Wim Van Gansbeke

A fast, comprehensive screening method for doping agents in urine by gas chromatography-triple quadrupole mass spectrometry

The use of performance enhancing drugs in sports is prohibited. For the detection of misuse of such substances gas chromatography or liquid chromatography coupled to mass spectrometry are the most frequently used detection techniques. In this work the development and validation of a fast gas chromatography tandem mass spectrometric method for the detection of a wide range of doping agents is described. The method can determine 13 endogenous steroids (the steroid profile), 19-norandrosterone, salbutamol and 11-nor-Δ9-tetrahydrocannabinol.9carboxylic acid in the applicable ranges and to detect qualitatively over 140 substances in accordance with the minimum required performance levels of the World Anti-Doping Agency in 1ml of urine. The classes of substances included in the method are anabolic steroids, β2-agonists, stimulants, narcotics, hormone antagonists and modulators and beta-blockers. Moreover, using a short capillary column and hydrogen as a carrier gas the run time of the method is less than 8min.

An improved gas chromatography screening method for doping substances using triple quadrupole mass spectrometry, with an emphasis on quality assurance

A GC-QqQ-MS method was developed for the detection of over 150 compounds from different classes (steroids, narcotics, stimulants, β-blockers, β-2-agonists and hormone antagonists) in a qualitative way. In the quantitative part, the traditional steroid profile with the most important endogenous steroids is expanded with six minor metabolites, which further improves the detection and identification of endogenous steroid abuse. In addition to these, norandrosterone, salbutamol and the major metabolite of cannabis are also quantified. Methods developed for anti-doping purposes should be subjected to the highest level of quality. Here, the addition of a combination of (deuterated) internal standards allows for an accurate quality control of every single step of the methodology: hydrolysis efficiency, derivatization efficiency and microbiological degradation are monitored in every single sample. Additionally, special attention is paid to the relationships between parameters indicating degradation by micro-organisms and the reliability of the steroid profile. The impact of the degradation is studied by evaluation of the quantities and percentages of 5α-androstane-3,17-dione and 5β-androstane-3,17-dione. The concept of measurement uncertainty was introduced for the evaluation of relative abundances of mass-to-charge ratios and the obtained ranges were compared with the World Anti-Doping Agency regulations on tolerance windows for relative ion intensities. The results indicate that the approaches are similar.

Development of a GC/C/IRMS method – Confirmation of a novel steroid profiling approach in doping control

In doping control, an athlete can only be convicted with the misuse with endogenous steroids like testosterone (T), if abnormal values of steroid metabolites and steroid ratios are observed and if the subsequent analysis with isotope ratios mass spectrometry (IRMS) confirms the presence of exogenously administered androgens. In this work, we compare the results of a novel steroid profiling approach with the performance an in-house developed IRMS method. The developed IRMS has the advantage over other methods to be relatively short in time and with target compounds androsterone, etiocholanolone, 5β-androstane 3α,17β-diol and 5α-androstane 3α,17β-diol. Pregnanediol was used as an endogenous reference compound (ERC). Reference limits for the IRMS values were established and applied as decision limits for the evaluation of excretion urine from administration with oral T, T-gel, dihydrotestosterone (DHT) - gel and dehydroepiandrosterone (DHEA). Results indicated the importance of both androstanediols as important IRMS markers where relative values compared to an ERC (Δδ(13)C) yielded better detection accuracy than absolute δ(13)C-values. The detection times of all administered endogenous steroids were evaluated using the proposed thresholds. The results of traditional steroid profiling and a new approach based upon minor steroid metabolites monitoring introduced in a longitudinal framework were evaluated with IRMS. With traditional steroid profiling methods, 95% of the atypical samples could be confirmed whereas an additional 74% of IRMS confirmed was provided by a new biomarkers strategy. These results prove that the other steroid profiling strategies can improve the efficiency in detection of misuse with endogenous steroids.

Fast quantification of 11-nor-Δ9-tetrahydrocannabinol-9-carboxylic acid (THCA) using microwave-accelerated derivatisation and gas chromatography–triple quadrupole mass spectrometry

A rapid and sensitive determination of cannabinoids in urine is important in many fields, from workplace drug testing over toxicology to the fight against doping. The detection of cannabis abuse is normally based on the quantification of the most important metabolite 11-nor-Δ9-tetrahydrocannabinol-9-carboxylic acid (THCA) in urine. In most fields THCA needs to be present at a concentration of exceeding 15ng/mL before a positive result can be reported. The method described in this paper, combines a 4min GC-MS/MS method with a fast sample preparation procedure using microwave assisted derivatisation in order to complete the quantification of THCA in urine in 30min, using only 1mL of urine. The method is selective, linear over the range 5-100ng/mL and shows excellent precision and trueness and hence, the estimated measurement uncertainty at the threshold level is small. The method also complies with applicable criteria for mass spectrometry and chromatography. Therefore the method can be used for rapid screening and confirmatory purposes.

