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 R o i g , = ~ ~ Núria M ~ n f o r t , ~ ~ ~ Pilar S á e ~ , ~ Rosa Bergesa and Jordi Seguraamb

aGrup de Recerca en Bioanalisis, IMIM-Hospital del Mar, Barcelona, Spain. E-mail: rventura@imim.es

'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, wi th gradient elution a t a 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 f rom 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 i t

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 groups of prohibited substances Ianabolic agents. hormones and related substances, beta-2-agonists, hormone antago- nists and modulators. diuret ics and other masking agents, st imulants. narcotics, cannabinoids, glucocort icosteroids], three prohibited methods [enhancement of oxygen transfer, chemical and physical manipulation and gene doping1 and two groups of substances prohibited in particular sports [alcohol,

beta-blockersl . ' The l ist is updated each year w i th new substances by the World Anti-doping Agency [WADA]. More than two hundred pharmacologically and chemically different substances are included in the present List of forbidden substances. The task of the doping control laboratories is to screen for the detection of the administration of a l l these banned substances by the analysis of the unchanged drugs andlor their metabolites in urine and. in case of suspicious

ISSN: 1469-0667 dni. l n 13C;Sloirnc 97n

O IM Publications LLP 2008 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- ysis for confirmation of the screening result. These analyses have to be performed in a short period of t ime according to the lnternational Standard for ~ a b o r a t o r i e s . ~ For this reason, laboratories are forced to develop high throughput screening and confirmation methods.

The conventional analytical strategy of doping control labo- ratories has been to use multi-analyte screens for groups of prohibited s u b ~ t a n c e s . ~ For long time, screening and confir- mat ion methods have been based mainly on gas chroma- tography coupled to mass spectrometry [GC-MS]. The polar nature of the metabolites of most of the banned substances and the low concentrations detected in urine lead to complex sample preparation procedures. including specific derivatisa- tions to fo rm suitable derivatives for GC-MS analysis of the different compounds. In recent years, the availability of robust and reliable mass spectrometr ic detectors for l iquid chro- matography [LCl based on electrospray ionisation [ESII has promoted the use of LC-MS or LC-tandem mass spectrometry [MS/MSl systems for screening and confirmation of most of the banned c o r n p ~ u n d s . " ~ ~ LC-MS/MS is currently the tech- nique of choice for the screening and confirmation of most of the groups of substances of doping interest.

Di i ferent s t ra tegies have been descr ibed , in o rder to reduce the complexity of the doping control analysis and the total analysis t ime. Fast GC-MS methods based on the use of shor ter co lumns and h igher ca r r ie r gas velocities wi th respect to conventional GC conditions have been described to reduce the chromatographic analysis time.24,25 The use of LC-MS/MS or LC-t ime-of-f l ight [ToFI/MS allowed the devel- opment of comprehensive screening procedures including different groups of substances with a single sample prepara- 'tion without the need of specific derivatisations as was needed for GC-MS-based m e t h o d ~ . ' ~ ' ~ , ~ ~ The direct analysis of conju- gated metabolites by LC-MS/MS has also been described to avoid the hydrolysis step during sample ~ r e ~ a r a t i o n . ' ~

I n recent years, new LC strategies have been developed with the aim of reducing the analysis t ime and increasing separa- tion efficiency, sensitivity and resolution. Ultraperformance liquid chromatography IUPLCI enables the use of columns packed w i th s m a l l part ic les [ < 2 p m ] coupled to chromato- graphic systems specially designed to r u n at the opt imum linear velocities Ihigh pressures and min imal system volumesl. UPLC allows the same resolution by using high Linear veloci- ties, which results in a reduction of the analysis time. or allows an improvement in chromatographic resolution using conven- tional l inear velocities. Fast data acquisition MS analysers are needed to obtain enough data points to define the narrow peaks obtained with the UPLC systems when running at high linear velocities. UPLC-MSIMS methods have been described for some doping agentsI9 and in other fields of analytical ~ h e m i s t r y . ~ ~ , ~ ~

I n th is paper, the re l iab i l i ty of UPLC-MS/MS for h igh- throughput screening methods in ant i-doping contro l has been tested. A fast and reliable screening method based on UPLC-MS/MS has been developed and validated. At present,

the m e t h o d a l lows the detect ion of d iuret ics and other masking agents [such as probenecid or 5cr-reductase inhib- i tor metabolites], some stimulants, efaproxiral [enhancer of oxygen t ransfer ] and metabol i te of esmolo l [beta-blocker drug].

