209 2009 c 1 (Schering-Plough,  Testing Drug Analysis and 2009 John Wiley & Sons, Ltd. c omics Division, Department of Plant oLab, UGent, Department of Clinical W. Van Thuyne g/mL was regarded as a doping violation 1 ary concentration of also exceeded µ amples had a concentration of above Bioinformatics and EvolutionarySystems Gen Biology, Technologiepark 30, B-9052 Gent, Belgium DoCoLab, UGent,Immunology, Department Technologiepark 30, B-9052 of Gent, Belgium Clinical Chemistry, Microbiology and Correspondence to: K. Deventer,Chemistry, DoC MicrobiologyZwijnaarde, and Belgium. E-mail: [email protected] Immunology, Technologiepark 30, B-9052 dicator to control pseudopehedrine intake. To control the O. J. Pozo, ∗ 2 1 tantially. Analysis of 116 in-competition samples containing g/mL. Statistical comparison of the two periods showed that cathine and pseudoephedrine concentrations only indicated and to compare thebefore incidence and of after elevated its PEPH-concentrations removalof from the the administration prohibited list.samples study containing The PEPH. were results then compared with routine Brussels) containing 240 mg PEPH.sulfate and 10 mg loratidine Experimental study The study was performed with sixand healthy volunteers five (one female male)and aged approved 23–39. by the TheHospital ethical study committee (UZ of protocol Gent, the wassigned Ghent Project University reviewed an EC informed UZGtablets 2006-404). consent of Each agreement CirrusPEPH.HCl volunteer and and 5 (UCB, mg .HCl was Brussels, each.volunteers Two given were weeks Belgium) given later, one the two tablet same containing of Clarinase 120 mg 2 µ prohibited list in 2004 the number of urine samples in which f a therapeutic daily dose (240 mg pseudoephedrine) in one eased significantly. Of the individual sports compared between reintroduce a threshold for pseudoephedrine. Copyright [11,12] g/mL can be exceeded. The same samples were also analysed for cathine, G. Baele, µ 1 [1] g/mL on the prohibited list. The maximum urin µ ). All extracts of these plants 1 with conflicting findings concerning P. Van Eenoo, , 209–213 Copyright ∗ sinica 1 1 , [2–10] 2009 g/mL is applied. Until December 2003, PEPH and g/mL and for and methylephedrine a µ µ doping; urine; pseudoephedrine; cathine; sports; ephedrine;

g/mL. This corresponded to 1.4% of all tested in competition samples in that period. In the period 2001–2003 only 0.18% g/mL, respectively. Hence, the actual situation is that PEPH