Development of a sensitive GC-C-IRMS method for the analysis of androgens

The administration of anabolic steroids is one of the most important issues in doping control and is detectable through a change in the carbon isotopic composition of testosterone and/or its metabolites. Gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS), however, remains a very laborious and expensive technique and substantial amounts of urine are needed to meet the sensitivity requirements of the IRMS. This can be problematic because only a limited amount of urine is available for anti-doping analysis on a broad spectrum of substances. In this work we introduce a new type of injection that increases the sensitivity of GC-C-IRMS by a factor of 13 and reduces the limit of detection, simply by using solvent vent injections instead of splitless injection. This drastically reduces the amount of urine required. On top of that, by only changing the injection technique, the detection parameters of the IRMS are not affected and there is no loss in linearity.

Profiling of urinary formestane and confirmation by isotope ratio mass spectrometry.

Formestane (F, androst-4-en-4-ol-3,17-dione) is an irreversible aromatase inhibitor with the ability to suppress the estrogen production from anabolic steroids. Consequently, F is mentioned on the World Anti-Doping Agency (WADA) prohibited list and because studies have shown that F is produced endogenously in small amounts, a threshold for urinary excreted F of 150 ng/mL was introduced. Lower concentrations could be due to endogenous production and need further investigation to prove the exact origin through determination of the carbon isotope ratio. However, because the current screening methods are a lot more sensitive, F is detected in practically every urine sample. A strict implementation of this WADA rule would imply that almost every urine sample needs additional investigation to verify an exogenous or endogenous origin. The main aim of this study was to propose and introduce a lower concentration limit of 25 ng/mL beneath which the detected F is considered as being endogenous and no further investigation is needed. The data presented in this paper suggests that this threshold provides a good balance between a sufficiently large detection window and not having to perform isotope ratio mass spectrometry (IRMS) analyses on negative urine samples.

Improved sensitivity by use of gas chromatography-positive chemical ionization triple quadrupole mass spectrometry for the analysis of drug related substances.

In 2013, the World Anti-Doping Agency (WADA) drastically lowered the minimum required performance levels (MRPLs) of most doping substances, demanding a substantial increase in sensitivity of the existing methods. For a number of compounds, conventional electron impact ionization gas chromatography tandem mass spectrometry (GC-EI-MS/MS) is often no longer sufficient to reach these MRPLs and new strategies are required. In this study, the capabilities of positive ion chemical ionization (PICI) GC-MS/MS are investigated for a wide range of drug related compounds of various classes by injection of silylated reference standards. Ammonia as PICI reagent gas had superior characteristics for GC-MS/MS purposes than methane. Compared to GC-EI-MS/MS, PICI (with ammonia as reagent gas) provided more selective ion transitions and consequently, increased sensitivity by an average factor of 50. The maximum increase (by factor of 500-1000) was observed in the analysis of stimulants, namely chlorprenaline, furfenorex and phentermine. In total, improved sensitivity was obtained for 113 out of 120 compounds. A new GC-PICI-MS/MS method has been developed and evaluated for the detection of a wide variety of exogenous doping substances and the quantification of endogenous steroids in urine in compliance with the required MRPLs established by WADA in 2013. The method consists of a hydrolysis and extraction step, followed by derivatization and subsequent 1μL pulsed splitless injection on GC-PICI-MS/MS (16min run). The increased sensitivity allows the set up of a balanced screening method that meets the requirements for both quantitative and qualitative compounds: sufficient capacity and resolution in combination with high sensitivity and short analysis time. This resulted in calibration curves with a wide linear range (e.g., 48-9600ng/mL for androsterone and etiochanolone; all r(2)>0.99) without compromising the requirements for the qualitative compounds.