Experimental Reagents and solvents Standards used were: acetazolamide, adrafinil, chlortalidone, e thacrynic ac id, furosemide. hydrochlorothiazide, inda- pamide, metolazone, r i tal inic acid and trichlormethiazide [Sigma-Aldrich, Madrid, Spain]; bendroflumethiazide degra- dation product ~4-amino-6-~trifluoromethyllbenzene-1.3-di- sulfonamide], canrenone and piretanide impurity [European Pharmacopoeia,Strasbourg. France1;amilorideand probenecid [Merck, Darmstadt. Germany]; bendroflumethiazide [Davur SA. San Sebastián de los Reyes, Madrid, Spainl; benzoylecgonine and methylphenidate [Cerilliant. Austin, TX, USA]; benzthiazide [A.H. Robbins Company, Richmond. Virginia, USA]; bumeta- nide [Boehringer Ingelheim, Ingelheim, Germanyl; buthiazide [Boehringer Mannheim. Mannheim, Germanyl; chlorothiazide [Acofarma. Terrassa, Barcelona, Spain]; clopamide [Sandoz, Barcelona. Spain), diclofenamide [Farmaquímica Española SA, Barcelona. Spain]: efaproxiral !Doping Control Laboratory, Cologne, Germany]; esmo lo l metabol i te , 4-[2-hydroxy-3- (11 -methy lethy l laminolpropoxylbenzene propanoic acid [SynFine Research, Ontario. Canada); finasteride metabolite,

u-carboxyf inaster ide [ N M l Austral ian Government. Sydney, Australia]; modafinil [Cephalon. Maisons-Alfort. France]; pire- tanide [Hoechst Ibérica SA, Barcelona, Spain]; polythiazide [Service Centra l d'Analyse, Vernaisson, Fancel; spironolac- tone [Searle Ibérica. Alcobendas, Madrid, Spainl; torasemide INational Doping Control Centre, Bangkok, Thailand]; triam- terene [Laborator ios Almira l l , Barcelona. Spain]; and xipa- mide [Laboratorios Lácer SA, Barcelona. Spainl. 7-propyltheo- phylline, synthesised in our laboratory, was used as interna\ standard.

Ethy l acetate [HPLC gradel , acetonitr i le and methanol [LC gradient gradel, formic acid ILC/MS grade] and sodium chloride, 25% ammonia, ammonium chloride [a l l analytical gradel were purchased f rom Merck [Darmstadt. Germanyl. Mi l l i Q water was obtained by a Mill i-Q purification system [Mill ipore Ibérica, Barcelona, Spain].

Organic layers were evaporated to dryness under nitrogen s t r e a m w i t h a Turbo-Vap LV evaporator f rom Zymark Corporation [Hopkinto, MA. USA].

Standards solutions Stock standard solutions of each analyte (1 mgmL- ' , in free base form] were prepared by dissolving 10 mg of the free base form in 1 0 m L of methanol. Working solutions of 100pgmL-'

- . 10pgmL- ' and 1 pgmL- ' were prepared by 1 : 10. 1 : 100 and 1 : 1000 dilutions of the 1 m g mL- ' stock solutions with meth- anol. Al1 solutions were stored at -20°C.

R.Ventura etal., Eur. J. Mass Spectrom. 14,191-200 12008)

Sample preparation procedure A modification of a sample preparation procedure previously described was used 30 Aliquots of ur ine samples (2 5 m L ] were added with a concentration of 100 ng mL - ' of 7-propyl- theophylline, used as in te rna l standard and adjusted to pH9 5 with ammonium chloride buffer 1100pLl. Then, sodium chloride (1 gl was added to promote salt ing-out effect and samples were extracted with 8 m L of ethyl acetate by shaking

at 40 movements p e r rnin for 20rnin. Af ter centr i fugation 11500g For 5rn,n), organlc layers were evaporated to d&ness under nitrogen stream in a water bafh at 10°C The extracts were reconstituted with 100pL of a mixture of deionised water:acetonitrile (90: 10, v l v l . and al iquots of 5 p L were analysed by UPLC-MSIMS.

Ultraperformance l iquid chromatography tandem mass spectrometry conditions

j Chromatographic separations were carried out on a Waters

Acquity UPLC system, equipped w i th a quaternary pump system using an Acquity BEH C,, column (100 rnrn x 2.1 m m

' ¡.d.. 1 .7pm particle sizel IWaters Corporation. Mil ford. MA, USA]. The column temperature was set to 45OC. Separation was performed with a binary mobile phase at a flow rate of 0.6mLmin- ' . The optimised separation conditions were as follows: solvent A. deionised waterwith 0.01 % formic acid; and solvent B. acetonitrile with 0.01% formic acid. The gradient elution was as follows: f rom Omin to 0 .6min , 5% B; f rom 0.6min to 3.8min, to 90% B; during 0.2min, 90% 6 ; frorn 4min to L l min. to 5% 6 ; from 4.1 min to 5min. 5% B. The mobile phases were filtered daily using fi lters of 0.22pm pore size. The sample volurne injected was 5 pL.

