Implementation and performance of the gas chromatography

Subscriber access provided by BOSTON UNIV

Article

Implementation and performance of the gas chromatography/ combustion/isotope ratio mass spectrometry-based method for the confirmatory analysis of endogenous anabolic steroids during the Rio de Janeiro Olympic and Paralympic Games 2016 Fábio Azamor de Oliveira, Alessandro Casilli, Thomas Piper, Thais Reis da Silva, Cristiane Abrantes da Silva, Raquel Vieira Santana da Silva, Marco Aurelio Dal Sasso, Gutierri Ricardo dos Santos Gonçalves Salgueiro, Monica Costa Padilha, Henrique Marcelo Gualberto Pereira, Mario Thevis, and Francisco Radler Aquino Neto Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b02341 • Publication Date (Web): 19 Aug 2019 Downloaded from pubs.acs.org on August 19, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 11 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Analytical Chemistry

Implementation and performance of the gas chromatography/combustion/isotope ratio mass spectrometry-based method for the confirmatory analysis of endogenous anabolic steroids during the Rio de Janeiro Olympic and Paralympic Games 2016 Fábio Azamor de Oliveira†,*, Alessandro Casilli†, Thomas Piper‡, Thais Reis da Silva†, Cristiane Abrantes da Silva†, Raquel Vieira Santana da Silva†, Marco Aurelio Dal Sasso†, Gutierri Ricardo dos Santos Gonçalves Salgueiro†, Monica Costa Padilha†, Henrique Marcelo Gualberto Pereira†, Mario Thevis‡, Francisco Radler de Aquino Neto† Federal University of Rio de Janeiro – UFRJ, Brazilian Doping Control Laboratory – LBCD, LADETEC, Av. Horácio Macedo, 1281 – Polo de Química – Bloco C – Cidade Universitária – Ιlha do Fundão – Rio de Janeiro, Brazil ‡ German Sport University Cologne, Center for Preventive Doping Research – Institute of Biochemistry, Am Sportpark Müngersdorf 6, 50933 Cologne, Germany †

ABSTRACT: Carbon isotope ratio (CIR) confirmation is one of the most complex and delicate analysis in doping control field, due to the nature of the molecules to be confirmed, normally present in urinary samples as a consequence of an endogenous production. The requirements for method validation established by the World Anti-Doping Agency (WADA) have been pushing the accredited laboratories to improve their methods. The choice of the method is always a cost benefit ratio involving a hard-working and timeconsuming analysis and the guarantee of reporting of reliable results. This work presents the method fully validated by the Brazilian Doping Control Laboratory as part of the preparation for the Rio de Janeiro Summer Olympic and Paralympic Games 2016. Sample preparation encompassed solid phase extraction, liquid-liquid extraction, enzymatic hydrolysis, acetylation and purification by preparative high-performance liquid chromatography, and analyses were performed by gas chromatography/combustion/isotope ratio mass spectrometry. This proved to be a robust method to CIR confirmation in a big event, as demonstrated by the analysis of 179 samples during the Games 2016, from clearly negative results and adverse findings for testosterone (T) and related substances, boldenone and metabolite, 19-norandrosterone and formestane. Two atypical findings were also reported for T and metabolites.

Since 2004, carbon isotope ratio (CIR) determination by gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) is required by the World Anti-Doping Agency (WADA) as the technique to unequivocally confirm suspicious cases of abuse of endogenous steroids by athletes1. Three years later, this technique was introduced to the laboratory accredited by WADA in Rio de Janeiro, Brazil, as part of the preparation to the Pan American Games 20072. The Technical Document (TD) from WADA then in force to regulate CIR confirmation was the first version, published in 20041. The second major event held in the Rio laboratory, then named Laboratório Brasileiro de Controle de Dopagem (LBCD), were the Rio 2016 Summer Olympic and Paralympic Games (O&PG) doping control3. The preparation for CIR analysis were then based on a more complete version of the TD4, which after two revisions represented a more robust although complex and challenging guidance for method validation. Another TD, dedicated to confirm 19-norsteroids abuse, had a new version coming into force in September 20165, between the O&PG, so the implementation of the modifications was anticipated during method validation. These TDs were updated later, without major changes compared with the former versions.

Urinary samples collected from athletes in or out of competition undergo screening procedures, among which the steroid profile evaluation, consisting of the estimation of absolute and relative concentrations of endogenous steroids involved in testosterone (T) metabolism6. These parameters are compared with population-based reference limits and CIR confirmation is required if any criterion is met, thus portraying a suspicious steroid profile (SSP). Additionally, the longitudinal evaluation of the steroid profiles taking into account subject-based threshold ranges has been elaborated in the last decades, culminating in the implementation of the steroidal module of the athlete’s biological passport (ABP) with steroid profiles reported on the Anti-Doping Administration and Management System (ADAMS). This strategy became compulsory since 2014 in order to detect atypical passport findings (ATPFs) and to trigger CIR confirmation7,8. So Rio 2016 was the first edition of the O&PG where the steroidal ABP was run in fullness. Apart from the steroid profile, screening procedures also detect exogenous prohibited substances or metabolites in athletes’ samples, estimate their concentration when required and indicate the necessary confirmatory procedure to each case.

