Rapid determination of anti-estrogens by gas chromatography/mass spectrometry in urine:Method valida

Journal of Pharmaceutical Analysis 2012; 2 (1): 1-11Contents lists available at Science directJournal of Pharmaceutical Analysiswww.elsevier.com/locate/jpawww.sciencedirect.comORIGINAL ARTICLERapid determination of anti-estrogens by gaschromatography/ mass spectrometry in urineMethod validation and application to real samplesE. Gerace"*,A. Salomone, G. Abbadessa, S. Racca, M. VincentiaecEntro Regionale Antidoping"A. Bertinaria Regione Gonzole 10/1, 10043 Orbassano, Turin, ItalydIpartimento di Chimica Analitica, Universita degli Studi di Torino, via P Giuria 5, 10125 Turin, tal *3 Orbassano, Turin, ItalyDipartimento di Scienze Cliniche e Biologiche, Universita degli Studi di Torino, Regione Gonzole 10/1, 100Received 29 July 2011 accepted 13 September 2011Available online 26 October 2011KEYWORDSAbstract A fast screening protocol was developed for the simultaneous determination of nine anti-Anti-estrogensestrogenic agents(aminoglutethimide, anastrozole, clomiphene, drostanolone, formestane, letrozole,Fast-GC/MSmesterolone, tamoxifen, testolactone)plus five of their metabolites in human urine. After an enzymaticUhydrolysis, these compounds can be extracted simultaneously from urine with a simple liquid-liquidvalidation:extraction at alkaline conditions. The analytes were subsequently analyzed by fast-gas chromatography/Breast cancermass spectrometry(fast-GC/MS)after derivatization. The use of a short column, high-flow carrier gasvelocity and fast temperature ramping produced an efficient separation of all analytes in about 4 min,allowing a processing rate of 10 samples/h. The present analytical method was validated according to UNIEN ISO/IEC 17025 guidelines for qualitative methods. The range of investigated parameters included themit of detection, selectivity, linearity, repeatability, robustness and extraction efficiency. High MSsampling rate, using a benchtop quadrupole mass analyzer resulted in accurate peak shape definitionunder both scan and selected ion monitoring modes, and high sensitivity in the latter mode. Therefore, theperformances of the method are comparable to the ones obtainable from traditional GC/MS analysis. Themethod was successfully tested on real samples arising from clinical treatments of hospitalized patients andcould profitably be used for clinical studies on anti-estrogenic drug administration9 2011 Xi'an Jiaotong University. Production and hosting by Elsevier B.V. All rights reservedorresponding author.Te:+3901190224249, mobile:+393393795125;fax:+39011924242E-mail address: enrico gerace@antidoping piemonte. it(E. Gerace).2095-1779 e 2011 Xi'an Jiaotong University. Production and hostingby Elsevier B v. All rights reservednsibility of Xi'an Jiaotong University.doi:l0.1016 j jha201l.09011中国煤化工CNMHGProduction and hosting by ElsevierE. Gerace et al1. Introductionsuccessfully analyzed to test the actual efficiency of theproposed methodAccording to their pharmacological properties, substances withti-estrogenic activity can be classified in aromatase inhibitors(aminoglutethimide, anastrozole, exemestane, formestane, letro- 2 Experimentalzole, testolactone), selective estrogen receptor modulators(raloxifene, tamoxifen, toremifene), substances with second2.1. Chemicals and reagentantiestrogenic substances (clomiphene, cyclofenil, fulvestrant). MethanoL, A-butyl methyl ether(T BME)and various inorganicThe therapeutic prescription for aromatase inhibitors is the salts were supplied by Riedel-de Hae]n(Seelze, Germany)treatment of metastatic breast cancer in post-menopausal women, B-glucuronidase(from Escherichia coli) and dithioerythritolbecause the inhibition of estrogens biosynthesis is supposed to were from Sigma-Aldrich(Milan, Italy). N-Methyl-N-tri-prevent the growth of tumor tissuc [1-3]. Selective estrogennethylsilyltrifluoroacetamide(MSTFA)was obtained fromreceptor modulators(SERMs) exhibit a mixed pharmacologiMacherey-Nagel(Duren, Germany)Aminoglutethimide. clomiphene citrate (57% cis form andand estrogen antagonist activity in others. SERMs have many 43%rans form), formestane and 17a-methyltestosterone werearmacological applications, and are currently pre purchased from SigmaAldrich (Mlan, Italy): drostanolonescribed