Yiannis S. Angelis

Preventive doping control screening analysis of prohibited substances in human urine using rapid‐resolution liquid chromatography/high‐resolution time‐of‐flight mass spectrometry

Unification of the screening protocols for a wide range of doping agents has become an important issue for doping control laboratories. This study presents the development and validation of a generic liquid chromatography/time-of-flight mass spectrometry (LC/TOFMS) screening method of 241 small molecule analytes from various categories of prohibited substances (stimulants, narcotics, diuretics, beta(2)-agonists, beta-blockers, hormone antagonists and modulators, glucocorticosteroids and anabolic agents). It is based on a single-step liquid-liquid extraction of hydrolyzed urine and the use of a rapid-resolution liquid chromatography/high-resolution time-of-flight mass spectrometric system acquiring continuous full scan data. Electrospray ionization in the positive mode was used. Validation parameters consisted of identification capability, limit of detection, specificity, ion suppression, extraction recovery, repeatability and mass accuracy. Detection criteria were established on the basis of retention time reproducibility and mass accuracy. The suitability of the methodology for doping control was demonstrated with positive urine samples. The preventive role of the method was proved by the case where full scan acquisition with accurate mass measurement allowed the retrospective reprocessing of acquired data from past doping control samples for the detection of a designer drug, the stimulant 4-methyl-2-hexanamine, which resulted in re-reporting a number of stored samples as positives for this particular substance, when, initially, they had been reported as negatives.

Gas chromatographic quantitative structure-retention relationships of trimethylsilylated anabolic androgenic steroids by multiple linear regression and partial least squares.

A quantitative structure-retention relationship (QSRR) study has been performed to correlate relative retention times (RRTs) of trimethylsilylated (TMS) anabolic androgenic steroids (AAS) with their molecular characteristics, encoded by the respective descriptors, for the prediction of RRTs of novel molecules, using gas chromatography time-of-flight mass spectrometry (GC-TOF-MS). The elucidation of similarities and dissimilarities among the data structures was carried out using principal component analysis (PCA). Successful models were established using multiple linear regression (MLR) and partial least squares (PLS) techniques as a function of topological, three-dimensional (3D) and physicochemical descriptors. The models are useful for the estimation of RRTs of designer steroids for which no analytical data is available.

External calibration in gas chromatography-combustion-isotope ratio mass spectrometry measurements of endogenous androgenic anabolic steroids in sports doping control.

An alternative calibration procedure for the gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) measurements of the World Antidoping Agency (WADA) Accredited Laboratories is presented. To alleviate the need for externally calibrated CO₂ gas for GC-C-IRMS analysis of urinary steroid metabolites, calibration using an external standard mixture solution of steroids with certified isotopic composition was investigated. The reference steroids of the calibration mixture and routine samples underwent identical instrumental processes. The calibration standards bracketed the entire range of the relevant δ¹³C values for the endogenous and exogenous steroids as well as their chromatographic retention times. The certified δ¹³C values of the reference calibrators were plotted in relation to measured m/z ¹³CO₂/¹²CO₂ (i.e. R(45/44)) mass spectrometric signals of each calibrator. δ¹³C values of the sample steroids were calculated from the least squares fit through the calibration curve. The effect of the external calibration on δ¹³C values, using the same calibration standards and set of urine samples but different brands of GC-C-IRMS instruments, was assessed by an interlaboratory study in the WADA Accredited Laboratories of Sydney, Australia and Athens, Greece. Relative correspondence between the laboratories for determination of androsterone, etiocholanolone, 5β-androstane-3α,17β-diacetate, and pregnanediacetate means were SD(δ¹³C)=0.12‰, 0.58‰, -0.34‰, and -0.40‰, respectively. These data demonstrate that accurate intralaboratory external calibration with certified steroids provided by United States Antidoping Agency (USADA) and without external CO₂ calibration is feasible and directly applicable to the WADA Accredited Laboratories for the harmonization of the GC-C-IRMS measurements.

Advances in the detection of designer steroids in anti-doping

The abuse of unknown designer androgenic anabolic steroids (AAS) is considered to be an issue of significant importance, as AAS are the choice of doping preference according to World Anti-doping Agency statistics. In addition, unknown designer AAS are preferred since the World Anti-doping Agency mass spectrometric identification criteria cannot be applied to unknown molecules. Consequently, cheating athletes have a strong motive to use designer AAS in order to both achieve performance enhancement and to escape from testing positive in anti-doping tests. To face the problem, a synergy is required between the anti-doping analytical science and sports anti-doping regulations. This Review examines various aspects of the designer AAS. First, the structural modifications of the already known AAS to create new designer molecules are explained. A list of the designer synthetic and endogenous AAS is then presented. Second, we discuss progress in the detection of designer AAS using: mass spectrometry and bioassays; analytical data processing of the unknown designer AAS; metabolite synthesis; and, long-term storage of urine and blood samples. Finally, the introduction of regulations from sports authorities as preventive measures for long-term storage and reprocessing of samples, initially reported as negatives, is discussed.

Comparison of multiple linear regression, partial least squares and artificial neural networks for prediction of gas chromatographic relative retention times of trimethylsilylated anabolic androgenic steroids.