ACS Paragon Plus Environment

Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Human body produces natural T and its precursors via cholesterol metabolism. This metabolic pathway involves steroids presenting widely known androgenic and anabolic properties9,10. T itself and its metabolites androsterone (A), etiocholanolone (Etio), 5α-androstane-3α,17β-diol (5α-diol) and 5β-androstane-3α,17β-diol (5β-diol) are the target compounds (TC) required to be analyzed in order to confirm whether a SSP or ATPF is due to an administration of a synthetic homologue of an endogenous steroid4. Epitestosterone (E) shall be analyzed whenever a steroid profile presents an elevated concentration of this substance, what might be a consequence of the intake of synthetic E combined with T or a prohormone, aiming to mask the T/E ratio, which triggers CIR confirmation when greater than 4.0. Boldenone (B) is an exogenous anabolic steroid, while 19-norandrosterone (19NA) is the main metabolite of anabolic 19-norsteroids, and formestane (F) is a steroidal aromatase inhibitor. None of these are commonly found in humans’ urine but, for various reasons, endogenous formation may occur, thus making mandatory CIR confirmation whenever one of these analytes is found in urine within a respective specific concentration range4,5,11–18. 5pregnane-3,20-diol (PD), 11-ketoetiocholanolone (11K), 11β-hydroxyandrosterone (OHA), 5α-androst-16-en-3α-ol and other substances are analyzed as endogenous reference compounds (ERCs), since they are not affected by an administration of steroids of synthetic origin4. A can be used as an ERC to 19NA analysis5, unless in case of co-administration of T (or its precursors). CIR determinations are expressed as 13C values, in per mil (‰), against the international standard Vienna Pee Dee Belemnite (VPDB)19. The CO2 used as a reference gas is calibrated against a secondary certified reference material (CRM) traceable to the VPDB scale, which mandatorily consists in a mixture of certified steroids4. Isotopic fractionation can occur during sample preparation, as a consequence of uncontrolled chemical reactions or due to losses during fraction collection in HPLC cleanup steps20,21. Thus, quality controls are mandatory in order to guarantee system stability and the overall suitability of the sample preparation. Furthermore, accurate CIR determinations rely on properly maintained GC/C/IRMS instruments22,23. CIR values of the endogenous steroids are strongly affected by eating habits, thus varying significantly from a population to another. In Piper’s et al.24 work, Finnish individuals produced endogenous steroids presenting the most negative CIR values (around -23 ‰), which means molecules relatively more depleted in 13C, while steroids from South American people presented the most enriched contents of 13C (and higher CIR values, around -17 ‰). The goal of this paper is to describe the procedures adopted for the full implementation of the method for CIR analysis used during Rio 2016 O&PG, discussing the results and outcomes. EXPERIMENTAL SECTION MATERIALS. Methanol (MeOH) GC grade, acetonitrile (ACN) HPLC grade, ethyl acetate GC grade, t-butyl methyl ether (TBME) HPLC grade and anhydrous pyridine were purchased from Tedia (Fairfield, OH, USA). Acetic anhydride ACS grade was from Spectrum Laboratory Products, Inc. (Gardena, CA, USA). Bond Elut C18 cartridges (500 mg, 3 mL) were from Agilent Technologies, Inc. (Folsom, CA, USA). -

Page 2 of 11

glucuronidase from Escherichia coli was from Roche Diagnostics GmbH (Mannheim, Germany). Steroid RMs PD, 11K, T and OHA were purchased from Steraloids Inc. (Newport, RI, USA). E, A, Etio, 5α-diol, 5β-diol, 19NA, 19NA glucuronide (19NA-G), F and 5-androst-1-en17-ol-3-one (boldenone-M1, BM1) were obtained from National Measurement Institute (North Ride, NSW, Australia). B and 17-Trenbolone (Tren) were from LGC GmbH (Luckenwalde, Germany). 5α-androstan-3β-ol (3-OH) was from Toronto Research Chemicals, Inc. (Toronto, ON, Canada). The CO2 tank gas (purity grade 4.8; White Martins, São Paulo, Brazil) was calibrated against the CRM CU/USADA 331 from the Cornell University (Ithaca, NY, USA). Helium 6.0 and oxygen 6.0 were from White Martins (São Paulo, Brazil). SAMPLE PREPARATION. The sample preparation protocol proposed by Piper et al.20,25 with few modifications is presented below. Ten to 25 mL of urine were centrifuged for 5 min at 1960 g. The supernatant was eluted through the SPE cartridge previously conditioned (5 mL of MeOH, 5 mL of water). After washing with 5 mL of water, the residue was eluted with 3 mL of MeOH and taken to dryness under a stream of N2. The residue was dissolved with 1.5 mL of sodium phosphate buffer (0.2 M, pH 7) and 4 mL of TBME were added. The mixture was shaken for 5 min at 300 rpm and centrifuged for 5 min at 1960 g. The organic layer was discarded. Then 100 µL of -glucuronidase were added and followed by incubation for 60 min at 50 °C. 500 µL of potassium carbonate buffer (20 %, pH 9-10) were added and the steroids were extracted twice by adding 4 mL of TBME, shaking and centrifuging as described above. Both organic layers were combined and taken to dryness under a stream of N2. The residue was reconstituted in 30 µL of an 80 ng/µL Tren standard solution in ACN, previously acetylated (as hereafter described), used as internal standard (IS) in HPLC cleanup and 30 µL of water were added. HPLC purification was performed on Dionex Ultimate 3000 (Thermo Scientific, Waltham, MA, USA) with diode array detector and fraction collection. The first HPLC purification was performed on XBridge™ Shield RP18 column and guard column (25 cm, 4.6 mm, 5 µm and 2 cm, 4.6 mm, 5 µm) from Waters (Milford, MA, USA). The column oven was kept at 25 oC. The injection volume was 50 µL and the flow rate 1 mL/min. A linear gradient was used increasing from 40/60 of ACN/water to 60 % ACN in 18 min, within 1 min to 98 % ACN, holding for 11 min, then re-equilibrating for 5 min. The wavelengths were 195 and 345 nm. An appropriate standard mix of free steroids was injected three times prior to the samples in order to check HPLC stability and establish the collection window to each analyte. The mixes were prepared in ACN and water was added at 50:50 immediately before injection. The mix to T confirmation contained T and E at 40 ng/µL (final concentration), 5-diol, A, Etio and 11K at 100 ng/µL, and PD at 400 ng/ µL. The mix for 19NA cleanup contained A at 100 ng/µL, 19NA at 200 ng/µL and PD at 400 ng/µL. The mix for B contained B and BM1 at 80 ng/µL, and PD at 400 ng/µL. The mix for F contained F at 80 ng/µL and PD at 400 ng/µL. All mixes also contained Tren previously acetylated (as hereafter described) at 40 ng/µL. Along the first HPLC step, 7 fractions were collected for T confirmation, which were: FI (containing 11K, with typical collection window from 8.2 to 9.0 min), FII (T, 10.8-11.5 min),