for women with breast cancer(tamoxifen, toremifene)or ( 17B-hydroxy-2a-methyl-Sa-androstan-3-one), mesterolone(17B-rom steraloidspausal women with a risk of asteoporosis (raloxifene)4.5 (Newport, RI, USA) drostanolone metaltabolite(3a-hydroxy-Clomiphene and cyclofenil have stimulating effects on the secre- 2a-methyl-Sat-androstan-17-one), mesterolone metabolitestion of hypophysial gonadotropic hormones and are mainly used (la-methyl-5x-androstan-3a, 17B-diol, 3a-hydroxy-la-methyl-.in the treatment of infertility (6,7.a-androstan-17-one)were from LGC Promochem (MilanDrostanolone and mesterolone are mainly consideredItaly ), testolactone was from US Pharmacopeia(Rockville,anabolic agents, but they also show some secondary anti. MD, USA). Tamoxifen citrate was kindly supplied by SOL-estrogenic effects [8]MAG(Garbagnate, Italy): letrozole was kindly provided fromMoreover, anti-estrogenic agents can be illicitly used in Novartis Pharma(Basel, Switzerland) and anastrozole wassport as they are able to treat and prevent the side effects kindly supplied by AstraZeneca(London, UK)produced from anabolic androgenic steroids misuse [9]Bis-4-cyanophenylmethanol (a letrozole metabolite),4-Accordingly, the use of anti-estrogenic agents is prohibited hydroxy-clomiphene (a clomiphene metabolite) and 4by World Anti-Doping Agency(WADA)regulations for both hydroxy-tamoxifen(a tamoxifen metabolite)were obtainedmale and female athletes in- and out-competitions [10).from"in vitro" phase I metabolism from rat liver microsomalDifferent conventional approaches, carried out on biologi- fraction(30-32 . All reagents were supplied by Sigma-Aldrichcal fluids(blood and urine ), were reported for the detection of (Milan, Italy)anti-estrogens, including immuno-analytical techniques [ll].Stock standard solutions were prepared in methanol at aand chromatographic methods [12, 13]). Since the consumption concentration of l000 ug/ml and were stored at -20C untilof these compounds by the athletes is prohibited in sport used. All solution and bufFers were prepared using deionizedevents, several screening procedures were developed for the water obtained from Milli-Q System(Millipore Corporatedetection of anti-estrogens in human urine for anti-doping Headquarters, Billerica, USA)purposes. Several GC/MS screening procedures applied toanti-estrogenic agents were developed [14-17 even if an 2.2. in vitro synthesis of phase I letrozole, clomiphene andincreasing number of procedures based on LC/MS have been tamoxifen metabolitespublished [18-22]. Anti-estrogenic agents are often screenedtogether with other class of substances, mainly using LC-MS/ 2. 2. 1. In vitro metabolic assaysMS procedures, usually more sensitive than GC/MS onesLiver microsomal fraction was prepared as described else-Fast GC/MS is gaining importance in routine analytical where [31]. Stock solutions( 500 uM)of each substrate testedlaboratories, since it improves sample throughput and global for in-vitro metabolic activity (letrozole clomiphene andproductivity, as is necessary in laboratories where a consider- tamoxifen)were prepared in DMSO. Reaction mixtures wereable number of daily samples have to be processed [23, 24]. The prepared in a test tube by adding 225 uL of 0.1 M Tris HCItime needed for instrumental determination can be drastically buffer(pH 7. 4), 225 uL of a microsomal suspension(approxi-shortened using short and narrow columns(5-10 m, i.d. 0.05- mately 2 mg of proteins), 2000 HL of NADPH generating0 I mm), fast temperature ramping and high carrier gas flow system(consisting of 0.32 mM NADPH, 6.4 mM glucose-6-rate as recently demonstrated [25-29)phosphate, 0.5 U/mL glucose-6-phosphate dehydrogenase,Aim of the present work was to develop an analytical 5 mM MgCl, solution and 0.8 mM of EDTA solution)andprocedure addressed to the fast screening of fourteen anti- 50 uL of 500 uM stock solution containing the tested subestrogenic drugs and metabolites(Fig. 1), using fast-GC stratemivtmree were inrubated at 37C underinstrumentation hyphenated to electron ionization mass spec- gentle中国煤化工 reaction was stoppedtrometry. Further"in vitro"metabolic experiments using the by czed substrate suspen-microsomal fraction from rat liver were carried out in order to sionsCNMHGd. The extraction ofsupply us the reference material for a larger panel of metabolism products from the resulting suspensions wasmetabolites. Lastly, urinary samples from patients under carred out following the same procedure used for the urinereatment with anti-estrogenic drugs were collected and samples and described as followsRapid determinatiourine by GC/MsdmiaamgCHQMesterolone metflame-Sa-anduatan-/po-on) (fa-mrinf- d-ndhoea /B-sod) tla-meth-a-untuatonBao-IFigure 1 Chemical structures of the anti-estrogenic agents considered in this study2. 