The UPLC instrument was coupled to a Quattro Premier XE triple quadrupole mass spectrometer [Micromass, Waters Corporation. Milford. MA. USA1 with an electrospray [Z-spray) ionisation source working in positive o r negative ionisation mode. Acquisition was perforrned in rnultiple reaction rnoni- toring [MRMI mode. The protonated o r deprotonated molec- u lar ion of each compound was selected as the precursor ion, depending on the ionisation mode. Source conditions in positive-ion mode were fixed as follows: capillary voltage, 3 kv; Lens voltage, 0.2V; source temperature 120°C; desol- vation temperature. 450°C; cone gas f low rate, 50 L h. '; desolvation gas flow-rate, 1200 L h.'. In negative mode. the conditions were the same. except that the capillary voltage 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 infusion of individual standard solutions of the compounds (10 pg m ~ - ' ] at 1 0 p ~ m i n - ' with mobile phase [50:50, A : B l at 200pLmin-l .

Cone energy voltages, transitions monitored and collision energy voltages were established for each analyte. and the optimised values are listed in Table 1. Data acquisition was performed in two acquisition groups [ for positive and nega- tive ions, respectively] with dwell t imes of 5rns. inter-channel

delays of 5ms and inter-scan times of 20ms. Al l data w acquired and processed using MassLynx 4.1 software.

Validation study The following parameters were evaluated: selectivity, l imits of detection [LODI. extraction recovery and intra-assay and intermediate precisions, according to a protoco[ previously d e ~ c r i b e d . ~ ' Selectivity and specificity were studied by the analysis of 10 different blank urine samples obtained from different healthy volunteers. The presence of any interfering

substance a t the refention t ime o f the compounds o f interest and the internal standard5 was verified.

The extraction recovery of each analyte was calculated by analysis of four replicates of a uririe sample spiked with the compound. The ratio of the peak areas of the analyte and the internal standard obtained from the extracted spiked samples were compared with the mean of the peak areas obtained when the analytes were added to extracted blank urine samples (representing 100% of extraction recovery].

The LOD was est imated by analysis of four replicates of blank urine samples spiked with the analyte at a concentration corresponding to the rninirnum required performance limits [MRPL] defined by WADA or l ~ w e r . ~ '

Intra-assay precision was assessed by analysis of four repli- cates of samples spiked at two different concentrations on the same day. lntermediate precision was estimated by analysis of one replicate of a quality control sample spiked with some of the compounds, on six different days and by two different analysts. The precisions were measured using the relative standard deviation [RSD] between the ratio of the areas of the compound and the ISTD in the different analyses.

Actual ur ine samples The screening method was applied to urine samples obtained in excretion studies involving the administration of therapeutic doses of the compounds to healthy volunteers and urine collection. The cl inical protocol was approved by the local Ethical Committee [CEIC-IMAS, lnstitut Minicipal dlssistencia Sanitaria, Barcelona. Spainl. For a l l the compounds, single doses were administered by oral route. Urine samples were collected before administration and up to 24h or 48h after administrat ion at different collection periods. The method was also applied to routine anti-doping control samples for a period of one year.

Results and discussion The compounds studied are listed in Tabie 1. Due to the differ- ences in the mechanism of diuretic a ~ t i v i t y , ~ ~ the diuretics group includes compounds with large differences in molecular structure and, thus. in physico-chemical properties. They may be classified according to their acid-base behaviour in different sub-groups: basic compounds, such as amiloride and t r iamterene [potass ium spar ing diuret ics]; neu t ra l 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 ~ y i ' ~ : ~ < ~ py ,~q . ~,y~a!~y;?n~~:i.q!~,~+~::.+y~~~r, ~xf,qaV;,y"<~;$~ .',<v.;íl:;(tarry ~.'m-:.*a;piin pmo?im-Tr$~~,z~t~~. ~ : ~ ~ i ~ r i ri rxNq : ? u < ~ , y C ~ i ~ ' ' , . . . ,' ' . " . , , . . , - . , , , . . . . . . .. , .,

..,. ... . , ., . . , ,. ..,. . .. ..'< .., < . L ' . . ' \ >

.c. , . , ,

, ,

230 2 5 171 Amiloride 229.05 POS 1.22 0.56

~ r o s e m i d f .thiazide etolazone

2nzoylecg talinic aci

j ra f in i l odafinil

Propyltec

Acetazolamide 221.99 neg 1 .48 0.69 221.99 1.48 0.69 Acetazolamide POS 294.95 1.49 0.69 Chlorothiazide neg 294.95 1.53 0.71 Chlorothiazide POS

Hydrochlorothiazide 296.97 neg 1.74 0.81 Triamterene 253.11 POS 1.81 0.84

2.03 0.94 Bt Di 2.05 0.95

2.16 1.00 CtII"! I O L I U U I

CI 1.01 Tc 1 .O3 Tricn~ormethiazide 1 .O3

1.1 o Ft POS L . J ~

Al POS 2.38 1.10 M 365 POS 2.40 1.11

365.06 2.49 1.15 lndapamide POS 430.98 2.49 1.15 Benzthiazide POS 353.03 2.54 1.18 Buthiazide POS