Gas chromatography/chemical ionization triple quadrupole mass spectrometry analysis of anabolic steroids: ionization and collision-induced dissociation behavior

RATIONALE The detection of new anabolic steroid metabolites and new designer steroids is a challenging task in doping analysis. Switching from electron ionization gas chromatography triple quadrupole mass spectrometry (GC/EI-MS/MS) to chemical ionization (CI) has proven to be an efficient way to increase the sensitivity of GC/MS/MS analyses and facilitate the detection of anabolic steroids. CI also extends the possibilities of GC/MS/MS analyses as the molecular ion is retained in its protonated form due to the softer ionization. In EI it can be difficult to find previously unknown but expected metabolites due to the low abundance or absence of the molecular ion and the extensive (and to a large extent unpredictable) fragmentation. The main aim of this work was to study the CI and collision-induced dissociation (CID) behavior of a large number of anabolic androgenic steroids (AAS) as their trimethylsilyl derivatives in order to determine correlations between structures and CID fragmentation. Clarification of these correlations is needed for the elucidation of structures of unknown steroids and new metabolites. METHODS The ionization and CID behavior of 65 AAS have been studied using GC/CI-MS/MS with ammonia as the reagent gas. Glucuronidated AAS reference standards were first hydrolyzed to obtain their free forms. Afterwards, all the standards were derivatized to their trimethylsilyl forms. Full scan and product ion scan analyses were used to examine the ionization and CID behavior. RESULTS Full scan and product ion scan analyses revealed clear correlations between AAS structure and the obtained mass spectra. These correlations were confirmed by analysis of multiple hydroxylated, methylated, chlorinated and deuterated analogs. CONCLUSIONS AAS have been divided into three groups according to their ionization behavior and into seven groups according to their CID behavior. Correlations between fragmentation and structure were revealed and fragmentation pathways were postulated.

Studies on the minor metabolite 6a-hydroxy-androstenedione for doping control purposes and its contribution to the steroid profile.

Recent publications have shown that the concentrations of minor metabolites such as formestane and 6a-hydroxy-androstenedione (6aOHADION) are import parameters, capable of increasing the specificity and efficiency of steroid abuse screening. The importance of such minor metabolites has been recognized for some time, but setting up concentration thresholds is not that straightforward with a single quadrupole gas chromatograph mass spectrometer (GC-MS) because of the low concentrations; this is especially the case for 6aOH-ADION. The main aim of this study was to propose a concentration threshold above which the detected 6aOH-ADION is considered suspicious and isotope ratio mass spectrometry (IRMS) is recommended. Routine doping control samples (2128) from athletes that entered our lab and were not found suspicious for the intake of any doping substance were used to determine the baseline concentrations of 6a-OH-ADION. For this purpose, the more sensitive gas chromatography-tandem mass spectrometry (GC-MS/MS) was used, capable of quantifying these low concentrations with high reliability. A urinary concentration threshold of 5 ng/mL was set. Concentrations above this threshold are considered suspicious and are forwarded to IRMS for confirmation in routine practice. In addition, an IRMS method was developed, capable of determining the 13C value of 6aOH-ADION. If a urine sample has an elevated 6aOH-ADION concentration and normal 13C values for the traditional IRMS target compounds, we are still able to check the 13C value of 6aOH-ADION. Six excretion studies were executed to stress the applicability of the threshold by visualizing the concentration and δ13C value time profiles of 6aOH-ADION.

Efficient approach for the detection and identification of new androgenic metabolites by applying SRM GC-CI-MS/MS: a methandienone case study.

Identification of anabolic androgenic steroids (AAS) is a vital issue in doping control and toxicology, and searching for metabolites with longer detection times remains an important task. Recently, a gas chromatography chemical ionization triple quadrupole mass spectrometry (GC-CI-MS/MS) method was introduced, and CI, in comparison with electron ionization (EI), proved to be capable of increasing the sensitivity significantly. In addition, correlations between AAS structure and fragmentation behavior could be revealed. This enables the search for previously unknown but expected metabolites by selection of their predicted transitions. The combination of both factors allows the setup of an efficient approach to search for new metabolites. The approach uses selected reaction monitoring which is inherently more sensitive than full scan or precursor ion scan. Additionally, structural information obtained from the structure specific CI fragmentation pattern facilitates metabolite identification. The procedure was demonstrated by a methandienone case study. Its metabolites have been studied extensively in the past, and this allowed an adequate evaluation of the efficiency of the approach. Thirty three metabolites were detected, including all relevant previously discovered metabolites. In our study, the previously reported long-term metabolite (18-nor-17β-hydroxymethyl,17α-methyl-androst-1,4,13-trien-3-one) could be detected up to 26 days by using GC-CI-MS/MS. The study proves the validity of the approach to search for metabolites of new synthetic AAS and new long-term metabolites of less studied AAS and illustrates the increase in sensitivity by using CI. Copyright