3. Characterization of"in-vitro"metabolites2.4. Sample preparationThe substrates obtained by"in vitro"metabolic assays were The sample preparation introduced minor modifications fromcharacterized by GC/ MS after solvent extraction and tri- the standard operating procedure described by Donike et al. [33]methylsilylation, as described in the subsequent paragraph. for the detection of anabolic steroids. Urine samples(3 mL)wereGC MS analyses were performed using an Agilent 6890N/ added with 15 uL of a 10 ug/mL methyltestosterone solution.5975 instrument(Agilent Technologies, Milan, Italy) with a used as the internal standard(IS), and then were buffered to pH17 m HP-I fused silica capillary column(J&W Scientific). with 7.4 with 2 mL of a 0.I M phosphate buffer (prepared byi.d. 0. 20 mm and film thickness 0. 1l um. The carrier gas was dissolving 4.63 g of KH POA and 11.75 g of Na2HPO4 2 H2Ohelium(fow rate I mL/min, split ratio 1: 10). The oven in I L of water). 30uL of B-glucuronidase from E. coli weretemperature program was: isothermal at 180C for 3 min, subsequently added and the mixture was incubated at 55C forheating rate of 5 C/min up to 240C, then 10C min tohne mixtures were270C, and lastly 40"C/min to 320"C (isothermal for 3 min).coo中国煤化工0 M carbonate buffThe temperatures of transfer line, injection port and Ms (prepdetector wereall set at 280C. Acquisition was carried out in ICNMHGand 6.72 g of NaHCOin full scan mode from m/z 50 to mj/ 550. The correct identity extraction( LLE)was performed by adding 10 mL of TBME andof metabolites was determined by both mass spectral inter- shaking in a multimixer for 10 min. After centrifugation atpretation and comparison with published spectra2200 rpm for 3 min, the organic layer was transferred into a testtube and dried under nitrogen at 70C. The dry residue was investigated compound, at the retention time interval expectedderivatized with 50uL of a MSTFA/NH,l,dithioerythritol for their elution(1000: 2: 4. v/w/w) solution for 30 min at 70C. An aliquot ofI uL was injected into the fast-GC MS system2.6.2. LinearityThe linear calibration model was checked by analyzing(three2.5. Fast-GC/MS analysisreplicates) negative urine samples spiked with standard solu-tions at six concentration levels for each analyte. The linearFast-GC/MS determinations were performed using a 6890N calibration parameters were obtained using the least squaresC,combined with a 5975 inert Mass Selective Detector regression method, while the correlation coefficients(R)were(Agilent Technologies, Milan, Italy), with electron ionization utilized to estimate linearity. Quantitative results from areaat 70 eV.A 5 m fused-silica capillary column(Supelco"Equity counts were corrected using the IS signal.1, with i.d. 0 I mm and film thickness 0. ll um was utilizedfor GC separation. The final analytical solution (I HL) was 2.6.3. Limit of detection(LOD)injected into the fast-GC system operating in the split mode LOd values were estimated as the analyte concentrations(5: 1).Helium was employed as carner gas at constant pressure whose response provided a S/N value equal to 3, as deterof 33.21 psi. The GC oven temperature was isothermally set mined from the least abundant among qualifier ions. LODat 180"C for 0.5 min, then initially raised to 220C with a numerical values were extrapolated from S/N values at the35'C/min heating rate and subsequently to 310C with a rate lowest concentration level (LCL)using the correspondingof 60C/min, followed by a isothermal period of I min at calibration curve. The noise was measured from -0.05 min310C. The total run time was 4. 