.68 1.24 Bumetanide 364.11 POS 2.74 1.27 Bendroflumethiazide 421.04 POS 2.81 1.30 Spironolactone 416.20 POS 2.87 1.33 Etacrynic acid 302.01 neg 2.89 1.34 Etacrynic acid 302.01 POS 2.89 1.34 Canrenone 340.20 POS 2.89 1.34 ~~~~~,~nmjp.c~"pi~-~~m~~~rn~~~.n~-~~~,~~i,(ui~.~~."~~~a~~m,r;n.~~~~~~~~~~ . , ...... , +',.. . . , .... Bi2nzoylecg POS 0.84 Ri talinic aci POS 0.85 Methylphen,wu,L nnc 0.91 Aijrafinil 1.05 M odafinil 1.06 y.'"'" ;,,.:. '-~,-~*..>?~-T'"'>
Esmolol metabolite Lu,.,u POS S .u , 0.84 Finasteride metabolite 402.25 POS 2.28 1 .O6 Efaproxiral. RSR 13 341.16 POS 2.80 1.30 Probenecid 285.10 POS 2.82 1.31
antagonistsl; weakly acidic diuretics. such as acetazolamide (loop diuretics]. Other compounds or diclofenamide lcarbonic anhydrase inhibitors] and thiazides procedure are some stimulants and related compounds, such as chLorthalidone. clopamide, cocaine metabolite; methylphen indapamide and metolazone; and strongly acidic diuretics. ritalinic acid; adrafinil and modafi such as furosemide, piretanide. bumetanide or etacrynic acid such as probenecid or 5n-reduct / R. Ventura etai.. Eur J. Marr Spcctram. 11,191-200 120081
rnetabolite. u-carbo~~finasteride1.3"enhancers of oxygen positive- and negative-ion modes and the fast scan speed of transfer [efaproxiral] and beta-blocker metabolites [meta- the instrument is crucial to allow the simultaneous detection bolite of esmolol, h-[2-hydroxy-3-[(l-methylethyllamino] of a high number of compounds. In the current conditions, propoxylbenzene propanoic a~idl.~"~~ the number of compounds detected in the screening method Electrospray ionisation working parameters were optimised could be increased by including additional ion transitions, or for all the compounds under study by using infusion of indi- using different time windows for different acquisition groups. vidual standard solutions. The optimised values are listed in Taking into account the mobile phase composition and Table 1. Positive- and negative-ion modes were tested. Higher flow-rate used, a high desolvation gas flow rate was used signal was obtained for most of the compounds in positive ion 11200 L h-'1 and desolvation and source temperatures were set mode, at Least one order of magnitude greater than in nega- at 450°C and 120°C in order to improve desolvation efficiency tive mode. Positive-ion mode was selected for most of the and analyte ionisation. compounds as indicated in Table 1. For some of the compounds Apart from formic acid, no other mobile phase additives [acetazolamide. chlorothiazide. etacrynic acid], both positive were needed either to promote ionisation or to improve and negative modes were used and, for a few compounds chromatographic behaviour of the different compounds. The only negative ionisation mode was used [hydrochorothiazide. gradient was optimised to obtain a reasonable short chrorna- diclofenamide, bendroflumethiazide degradation product]. tographic time [Table 1). Although not needed for chromato- The higher signals obtained in.positive mode for most of graphic separation. the final content of organic solvent in the the analytes compared to previous ~orks~~~~'"~~are probably mobile phase was increased to 90% in order to have a good explained by the composition of the mobile phase and the pres- clean-up of the chromatographic column. to avoid problems of ente of functional groups with high proton affinity in most of blocking and to extend the Life of the columns. In addition, the the compounds under study. Using mobile phases containing sample extraction and the small volume of sample injected acetonitrile and water or water with acetic acid or ammoniurn contributed to extend the Life of the column. According to 'acetate, positive-ion mode was favoured for basic and neutral our experience more than 3500 analyses per column can be compounds. however negatively charged ions were mainLy performed. The last compound eluted at approximately 3min. formed for acidic diureti~s.'~~.'"~~~However, using mobile The stabilisation of the column at initial mobile phase condi- phases at more acidic pH [water with formic acidl. positive tions was performed in only 0.9min and the total run time was ion mode was preferred for most of the compounds. including 5min per sarnple. In these conditions, a reduction between most of the acidic diuretics. as described by Goebel et al." two and four-fold in the analysis time was achieved compared Both modes of ionisation must be used for a comprehensive to previous methods using columns packed with larger sized screening for diuretics and the mass spectrometer should be particles.6,8~'2~'"~21~30The reproducibility in retention times was able to switch between positive and negative ionisation modes also good in both intra- and inter-assay conditions. in a single analysis. Otherwise, two analyses per sample have The selectivity/specificity was evaluated by analysis of ten to be performed as described by some auth~rs.