µ µ The aim of this study was to investigate urinary concentrations Analysis of excretion urine samples after administration o Ephedrine-type are applied therapeutically for There has been much discussion concerning the effect of are still used in nutritional supplements. administration showed that thewhich threshold has currently of a threshold 25 of 5 the threshold for some volunteers.a Comparison poor of the correlation measured between(ab)use them. of Hence, in cathine sports it is is recommended not that WADA a good in Keywords: John Wiley & Sons, Ltd. the ergogenity of PEPH.International Olympic Committee To (IOC) and the control World Anti the Doping Agency(WADA)putthem use onthelistof of prohibitedsubstances. ephedrines, the Drug Test. Analysis Because of the frequent therapeutic applicationergogenic potency, and the the regulations debated are complicated:threshold for of cathine a 5 has been removed butprohibited list. its metabolite, cathine, remains on the of PEPH and cathine after administration of different preparations threshold of 10 NEPH were also prohibited at10 concentrations higher than 25 and Stimulants are synthetic derivates ofpharmacological and effects have similar producing on excitement and euphoria mental and increased motorThe function activity. ephedrines are and oneapplied behaviour, of stimulants. the These oldest substancesephedra groups or can of ma be huang therapeutically ( found naturally in Introduction of all analysed in competition samples contained more than 25 Until the end ofby 2003 the a World urinary Anti-Doping concentrationpseudoephedrine Agency. of was Since pseudoephedrine detected its in exceeding removal ourpseudoephedrine 25 from laboratory in increased the 2007 subs and25 2008, revealed that 66% of theseafter the s removal of pseudoephedrinethe from two the periods, list only its cycling use is incr shown to yield a significant increase. in relation to doping control K. Deventer, and F. T. Delbeke pseudoephedrine and its metabolite cathine (www.drugtestinganalysis.com) DOI 10.1002/dta.31 Interpretation of urinary concentrations of Research Article Received: 30 March 2009 Revised: 20 April 2009 Accepted: 21 April 2009 Published online in Wiley Interscience: 26 June 2009 decongesting the respiratory tract. For example, pseudoephedrine (PEPH) is frequently usedcontaining for ephedrine (EPH) allergic are used in while the treatment preparations of coughs. ephedrines in sports, . [15] et al g/mL g/mL. g/mL µ µ [16,19,20] asingle µ , 209–213 43 25 1 . [19] , > samples with Percentage of 2009 K. Deventer PEPH g/mL and the total µ . administered 360 mg g/mL for Vasocedine, The maximum urinary µ g/mL. found maximum urinary et al g/mL µ [15] µ [18] varied between 4 h and 24 h. . 25 with PEPH > Drug Test. Analysis g/mL. In another study No. of samples µ max et al g/mL.Thetotalamountofunchanged µ g/mL for Clarinase, 2.3 and 7 g/mL after administration of a single µ µ g/mL. These observations were supported 39 µ . No. of samples g/mL (Figure 1) is possible after administration analysed µ Routine samples with concentrations of PEPH above g/mLintheperiodOctober2007–December2008inourlaboratory [16,17] µ Cathine was also detected in the urine samples for all After Clarinase intake, PEPH could already be detected one For Vasocedine maximum concentrations of PEPH were reached The result of this excretion study show that urinary PEPH Table 2. 25 Sport AthleticsCyclingField hockeyHandballIce hockey 447Indoor football 116 1548Martial artsJudo 153 177Rugby 74 2Volleyball 60 2Other 73 sportsTotal 1 1 3 158 2878 295 29 0.4 1 3.9 1.7 5948 1 0.7 0 5 0.6 1 4.1 1.4 77 0.6 0 1.7 3.4 1.3 for Cirrusrespectively. and Excreted amounts between were betweenthe dose. 3.3 0.4% This low and percentage and 2.6% is in of stating accordance with 12 that previous work cathine is only a minor metabolite of PEPH. preparations (Figure 1). Maximumbetween urinary 3.66 concentrations and varied 10 hour after intake (Figure 1). T dose of 120 mgurinary PEPH.HCl concentration of was PEPH administered was 91 and the maximum Nevertheless, the results inthe threshold this of 5 work also show that exceeding This variation can be assignedflow. to the urinary pH and the urinary Results and Discussion Excretion study Large individual differences were foundPEPH in and the concentrations cathine. of in A a similar previous variation study has measuring also urinary been PEPH observed concentrations. concentrations of 114 dose of 30 mg of PEPH.HCl. Chester of PEPH.HCl over a 36 h period. concentration of PEPH was 215 The total amount of12 hours unchanged varied between drug 21% and excreted 29% during of the the administered dose. first between4and12 hafteradministration.Maximumconcentrations variedbetween95and229 drug excreted during the first 12 hours wasof between the 27% administered and 50% dose. After intakeconcentrations of Cirrus, were maximum urinary between 98 and 163 excreted amount of unchanged41%. drug varied between 19% and concentrations in an individual,daily taking the dose maximal therapeutic (24used h threshold of 25 period),by can previous greatly studies. exceed Tseng the previously Maximum concentrations ranged between 65 and 233 2009 John Wiley & Sons, Ltd. g/mL µ c 25 > samples with Percentage of PEPH Copyright (Qualiphar,Bornem, and compared with g/mL is equal in both  ) was used to compute µ 8 [14] 10 g/mL) were collected from µ × g/mL g/mL is equal in both periods µ µ .HCl each. In all cases PEPH was 25 with PEPH > [13] No of samples g/mL) in both periods (Table 1 and µ C awaiting analysis. Volume and density were No. of ◦ samples analysed Routine samples with concentrations of PEPH above values. Second, the common sports between both periods g/mL in the period 2001–2003 in our laboratory µ g/mL, are present for cycling in both periods, a chi-square test p µ First, the association between sports type and PEPH concen- Urine samples were collected before (0h) and quantitatively at AthleticsCyclingTennisSwimmingIce 954 hockeyFootball 3531Basketball 309 134Tabletennis 190Volleyball 1 1070Other sports 6 388 132Total 1 1 2 4216 441 3 3 0.1 1 11 365 0.2 0 2 0.3 0.7 1.1 20 0.3 0.8 0.8 0.0 0.5 0.2 Sport Table 1. 25 Drug Testing Drug Analysis and Table 2) was determined. Fisher’sditions exact test for was performing used a as chi-squaresmall the test con- cell were values. not fulfilled Givennumber due of Monte the to Carlo simulations dimensions (5 of both tables, a large tration (above and below 25 concentrations of PEPH in(before samples removal of from PEPH the from the period list). 2001–2003 www.drugtestinganalysis.com the fourth quarter of 2007 until the endof of 2008. The PEPH concentration andpreviously its described metabolite GC-NPD cathine method were determined using a To investigate the use of PEPH inurine sports, samples containing all PEPH in-competition (LOD routine 1 Routine samples each. Another two weeksfourtabletsofVasocedinePseudoephedrine later the same volunteers were given 1, 2, 3, 6, 9, 12 hourswere after collected intake after of 24, the 36, tablets. 48, Additionalwere 72, samples stored 96 at and -20 120 h. All urine samples Belgium) containing 60 mg PEPH administered in one administrationdose over of a the 24-h period. maximal therapeutic measured and all sampleswere analysed were using a analysed previously described in LC-MS/MSthe method duplicate. quantitation for of Samples ephedrines. periods. Because of small cell values,hypergeometric Fisher’s distribution) exact was test used (using for the theFinally, a three chi-square test other was sports. used to test whetherPEPH the concentrations proportion of exceeding 25 for all sports combined. CRAN R versionthe 2.8.1 statistical was analyses. used to perform (athletics, cycling, and volleyball) were compared. Since sufficient samples, for both PEPH concentrations above and25 below was used to test thewith null a hypothesis PEPH that concentration the higher than number 25 of samples the