14 min, while the overall before the peak onset till the beginning of the GC peak forcycle time, including oven cooling and thermal equilibration each analyte. Extrapolated LOD values were experimentallywas about 6 min. The GC injector and transfer line were confirmed by studying urine samples spiked with all analytesmaintained at 280C. Data were acquired in the selected-ion at LODs concentrationsmonitoring(SIM)mode at dwell time of 15 ms, using for eacompound detection the diagnostic ions listed in Table I2.6.4. Extraction recoveryExtraction recoveries were calculated by comparing two26. Method validationexperimental sets of data. In the first set, ten blank urinespiked before the extraction2.6.1. Selectivityanalytes at 50, 250 and 500 ng/mL final concentration, exceptTen different blank urine samples were deconjugated, for testolactone added at 250, 500 and 1000 ng/mL concentra-extracted, derivatized and analyzed as described above. The tion. In the second set, ten blank urine samples were spikedoccurrence of possible interferences from endogenous sub- after the extraction step, with standard working solutions. atstances or derivatization byproducts was tested by monitoring the same final concentration of 50, 250 and 500 (or 1000)ng/mLthe selected-ion chromatograms, characteristic for each Recovery (%)was calculated as the ratio between theTable 1 Molecular weights of the 14 analytes and their TMS-derivatives, GC retention times, characteristic ions(El mode)andesponding retention time windows used in SIM experimentsAnalyte No, of TMs TMS derivatives Rt (min) Ions(m/z)ETRT timemolecular groupsmolecular weightwindow(min)weightTarget Others1.90293,2661.80240Letrozole metabolite194217291,190180-240Aminoglutethimide520,5051.80-240448,343240254Mesterolone met (3a-oI-17-one)448,343240-2.54Mesterolone met(3a, 17B-diol)3712.595872,372.54266Mesterolone157,4332.54266Drostanolone405.1412542.66ISTD446301,431Testolactone中国煤化工2028Clomiphene(two isomers)CNMHG 2884 13Clomiphene metabolite3.33100,493288413Reference material obtained by in vitro metabolism experimentsapid determination of anti-estrogens in urine by GC/MSresponse(peak area)obtained from the two separate series of ion of main clomiphene metabolite, namely 4-hydroxy-clomiphene, is detected from its mono-tMs derivative at m/z 493(2%): cleavage of the C-C bond in a-position to the amino2.6.5. Repeatability (intra-assay precision)group leads to the fragment at m/z 86(base peak), whileIntra-assay precision (repeatability), expressed as percent cleavage of the C-C bond in B-position to the amino groupvariation coefficient (CV%), was assessed by extracting and yields the fragment at m/z 100(8%)analyzing ten replicates of negative urine samples, spiked withHydroxylation of the aromatic ring also occurs in thethe standard solutions at two concentration levels (final tamoxifen metabolism, yielding 4-hydroxy-tamoxifen [36, 37concentrations of 50 ng/mL and 500 ng/mL for each analyte), The major fragmentation of its mono-TMS derivative isperformed by the same operator Standard criteria taken from determined by the localization of the positive charge on thethe literature [34] designate satisfactory intra-assay precision amino-group, yielding both the base peak at m/z 58(basefor qualitative screening methods when CV% values are below peak)and the major fragment at m/z 72(39%)resulting from5% at 500 ng/mL concentrations and below 25% at 50 ng/mL the cleavage in a- and p-position with respect to the aminoconcentrationsgroup. The presence of the hydroxyl group on the tamoxifenstructure is testified by the metabolite molecular ion at m/z2.6.6. Robustness459(8%)The method robustness was evaluated by changing theoperators involved in both preparative and instrumental 3. 2. Derivatization productsphases of analysis, as well as the batch of chemicals andreagents employed in the sample treatment and the spiking Most anti-estrogenic agents and their metabolites listed invel of several urine samples. For each analyte, compliance of Fig. I contain polar groups in their structures, that make themtion. Therefore, the development of an efficient GC/MSprotocol, for identifying anti-estrogen in biological matrices2. 