~' different blank urine samples and no matrix interferenceswere Cone voltage was optimised to obtain maximum signal for detected at the retention times of the compounds of interest. either the protonated or deprotonated molecular ions. [M+H]' Figure 1 shows the chromatograms obtained after analysis of or [M-HI-, depending on the compound. Data acquisition was a blank urine sample and those obtained after analysis of a performed in MRM mode and different collision energies were urine sample spiked with some of the compounds. studied in order to obtain the maximum response for each Results of LOD, repeatability at two concentration levels. transition. For screening purposes, only one ion transition was extraction recovery [calculated at the high concentration]. and used for most of the compounds [Table 11. After a suspicious intermediate precision are listed in Table 2. Regarding the screening result, the extract of the sample was re-analysed by LOD, the concentration listed in the table is the lowest concen- using an specific MS/MS method for unequivocal identification tration tested and, for most of the compounds, lower concen- of the suspected compound, including at least three specific trations could be detected. For all the compounds in the study, ion transitions, according to the WADA criteria for identifica- the LOD listed was lower than MRPL defined by WADA.32 For ti~n.~'If the result of this pre-confirmation analysis was posi- diuretics, the LOD obtained were always lower than those tive, a new aliquot of the sample was analysed for confirmation obtained by GC-MS. purposes by using the specific MS/MS method. The extraction recoveries ranged from less than 10% to ln the screening step, simultaneous acquisition of positive 10O0/0 [Table 21. The extraction procedure was firstly optimised and negative ions was performed. A total of 33 transitions were to the detection of diuretic compounds3" and. in spite of using measured in positive mode and six transitions were measured extraction at basic pH, it was appropriate even for the extrac- in negative mode. The peak widths obtained were around tion of more acidic diuretics. due to the use of a polar organic 5-6s. In the conditions of data acquisition used [short dwell, solvent [ethyl acetate] and salting-out effect [Table 21. The interchannel and interscan times]. a total of 10-12 data points extraction of other compounds without carboxylic acid func- per peak can be acquired. allowing an adequate definition of tions [adrafinil, modafinil, methylphenidate. efaproxiral] was the chromatographic peak [optimum number of data points: always higher than 7O0/0 [Table 21. However, the extraction between 10 and 151. The possibility of a fast switching between of some compounds with free carboxylic acid functions was . 196 Screening of Diuretics and Other Doping Agents using UPLC-MS/MS
~roolmdiiks cnloiahba. o*ome-d10a II Y"0<*.01r(l,lL5* ,:Yua6omi,a SI- II YIYO~:~UIY~.C% 29- lb, 9.-111 tvaL:l W-iiP
.m .m ." :m '.m .. 1 irm 0 ,m
HyOmNoidhniin E-Id m* Bruel(.rqwne **oQ**r.,,hN,d. E*m "" .. l..* ,m 2x4 ;*m iim ,m ,.m ?m 1riam.rinr Rt.Ic"eA~d FinaSlt.ld. M 1r,."d*r.* R,t,i,"~A~d F4"l.nd.m.t Il."YO":.,.,<,l* I,"I"O,IOii<...Bi. '1 Y"Y.,,.C,~".15. PI Y"Y.I1IS,.I".I* S, "I.O,ES I,,,,, ES. ,,.."a,,:c,,,"*Fs 13 - :n IP -2, -.o-?. 01-DI o .. .. lam cm irm zmo :.w :,m Ibm i tnl 1630 :m 2.- :ti0 F7oMn.m RSR 13 B>.mr>ixlm. Fir.. Figure 1. LC-MS/MS chromatograms obtained after analysis of a blank urine [leftl and a urine spiked with most of the compounds lrightl. All the compounds were spiked at concentration of 200ng rn~-',except acetazolamide, adrafinil, etacrynic acid, hydrochloro- thiazide, methylphenidate, modafinil and ritalinic acid 1.400 ng m~-'];esmolol and finasteride metabolites, efaproxiral. canrenone and torasemide were not added to this urine. Table 2. Validation data: Lirnits of detection (LODI, repeatability, extraction recoveries and intermediate precision. RSD. relative standard deviation. Acetazolamide 13.5 LUU Adrafinil 13.3 400 Althiazide 24.5 200 Amiloride 12.7 200 Bedroflumethiazide 7.3 200 Benzoylecgonine 12.8 200 Benzthiazide 10.2 200 Bumetanide 9.3 Buthiazide 12.3 Canrenone 6.0 Chlorothiazide 5O LUU Chlortalidone 5O 200 Clopamide 5O 200 Diclofenamide 200 Esmolol metabolite 400 Etacrynic acid 200 Finasteride ! metaboli I UU IUU 1J.U 200 Furosemidi e 50 50 13.9 200 Hydrochlor .othiazide 50 5 O 20.7 200 . . - Indapamidt 50 200 Methylpher 1O0 400 Metolazone 50 200 hilodafinil IUU 400 Piretanide 50 200 P iretanide impurity 5 0 200 ., LL'--'A rn Poiyinld~lu 3U 3 u 200 Probenecic 