210 211 volunteer4 volunteer5 volunteer6 volunteer1 volunteer2 volunteer3 volunteer1 volunteer2 volunteer3 volunteer4 volunteer5 volunteer1 volunteer2 volunteer3 volunteer4 volunteer5 g/ml), former threshold µ Drug Testing Drug Analysis and www.drugtestinganalysis.com Time (h) Time (h) Time (h) d line: threshold for cathine (5 Cathine Cathine CLARINASE Cathine VASOCEDINE CIRRUS 0 5 10 15 20 25 30 35 40 45 50 0 5 10 15 20 25 30 35 40 45 50 0 5 10 15 20 25 30 35 40 45 50 8 6 4 2 0 8 6 4 2 0

8 6 4 2 0

14 12 10 14 12 10

tration of (A) Clarinase (240 mg pseudoephedrine sulphate), (B) Cirrus

14 12 10 g/ml) ( Conc g/ml) ( Conc g/ml) ( Conc µ µ µ 2009 John Wiley & Sons, Ltd. c 60 mg pseudoephedrine.HCl). Dotte volunteer1 volunteer2 volunteer3 volunteer4 volunteer5 × volunteer1 volunteer2 volunteer3 volunteer4 volunteer5 volunteer6 volunteer3 volunteer4 volunteer5 volunteer1 volunteer2 Time (h) Time (h) Time (h) , 209–213 Copyright g/mL). 1 µ , CIRRUS 2009 CLARINASE VASOCEDINE Pseudoephedrine Pseudoephedrine Pseudoephedrine 0 5 10 15 20 25 30 35 40 45 50 0 5 10 15 20 25 30 35 40 45 50 0 5 10 15 20 25 30 35 40 45 50 0 0 Urinary excretion curves for pseudoephedrine and cathine after adminis 0 50 50

50 250 200 150 100

250 200 150 100

250 200 150 100

g/ml) ( Conc Conc ( Conc g/ml) Conc ( Conc g/ml)

µ µ µ 120 mg pseudoephedrine.HCl) and (C) Vascosedine (4 (c) (b) (a) × Urinary concentrations of pseudoephedrine and cathine for pseudoephedrine (25 Drug Test. Analysis Figure 1. (2 . was et al 20 33) was − . , 209–213 0 10 1 , = × 2 1 . 2009 K. Deventer value of 2 p Drug Test. Analysis g/mL) µ C ( [21] g/mL previously used by WADA. µ 38) making it difficult to associate concentrations . 0 123456789 = 2 0 75 50 25