7. Real urine samplesrequires preliminary derivatization of these polar groups.Trimethylsilylation makes the analytes more volatile andThree urine samples from women suffering from breast produces a molecular weight increase of 72 u, for each reactivemetastatic cancer and under treatment with Arimidex"(ana- hydrogen. For steroids and anti-estrogenic agents such asstrozole), Femara"(letrozole)and Nolvadex(tamoxifen drostanolone and mesterolone containing more than one polarcitrate)were collected and analyzed. Aliquots of 30 mL of group MSTFA is proved to be the most efficient agent to giveurine were collected and stored at-20C until analysis. All TMS ethers, since it produces single fully substituted deriva-selected patients provided a fully informed permission for the tives nearly without side-products, whose significant massscientific use of these analyses.spectra contain diagnostic target ions, ideal for screeningpurposes [15, 16, 25,3513. Results and discussionMSTFA reacts with all hydroxyl and keto groups of anti-estrogenic agents, and amine anorogens3. 1. GC/MS characterization of phase I letrozole, clornoglutethimide, to give their tris-TMS and tetrakis-TMSand tamoxifen metabolitesderivatives, respectively. The absence of exchangeable hydro-gens on anastrozole, clomiphene and tamoxifen does notThe derivatized extracts obtained from " in-itro metabolic cular weights of the original anti-estrogenic agents and theirassays yielded relatively clean chromatographic profiles from derivatives, together with the number of TMS-groups intro.which the identification of the starting drugsetabolic products was straightforward. However, the experi-duced by the derivatization, are reported in Table Imental mass spectra obtained from these metabolites werecompared with literature spectra, for a correct structural 3.3. Fast GC/MS characterizationdentity assignment [15,351Fig. 2 shows the total-ion chromatograms, mass spectra Full scan electron ionization mass spectra of all parent drugsand fragmentation patterns for the derivatized metabolites were recorded from reference standards. in order to determineof letrozole(bis-4-cyanophenyl-methanol ), clomiphene (4- the most appropriate SIM protocol for accurate and sensitivehydroxy-clomiphene)and tamoxifen(4-hydroxy-tamoxifen), detection of these target analytes. Likewise, full scan massobtained from"in vitro"metabolic experimentsspectra for target metabolites were recorded from in vitrohe bis-4-cyanophenyl-methanol is the main inactive meta- metabolic mixturesbolite of letrozole[15]. The mono-TMS derivative shows the A previous study [15] including GC/MS experiments onmolecular ion at m/z 306(relative abundance of 2%)and a letrozole reference standards found that the detection of thishigh-mass fragment m/ 291(56%), due to the loss of a methyl compound is difficult, due to its poor chromatographicradical from the TMS residue. The fragment at m/z 217(base properties using a standard polysiloxane column. We encoun-k)isted by the trimethylsiloxy-radical climination, tereroblem and decided to detect letrozole fromhile a subsequent rearrangement leads to the m/z 190 ion its中国煤化T: yanophenylmethanol(44%)by HCN loss-difficultiesHydroxylation of the aromatic ring represents the maCN MH Gracteristic ions(quali-hase I metabolic route for clomiphene [31]. The molecular fier ions vS. target ion)along the gC peak were used for theE. Gerace et aLA6070809010011012013014015.016.0170BTime(min)TMSO17Time(minC中国煤化工CNMHGFigure 2 GC/MS-SIM chromatograms of the derivatized extracts obtained from"in-vitro"metabolic assays, mass spectra and proposedfragmentations pattern of letrozole(A), clomiphene( B)and tamoxifen( C)metabolitesRapid determination of anti-estrogens in urine by GC/Ms7positive identification of each target compound together with 3. 4.2. Linearity and limit of detectionthe coincidence of its retention time with the expected one. Al The range of concentration studied was planned according to thetarget analytes were easily characterized by different frag- approximate response factors obtained from the preliminaryments. The SIM protocol developed proved efficient in experiments with standard solutions. All calibration curvesdiscriminating the interferencesproved linear as demonstrated by correlation values comprisedTable I reports retention times and selected ions, used for between 0.990 and 0.999. Calibration curves are not available forthe identification of anti-estrogenic agents or their trimethyl- clomiphene, letrozole and tamoxifen metabolites because theirsilyl derivatives in the El modepurity and exact concentration are not known with certainty. TheFig. 