2.58 Polythiazide 438.97 POS 1.19 362.09 2.64 1.22 Piretanide POS

Xipamide 354.04 POS 2.68 1.24 Bumetanide 364.11 POS 2.74 1.27 Bendroflumethiazide 421.04 POS 2.81 1.30

416.20 2.87 1.33 Spironolactone POS 302.01 2.89 1.34 Etacrynic acid neg

Etacrynic acid 302.01 POS 2.89 1.34 Canrenone 340.20 POS 2.89 1.34

~ ~ ~ ~ ~ , ~ n m j p . c ~ " p i ~ - ~ ~ m ~ ~ ~ r n ~ ~ ~ . n ~ - ~ ~ ~ , ~ ~ i , ( u i ~ . ~ ~ . " ~ ~ ~ a ~ ~ m , r ; n . ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ . + ' , . . . . , . . . .. .,

, . . . .

Bi POS 0.84 Ri POS 0.85 Methylphen,wu,L nnc 0.91 Ai 1.05 M 1.06

y.'"'" ;,,.:. '-~,-~*..>?~-T'"'><F~,~.I.~RR~ ,iii," :yTZ;,T\.!,..'i;,~T, WW J!.,J!7<~,~p., "p.z.r;.-* vs,rr..;y r;. -.y:T>*<.., 1111411*41.."<. .. . ,, . ,.' ' , , ,. ... 1 . . . . ' ., I' , , .

7- I" . ., . . . . . , -. , . , . . . ,!l.. , , 'i

0.84 Esmolol metabolite L u , . , u POS S .u , 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 s t imu lan ts and related compounds, such as chLorthalidone. clopamide, cocaine metabol i te ; methy lphen indapamide and metolazone; and strongly acidic diuretics. ritalinic acid; adrafinil and modafi such as furosemide, piretanide. bumetanide o r etacrynic acid such as probenecid o r 5n-reduct

/ R . Ventura etai.. Eur J. Marr Spcctram. 11,191-200 120081

rnetabolite. u - c a r b o ~ ~ f i n a s t e r i d e 1 . 3 " enhancers of oxygen transfer [efaproxiral] and beta-blocker metabol i tes [meta - bolite of esmolol, h-[2-hydroxy-3-[( l-methylethyl lamino] propoxylbenzene propanoic a ~ i d l . ~ " ~ ~

Electrospray ionisation working parameters were optimised for a l l the compounds under study by using infusion of indi- vidual standard solutions. The optimised values are listed in Table 1. Positive- and negative-ion modes were tested. Higher signal was obtained for most of the compounds in positive ion mode, at Least one order of magnitude greater than in nega- tive mode. Positive-ion mode was selected for most of the compounds as indicated in Table 1. For some of the compounds [acetazolamide. chlorothiazide. etacrynic acid], both positive and negative modes were used and, for a few compounds only negative ionisation mode was used [hydrochorothiazide. diclofenamide, bendroflumethiazide degradation product].

The higher signals obtained in.positive mode for most of the analytes compared to previous ~ o r k s ~ ~ ~ ~ ' " ~ ~ are probably explained by the composition of the mobile phase and the pres- ente of functional groups with high proton affinity in most of the compounds under study. Using mobile phases containing acetonitrile and water or water with acetic acid o r ammoniurn

'acetate, positive-ion mode was favoured for basic and neutral compounds. however negatively charged ions were mainLy formed for acidic d i u r e t i ~ s . ' ~ ~ . ' " ~ ~ ~ However, us ing mobi le phases at more acidic pH [water with formic acidl. positive ion mode was preferred for most of the compounds. including most of the acidic diuretics. as described by Goebel e t al." Both modes of ionisation must be used for a comprehensive screening for diuretics and the mass spectrometer should be able to switch between positive and negative ionisation modes in a single analysis. Otherwise, two analyses per sample have to be performed as described by some a u t h ~ r s . ~ '

Cone voltage was optimised to obtain maximum signal for either the protonated or deprotonated molecular ions. [M+H]' or [M-HI-, depending on the compound. Data acquisition was performed in MRM mode and different collision energies were studied in order to obtain the maximum response for each transition. For screening purposes, only one ion transition was used for most of the compounds [Table 11. After a suspicious screening result, the extract of the sample was re-analysed by using an specific MS/MS method for unequivocal identification of the suspected compound, including at least three specific ion transitions, according to the WADA criteria for identifica- t i ~ n . ~ ' If the result of this pre-confirmation analysis was posi- tive, a new aliquot of the sample was analysed for confirmation purposes by using the specific MS/MS method.

ln the screening step, simultaneous acquisition of positive and negative ions was performed. A total of 33 transitions were measured in positive mode and six transitions were measured in negative mode. The peak widths obtained were around 5-6s. In the conditions of data acquisition used [short dwell, interchannel and interscan times]. a total of 10-12 data points per peak can be acquired. allowing an adequate definition of the chromatographic peak [opt imum number of data points: between 10 and 151. The possibility of a fast switching between

positive- and negative-ion modes and the fast scan speed of the instrument is crucial to allow the simultaneous detection of a high number of compounds. In the current conditions, the number of compounds detected in the screening method could be increased by including additional ion transitions, or using different time windows for different acquisition groups.