Page 3 of 11 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


FIIIA (E, 12.2-13.0 min), FIIIB (5-diol, 13.0-14.1 min), FIV (Etio and 5-diol, 14.5-16.0 min), FV (A, 16.4-17.9 min) and FVI (PD, 19.2-19.9 min). FIIIA and FIIIB were combined to a unique vial after collection. For 19NA analysis, 3 fractions were collected in this step: FI (19NA, 14.8-15.75 min), FII (A) and FIII (PD). For B, 3 fractions: FI (B, 8.2-9.1 min), FII (BM1, 12.2-13.1 min) and FIII (PD). For F, 2 fractions: FI (F, 13.014.0 min) and FII (PD). After evaporation to dryness in vacuum concentrator at 60 oC and 5.1 mbar of nominal vacuum, followed by storage in vacuum oven for 1 h at room temperature, 50 µL of anhydrous pyridine and 50 µL of acetic anhydride were added, and the sample was incubated for 1 h at 70 oC. The reagents were taken to dryness in vacuum concentrator as described above. The fractions containing acetylated 11K (11K_Ac), PD_Ac and A_Ac were stored until CIR analysis. The residues of the others were reconstituted in 30 µL of a 40 ng/µL Tren standard solution in ACN, previously acetylated, used as IS in HPLC cleanup and 30 µL of water were added. The second HPLC purification for 19NA confirmation was performed on XBridge™ C18 column and guard column (15 cm, 4.6 mm, 5 µm and 2 cm, 4.6 mm, 5 µm) from Waters (Milford, MA, USA). For the other substances, the second HPLC step was performed using the same column already described for the first step. For all of them, oven temperature, injection volume, flow rate and wavelengths for detection were kept the same as in the first cleanup step. For B, also the solvent gradient used was the same. For the others (including BM1 and 19NA), a linear gradient was used increasing from 60/40 of ACN/water to 98 % ACN in 16 min, holding for 9 min, then decreasing to 60 % ACN in 0.5 min and re-equilibrating for 5 min. The mixes for the second HPLC cleanup were prepared with free standards and were acetylated prior to reconstitution with ACN. Water was added at 50:50 immediately before injection. The mix for purification of all acetylated fractions from T suspicious samples contained T and E at 40 ng/µL, and 5-diol, 5-diol and Etio at 100 ng/µL. The mix for 19NA_Ac cleanup contained this analyte at 200 ng/µL. The mix for B_Ac contained B at 80 ng/µL and OHA at 200 ng/µL. The mix for BM1_Ac contained this analyte at 80 ng/µL. The mix for F_Ac contained F at 80 ng/µL. All mixes also contained Tren_Ac (IS) at 20 ng/µL. Along the second HPLC step, T_Ac was purified from FII (typical collection window from 8.9 to 9.5 min), E_Ac (8.29.0 min) and 5-diol_Ac (15.0-15.8 min) were purified from FIII, Etio_Ac (10.1-11.5 min) and 5-diol_Ac (15.4-16.2 min) were purified from FIV. For 19NA analysis, 19NA_Ac was purified from FI (8.2-8.8 min). For B analysis, B_Ac was purified from FI (18.0-18.9 min) and BM1_Ac from FII (9.510.3 min). Finally, for F analysis, F_Ac was purified from F1 (5.1-5.8 min). All the fractions were taken to dryness as described above. STEROID PROFILE DETERMINATIONS. All urinary steroid concentrations were determined following established routine protocols26 and are herein briefly described. Sample preparation encompassed the spike of 2 mL of urine with IS, enzymatic hydrolysis with β-glucuronidase, liquid-liquid extraction with TBME and derivatization with a MSTFA/NH4I/2-mercaptoethanol mixture. The analysis was

performed in a GC-MS/MS in selection reaction monitoring (SRM) mode. GC/C/IRMS SETUP. Sample measurements were performed on a Trace 1310 gas chromatograph with a TriPlus RSH autosampler (Thermo Scientific, Waltham, MA, USA) coupled to a Delta V Plus isotope ratio mass spectrometer (ThermoFisher Scientific, Bremen, Germany). Both systems were connected via GC IsoLink II and ConFlo IV (ThermoFisher Scientific, Bremen, Germany). To one of the three instruments a Thermo ISQ LT single quadrupole mass spectrometer (Thermo Scientific, Waltham, MA, USA) was connected to the end of the GC column, in order to simultaneously evaluate peak purity and specificity. The column was an Agilent J&W Scientific VF-17ms (length 30 m, 0.25 mm i.d., 0.25 μm film thickness). Fractions were reconstituted in convenient volumes of ethyl acetate. Injections were performed in splitless mode at 280 oC with 1 to 3 µL. A double injection method was used with the injection of 0.2µL of 3-OH at 25 ng/µL previously acetylated (3β-OH_Ac) as IS. The GC temperature started at 100 oC (held for 2 min), then increasing at 25 oC/min to 270 oC, then at 2 oC/min to 290 oC, then at 30 oC/min to 300 oC (held for 2 min). Carrier gas was He 6.0 with a constant flow of 1.4 mL/min. The combustion furnace was operated at 950 °C and the combustion reactor was regenerated for 1 h before each sequence of analysis. Data were acquired by use of Isodat 3.0 Gas Isotope Ratio MS Software (Thermo Scientific). GAS TANK CALIBRATION. Five-fold injection of 1 µL of a CU/USADA 33-1 solution at approximately 25 ng/µL was performed and all the results for each analyte were considered to evaluate the CO2 gas tank calibration and re-calibrate it when needed. Standard deviation between the results for each analyte was considered to evaluate system stability. In-house RMs at 25 ng/µL of each TC and ERC were injected before and after each sequence of samples in order to ensure instrument performance, stability and calibration. NEGATIVE QUALITY CONTROL FOR T AND RELATED SUBSTANCES (QCNT). Two healthy male volunteers (22 and 34 years old), who declared not to have used any prohibited substance or dietary supplement, were previously selected for excreting endogenous steroids in convenient amounts in urine, i. e. at least 30 ng/mL (mainly of T, E and -diol). NEGATIVE QUALITY CONTROL FOR 19NA (QCN19NA). About 10 mg of 19NA standard were solubilized in ACN/water 50:50 and the saturated solution was injected around a hundred times in HPLC, until the solution was exhausted. The instrumental conditions were those used in the first cleanup step described above. The first half of the peak width, more enriched in 13C, was collected from each injection and these fractions were combined. This procedure was repeated with this mixture until 19NA with a 13C value similar to the ERCs in the QCNT was obtained. This enriched standard was reconstituted in methanol and 25 mL of the QCNT was spiked at 10 ng/mL during the sample preparation, right after hydrolysis. POSITIVE QUALITY CONTROL FOR T AND RELATED SUBSTANCES (QCPT). Three healthy male volunteers (24, 36 and 40 years old), who declared not to have used any prohibited substance or dietary supplement, administered 1 tablet (each) of DHEA (Puritan’s Pride, USA) containing 50 mg of the prohormone. Three other volunteers (23, 24 and 32 years old),



