Doping control container for urine stabilization: a pilot study.

Urine collection containers used in the doping control collection procedure do not provide a protective environment for urine, against degradation by microorganisms and proteolytic enzymes. An in-house chemical stabilization mixture was developed to tackle urine degradation problems encountered in human sport samples, in cases of microbial contamination or proteolytic activity. The mixture consists of antimicrobial substances and protease inhibitors for the simultaneous inactivation of a wide range of proteolytic enzymes. It has already been tested in lab-scale, as part of World Anti-Doping Agency's (WADA) funded research project, in terms of efficiency against microbial and proteolytic activity. The present work, funded also by WADA, is a follow-up study on the improvement of chemical stabilization mixture composition, application mode and limitation of interferences, using pilot urine collection containers, spray-coated in their internal surface with the chemical stabilization mixture. Urine in plastic stabilized collection containers have been gone through various incubation cycles to test for stabilization efficiency and analytical matrix interferences by three WADA accredited Laboratories (Athens, Ghent, and Rome). The spray-coated chemical stabilization mixture was tested against microorganism elimination and steroid glucuronide degradation, as well as enzymatic breakdown of proteins, such as intact hCG, recombinant erythropoietin and small peptides (GHRPs, ipamorelin), induced by proteolytic enzymes. Potential analytical interferences, observed in the presence of spray-coated chemical stabilization mixture, were recorded using routine screening procedures. The results of the current study support the application of the spray-coated plastic urine container, in the doping control collection procedure. Copyright © 2016 John Wiley & Sons, Ltd.

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.

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.