Taking into account the mobi le phase composition and f low-rate used, a high desolvation gas flow rate was used 11200 L h-'1 and desolvation and source temperatures were set at 450°C and 120°C in order to improve desolvation efficiency and analyte ionisation.

Apart f rom formic acid, no other mobile phase additives were needed e i ther to promote ionisation or to improve chromatographic behaviour of the different compounds. The gradient was optimised to obtain a reasonable short chrorna- tographic t ime [Table 1). Although not needed for chromato- graphic separation. the final content of organic solvent in the mobile phase was increased to 90% in order to have a good clean-up of the chromatographic column. to avoid problems of blocking and to extend the Life of the columns. In addition, the sample extraction and the sma l l volume of sample injected contr ibuted to extend the Life of the co lumn. According to our experience more than 3500 analyses per column can be performed. The last compound eluted at approximately 3min. The stabilisation of the column at initial mobile phase condi- tions was performed in only 0.9min and the total run time was 5 m i n per sarnple. In these conditions, a reduction between two and four-fold in the analysis t ime was achieved compared to previous methods using columns packed with larger sized part ic les.6,8~'2~'"~21~30 The reproducibility in retention times was also good in both intra- and inter-assay conditions.

The selectivity/specificity was evaluated by analysis of ten different blank urine samples and no matrix interferenceswere detected at the retention times of the compounds of interest. Figure 1 shows the chromatograms obtained after analysis of a blank urine sample and those obtained after analysis of a urine sample spiked with some of the compounds.

Results of LOD, repeatabil ity at two concentration levels. extraction recovery [calculated at the high concentration]. and intermediate precision are l isted in Table 2. Regarding the LOD, the concentration listed in the table is the lowest concen- tration tested and, for most of the compounds, lower concen- trations could be detected. For a l l the compounds in the study, the LOD listed was lower than MRPL defined by WADA.32 For diuretics, the LOD obtained were always lower than those obtained by GC-MS.

The extraction recoveries ranged f rom less than 10% to 10O0/0 [Table 21. The extraction procedure was firstly optimised to the detection of diuretic compounds3" and. in spite of using extraction at basic pH, it was appropriate even for the extrac- tion of more acidic diuretics. due to the use of a polar organic solvent [ethyl acetate] and salt ing-out effect [Table 21. The extraction of other compounds without carboxylic acid func- tions [adrafinil, modafinil, methylphenidate. efaproxiral] was always higher than 7O0/0 [Table 21. However, the extraction of some compounds with free carboxylic acid functions was

. 196 Screening of Diuretics and Other Doping Agents using UPLC-MS/MS

HyOmNoidhniin E-Id m* Bruel(.rqwne ,:"LY.,II>.,,<..E- <, ".YOIS>',.,,...ISI ~ I Y . . Y < , : L I I I " . I ~

=:--O: 1 0 - 168 1 m 1011031

"" .. ,m 2 x 4

1riam.rinr Rt.Ic"eA~d FinaSlt.ld. M I, "I"O,IOii<...Bi. '1 Y"Y.,,.C,~".15. P I Y"Y.I1IS,.I".I*

1 3 - :n

.. Ibm i tnl 1630 :m 2.- :ti0

F7oMn.m RSR 13 B>.mr>ixlm. F i r . . < C r l r . i . 1 S . I I . " Y O < C ~ I I . . F C ,,"""or,:',a,".r*

! ! ~1 ;hk,.,

1040 >:m :M0 28- >dm >m >m

~roolmdiiks cnloiahba. o*ome-d10a I I Y"0<*.01r(l,lL5*

2 9 - lb, , : Y u a 6 o m i , a SI- I I Y I Y O ~ : ~ U I Y ~ . C %

9 . - 111 tvaL:l W - i i P

:m '.m ..

1 irm .m

0 ,m ."

**oQ**r.,,hN,d. E*m<iW m. Bmro,l.rgon". . . i I i".*,:<iur..csi - 8 "P.m,:~,..".f*

;*m i i m ,m ,.m l..* ?m

1r,."d*r.* R, t , i , "~A~d F4"l.nd.m.t S , "I.O,ES I,,,,, ES. Il."YO":.,.,<,l* ,,.."a,,:c,,,"*Fs

IP -2, -.o-?. 0 1 - D I

o i r m zmo :.w :,m

.. lam cm

Figure 1. LC-MS/MS chromatograms obtained after analysis of a blank urine [leftl and a urine spiked with most of the compounds lrightl. Al l 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 Adrafinil Althiazide Amiloride Bedroflumethiazide

Benzoylecgonine Benzthiazide Bumetanide Buthiazide Canrenone

13.5 L U U

13.3 400 24.5 200 12.7 200 7.3 200

12.8 200 10.2 200 9.3

12.3 6.0

Chlorothiazide 5 O Chlortalidone 5 O Clopamide 5 O Diclofenamide Esmolol metabolite

Etacrynic acid F F H Indapamidt

Methylpher Metolazone hi P P

Po iy in ld~ lu Probenecic Ritalinic ac RSR 17

S tone

TI 2

TI d l ~ l l L ~ l elie Trichlorme Xipamide

LUU

200 200 200 400

200 I UU IUU 1J.U 200 50 50 13.9 200 50 5 O 20.7 200 50

- 200

1 O0 400 5 0 200

400 200 200

200 200 400 400 400

200 200

inasteride urosemidi ydrochlor

. .