in equal conditions, administered 1 capsule (each) of Restandol® Testocaps™ (MSD, UK) containing 40 mg of T undecanoate. All of them collected urine for 36 h after administration. An informed consent was signed by each volunteer and the study was approved by the local ethical committee (Hospital Universitário Clementino Fraga Filho – Universidade Federal do Rio de Janeiro – protocol number 020/00). Each collection was analyzed separately in order to select those which presented altered CIR values for all the TCs. The selected spot urines were than combined to generate QCPT. POSITIVE QUALITY CONTROLS FOR E, 19NA, B (AND BM1) AND F (QCPE, QCP19NA, QCPB and QCPF). During the sample treatment, 10 mL of the QCPT were spiked right after hydrolysis with 12.5 µL of a standard solution containing E at 50 ng/µL in order to produce QCPE. The same procedure was carried out to generate QCPs for 19NA, B and BM1, and F, where 25 mL of the QCNT were spiked with 10 µL, 15 µL and 15 µL, or 50 µL (respectively) of standard solutions at 25 ng/µL, as appropriate. METHOD VALIDATION. Standard solutions of each TC and ERC previously acetylated were prepared at different amounts and injections of 1 µL were performed. The instrumental linearity was defined as the range of peak intensities that gave a consistent 13C value for each steroid4. QCNT was diluted with water at different rates, up to 40-fold dilution, and 20 mL of each were prepared as described above. One aliquot of the neat QCNT was also prepared. Twenty-five mililiters of QCPs for 19NA, B and BM1, and F were prepared at five concentration levels, to know: 19NA at 2.5, 5.0, 7.5, 10 and 15 ng/mL; B and BM1 at 2.5, 5.0, 10, 20 and 30 ng/mL; and F at 25, 50, 85, 120 and 150 ng/mL. The final residues were reconstituted in adequate solvent volumes to produce peak intensities within the respective instrumental linear ranges. The linear range of the method was defined as the range of concentrations in urine whose CIR measurements presented a standard deviation lower than 0.5 ‰. LOQ was considered as the minimum concentration within the linearity of the method that, prepared in a triplicate, produced a standard deviation lower than 1.0 ‰. Precision was calculated as the standard deviation between the CIR value determinations for each steroid in the QCNT and in the QCP for B and BM1, 19NA and F. For within assay precision, 5 aliquots of the respective urinary controls were prepared by the same analyst on the same day. For T and metabolites between assay precision, 20 aliquots of the control were prepared by 3 different analysts in different days, whereas for B and BM1, 19NA and F between assay precision, 15 aliquots of the control were prepared by 3 analysts on different days. Linear mixing models (LMM) are presented in the literature as a consistent statistical tool to estimate the combined uncertainty (uc) in CIR determination of endogenous steroids and its principle is thoroughly presented by Piper et al.20,21,27. The protocol is herein summarized. Aliquots containing 10 mL of the QCNT were spiked with standards of T, E, 5α-diol, 5βdiol, A, Etio, PD and 11K showing remarkably different isotopic signatures compared to the correspondent endogenous steroid in the urine. A mass balance is applicable to this two pool mixing model, as follows:

cm = ce + ca

(1)

Page 4 of 11

cm × δ13Cm = ce × δ13Ce + ca × δ13Ca

(2)

where ce represents the endogenous concentration of the steroid under investigation, ca the variable amount added and cm the resulting concentration of the mixture, while 13Ce and 13Ca represent the CIR values of the endogenous and the added portion, respectively, and 13Cm the CIR value of the mixture. Combining and rearranging the equation lead to: ce

δ13Cm = (δ13Ce ― δ13Ca)cm + δ13Ca

(3)

Equation 3 corresponds to a basic linear equation (y = ax + b) in which the measured CIR of the mixture is plotted against the concentration ratio ce/cm. In this method validation, three batches of six aliquots were prepared and the concentration ratio ce/cm covered a range from around 0.3 to 1.0. The intercept of the line of best fit experimentally obtained for each steroid was compared to the measured CIR value, i. e., the CIR value obtained by the direct injection and analysis of each standard by GC/C/IRMS. The difference between both values indicated bias. Following WADA requirements4, urine samples from 24 male and 28 female volunteers were selected for presenting adequate concentrations of the TCs and ERCs. The volunteers declared not to have used synthetic anabolic hormones. The method performance was assessed as required in the TD2016IRMS4. SAMPLE EVALUATION. In negative samples, the CIR values of the ERCs did not differ significantly from those of the TCs, whereas samples in which that difference exceeded the relevant thresholds defined by WADA4,5 were considered Adverse Analytical Findings (AAFs). Samples partially matching a positivity criterion were considered as Atypical Findings (ATF). The difference between the CIR (δ13C) values is expressed as Δδ13C and calculated as follows:

Δδ13C = δ13CERC ― δ13CTC

(4)

O&PG SAMPLES. From the 4071 urinary samples received during the Olympics3, 63 arose as SSPs, 45 of those due to T/E ratio greater than 4.0 and 18 due to other parameters. Also, the Athlete Passport Management Unit (APMU) or ADAMS requested the analysis of 71 ATPFs. One sample with SSP also presented B and BM1 at 1.2 and 3.8 ng/mL respectively, thus requiring this additional CIR confirmation. Another sample presented 19NA at 3.8 ng/mL and another one presented F at 81.7 ng/mL, both requiring such a confirmatory analysis. From the 1396 urinary samples from the Paralympics3, 11 samples presenting elevated T/E ratio, 10 presenting other suspicious steroidal parameters, 21 ATPFs and one presenting 19NA at 4.1 ng/mL demanded CIR confirmation. All the above mentioned concentrations were adjusted to a specific gravity of 1.020 g/L4,5. RESULTS AND DISCUSSION The method for CIR determination was fully implemented for the confirmatory analysis of T and related substances, 19NA, B and its main metabolite BM1, and F, in compliance with the WADA requirements4,5, as discussed below. HPLC PURIFICATION. The sample treatment protocol herein discussed was tightly anchored in two steps of HPLC


Page 5 of 11 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


purification with an acetylation step in between, resulting in a quite time-consuming procedure. But it allowed to isolate each analyte in a single fraction, free from relevant amounts of matrix interferences, even for those present in very low concentration, thus demanding the reconstitution of the final residues in very low solvent volumes in order to obtain suitable peak intensities for CIR analysis. In preliminary experiments of CIR analysis of different samples after the full sample preparation procedure, an interference was observed entirely coeluting with T_Ac by GC/C/IRMS. The collection windows then applied to the HPLC purification were 1.50 min width. Thus, shorter collection windows were applied to both HPLC steps, along with collections before and after the fraction of interest. The analysis of each fraction allowed to conclude that, differently from GC analysis, the interference eluted slightly before T in both HPLC steps. The interference was isolated by HPLC and analyzed by GC-MS after acetylation and silylation for characterization, and the obtained mass spectra corresponded to those presented by Polet et al.28 from an interference also observed in T fractions. Therefore, it is very likely that the interference herein observed in all samples was the same previously reported by those authors. In our validation, as mentioned above, this interference was eliminated by shortening the collection windows of T in both purification steps. A 0.10 min tolerance was applied before and after the T peak in the standard mix in order to establish thin collection windows (circa 0.80 min width) for this analyte for both HPLC steps. No losses of T amounts were observed by analyzing the fractions before and after the collection of T. Shortened collection windows were also applied to other analytes during HPLC purification in order to avoid transferring big amounts of borderer peaks that, although not coeluting with the substances of interest, could disturb the stability of the combustion reactor during conversion into CO2 or diminish its lifetime for CIR analysis. This is notably the case of 19NA_Ac and B_Ac, that once collected in large windows embody significant amounts of Etio_Ac and OHA_Ac, respectively. In order to avoid this disturbance, the collection windows were established by applying a 0.10 min tolerance before and after the peaks in the standard mix. CALIBRATION. The CO2 reference gas calibration with CU/USADA 33-1 is reported in the literature as presenting noteworthy offsets with regards to the certified CIR values, mainly for A_Ac and 11K_Ac measurements22,29. Those offsets can be minimized by properly assembling the combustion reactor and its connection to the fused silica capillary, which ensures the elution of the compounds into the hot zone and its adequate conversion into CO222,23. A description on assembling and positioning the reactor is presented in Supporting Information. Table 1. Linearity of the instrument.