Comparison of urinary PEPH concentrations in samples, col- These results are remarkable when compared with a study N etes tested for doping control purposes in the period from October 2007 found when PEPH and cathine concentrationswere in compared. routine samples of PEPH with cathine. A similar weak correlation (R Comparison of excretion studysamples data and data from routine To further understand thethe relationship kinetics between PEPH of and cathine PEPH wasparticular investigated regard and with to its the question metabolite, of of PEPH could whether yield high high concentrations concentrations of cathine. Concentrations of PEPH and cathine ofeach the other excretion (Figure 3a). study Only werefound a plotted (R weak against positive correlation could be Conclusions The administration experiments in this work showtions that of concentra- cathine and PEPH areis difficult to not correlate. a Hence, cathine goodsports. indicator for The the excretion control studyuse of also of the PEPH demonstrated (ab)use can that of leadthreshold PEPH therapeutic to of in 25 positive testing, taking into account the lected before and after removalshows of that PEPH the use from of the PEPH prohibitedreintroduction has list, increased of significantly. Hence, a the thresholdsports, for is highly PEPH, recommended. to control its (ab)use in obtained, thereby causing theHence rejection a of significant thisperiods difference null for was all hypothesis. sports observed combined andthe between it use of can PEPH the has thus increased be since two its concluded removallist. that from the prohibited performed after was removed from the list,no which increase showed in its use. for ice hockey andthe 0.12 null for hypothesis for volleyball, each of resulting thesetwo in sports. Finally, periods acceptance comparing for the of all sports combined, a p 2009 John Wiley & Sons, Ltd. c g/mL is shown µ Copyright g/mL. 66% of these µ was obtained, yielding g/mL) 5 µ − C ( 10 for the period from October 9 × value and in the period from − Pseudoephedrine Cathine 6 . p 10 × g/mL of PEPH. These positive findings 25 50 75 100 125 150 175 µ value of 2 g/mL (Table 2). Taking into account the for cycling, clearly leading to the rejection p µ value. When the four common sports were 26 value of 2 p

0 [19] −

p 10 20 30 40 N 10 × g/mL. 0 µ . Distribution of PEPH and cathine levels in routine samples of 116 athl g/mL. Since the fourth quarter of 2007, 116 samples containing Testing the association between PEPH concentration and type value of 3 µ Drug Testing Drug Analysis and www.drugtestinganalysis.com of sports yielded a Routine samples Before the removal of PEPH from thewere WADA-list, declared only few positive samples forlaboratory. PEPH Only in the 20contained year more samples than 2001–2004 25 out in our of 11 365 (0.18%) (Table 1) samples contained a concentration of PEPHused above threshold the previously ofpreviously 25 applied threshold, the highestanalytical percentages findings of (AAFs) adverse would be observed inice cycling, rugby hockey. and A total of 1.4% of(5948) the would in result competition-samples in analysed an AAF. of high therapeutic dosesonly of published PEPH. scientific This paperthe is excretion describing of in quantitative cathine contrast after datathat PEPH with on after intake the so 120 far, mgexceeded which PEPH.HCl 1 indicates the maximum concentration never Figure 2. were always in combination7 with cathine concentrations below PEPH were collected. The distributionthese samples of is PEPH presented concentrations in Figure in these 2. samples Concentrations of varied PEPH between in 5.2 and 197 to December 2008. October 2007 until Decembercontribution 2008 to cycling the yielded the highest to differ significantly betweenthe sports period types January inthe 2001 highest both until contribution periods. December to In 2003 this ice hockey had the same conclusion. In othercontaining words, a PEPH the concentration percentage higher of than samples 25 2007 until December 2008,null thereby causing hypothesis the that rejection PEPHindependent of concentration from the one and another. For type the ofuntil period December from 2003, sports a January 2001 are values were obtained for the other sports: 0.24 for athletics, 0.14 of the null hypothesis. Using the Fischer’s test, the following compared between the two periods, thep chi-square test yielded a