! metaboli e .othiazide

lodafinil iretanide iretanide ., L L ' - - ' A

IUU

50 5 0 r n

impuri ty

3U

50 100 inn

3 u 5 O

100 inn

- 198 Screening of Diuretics and Other Doping Agents using UPLC-MS/MS '

iower than 10% (ben~o~ lecgon ine . esmolol metabolite, finas- teride metabolite, ritalinic acid). As for other methods using LC-MSIMS, a substantial reduction in the sarnple preparation procedure was achieved cornpared to previous methods using GC-MS. due to the elimination of the derivatisation. which is a time-consuming step for diuretic c o m p ~ u n d s . ~ ~

Owing to the high sensitivity of the equiprnent, in spite of the low extraction recoveries for some compounds. the method allowed the detection of a l l the compounds at concentra- tions lower than the MRPL defined by WADA. Appropriate repeatabilities were obtained, even for compounds with Low extraction recoveres. The RSD were in general Lower than 15% for the highest concentration studied and lower than 20% for the lowest concentration. lntermediate precision was esti- rnated using the quality control sample spiked with some of the compounds analysed during routine work. For most of the compounds, RSD Lower than 25% were obtained.

As a f inal validation. samples obtained after administra- tion of some of the compounds to healthy volunteers were analysed. In Figure 2. chromatograms obtained after analysis of blank urine samples are compared with those obtained after analysis of samples collected after administration of

lSTD E Benzoylecgonine ES+ 223>181 ES+ 290>168

ISTD ES+ 223481

Finasteride met ES+ 403>335

finasteride and furosemide and a sample detected positive to benzoylecgonine [cocaine metabolite] in routine work.

In order to evaluate the reliability for routine work, the devel- oped rnethod was applied to the analysis of doping control routine samples during one year. In our hands. the system has proved to be stable and reliable with simple maintenance operations [daily fi ltration of the rnobile phase components, change of the column pre-fi lter and cleaning of the cone and the ion source, when necessaryl. The system can work for 24 h a day and, taking into account the total analysis time per sample, more than 200 samples per day can be analysed.

In surnmary, the work presented demonstrates the poten- tial of UPLC-MS/MS with simultaneous detection in positive and negative modes for high-throughput and comprehensive screening procedurec in anti-doping control. At present. the method is applied to the analysis of 34 compounds [mainly diuretics and other acidic drugs]; however, due to the char- acteristics of the equipment used. it can easily be extended to the analysis of other compounds. The short analysis time resulting from simple sample preparation and rapid instru- mental analysis allow a fast turn-around time and makes it of great interest for routine anti-doping control purposes.

ISTD Benzoylecgonine ES+ 223>181 ES+ 290>168 1

ISTD ES+ 223s181

Finasteride met ES+ 403~335

ISTD Furosemide ES+ 223>181 ES+ 331 >81

ISTD Furosemide 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 -- ~

Acknowledgements chromatographyltandem rnass spectrometry". Rapid 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 ,,

. i for beta2-agonists in human or equine urine", J. Mass

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 lnternat ional electrospray tandem mass spectrometry". Anal. Chim. : 4.

Standard. http://www.wada-ama.org/rtecontent/docu- i !l Acta 502, 65 (20041. do¡: 10.1016/j.aca.2003.09.062

i f ment/2008-List-En.pdf (20071. 13. A . Leinonen, T. Kuuranne, T. Kotiaho and R. Kostiainen, 1 ' 2 2. World Anti-doping Agency [WADA]. The World Anti- ! , . . 1 Doping Code. lnternational Standard for Laboratories !, [version 5.01. http://www.wada-ama.org/rtecontent/ 1 .: 1 7 . document/lab-08-V-05.pdf 120081. 1. 3. G.J. Trout and R. Kazlauskas, "Sports drug testing-an

analyst's perspective". Chern. Soc. Rev. 33, l(20041. doi: ! ' 10.103P/bZ01476a .i! 7: 4. S.D. Garbis. L. Hanley and S. Kalita, "Detection of

1;; 9 thiazide-based diuretics in equine urine by liquid t chromatography/rnass spectrometry". J. Assoc. Offic. $ Anal. Chem. Int. 81, 948 119981.