The CIR standard deviation (SD) to each steroid between the five injections was not greater than 0.40 ‰ and CIR offsets were not greater than 0.50 ‰ compared to the certified values. Figure 1 shows the QC charts for 3-OH_Ac, 5-cholestane (5-chol), A_Ac and 11K_Ac measurements in the hybrid system all-over 2016. The A_Ac CIR offsets evidently contributed to a certain extent to the calibration of the CIR value of the reference gas. However, it did not impact significantly the steroids CIR measurement, as depicted in the External Quality Assessment Scheme (EQAS) reports of WADA (data not shown) all over the last years and the analysis of the CRM MX016 presented hereafter. The combustion reactor was replaced three times during 2016, one of those before the beginning of the O&PG as part of the preventive maintenance. The CIR value of the reference gas throughout 2016 was from -32.74 ‰ to -32.16 ‰.

Figure 1. Control charts of CIR measurements of the constituents of the CRM CU/USADA 33-1 in the hybrid system all-over 2016. (A) 3-OH_Ac. (B) 5-chol. (C) A_Ac. (D) 11K_Ac. The solid black lines are the certified values; the solid gray lines are the ranges established as the certified values ± 0.50 ‰; the dashed black lines are the mean values.

INSTRUMENTAL LINEARITY, METHOD LINEARITY AND LOQ. The linear range of the instrument was assessed by the analysis of standard solutions of steroids previously acetylated over a range of approximately 10 ng to 90 ng of steroid on column. The linear range of the instrument was defined as the range of peak intensities (in mV) that gave CIR values within 0.5 ‰ of the mean values. The results obtained are depicted in Table 1 and Figures are presented in Supporting Information. The SD of the CIR values ranged from 0.1 ‰ to 0.3 ‰ for the steroids. The linear ranges were considered suitable for appropriate CIR measurements.

PD

11K

T

5-diol 5-diol

A

Etio

E

19NA

B

BM1

F

Injections

16

14

11

15

20

13

14

10

12

22

22

12

Linear range (mV)

6273736

6803774

6344435

5475825

4595904

7333918

11654089

6912749

3103597

1723415

2828867

10554143

Mean δ13C (‰)

-20.6

-16.3

-33.0

-32.1

-33.4

-32.4

-29.2

-30.6

-30.7

-29.3

-30.8

-31.6

SD

0.2

0.2

0.3

0.1

0.3

0.3

0.2

0.3

0.2

0.3

0.2

0.3

Analysis of standard solutions over a range of approximately 10 ng to 90 ng of steroid on column.



Page 6 of 11

QCs AND METHOD PRECISION. QCNT was obtained as a Table 2 summarizes the results obtained for linearity of the pool of 27 urinary specimens from the two volunteers totalizing method and LOQ. The linear ranges of the method to all steroids 10 L. Table 3 presents concentrations and δ13C values to all the fit the expected concentrations in suspicious samples. For ERCs and TCs in QCNT. Overall the results obtained were self19NA, B, BM1 and F, the method linearity covered the full consistent, with the δ13C values of the steroids differing not concentration ranges in which CIR confirmation is required. For 19NA, LOQ corresponded to the minimum concentration more than 2.2 ‰ from each other. that triggers CIR confirmation, whereas for B, BM1 and F, it corresponded to half of the lowest suspected concentration. Table 2. Linearity of the method and limit of quantification (LOQ). PD

11K

T

5-diol 5-diol

A

Etio

E

19NA

B

BM1

F

Linearity of the method Linear range (ng/mL)

9.0359.5

22.1885.5

12.3122.9

14.3285.6

34.7694.8

138.05520.4

128.82576.3

10.987.4

2.515.0

2.530.0

2.530.0

25.0150.0

Mean δ13C (‰)

-18.0

-18.4

-18.9

-18.7

-18.3

-18.7

-18.7

-19.4

-29.3

-27.4

-29.7

-29.0

SDOverall(‰)

0.1

0.5

0.5

0.3

0.4

0.2

0.4

0.5

0.4

0.4

0.1

0.4

0.03

0.1

0.4

0.3

0.04

0.2

0.6

0.3

0.4

0.3

0.5

0.4

LOQ (n = 3) SDLOQ (‰)

For T, T metabolites and ERCs, the QCNT was diluted with water at different rates up to 40-fold and 20 mL of each dilution were prepared. For 19NA, B, BM1 and F, 25 mL aliquots of the QCNT were spiked at concentration levels from around half of the minimum concentration up to the maximum concentration in which CIR confirmation is required.

Table 3. Negative Quality Control for T (QCNT), within assay and between assay method precision. PD

11K

T

5-diol

5-diol

A

Etio

E

359.5

885.5

122.9

285.6

694.8

5520.4

2576.3

87.4

Mean δ13C (‰)

-17.9

-18.3

-19.1

-18.6

-18.7

-17.6

-19.3

-19.8

Precision (SD)

0.2

0.1

0.2

0.2

0.1

0.4

0.3

0.2

Mean δ13C (‰)

-17.6

-17.6

-19.1

-18.1

-18.2

-17.9

-19.6

-19.7

Precision (SD)

0.6

0.5

0.2

0.4

0.5

0.6

0.7

0.2

Concentration (ng/mL) Within assay (n = 5)

Between assay (n = 20)

QCPT was obtained by selecting and combining specimens collected for around 36 h after a single intake of DHEA by each of 3 volunteers and T undecanoate by 3 others. The Δδ13C values after the administration of DHEA and T undecanoate by one of the volunteers each are shown in Figure 2. Following DHEA administration, CIR of T remained affected only for around 13 h, corresponding to the 3 first collections. For the other TCs, notably for the 5β-metabolites (5β-diol and Etio), the steroid administration could be detected in a wider detection window. Following T undecanoate administration, only T was significantly affected. Combining the selected specimens from the 6 volunteers allowed to produce 3 L of QCPT, a larger volume than if using only DHEA excretion. In this pool, all the TCs affectable by the intake of T or a related substance presented CIR values (‰) compatible with a positive urine: PD = -17.3, 11K = -17.6, T = -21.6, 5α-diol = -21.8, 5β-diol = -26.5, A = -25.3, Etio = -28.5. Therefore, the Δδ13C values ranged from 4.3 ‰ for T to 11.2 ‰ to Etio, taking PD as ERC.QCPE was obtained by spiking QCPT with a standard solution of E after hydrolysis, thus presenting a CIR value equal to -26.2 ‰ and a Δδ13C value equal to 8.8 ‰ against PD. QCN19NA was produced by spiking QCNT with a standard solution of 19NA after hydrolysis. This standard was previously prepared by isotopic fractionation in HPLC until a 13C value

equal to -19.8 ‰ was obtained. The amount of 19NA standard with “like endogenous” CIR value was circa 1.2 mg.

