Screening of Doping Substances in Human Urine with Gas and Liquid Chromatography Coupled to High-Resolution Mass Spectrometry

The unification of different analytical screening procedures has become an important issue for the World Anti-Doping Agency (WADA) Accredited Laboratories and can be cost-effective in terms of saving laboratory resources, reducing reporting time and laboratory infrastructure. The use of the unknown designer drugs (new drugs circulated without official approval of national pharmaceutical organizations) by athletes is considered a serious threat for the antidoping system and directs the analytical technology to acquire sensitive (especially for the anabolic agents), specific and accurate data for as many prohibited known and designer molecules as possible through full scan acquisition mass spectrometry. Moreover, the long-term storage of the antidoping samples and reanalysis of samples and acquired data with negative antidoping outcome, e.g. after the circulation of unknown designers is being known by antidoping authorities or the detection of new long-term metabolites that change the detection times in urine, would create an antidoping framework where the new information can be applied not only to current and future tests, but also to previously negative reported samples. The overall aim of this PhD project is to study the use of high-resolution full scan (FS/HR) liquid chromatography mass spectrometry (LC/MS) and gas chromatography mass spectrometry (GC/MS) small molecule platforms for comprehensive antidoping screening. FS/HR allows to accurately identify and quantify the endogenous anabolic steroids included in the Athlete Biological Passport (ABP) measured with GC/MS and to consider new steroidal ABP biomarkers such as intact sulfate phase II metabolites and to apply new mass spectrometry data processing approach for the detection of unknown designer doping agents. The main conclusion is that the new FS/HR GC/MS and LC/MS methods presented in this PhD research are validated for high-throughput antidoping screening and are ready to be applied as routine procedures in any WADA Accredited Laboratory equipped with the respective analytical technologies

Quantification Of Endogenous Anabolic Androgenic Steroids Using Gas Chromatography Tandem Mass Spectrometry: An Application Of Steroid Profile From Eurycoma Longifolia Administration

A method was developed using gas chromatography tandem mass spectrometry (GC/MS/MS) to quantify the levels of endogenous androgenic anabolic steroids (EAAS) including testosterone, dehydroepiandrosterone, 4-andro -stenedione, 5-androstenediol, dihydrotestosterone and other related metabolites like androsterone, etiocholanolone, 5-α-androstandiol and 5-β-androstandiol in urine samples. The steroids were extracted using SPE method followed by LLE and analyzed by GC/MS/MS. Multiple Reaction Monitoring (MRM) was adopted for the analysis, using two MRM transitions as the qualifier ions and one MRM transition as the quantifier ion for each analyte. The linearity, extraction recovery, LOD, intra- and inter–day precision and accuracy as well as robustness of the method were validated. The correlation coefficients (r2) obtained was higher than or equal to 0.993 for all the compounds. The recoveries achieved using the method were higher than 70% for all analytes The limits of detection that was obtained using the method described in the present study ranged from 0.1 to 0.2 ng/mL. The limits of quantification ranged from to 0.5 to 1 ng/mL. The results showed good precision; where CV < 8% and accuracy ≥ 80% for all compounds in all assayed levels. The validation results showed that the analytical method used in this study to quantify the investigated anabolic steroids in urine has complied with the criteria of the validation for quantitative methods according to WADA ISL and NATA Technical Note

Ultra-Fast Retroactive Processing by MetAlign of Liquid-Chromatography High-Resolution Full-Scan Orbitrap Mass Spectrometry Data in WADA Human Urine Sample Monitoring Program

Rationale: The World Antidoping Agency (WADA) Monitoring program concentrates analytical data from the WADA Accredited Laboratories for substances which are not prohibited but whose potential misuse must be known. The WADA List of Monitoring substances is updated annually, where substances may be removed, introduced or transferred to the Prohibited List, depending on the prevalence of their use. Retroactive processing of old sample datafiles has the potential to create information for the prevalence of use of candidate substances for the Monitoring List in previous years. MetAlign is a freeware software with functionality to reduce the size of liquid chromatography (LC)/high-resolution (HR) full-scan (FS) mass spectrometry (MS) datafiles and to perform a fast search for the presence of substances in thousands of reduced datafiles. Methods: Validation was performed to the search procedure of MetAlign applied to Anti-Doping Lab Qatar (ADLQ)-screened LC/HR-FS-MS reduced datafiles originated from antidoping samples for tramadol (TRA), ecdysterone (ECDY) and the ECDY metabolite 14-desoxyecdysterone (DESECDY) of the WADA Monitoring List. Searching parameters were related to combinations of accurate masses and retention times (RTs). Results: MetAlign search validation criteria were based on the creation of correct identifications, false positives (FPs) and false negatives (FNs). The search for TRA in 7410 ADLQ routine LC/HR-FS-MS datafiles from the years 2017 to 2020 revealed no false identification (FPs and FNs) compared with the ADLQ WADA reports. ECDY and DESECDY were detected by MetAlign search in approximately 5% of the same cohort of antidoping samples. Conclusions: MetAlign is a powerful tool for the fast retroactive processing of old reduced datafiles collected in screening by LC/HR-FS-MS to reveal the prevalence of use of antidoping substances. The current study proposed the validation scheme of the MetAlign search procedure, to be implemented per individual substance in the WADA Monitoring program, for the elimination of FNs and FPs.</p

Population Reference Ranges of Urinary Endogenous Sulfate Steroids Concentrations and Ratios as Complement to the Steroid Profile in Sports Antidoping