1 5. V. Sani-Nebot. l . Toro. R . Berges. R . Ventura. J. Segura and J. Barbosa. "Determination and characterization of diuretics in human urine by liquid chromatography cou- pled to pneumatically assisted electrospray ionization mass spectrometry", J. Mass Spectrom. 36, 652 (20011. do¡: 10.1002/jms.166

6. D. Thieme. J. Grosse. R. Lang. R.K. Mue l le rand A. Wahl, "Screening, confirmation and quantif ication of diuretics in urine for doping control analysis by high-performance liquid chromatography-atmospheric pressure ionisation tandem mass spectrometry". J. Chromatogr. B 757, 49 120011. do¡: 10.1016/S0378-4347101I00058-5

7. M. Thevis, G. Opfermann and W. Schanzer. "High speed determination of beta-receptor blocking agents in human urine by liquid chromatography/tandem mass spectrometry", Biorned Chromatogr 15, 393 l20011. doi: 10.1002/bmc.87

8. K. Deventer, F.T. Delbeke, K. Roels and P. van Eenoo, "Screening for 18 diuretics and probenecid in doping

1

1 anaiysis by liquid chromatography-tandem mass spec- trometry", Biomed. Chromatogr. 16, 529 (20021. doi: 10.1002/bmc.201

9. A. Leinonen, T. Kuuranne and R. Kostiainen. "Liquid chromatography/mass spectrometry in anabolic steroid analysis-optimization and comparison of three ioniza- tion techniques: electrospray ionization, atmospheric pressure chemical ionization and atmospheric pressure photoionization", J. Mass Spectrorn. 37, 693 I20021. doi: 10.1002/jms.328

; 10. K. Deventer and F.T. Delbeke, "Validation of a screening

I method for corticosteroids in doping analysis by l iquid

"Screening of free 17-alkyl-substituted anabolic steroids in human urine by liquid chromatography-electrospray ionization tandem mass spectrometry", Steroids 69, 101 [2004]. do¡: 10.1016/j.steroids.2003.10.007

14. K. Deventer, P. van Eenoo and F.T. Delbeke, "Simultaneous determination of beta-blocking agents and diuretics in doping analysis by liquid chromatography/mass spectrometry wi th scan-to-scan polarity switching", Rapid Commun. Mass Spectrorn. 19, 90 (20051. doi: 10.1002/rcm.1744

15. M. Thevis, H. Geyer, U. Mareck and W. Schanzer, "Screening for unknown synthetic steroids in human urine by liquid chromatography-tandem mass spectrometry". J. Mass Spectrom. 40, 955 120051. doi: 10.1002/jms.873

16. K. Deventer, P. van Eenoo and F.T. Delbeke. "Screening for anabolic steroids in doping analysis by liquid chromatography/electrospray ion trap mass spectrom- etry", Biomed. Chromatogr. 20,429 (20061. do¡: 10.1002/ bmc.579

17. K. Deventer, P. van Eenoo and F.T. Delbeke. "Screening for amphetamine and amphetamine-type drugs in dop- ing analysis by liquid chromatography/mass spectrom- etry", Rapid Commun. Mass Spectrom. 20,877 l2006l. doi: 10.1002/rcm.2372

18. M.H. Spyridaki, P. Kiousi, A. Vonaparti, P. Valavani, V. Zonaras, M. Zahariou, E. Sianos. G. Tsoupras and C. Georgakopoulos. "Doping contro l analysis in human urine by liquid chromatography-electrospray ioniza- tion ion trap mass spectrometry for the Olympic Garnes Athens 2004: Determination of corticosteroids and quantification of ephedrines. salbutamol and morphine", Anal. Chim. Acta 5731574, 242 12006). do¡: 10.1016/j. aca.2006.04.042

19. M. Mazzarino and F. Botre, "A fast Liquid chromatographic/mass spectrometric screening method for the simultaneous detection of synthetic glu- cocorticoids, some st imulants. anti-oestrogen drugs and synthetic anabolic steroids" Rapid Commun. Mass Spectrom. 20, 3465 120061. doi: 10.1002/rcm.2729

20. U. Mareck, H. Geyer. S. Guddat. N. Haenelt, A. Koch. M. Kohler, G. Opfermann, M. Thevis and W. Schanzer.

Screening of Diuretics and Other Doping Agents using UPLC-MSIMS

"ldentification of the aromatase inhibitors anastro- zole and exemestane in human urine using liquid chromatography/tandem mass spectrometry", Rapid Cornrnun. Mass Spectrorn. 20, 1954 í20061. doi: 10.1002/ rcm.2545

21. L. Polit i, L . Morini and A. Polettini, "A direct screen- ing procedure for diuretics in hurnan urine by liquid chromatography-tandem mass spectrometry with infor- mation dependent acquisition", Clin. Chim. Acta 386,46 (20071. doi: 10.1016/j.cca.2007.07.018

22..K. Deventer, J.O. Pozo, P. van Eenoo and F.T. Delbeke, "Development of a q;alitative l iquid chromatography/ tandern mass spectrometr ic method for the detection of narcotics i n ur ine relevant to doping analysis", Rapid Comrnun. Mass Spectrom. 21,3015 120071. doi: 10.1002/ rcrn.3142