Figure 2. Δδ13C values following a single intake of (A) DHEA by a volunteer and of (B) T undecanoate by another. The open circles are T, the open triangles are A, the black triangles are Etio, the open diamonds are 5α-diol and the black diamonds are 5β-diol. The thin


Page 7 of 11 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


gray line at 3.0 ‰ represents the threshold applied to select the suitable points.

respectively (-32.9 ‰ and -32.4 ‰). The open triangles and the open diamonds represent the LMMs of T and A, respectively.

The CIR value of 19NA obtained in the QCN19NA was -19.7 ‰. QCP19NA, QCPB and QCPF were also obtained by spiking QCNT with proper standard solutions, containing the respective analytes, after hydrolysis. The CIR values (‰) in the QCs were: 19NA = -29.5, B = -27.6, BM1 = -29.7 and F = -29.0. BIAS AND uc DETERMINATION. For the ERCs, T and metabolites, bias was determined by means of LMMs. The measured CIR values were those determined for the acetylated standards in the experiment of linearity of the instrument, as the standards were the same. These measured values were compared with the experimental values obtained by LMMs and the difference was considered as bias. Figure 3 depicts the LMM experiments by presenting the graphics obtained for T and A, whose bias were estimated as 0.5 ‰ and 0.9 ‰, respectively. For all analytes, the points in triplicate were perfectly overlapped and the curve of best fit presented good coefficient of determination.

As no urine containing endogenous 19NA, B, BM1 or F was available, bias was estimated for these TCs as the difference between the CIR values of the respective standards used for instrumental linearity determination and CIR determination of the same standards after addition into urine and analysis of the respective QCPs. The uc was estimated in all cases as the square root of the sum of the squares of intermediate precision and bias. Table 4 summarizes the results obtained for bias and uc. The uc were estimated from 0.4 ‰ (for E and B) to 1.0 ‰ (for A). A possible explanation for the higher estimated uc for A is presented in Supporting Information. In parallel, the available CRM MX016 containing 19NA-G with a certified CIR value was spiked into QCNT in two different days, before the full sample preparation at three levels, 2.5, 5.0 and 10 ng/mL, in order to cross-check uc estimation. For MX016, intermediate precision was 0.2 ‰, bias (calculated against the certified CIR value) was 0.5 ‰ and uc, 0.6 ‰. As the first uc estimation for 19NA was 0.5 ‰, we concluded that no significant drifts are expected to occur between the two approaches, what means that the contribution of the reference gas calibration and of the initial sample preparation steps to bias is negligible. METHOD PERFORMANCE EVALUATION. The method performance was assessed by analyzing negative urines from volunteers. Table 5 summarizes the method performance evaluation for analysis of T and metabolites. The Δδ13C values of all combinations of ERCs and TCs in the steroid profile were assessed. The SD in the Δδ13C determination of all pairs of ERC and TC were lower than 1.2 ‰, what ensured robustness of the method. The mean value + 2 SD to all pairs were lower than 3.0 ‰ (or lower than 4.0 ‰ for Etio and E), which guaranteed Figure 3. LMMs for T and A. The gray triangle and the gray that negative samples are properly distinguished by this diamond are the measured CIR values for T and A standard, method. Table 4. Combined uncertainties (uc) estimated from intermediate precision and bias. PD

11K

T

5-diol 5-diol A

Etio

E

19NA

B

BM1

F

Intermediate precision (‰) 0.6

0.5

0.2

0.4

0.5

0.6

0.7

0.2

0.4

0.4

0.6

0.4

Bias (‰)

0.3

0.1

0.6

0.3

0.3

0.9

0.4

0.4

0.3

0.1

0.4

0.5

uc(‰)

0.7

0.5

0.6

0.5

0.6

1.0

0.8

0.4

0.5

0.4

0.7

0.6

Table 5. Negative sample’s analysis to evaluate method performance. PD Number of males / females 24 / 28 Mean

δ13C

(‰)

-18.0

ERC

11K

T

5-diol

5-diol

A

Etio

E

24 / 28

23 / 17

22 / 20

23 / 25

24 / 27

24 / 25

23 / 17

-18.2

-19.5

-19.0

-18.9

-18.1

-18.8

-20.3

PD

11K

PD

11K

PD

11K

PD

11K

PD

11K

PD

11K

1.4

1.3

1.1

0.9

0.9

0.7

0.1

-0.1

0.8

0.6

2.3

2.2

SD (‰)

0.6

0.7

0.5

0.6

0.7

0.9

0.5

0.7

0.6

0.9

0.6

0.7

Mean + 2 SD (‰)

2.6

2.8

2.2

2.2

2.4

2.5

1.2

1.4

2.1

2.4

3.5

3.6

Mean

∆δ13C

(‰)

ERC: Endogenous reference compound.

ATHLETES’ SUSPICIOUS SAMPLES CONFIRMATION. PD, 5α-diol, 5β-diol and T were analyzed in all suspicious cases related to steroid profiles, unless any of them was suppressed in a sample. A and Etio were analyzed whenever the suspicious parameter was related to them, or the diols were suppressed in

a sample. Also, in case of AAFs or ATFs, the sample was reprepared and all the TCs were analyzed before reporting the results. 11K was isolated to be analyzed in case of any problem related to PD, but such a case did not arise, neither does