3 shows the fast-GC/MS chromatogram of a mixture experimental data relative to calibration curves. limits of detectionof analytes at the calibration level concentration of 250 ng/ mL and S/N values for all target analytes are reported in Table 3.obtained in the SIM mode. It is noteworthy that the entire LOd values obtained for most target compound were found atGC/MS run was completed in about 3 min. Two more minutes 10 ng mL, including the ones for formestane, tamoxifen, drostawere needed for oven cooling, temperature equilibration and noone, mesterolone(and their metabolites). while few LODinjection. Satisfactory chromatographic separation of all values(15 45 ng/ mL) slightly exceeded this limit except the onetarget analytes was obtained including clomiphene isomers. for testolactone that was 200 ng/mL.Even if a fast gradient was applied for the chromatographic a blank urine sample, spiked with the target analytes at theseparation,the high MS-sampling rate, using a modern LOD concentrations indicated in Table 3, was analyzed inbenchtop quadrupole mass analyzer, guaranteed a minimum triplicate. All experimental S/N values observed exceeded theof 10 points across the chromatographic peak without sig. critical value of 3, as expectednificant loss of information. The peaks were adequatelydescribed allowing a good integration reproducibility, as 3.4.3. Extraction recoverydemonstrated by satisfactory CV% values reported in Table 2. Extraction recoveries were calculated by comparing the resultsThe SIM protocol described in Table I was used to build from blank urine samples spiked before the extraction stepthe calibration graphs for all analyteswith the ones from negative urine samples, which were first3.4. Method validationextracted and then spiked, before the analysis. The resultsobtained at 50 ng/mL concentration are reported in Table 2.Most recovery values ranged from 102.7%(mesterolone)toAccording to Iso 17025 requirements and ICH guidelines 83. 7%(mesterolone metabolite-3a, 17B-diol ) with the excep.18]. all validation parameters useful to evaluate the overall tion of testolactone and drostanolone metabolite(3a-o1-17.performance of a qualitative analytical method were investi- one)showing average recovery efficiencies of 40.6%andgated, including selectivity, linearity, limit of detection, recov- 35.3%, respectively. For the latter. the limited extractionery. repeatability and robustnessrecovery that we observed is refected in literature data [34].while no previous data are available for liquid-liquid testo3.4.1. Selectivitylactone extraction efficiency. It should be noted that thelon-chromatograms from 10 negative urine samples showed extraction recoveries were quite stable, even for the twono interfering signals (i. e. S/N ratio minor than 3) at the compounds with lower efficiency, as is demonstrated byretention time where each analyte is expected to elute. This CV% values, all ranging between 10% and 18%. Taking intodemonstrated that the method is selective for all 14 anti- account that a screening method for multiple target analytes isestrogenic agents and free from positive interference from not likely to provide homogeneously high extraction recov-urine components and column bleedingeries and that a 35% recovery is acceptable for a qualitativescreening, provided good extraction repeatability is obtained,it can be concluded that the observed extraction efficienciesare perfectly compatible with the screening purpose of thepresent method. Extraction efficiency data obtained at higherconcentrations(250 and 500 ng/mL) confirmed the results presented in Table 3, with a slight decrease of most recovery values.For example, at the concentration of 500 ng/mL, recoveryefficiency values were comprised between 101. 