Nik De Brabanter

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.

In vivo and in vitro metabolism of the synthetic cannabinoid JWH‐200

RATIONALE The synthetic cannabinoid JWH-200 (1-[2-(4-morpholinyl)ethyl]-3-(1-naphthoyl)-indole) appeared on the market around 2009. In order to identify markers for misuse of this compound and allow for the development of adequate routine methods, the metabolism of this compound was investigated using two models. METHODS In vitro and in vivo (both with and without enzymatic hydrolysis) samples were purified by solid-phase extraction and analyzed using liquid chromatography. Electrospray ionization high-resolution Orbitrap mass spectrometry was used for the identification of the metabolites. To confirm the results in vivo, triple-quadrupole mass spectrometry was employed RESULTS In the in vitro model, using human liver microsomes, 22 metabolites were detected which could be divided into 11 metabolite classes. By using the chimeric mouse model with humanized liver, most of these metabolites were confirmed in vivo. It was found that all metabolites are excreted in urine as conjugates, mostly as glucuronides with varying conjugation rates. CONCLUSIONS The metabolite formed by consecutive morpholine cleavage and oxidation of the remaining side chain to a carboxylic group was detected in the highest amounts with the longest detection time. Therefore, it is the best candidate metabolite to detect JWH-200 abuse in urine.

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.

Current status and bioanalytical challenges in the detection of unknown anabolic androgenic steroids in doping control analysis

Androgenic anabolic steroids (AAS) are prohibited in sports due to their anabolic effects. Doping control laboratories usually face the screening of AAS misuse by target methods based on MS detection. Although these methods allow for the sensitive and specific detection of targeted compounds and metabolites, the rest remain undetectable. This fact opens a door for cheaters, since different AAS can be synthesized in order to evade doping control tests. This situation was evidenced in 2003 with the discovery of the designer steroid tetrahydrogestrinone. One decade after this discovery, the detection of unknown AAS still remains one of the main analytical challenges in the doping control field. In this manuscript, the current situation in the detection of unknown AAS is reviewed. Although important steps have been made in order to minimize this analytical problem and different analytical strategies have been proposed, there are still some drawbacks related to each approach.