Page 8 of 11

20.1 ‰; B = -21.9 ‰; BM1 = -21.9 ‰), as well as one case of suspicious cases of E. For suspicious cases of 19NA, B (and F (CIR values: PD = -20.5 ‰; F = -19.1 ‰). BM1) and F, the respective TCs and PD were analyzed. Most of SSPs and ATPFs confirmed by IRMS were found unsuspicious. From the Olympics, 131 of 134 samples presented CIR values compatible with an endogenous production, while 40 of 42 from Paralympics presented not altered CIR values. Figure 4 presents the Δδ13C distribution of the Olympic and the Paralympic athletes to 5α-diol, 5β-diol and T against PD as ERC. No marked differences were observed between the two groups in terms of Δδ13C values. Also for A and Etio (for which less results were obtained), no significant differences were observed. Between the Olympic samples, the Δδ13C for A varied from 0.2 ‰ to 1.8 ‰ and for Etio it varied from 0.4 ‰ to 2.4 ‰. Between the Paralympic samples, the Δδ13C varied from 0.0 ‰ to 2.0 ‰ and from 1.2 ‰ to 2.7 ‰ for A and Etio, respectively. One Olympic sample was considered as an outlier for the three TCs in Figure 4. This sample presented the following absolute CIR values: PD = -21.9 ‰, 5αdiol = -20.9 ‰, 5β-diol = -19.7 ‰ and T = -20.3 ‰. The Figure 4. Δδ13C distribution of 5α-diol, 5β-diol and T (against PD absolute CIR values for PD varied from -22.6 ‰ to -16.7 ‰ in as ERC) in athletes’ negative samples. The white boxes represent the Olympic samples and from -24.0 ‰ to 17.1 ‰ in the the Olympic athletes, while the gray boxes represent the Paralympic samples. Paralympic athletes. One suspicious case of B and BM1 was confirmed as consistent with endogenous production (CIR values: PD = Table 6. AAFs and ATFs resulting from CIR analysis and reported during the O&PG 2016. Event

Gender

Modality

Request –Suspicious concentration (ng/mL) or ratio

δ13C values supporting the findings (‰)

Conclusion

Olympics

M

Weightlifting

19NA = 3.8

PD = -21.2; 19NA = -30.0

AAF

Olympics

M

Weightlifting

ATPF

PD = -20.8; T = -26.2; 5α-diol = -27.2; 5β-diol = -27.0

AAF

Olympics

M

Weightlifting

SSP – T/E = 9.0

PD = -18.9; T = -24.8; 5α-diol = -27.5; 5β-diol = -26.1

AAF

Olympics

M

Weightlifting

ATPF

PD = -20.7; T = -24.0; 5α-diol = -23.3; 5β-diol = -21.3

ATF

Throws

19NA = 4.1

PD = -19.9; 19NA = -30.5

AAF AAF ATF

Paralympics M Paralympics M

Swimming

SSP – T/E = 33.8

PD = -19.7; T = -27.6; 5α-diol = -26.3; 5β-diol = -27.0; A = -23.8; Etio = -25.2

Paralympics M

Powerlifting

ATPF

PD = -18.2; T = -22.7; 5α-diol = -20.7; 5β-diol = -20.7; Etio = -21.5

SSP: Suspicious steroid profile. ATPF: Atypical passport finding. AAF: Adverse analytical finding. ATF: Atypical finding.

Table 6 presents the five AAFs and two ATFs found during the O&PG. All cases involved male athletes. For the Olympics, two AAFs were reported for T and metabolites, being one of them triggered by a SSP (T/E = 9.0) and another by an ATPF. In both cases, T and both diols were affected. Besides this, an ATF was found in a sample whose suspicion relied on an ATPF. This ATF presented an altered CIR value for T (Δδ13C = 3.3 ‰), while 5α-diol and 5β-diol presented CIR values consistent with endogenous production (Δδ13C = 2.6 ‰ and 0.6 ‰, respectively). Also, an AAF was reported for 19NA. For the Paralympics, one AAF triggered by a SSP (T/E = 33.8) was reported for T and metabolites, in which T, both diols, A and Etio were affected, and another AAF was found for 19NA. Also, an ATF was reported with altered CIR value for T (Δδ13C = 4.5 ‰), a weakly suspicious CIR value for Etio (Δδ13C = 3.3 ‰) and unsuspicious values for 5α-diol and 5β-

diol (Δδ13C = 2.5 ‰ each). This confirmation was triggered by an ATPF. The validated method allowed the analysis of 179 samples and delivery of the results within 34 h for negative samples and 72 h whenever a re-preparation was needed (including AAFs and ATFs). Working in a 24 h / 7 days routine, significant problems related to dirtiness into injectors or columns, elevated organic backgrounds, leaks, bad combustion reactor performance, dirty ion sources or drifts in quality controls CIR determinations were not observed, proving the robustness of the method. CONCLUSION The validation parameters of the method presented in this work fully met WADA requirements, comprising adequate


Page 9 of 11 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


linear ranges, suitable LOQs and uc not greater than 1.0 ‰ to each of the 12 steroids (TCs and ERCs). The analysis of volunteers’ negative samples proved the good method performance. The analysis of 179 samples during the O&PG 2016 in a very tight routine, including one negative case of B and BM1, one negative case of F, two AAFs for 19NA, three AAFs and two ATFs for T and related substances, confirmed the robustness of the method.

ASSOCIATED CONTENT Supporting Information HPLC purification of testosterone, gas calibration, Quality Control charts, internal standards evaluation, linearity of the instrument, androsterone bias, reference population (PDF).

AUTHOR INFORMATION Corresponding Author * E-mail: [email protected]; [email protected]. Tel.: +55 21 3938-3791 / + 55 21 3938-3726.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT The authors are gratefully indebted to SENS, Nova Analitica and Thermo Scientific for their technical support; to the Brazilian Federal Government and its Ministries of Sport and Education, UFRJ and its Chemistry Institute; to WADA, WAADS and the WADA-accredited laboratories for the technical and scientific support, and for the release of their experts to work during the Games.

REFERENCES (1) World Anti-Doping Agency. TD2004EAAS – Reporting and evaluation guidance for testosterone, epitestosterone, T/E ratio and other endogenous steroids, Montreal, s.n., 2004. (2) Aguilera, R.; Chapman, T. E.; Pereira, H.; Oliveira, G. C.; Illanes, R. P.; Fernandes, T. F.; Azevedo, D. A.; Aquino Neto, F. R. Drug testing data from the 2007 Pan American Games: δ13C values of urinary androsterone, etiocholanolone and androstanediols determined by GC/C/IRMS. J. Steroid Biochem. Molec. Biol. 2009, 115, 107–114. (3) Pereira, H. M. G.; Sardela, V. F.; Padilha, M. C.; Mirotti, L.; Casilli, A.; Oliveira, F. A.; Cavalcanti, G. A.; Rodrigues, L. M. L.; Araujo, A. L. D.; Levy, R. S.; Teixeira, P. A. C.; Oliveira, F. A. G.; Duarte, A. C. G.; Carneiro, A. C. D.; Evaristo, J. A. M.; Santos, G. R. C.; Costa, G. C. V.; Castro, F. L.; Nogueira, F. C. S.; Scalco, F. B.; Pizzatti, L.; Aquino Neto, F. R. Doping control analysis at the Rio 2016 Olympic and Paralympic Games. Drug Test. Analysis 2017, 9, 1658– 1672. (4) World Anti-Doping Agency. TD2016IRMS. Available at: https://www.wada-ama.org/sites/default/files/resources/files/wadatd2016irms-detection_synthetic_forms_eaas_by_irms-en.pdf [09 May, 2019]. (5) World Anti-Doping Agency. TD2016NA. Available at: https://www.wadaama.org/sites/default/files/resources/files/td2016na_eng.pdf [09 May, 2019]. (6) World Anti-Doping Agency. TD2018EAAS. Available at: https://www.wadaama.org/sites/default/files/resources/files/td2018eaas_final_eng.pdf [09 May, 2019]. (7) Sottas, P.-E.; Saudan, C.; Schweizer, C.; Baume, N.; Mangin, P.; Saugy, M. From population- to subject-based limits of T/E ratio to detect testosterone abuse in elite sports. Forensic Sci. Int. 2008, 174, 166–172.