4% of formestaneand 72.9% of aminoglutethimide as tris-TMs derivativesRecovery value for drostanolone metabolite(3a-ol-17-onespiked at the highest concentration dropped to 20.3%, while儿for testolactone no important difference with the result obtained12022242620at lower concentration was observed (40.0%)Figure 3 Fast GC/MS-SIM chromatogram of a blank urine 3.4.4.sample fortified with all target compounds at the concentration of The中国煤化工 dence of250 ng/mL:(I)anastrozole. (2)aminoglutethimide. (3)drostano. tionCNMH Gof characteristic ionslone metabolite(3a-o1-17-one ), (4)mesterolone metabolite(3a-ol- weretatu tests. The repeatability17-one),(5)mesterolone metabolite(3a, 17bdiol), (6)mesterolone, proved satisfactory for all the target analytes. as the coefficient(7)tamoxifen (8)drostanolone, (9)testolactone, (10)formestane, of variation percentage(CV% )was always abundantly lower(ll)clomiphene(two isomers).than 25% for the samples spiked at 50 ng/mL, and lower thanE. Gerace et alTable 2 Percentage recovery (%)and repeatability (intra-assay precision) (Cv%)for each analyte tested. The metabolites fromin-vitro assays are not includedDetected analytesExtraction efficiencyRepeatabilityConc (ng/mL)Recovery (%)CV%时tCv% at50 ng/mL500 ng/mL89.2Aminoglutethimide(3TMS)Aminoglutethimide(4TMS)0000000l1920.292817.7102Drostanolone met(3a-ol-17-one)120Mesterolone met(a-ol-17-one)85813.0Mesterolone met(3a, 17B-diol12.3TamoxifenMesteroloneDrostanolone22Testolactone40697.91.5Clomiphene(two isomers)9398.791512.28.195Table 3 Calibration intervals: gradients, intercepts and R values obtained for calibration curves; LOD and S/N atCalibration Level(LCL)values for the TMs derivatives of the investigated 1l anti-estrogenic agents. The metabolitesfrom in-vitro assays are not includedCalibration levellope intercept RLOD (ng/mL) S/N at LCLconcentration(ng/mL)10010000.595Aminoglutethimide(3TMS)50-10000.0401099725Aminoglutethimide(4TMS)00413001790990Drostanolone met(32-0l-1725-100003440.123Mesterolone met (32-oF-17-one25-100004850.122099710007250.9979.01.22-0.0466099900007.5Mesterolone7.5DrostanoloneTestolactone0004600106099Formestane0.10209Clomiphene(two isomers)25-1000099925-10000.l25212% for the samples spiked at 500 ng/mL. The repeatability drug. Chromatograms obtained by fast-GC/MS analysis offor testolactone was obviously tested only at 500 ng/mL.three real urine samples from patients under treatment withNo significant variation of retention times, relative ion different anti-estrogenic agents are reported in Fig. 4. In theabundance or limits of detection were observed by changing first urine sample(Fig. 4A) the estimated concentration ofthe operator the instrument (same model), the chemical anastrozole was 98 ng/mL. GC plots in the first column showbatches, the stock standard solutions and the sample volume. the superimposed profiles of the selected ions used for theIn all experiments. no statistically significant variations of different drugs identification. Comparison with the GC graphsdetected concentrations were obtained (data not shown)represented in the third column, and relative to the real sampies,provide clear evidence that the relative ion abundances areerfectly reproduced throughout each GC peak profil3.35. Real urine samplesselecte renter in the central column中国煤化工 ed is selective and noReal urine samples were collected during outpatient medical interfexamination, not allowing a strict control of the time elapsed ThCNMHGdjor their metabolitesfrom the last drug administration. The patients were being were positively detected in real urine samples from patientstreated with tamoxifen, anastrozole and letrozole. All the under pharmacological treatment, showing that the presentedsamples examined turned out positive for the administered method can find practical application for clinical purposesRapid determination of anti-estrogens in urine by GC/MSReference standardBlank urIneRealRT1904sampleM nTime(min)Tnm的Time(min)BReference standardBlank urineReal samp1.05227m/217mimI22?14Time(min)Reference standard130000Real sampleRT27452752mh 5.m459Figure 4 Selected-ion chromatograms of extracted urine samples obtained from patients treated with(A)anastrozole. B)letrozole and中国煤化工CNMHGInstrIse from the introduc.tion of fast-gas chromatography. Although the anaThe continuous cffort to increase the analytical throughput in methods based on liquid chromatography combined withbiomedical determinations and reduce the time needed for the multiple stage mass spectrometry are gaining increasingE. Gerace ct alpopularity with respect to GC/MS, just because they allow (6]S. Goto, K. Takakura, K. Nakanishi, et al. Efficacy of clomi-more rapid determinations, the extensive application of fastphene citrate and cyclofenil for infertile women with normalGC/ MS procedures is likely to hold back this trend, byulatory cycles, Fertil. SteriL. 