212 213 , , 68 , , 139. 1980 , 149. Sept 162 1979 , 157 . , 745. 20 , 27 , 1968 J. Chromatogr. B 2001 J. Chromatogr. B 2006 Final , 297. 2006 Br.J.Clin.Pharmacol. 222 ENG , J. Pharm. Sci. Drug Testing Drug Analysis and List Clin.Pharmacol.Ther. 1981 ume 44: and Ergogenic (Eds: C. E. Cooper, R. Beneke), , http://www.wada-ama.org/ Forensic. Sci. Int. ommittee,ListofProhibitedClasses www.drugtestinganalysis.com Int. J. Sports Med. 2009. , pp. 109–123. Prohibited J. Pharmacol. Exp. Ther. , 387. 2008 , 2611. 12 J. Chromatogr. , am, T. Reilly, M. Powell, 81 , 2002 = 0.3809 2 1996 R Essays in Biochemistry, Vol = 0.3308 , 369. , 255. 2 , 62. R 877 760 57 , , , , 690. 1243. Clin. J. Sport Med. J. Appl. Physiol. rtecontent/document/2009 08.pdf (accessed 4th June 2009). of Substances and Prohibited Methods, Lausanne, 2009 2001 Aids to Improve SportPortland Press: Performance, London, hibited List, Montreal, 2004 R. C. Morris, E. J. Mcsherry,28 K. L. Melmon, g/ml) g/ml) µ µ [8] K.S.Chu,T.J.Doherty,G.Parise,J.S.Milheiro,M.A.Tarnopolsky, [9] H. Gillies, W. E. Derman, T. D. Noakes, P. Smith, A. Evans, G. Gabriels, ( ( [20] C. M. Lai, R. G. Stoll, Z.[21] M. Look, W. A. Van Yacobi, Thuyne, F. T. Delbeke, [10] G. Jones, in [12] IOC,TheInternationalOlympicC [13] K. Deventer, O. J. Pozo, P. Van Eenoo, F.[14] T. Delbeke, P. Van Eenoo, F. T. Delbeke, K. Roels,[15] P. De N. Backer, Chester, D. R. Mottr [18] Y. L. Tseng,[19] M. H. Shieh, L. F. Y. H. Lo, Kuo, G. Land, A. Bye, [11] WADA. The World Anti-Doping Agency, The 2009 Pro- [16] D. C. Brater, S. Kaojarern,[17] L. Z. Benet, G. R. Wilkinso, A. H. E. Beckett, T. Lin, T. Lockwood, , , , , 19 12 35 , , , 2009 John Wiley & Sons, Ltd. Br. J. 2002 ,3. c pseudoephedrine pseudoephedrine 24 C C 2006 , 2002 2003 ne samples (a) and excretion urine samples (b). 2003 Nutr. Res. Rev. 0 50 100 150 200 250 Med. Sci. Sports Exerc. 0 50 100 150 200 250 Clin.J.SportMed. 8 6 4 2 0

6 5 4 3 2 1 0

14 12 10

cathine cathine

g/ml) ( C g/ml) ( C µ µ Int. J. Sports Med. Med. Sci. Sports Exerc. (b) (a) , 329. 38 , , 205. 50 , 2006 , 209–213 Copyright 1 , 2000 2009 Correlation between PEPH and cathine in the routi , 344. 34 Med. Sci. Sports Exerc. 147. 209. 987. Clin. Pharmacol. [1] W. Van Thuyne, P. Van Eenoo, F. T. Delbeke, 7 .ogs .acc,KCrel .aitn A.E.Jeukendrup, B.Hamilton, K.Currell, S.Hancock, K.Hodges, [7] [3] R. Bouchard, A. R. Weber, J. D. Geiger, [2] D. G. Bell, T. M. McLellan, C. M. Sabiston, [4] I. Jacobs, H. Pasternak, D. G. Bell, [5] N. D. Gill, A. Shield, A. J. Blazevich, S. Zhou, R. P. Weatherby, [6] N. Chester, T. Reilly, D. R. Mottram, Urinary concentrations of pseudoephedrine and cathine Drug Test. Analysis References Acknowledgements Postdoctoral grants bySports the and Brussels Flemish (PVE, WVT Ministry andde KD) of and Educacion the Culture, Spanish y Ministerio Youth, Cienciadevelopmentofthemethodtoanalysetheexcretionurinesamples (OJP) are gratefullywas acknowledged. funded The by WADA. TheWim technical Van assistance of Gansbeke KrisDemey Roels were and greatly and appreciated. the administrative assistance of Trui Figure 3.