(8) Geyer, H.; Schänzer, W.; Thevis, M. Anabolic agents: recent strategies for their detection and protection from inadvertent doping. Br. J. Sports Med. 2014, 48, 820–826. (9) Gosetti, F.; Mazzucco, E.; Gennaro, M. C.; Marengo, E. Ultra high performance liquid chromatography tandem mass spectrometry determination and profiling of prohibited steroids in human biological matrices, A review. J. Chromatogr. B 2013, 927, 22– 36. (10) Kempná, P.; Flück, C. E. Adrenal gland development and defects. Best Pract. Res. Clin. Endocrinol. Metab. 2008, 22, 77–93. (11) Palonek, E.; Ericsson, M.; Gårevik, N.; Rane, A.; Lehtihet, M.; Ekström, L. Atypical excretion profile and GC/C/IRMS findings may last for nine months after a single dose of nandrolone decanoate. Steroids 2016, 108, 105–111. (12) Hebestreit, M.; Flenker, U.; Fußhöller, G.; Geyer, H.; Güntner, U.; Mareck, U.; Piper, T.; Thevis, M.; Ayotte, C.; Schänzer, W. Determination of the origin of urinary norandrosterone traces by gas chromatography combustion isotope ratio mass spectrometry. Analyst 2006, 131, 1021–1026. (13) de la Torre, X.; Colamonici, C.; Curcio, D.; Molaioni, F.; Pizzardi, M.; Botrè, F. A simplified procedure for GC/C/IRMS analysis of underivatized 19-norandrosterone in urine following HPLC purification. Steroids 2011, 76, 471–477. (14) Piper, T.; Geyer, H.; Gougoulidis, V.; Flenker, U.; Schänzer, W. Determination of 13C/12C ratios of urinary excreted boldenone and its main metabolite 5β-androst-1-en-17β-ol-3-one. Drug Test. Analysis 2010, 2, 217–224. (15) Toledano, R. M.; Díaz-Plaza, E. M.; Cortes, J. M.; Aragón, A.; Vázquez, A. M.; Villén, J.; Muñoz-Guerra, J. Development of an analytical method for the determination of the misuse in sports of boldenone through the analysis of urine by on-line coupling liquid chromatography-gas chromatography-combustion-isotope ratio mass spectrometry. J. Chromatogr. A 2014, 1370, 171–178. (16) Piper, T.; Fusshöller, G.; Emery, C.; Schänzer, W.; Saugy, M. Investigations on carbon isotope ratios and concentrations of urinary formestane. Drug Test. Analysis 2012, 4, 942–950. (17) de la Torre, X.; Colamonici, C.; Curcio, D.; Jardines, D.; Molaioni, F.; Parr, M. K.; Botrè, F. Detection of formestane abuse by mass spectrometric techniques. Drug Test. Analysis 2014, 6, 1133– 1140. (18) Polet, M.; Van Renterghem, P.; Van Gansbeke, W.; Van Eenoo, P. Profiling of urinary formestane and confirmation by isotope ratio mass spectrometry. Steroids 2013, 78, 1103–1109. (19) Craig, H. Isotopic standards for carbon and oxygen and correction factors for mass-spectrometric analysis of carbon dioxide. Geochim. Cosmochim. Acta 1957, 12, 133–149. (20) Piper, T.; Mareck, U.; Geyer, H.; Flenker, U.; Thevis, M.; Platen, P.; Schänzer, W. Determination of 13C/12C ratios of endogenous urinary steroids: method validation, reference population and application to doping control purposes. Rapid Commun. Mass Spectrom. 2008, 22, 2161–2175. (21) Piper, T.; Thevis, M. Applications of isotope ratio mass spectrometry in sports drug testing accounting for isotope fractionation in analysis of biological samples. Methods Enzymol. 2017, 596, 403– 432. (22) Piper, T.; Emery, C.; Thomas, A.; Saugy, M.; Thevis, M. Combination of carbon isotope ratio with hydrogen isotope ratio determinations in sports drug testing. Anal. Bioanal. Chem. 2013, 405, 5455–5466. (23) Cawley, A. T.; Flenker, U. The application of carbon isotope ratio mass spectrometry to doping control. J. Mass Spectrom. 2008, 43, 854–864. (24) Piper, T.; Flenker, U.; Mareck, U.; Schänzer, W. 13C/12C Ratios of endogenous urinary steroids investigated for doping control purposes. Drug Test. Analysis 2009, 1, 65–72. (25) Piper, T.; Thomas, A.; Thevis, M.; Saugy, M. Investigations on hydrogen isotope ratios of endogenous urinary steroids: referencepopulation-based thresholds and proof-of-concept. Drug Test. Analysis 2012, 4, 717–727. (26) Nicolich, R. S.; Padilha, M. C.; Aquino Neto, F. R. Study of the endogenous steroid profile of male athletes from the Brazilian


9


Page 10 of 11

National Soccer Championship 2009. Drug Test. Analysis 2010, 2, 599–602. (27) Piper, T.; Thevis, M.; Flenker, U.; Schänzer, W. Determination of the deuterium/hydrogen ratio of endogenous urinary steroids for doping control purposes. Rapid Commun. Mass Spectrom. 2009, 23, 1917–1926. (28) Polet, M.; Van Gansbeke, W.; Deventer, K.; Van Eenoo, P. Development of a sensitive GC-C-IRMS method for the analysis of androgens. Biomed. Chromatogr. 2013, 27, 259–266. (29) Polet, M.; Van Eenoo, P. GC-C-IRMS in routine doping control practice: 3 years of drug testing data, quality control and evolution of the method. Anal. Bioanal. Chem. 2015, 407, 4397–4409.


10

Page 11 of 11 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


For Table of Contents Only


11

Implementation and performance of the gas chromatography

Recommend Documents