The population based Steroid Profile (SP) ratio of testosterone (T) and epitestosterone (E) has been considered as a biomarker approach to detect testosterone abuse in '80s. The contemporary Antidoping Laboratories apply the World Antidoping Agency (WADA) Technical Document (TD) for Endogenous Androgenic Anabolic Steroids (EAAS) in the analysis of SP during their screening. The SP Athlete Biological Passport (ABP) adaptive model uses the concentrations of the total of free and glucuronide conjugated forms of six EAASs concentrations and ratios measured by GC/MS. In the Antidoping Lab Qatar (ADLQ), the routine LC/MS screening method was used to quantitatively estimate the sulfate conjugated EAAS in the same analytical run as for the rest qualitative analytes. Seven sulfate EAAS were quantified for a number of routine antidoping male and female urine samples during screening. Concentrations, statistical parameters and selected ratios for the 6 EAAS, the 6 sulfate EAAS and 29 proposed ratios of concentrations from both EAAS and sulfate EAAS, which potentially used as SP ABP biomarkers, population reference limits and distributions have been estimated after the GC/MSMS analysis for EAAS and LC/Orbitrap/MS analysis for sulfate EAAS

Comparison of gas chromatography/quadrupole time-of-flight and quadrupole Orbitrap mass spectrometry in anti-doping analysis : I. Detection of anabolic-androgenic steroids

Rationale: The World Anti-Doping Agency (WADA) encourages drug-testing laboratories to develop screening methods that can detect as many doping substances as possible in urine. The use of full-scan high-resolution acquisition (FS/HR) with gas chromatography/mass spectrometry (GC/MS) for the detection of known and unknown trimethylsilyl (TMS) derivatives of anabolic-androgenic steroids (AAS) provides anti-doping testing bodies with a new analytical tool. Methods: The AAS were extracted from urine samples by generic liquid–liquid extraction, after enzymatic hydrolysis, and TMS derivatization. The extracted urine was analyzed by GC/Q-TOF and GC/Q-Orbitrap to compare the performance of the two instrument types for the detection of 46 AAS in human urine. The quantitation of endogenous anabolic steroids and the ability of the two analytical platforms to comply with the requirements for testing as part of the WADA Athlete Biological Passport (ABP) were also assessed. Results: The data presented show that the analytical performance for both instruments complies with the WADA specifications. The limits of detection (LODs) for both instruments are well below the WADA 50% Minimum Required Performance Levels. The mass errors in the current study for the GC/Q-Orbitrap platform are lower than those obtained for the GC/Q-TOF instrument. Conclusions: The data presented herein proved that both molecular profiling platforms can be used for antidoping screening. The mass accuracies are excellent in both instruments; however, the GC/Q-Orbitrap performs better as it provides higher resolution than the GC/Q-TOF platform.</p

Gas chromatographic quadrupole time-of-flight full scan high resolution mass spectrometric screening of human urine in antidoping analysis

This paper presents the development and validation of a high-resolution full scan (FS) electron impact ionization (EI) gas chromatography coupled to quadrupole Time-of-Flight mass spectrometry (GC/QTOF) platform for screening anabolic androgenic steroids (AAS) in human urine samples. The World Antidoping Agency (WADA) enlists AAS as prohibited doping agents in sports, and our method has been developed to comply with the qualitative specifications of WADA to be applied for the detection of sports antidoping prohibited substances, mainly for AAS. The method also comprises of the quantitative analysis of the WADA's Athlete Biological Passport (ABP) endogenous steroidal parameters. The applied preparation of urine samples includes enzymatic hydrolysis for the cleavage of the Phase II glucuronide conjugates, generic liquid liquid extraction and trimethylsilyl (TMS) derivatization steps. Tandem mass spectrometry (MS/MS) acquisition was applied on few selected ions to enhance the specificity and sensitivity of GC/TOF signal of few compounds. The full scan high resolution acquisition of analytical signal, for known and unknown TMS derivatives of AAS provides the anti doping system with a new analytical tool for the detection designer drugs and novel metabolites, which prolongs the AAS detection, after electronic data files' reprocessing. The current method is complementary to the respective liquid chromatography coupled to mass spectrometry (LC/MS) methodology widely used to detect prohibited molecules in sport, which cannot be efficiently ionized with atmospheric pressure ionization interface

High resolution full scan liquid chromatography mass spectrometry comprehensive screening in sports antidoping urine analysis

The aim of this paper is to present the development and validation of a high-resolution full scan (HR-FS) electrospray ionization (ESI) liquid chromatography coupled to quadrupole Orbitrap mass spectrometer (LC/Q/Orbitrap MS) platform for the screening of prohibited substances in human urine according to World Antidoping Agency (WADA) requirements. The method was also validated for quantitative analysis of six endogenous steroids (epitestosterone, testosterone, 5α-dihydrotestosterone, dehydroepiandrosterone, androsterone and etiocholanolone) in their intact sulfates form. The sample preparation comprised a combination of a hydrolyzed urine liquid–liquid extraction and the dilute & shoot addition of original urine in the extracted aliquot. The HR-FS MS acquisition mode with Polarity Switching was applied in combination of the Quadrupole-Orbitrap mass filter. The HR-FS acquisition of analytical signal, for known and unknown small molecules, allows the inclusion of all analytes detectable with LC–MS for antidoping investigations to identify the use of known or novel prohibited substances and metabolites after electronic data files’ reprocessing. The method has been validated to be fit-for-purpose for the antidoping analysis