76(2001)409-411considering the large difference in price, the diffuse experien[7 H.R. Maeda, M. Seki, K. Seki, et al, Effects of clomipheneexisting in the GC-based analytical methods and the absencefeil, epimestrol and ro 48347 on pituitary and serumof signal suppression/enhancement phenomena, occasionallyprolactin in mature female rats, Endocrinol. Jpn. 19(1972)occurring in electrospray ionization when complex biolo525-532.ed by lc/M[8]L. Marinov, V. Tsekova, K. Koino, et aL, Drostanolonepropionate(materil) in disseminated breastIn womenAnalytical methods have to respond to the requirementsImmediate results, Khirurgiia(Sofia)40( 1987)80-86established by the specific inquiry under way. While in anti- F9)DJ. Handelsman, Clinical review: the rationale fordoping screening analysis a common goal is to identify the largesten blockersnumber of prohibited substances with the lowest number ofJ Clin Endocrinol Metab. 91(2006)1646 1653analytical methods, independently from their pharmacological [10] World Anti-Doping Agency. The 2010 Prohibited List, Availableeffects, epidemiology studies are frequently addressed to theat(http://www.wada-ama.org/documEnts/world_anti-doping.detection of a certain class of drugs. Such inquires may takeProgram/WADP-Prohibited-list/WADA Prohibited_List_ 2010advantage of analytical methods specifically designed tor the class C.U. Pister. M. Duval, Godbillon, et al. Development,ofepidemiologic study is possibly of interest, as these compoundsethod for the deterare daily employed for clinical purposes(mainly in the treatmentmination of the aromatase inhibitor CGS 20, 267 in biologicalof breast cancer or masculine and feminine infertility) or illicitlyfuids, J Pharm. Sci. 83(1994)520-524used by athletes in order to treat or prevent the side effects [12F Marfil, V Pineau, A. Siouf, et al, High-performance liquidassociated with anabolic androgenic steroids abuschromatography of the aromatase inhibitor, letrozole, and itsFast-GC/MS analysis, performed on narrow bore columns atmetabolite in biological fluids with automated liquid-solid extrac-igh heating rates, provides significant reduction of the timetion and fluorescence detection, J. Chromatogr. B: Biomed. Sci.required for the analysis of these anti-estrogenic agents, whileAppl.683(1996)2581-2588maintaining chromatographic separation, limits of detection. and [13]B. Beere, Bert, A Oberguggenberger et a, Developmentrepeatability comparable with those obtained by conventionalnd validation of a liquid chromatography-tandem mass spectro-GC. This makes the method particularty suited for routinemetry method for the simultaneous quantification of tamoxifen,anastrozole and letrozole in human plasma and its application toanalysis, especially when a considerable number of samples haveto be analyzed, as about 10 injections/h could be performed. [14]H. Baez, C Camargo, H Osorio, et al, Detection of tamoxifenThe method proposed in the present study is selective formetabolites by GC MSD, J Chromatogr. Sci. 42(2004)551-553.the detection of anti-estrogenic agents in human urine. The [15] U. Mareck. G. Sigmund, G Opfermann. et al. Identification ofmethod appears to be sufficiently fexible to include newthe aromatase inhibitor letrozole in urine by gas chromatogra-substances(and metabolites) of the same class that mayphy/mass spectrometry, Rapid Commun. Mass Spectrom. 19appear on the market in the future. Moreover, since fast-GC/MS technique allows to lower the time needed for method[16]U. Mareck, G. Sigmund, G Opfermann, et al, Identification ofvalidation, a future extension of the present method on bloodthe aromatase inhibitor aminoglutethimide in urine by gassamples could be rapidly performedchromatography/mass spectrometry, Rapid Commun. MassSpectrom16(2002)22092214[17 P. Gartner, K. Hofbauer, C. Reichel, et al., Synthesis andAcknowledgmentsidentification of hydroxylated metabolites of the anti-estrogenicgent cyclofenil, J. Mass Spectrom. 43(2008)958-964We thank Astra Zeneca(London, UK)for providing anastro[18]M. Kolmonen. A. Leinonen, A. 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