50 5 O 200 Ritalinic ac 100 100 400 RSR 17 inn inn 400 S tone 400 TI 2 200 TI dl~llL~lelie 200 Trichlorme Xipamide - 198 Screening of Diuretics and Other Doping Agents using UPLC-MS/MS ' iower than 10% (ben~o~lecgonine.esmolol metabolite, finas- finasteride and furosemide and a sample detected positive to teride metabolite, ritalinic acid). As for other methods using benzoylecgonine [cocaine metabolite] in routine work. LC-MSIMS, a substantial reduction in the sarnple preparation In order to evaluate the reliability for routine work, the devel- procedure was achieved cornpared to previous methods using oped rnethod was applied to the analysis of doping control GC-MS. due to the elimination of the derivatisation. which is a routine samples during one year. In our hands. the system time-consuming step for diuretic comp~unds.~~ has proved to be stable and reliable with simple maintenance Owing to the high sensitivity of the equiprnent, in spite of the operations [daily filtration of the rnobile phase components, low extraction recoveries for some compounds. the method change of the column pre-filter and cleaning of the cone and allowed the detection of all the compounds at concentra- the ion source, when necessaryl. The system can work for tions lower than the MRPL defined by WADA. Appropriate 24 h a day and, taking into account the total analysis time per repeatabilities were obtained, even for compounds with Low sample, more than 200 samples per day can be analysed. extraction recoveres. The RSD were in general Lower than In surnmary, the work presented demonstrates the poten- 15% for the highest concentration studied and lower than 20% tial of UPLC-MS/MS with simultaneous detection in positive for the lowest concentration. lntermediate precision was esti- and negative modes for high-throughput and comprehensive rnated using the quality control sample spiked with some of screening procedurec in anti-doping control. At present. the the compounds analysed during routine work. For most of the method is applied to the analysis of 34 compounds [mainly compounds, RSD Lower than 25% were obtained. diuretics and other acidic drugs]; however, due to the char- As a final validation. samples obtained after administra- acteristics of the equipment used. it can easily be extended tion of some of the compounds to healthy volunteers were to the analysis of other compounds. The short analysis time analysed. In Figure 2. chromatograms obtained after analysis resulting from simple sample preparation and rapid instru- of blank urine samples are compared with those obtained mental analysis allow a fast turn-around time and makes it of after analysis of samples collected after administration of great interest for routine anti-doping control purposes. lSTD E Benzoylecgonine ISTD Benzoylecgonine ES+ 223>181 ES+ 290>168 ES+ 223>181 ES+ 290>168 1 ISTD Finasteride met ISTD Finasteride met ES+ 223481 ES+ 403>335 ES+ 223s181 ES+ 403~335 ISTD Furosemide ISTD Furosemide ES+ 223>181 ES+ 331 >81 ES+ 223>181 ES+ 331>81 1 ! i ; Figure 2. LC-MSIMS results of positive sarnples to: A, benzoylecgonine, cocaine metabolite; B. finasteride metabolite; C, furosemide. 1 ion chromatograms of the ISTD and the compound obtained after analysis of a blank sample lleftl. and ion chrornatograms of the iSTD 1 and the compound obtained after analysis of a positive sample [right). 1 ~ . ~~- ...... ~.... - . ~ ~ . ~~ .. ~...~ ~ ~ . . - . .- . . . . ~~. R. Ventura et al., Eur. J. Mass Spectrom. 14,191-200 IZO081 199 -- ~ chromatographyltandem rnass spectrometry". Rapid Acknowledgements Commum. Mass Spectrom. 17, 2107 120031. doi: 10.1002/ ' The financial support received frorn Concell Catala de I'Esport. rcm.1157 Generalitat de Catalunya [Spainl and Ministerio de Educación 11. M. Thevis, G. Opfermann and W. Schanzer, "Liquid '.: y Ciencia [Spainl Iproject number DEP2006-56177-C03-01) is chromatography/electrospray ionization tandem mass acknowledged. spectrometric screening and confirmation methods 1 ,, for beta2-agonists in human or equine urine", J. Mass . i Y 1, Spectrom. 38, 1197 120031. do¡: 10.1002/jms.512 References 12. C. Goebel. G. Trout and R. Kazlauskas, "Rapid screening method for diuretics in dopinq. - control usinq automated ' .>, . 1. World Anti-doping Agency [WADA]. The World Anti- solid phase extraction and Liquid-chromatography- ' '5 Doping Code. The 2008 Prohibited List lnternational electrospray tandem mass spectrometry". Anal. Chim. : 4. Standard. http://www.wada-ama.org/rtecontent/docu- Acta 502, 65 (20041. do¡: 10.1016/j.aca.2003.09.062 i!l if ment/2008-List-En.pdf (20071. 13. A. Leinonen, T. Kuuranne, T. Kotiaho and R. Kostiainen, 1'2 2. World Anti-doping Agency [WADA]. 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