Generic sample preparation combined with high-resolution liquid chromatography- time-of-flight mass spectrometry for unification of urine screening in doping-control laboratories

A unification of doping-control screening procedures of prohibited small molecule substances—including stimulants, narcotics, steroids, ß2-agonists and diuretics—is highly urgent in order to free resources for new classes such as banned proteins. Conceptually this may be achieved by the use of a combination of one gas chromatography–time-of-flight mass spectrometry method and one liquid chromatography–time-of-flight mass spectrometry method. In this work a quantitative screening method using high-resolution liquid chromatography in combination with accurate-mass time-of-flight mass spectrometry was developed and validated for determination of glucocorticosteroids, ß2-agonists, thiazide diuretics, and narcotics and stimulants in urine. To enable the simultaneous isolation of all the compounds of interest and the necessary purification of the resulting extracts, a generic extraction and hydrolysis procedure was combined with a solid-phase extraction modified for these groups of compounds. All 56 compounds are determined using positive electrospray ionisation with the exception of the thiazide diuretics for which the best sensitivity was obtained by using negative electrospray ionisation. The results show that, with the exception of clenhexyl, procaterol, and reproterol, all compounds can be detected below the respective minimum required performance level and the results for linearity, repeatability, within-lab reproducibility, and accuracy show that the method can be used for quantitative screening. If qualitative screening is sufficient the instrumental analysis may be limited to positive ionisation, because all analytes including the thiazides can be detected at the respective minimum required levels in the positive mode. The results show that the application of accurate-mass time-of-flight mass spectrometry in combination with generic extraction and purification procedures is suitable for unification and expansion of the window of screening methods of doping laboratories. Moreover, the full-scan accurate-mass data sets obtained still allow retrospective examination for emerging doping agents, without re-analyzing the samples

Quantitative structure-retention relationship study of α-, β1-, and β2-agonists using multiple linear regression and partial least-squares procedures

A quantitative structure-retention relationship (QSRR) study has been performed for the correlation of the gas chromatographic-mass spectrometry (GC-MS) relative retention times (RRT) of α-, β1-, and β2-agonists with their molecular characteristics in order to create models for the prediction of RRT values of unanalyzed molecules. The data structure was investigated by principal component analysis (PCA). Modeling of the RRT of α-, β1-, and β2-agonists, as a function of many diverse descriptors, was established by means of the multiple linear regression (MLR) and partial least-squares projections to latent structures (PLS) methods. © 2004 Published by Elsevier B.V

Quantitative structure-retention relationship study of α-, β1-, and β2-agonists using multiple linear regression and partial least-squares procedures

A quantitative structure-retention relationship (QSRR) study has been performed for the correlation of the gas chromatographic-mass spectrometry (GC-MS) relative retention times (RRT) of α-, β1-, and β2-agonists with their molecular characteristics in order to create models for the prediction of RRT values of unanalyzed molecules. The data structure was investigated by principal component analysis (PCA). Modeling of the RRT of α-, β1-, and β2-agonists, as a function of many diverse descriptors, was established by means of the multiple linear regression (MLR) and partial least-squares projections to latent structures (PLS) methods. © 2004 Published by Elsevier B.V

Stabilization of human urine doping control samples: II. Microbial degradation of steroids

The transportation of urine samples, collected for doping control analysis, does not always meet ideal conditions of storage and prompt delivery to the World Anti-Doping Agency (WADA) accredited laboratories. Because sample collection is not conducted under sterile conditions, microbial activity may cause changes to the endogenous steroid profiles of samples. In the current work, funded by WADA, a chemical mixture consisting of antibiotics, antimycotic substances and protease inhibitors was applied in urine aliquots fortified with conjugated and deuterated steroids and inoculated with nine representative microorganisms. Aliquots with and without the chemical mixture were incubated at 37 °C for 7 days to simulate the transportation period, whereas another series of aliquots was stored at -20 °C as reference. Microbial growth was assessed immediately after inoculation and at the end of the incubation period. Variations in pH and specific gravity values were recorded. Gas chromatography-mass spectrometry (GC-MS) analysis was performed for the detection of steroids in the free, glucuronide, and sulfate fractions. The addition of the chemical stabilization mixture to urine samples inhibited microorganism growth and prevented steroid degradation at 37 °C. On the other hand, four of the nine microorganisms induced alterations in the steroid profile of the unstabilized samples incubated at 37 °C. © 2009 Elsevier Inc. All rights reserved

Stabilization of human urine doping control samples: IV. Human chorionic gonadotropin

The presence of proteolytic enzymes in urine samples, coming from exogenous or endogenous sources, enhances the cleavage of human chorionic gonadotropin (hCG). Moreover, elevated temperatures occurring occasionally during the delayed transportation of sport urine samples, favor the nicking of the hCG molecule. The aim of the current study, funded by the World Anti-Doping Agency (WADA), was the application of a stabilization mixture in athletes' urine samples to chemically inactivate proteolytic enzymes coming from exogenous or endogenous sources so as to prevent the degradation of hCG. The stabilization mixture applied, already tested for the stabilization of endogenous steroids and recombinant erythropoietin (rEPO), was a combination of antibiotics, antimycotic substances, and protease inhibitors. Incubation experiments were conducted in the presence or absence of the stabilization mixture in urine aliquots spiked with six proteases (first series of experiments) and one microorganism associated with urinary tract infections (UTI) (second series of experiments). Intact hCG levels were evaluated by using the EIAgen Total hCG kit. In the first series of experiments, hCG levels were reduced in the untreated aliquots following incubation at 37 °C. The addition of the chemical stabilization mixture prevented degradation of hCG induced by four of the proteases applied. In the second series of experiments, no significant difference was found in urine inoculated with E. coli, between aliquots treated with chemical mixture and the untreated aliquots. The addition of the proposed chemical stabilization mixture improves the quality of athletes' urine samples against possible deterioration due to high temperatures or attempts of proteolytic manipulation. © 2010 Springer-Verlag