23. M. Mazzarino, S. Turi and F. Botre, "A screening method for the detection of synthetic glucocorticosteroids in human urine by l iquid chromatography-rnass spec- t rometry based on class-characteristic fragmentation pathways", Anal. Bioanal. Chern. 390, 1389 120081. doi: 10.1007/~00216-007-1802-1

24. V. Morra. P. Davit. P. Capra, M. Vincenti, A. Di Stilo and F. Botre. "Fast gas chromatographic/mass spec- l rometr ic determination of diuret ics and masking agents in human urine: Development and validation of a productive screening protocol for anti-doping analy- sis", J. Chromatogr. % 1135, 219 120061. doi: 10.1016/j. chroma.2006.09.034

25. M. Mazzarino, M. Orengia and F. Botre, "Application of fast gas chromatography/mass spectrometry for the rapid screening of synthetic anabolic steroids and other drugs in anti-doping analysis", Rapid Comrnun. Mass Spectrom. 21, 4117 120071. doi: 10.1 002/rcm.3326

26. M. Kolmonen, A. Leinonen. A. Pelander and l. Ojanpera, "A general screening method for doping agents in human urine by solid phase extract ion and liquid chromatography/time-of-flight mass spectrom- etry", Anal. Chirn. Acta 585, 94 (20071. doi: 10.1016/j. aca.2006.12.028

27. J.O. Pozo, P. van Eenoo, W. van Thuyne. K. Deventer and F.T. Delbeke. "Direct quantif ication of steroid glucuro- nides in hurnan urine by l iquid chromatography-elec- trospray tandern mass spectrometry", J. Chrornatogr. A 1183, 108 120081. doi: 10.1016/j.chroma.2008.01.045

28. M. Huerta-Fontanela. M. Galcerán and F. Ventura, "Ultraperforrnance l iquid chromatography-tandem mass spectrometry analysis of s t imulatory drugs of abuse in wastewater and sur face waters", Anal. Chem. 79,3821 120081. doi: 10.1021/~c062370x

29. T. Kovalczuk, O. Lacina, M. Jech, J. Poustka and J. Hajslova, "Novel approach to fast determination of mult iple pesticide residues using ultra-performance liquid chromatography-tandem mass spectrometry [UPLC-MS/MSl", Food Addit. Contam. 25, 444 120081. doi: 10.1080/02652030701570156

30. R. Ventura, T. Nadal, P. Alcalde. J.A. Pascual and J. Segura, "Fast screening method for diuretics. probenecid and other compounds of doping interest". J. Chromatogr. A 655, 233 (19931. doi: 10.1016/0021- 9673193183228-K

31. C. Jimenez, R. Ventura and J. Segura. "Validation of qualitative chrornatographic methods: strategy in anti- doping contro l laboratories", J. Chromatogr. B 767,341 I20021. doi: 10.1016/S1570-0232[01]00593-1

32. World Anti-doping Agency [WADA]. Minimum required performance l imi ts for detection of prohibited sub- stances. WADA Technical document TD2004MRPL. http://www.wada-ama.org/rtecontent/document/perf~ lirnits-2.pdf 120041.

33. E. Jackson. in Goodrnan & Gilrnan's The Pharmacological Basis of Therapeutics. Ed by L. Brunton, J. Lazo and K. Parker. McGraw-Hill. Medical Publishing Division, New York. USA, p. 737 120051.

34. M. Thevis. H. Geyer, U. Mareck, U. Flenker and W. Schanzer, "Doping-control analysis of the 5alpha- reductase inhibitor finasteride: determination of i ts influence on ur inary steroid profiles and detection of i ts major ur inary metabolite", Ther. Drug Monit. 29, 236 120071. doi: 10.1097/FTD.Ob013e31803bb85d

35. A. Achari. D. Dr isse l and J. Hulse. "Liquid- chromatographic analysis for esmolol and i ts major metabolite in urine", Clin. Chem. 32, 374 (19861.

36. A. Achari, D. Drissel. W. Matier and J. Hulse, "Metabolism and ur inary excretion of esmolol in humans", J. Clin. Pharrnacol. 26,44 (19861.

37. M. Thevis. H . Schmickler and W. Schanzer, "Mass spectrometr ic behaviour of thiazide-based diuretics after electrospray ionization and coll ision-induced dis- sociation" Anal. Chern. 7 4 3802 120021. doi: 10.1021/ ac020020e

38. World Anti-doping Agency (WADAI. ldentification crite- r ia for qualitative assays. WADA Technical Document TD20031DCR. http://www.wada-ama.org/rtecontent/ document/criteria-1-2,pdf [2004].

39. R. Ventura and J. Segura. "Detection of diuretic agents in doping control". J. Chrornatogr. B 687, 127 (1996). doi: lO.lOl6/S0378-4347196100279-4