Stabilization of human urine doping control samples: A current opinion

Transportation of doping control urine samples from the collection sites to the World Anti-doping Agency (WADA) Accredited Laboratories is conducted under ambient temperatures. When sample delivery is not immediate, microbial contamination of urine, especially in summer, is a common phenomenon that may affect sample integrity and may result in misinterpretation of analytical data. Furthermore, the possibility of intentional contamination of sports samples during collection with proteolytic enzymes, masking the abuse of prohibited proteins such as erythropoietin (EPO) and peptide hormones, is a practice that has already been reported. Consequently, stabilization of urine samples with a suitable method in a way that protects samples' integrity is important. Currently, no stabilization method is applied in the sample collection equipment system in order to prevent degradation of urine compounds. The present work is an overview of a study, funded by WADA, on degradation and stabilization aspects of sports urine samples against the above threats of degradation. Extensive method development resulted in the creation of a mixture of chemical agents for the stabilization of urine. Evaluation of results demonstrated that the stabilization mixture could stabilize endogenous steroids, recombinant EPO, and human chorionic gonadotropin in almost the entire range of the experimental conditions tested. © 2011 Springer-Verlag

Stabilization of human urine doping control samples: III. Recombinant human erythropoietin

Background: The tampering of athlete's urine samples by the addition of proteolytic enzymes during the doping control sampling procedure was reported recently. The aim of the current study, funded by the World Anti-Doping Agency (WADA), was the application of a stabilization mixture in urine samples to chemically inactivate proteolytic enzymes and improve the electrophoteric signal of erythropoietin (EPO) in human urine. Methods: The stabilization mixture applied was a combination of antibiotics, antimycotic substances and protease inhibitors. A series of incubation experiments were conducted under controlled conditions in the presence and absence of the stabilization mixture in urine aliquots spiked with six proteases. Two different analytical techniques were applied for the qualitative and quantitative EPO measurement: isoelectric focusing (IEF) and chemiluminescent immunoassay respectively. Results: The addition of the chemical stabilization mixture into urine aliquots substantially improved EPO detection in the presence of proteolytic enzymes following incubation at 37 °C or storage at - 20 °C. Conclusions: The results of this study indicated that the stabilization of urine prior to the sample collection procedure with the proposed chemical mixture might prove to be a useful tool for the preservation of anti-doping samples. © 2009 Elsevier B.V. All rights reserved

Study of excretion of ecdysterone in human urine

A study of excretion in human urine of ecdysterone, which is the active component of several over-the-counter supplements such as 'Ecdysten', reportedly used by athletes, is presented. The study was performed after oral administration of 20 mg of ecdysterone. The collected urine samples were prepared using the standard screening extraction procedure for the free and conjugated fraction of anabolic steroids, and analyzed by gas chromatography (GC) coupled with quadrupole mass spectrometry (MS) and also with high-resolution mass spectrometry (HRMS). Two ecdysterone metabolites were identified and detected along with unchanged ecdysterone. Accurate mass measurements were made for diagnostic ions, including the molecular ion of the main metabolite of ecdysterone, deoxyecdysone, which, to our knowledge, has not previously been reported in the literature. These accurate mass measurements support the proposed fragmentation scheme. Copyright © 2001 John Wiley & Sons, Ltd

A generic screening methodology for horse doping control by LC-TOF-MS, GC-HRMS and GC-MS

In the present study a general screening protocol was developed to detect prohibited substances and metabolites for doping control purposes in equine sports. It was based on the establishment of a unified sample preparation and on the combined implementation of liquid and gas chromatographic MS analysis. The sample pretreatment began with two parallel procedures: enzymatic hydrolysis of sulfate and glucuronide conjugates, and methanolysis of the 17β-sulfate steroid conjugates. The extracts were treated for LC-TOF-MS, GC-HRMS and GC-MS assays. The majority of the prohibited substances were identified through a high mass accuracy technique, such as LC-TOF-MS, without prior derivatization. The sample preparation procedure included the formation of methylated and trimethylsilylated derivatives common in toxicological GC-MS libraries. The screening method was enhanced by post-run library searching using automated mass spectral deconvolution and identification system (AMDIS) combined with deconvolution reporting software (DRS). The current methodology is able to detect the presence of more than 350 target analytes in horse urine and may easily incorporate a lot of new substances without changes in chromatography. The full scan acquisition allows retrospective identification of prohibited substances in stored urine samples after reprocessing of the acquired data. Validation was performed for sixty representative compounds and included limit of detection, matrix interference - specificity, extraction recovery, precision, mass accuracy, matrix effect and carry over contamination. The suitability of the method was demonstrated with previously declared positive horse urine samples. © 2013 Elsevier B.V