The comparison among different modelling techniques, such as multiple linear regression, partial least squares and artificial neural networks, has been performed in order to construct and evaluate models for prediction of gas chromatographic relative retention times of trimethylsilylated anabolic androgenic steroids. The performance of the quantitative structure-retention relationship study, using the multiple linear regression and partial least squares techniques, has been previously conducted. In the present study, artificial neural networks models were constructed and used for the prediction of relative retention times of anabolic androgenic steroids, while their efficiency is compared with that of the models derived from the multiple linear regression and partial least squares techniques. For overall ranking of the models, a novel procedure [Trends Anal. Chem. 29 (2010) 101-109] based on sum of ranking differences was applied, which permits the best model to be selected. The suggested models are considered useful for the estimation of relative retention times of designer steroids for which no analytical data are available.

Two-step silylation procedure for the unified analysis of 190 doping control substances in human urine samples by GC–MS

BACKGROUND While a number of different derivatization procedures for screening GC-MS analysis of prohibited substances are followed by doping control laboratories, a unified derivatization procedure for the GC-MS analysis of 190 different doping agents was developed. RESULTS Following preliminary experiments, a two-step derivatization procedure was selected. The evaluation of various silylation parameters, such as reagent composition, reaction time, reaction temperature, catalysts and microwave oven reaction time, for this procedure was carried out. CONCLUSION The suitability of the developed procedure was demonstrated through application on urine samples at concentration levels of the minimum required performance limit for all tested substances. This new derivatization procedure, which significantly decreases time and cost, is suitable for a routine basis application.

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.

Examination of the kinetic isotopic effect to the acetylation derivatization for the gas chromatographic-combustion-isotope ratio mass spectrometric doping control analysis of endogenous steroids

In gas chromatographic-combustion-isotope ratio mass spectrometry (GC-C-IRMS) doping control analysis, endogenous androgenic anabolic steroids and their metabolites are commonly acetylated using acetic anhydride reagent, thus incorporating exogenous carbon that contributes to the measured isotope ratio. Comparison of the endogenous δ(13)C of free, mono-, and di-acetylated steroids requires application of corrections, typically through straightforward use of the mass balance equation. Variability in kinetic isotope effects (KIE) due to steroid structures could cause fractionation of endogenous steroid carbon, resulting in inaccurate results. To test for possible KIE influence on δ(13)C, acetic anhydride of graded isotope ratio within the natural abundance range was used under normal derivatization conditions to test for linearity. In all cases, plots of measured steroid acetate δ(13)C versus acetic anhydride δ(13)C were linear and slopes were not significantly different. Regression analysis of the Δδ(13)C of enriched acetic anhydrides versus Δδ(13)C of derivatized steroids shows that KIE are similar in all cases. We conclude that δ(13)C calculated from the mass balance equation is independent of the δ(13)C of the acetic anhydride reagent, and that net KIE under normal derivatization conditions do not bias the final reported steroid δ(13)C.

Two-step derivatization procedures for the ionization enhancement of anabolic steroids in LC–ESI-MS for doping control analysis

BACKGROUND Two-step derivatization procedures were developed for the enhancement of the positive ESI in LC-MS detection of anabolic androgenic steroids, a class of prohibited substances with limited ionization efficiency in atmospheric pressure interfaces. The developed procedures are based on the esterification of hydroxyl groups of anabolic steroids with picolinic acid, followed by conversion of carbonyl groups to Schiff bases by either Girards reagent T or 2-hydrazino pyridin. RESULTS Ionization efficiency for the model derivatized compounds 19-norandrosterone (nandrolone main metabolite) and methasterone was higher by almost two orders of magnitude compared with the respective efficiency of the underivatized compounds. CONCLUSION The obtained derivatives provided a significant improvement in the ESI sensitivity, compared with those of underivatized molecules in positive LC-ESI-ion trap-MS full-scan mode.

Gas chromatographic-mass spectrometric quantitation of busulfan in human plasma for therapeutic drug monitoring: a new on-line derivatization procedure for the conversion of busulfan to 1,4-diiodobutane.

A simplified gas chromatographic-mass spectrometric (GC-MS) analytical method, involving a novel derivatization procedure was developed for monitoring busulfan (Bu) plasma concentrations in populations undergoing bone marrow transplantation. Plasma samples (500 μL) containing Bu-d8 as internal standard were extracted with ethyl acetate (2 mL) followed by centrifugation (1800 rpm, 5 min) and evaporation of the organic layer under nitrogen flow (50 °C). The dry residue was reconstituted with 100 μL iodine solution in acetonitrile (0.25%, w/v) and 3 μL were injected into the GC-MS system at 250 °C. Conversion of Bu to 1,4-diiodobutane was accomplished on-line without the need of an extra derivatization step. MS was operated at selected ion monitoring mode at m/z 183 and 191 corresponding to Bu and Bu-d8 derivatives. Total analysis time was 11.5 min. Calibration curves were linear (mean r=0.9996) over a concentration range of 25-3651 ng/mL using a (1/x)-weighted scheme. Limit of detection and lower limit of quantitation were 10.6 and 25 ng/mL, respectively. Overall accuracy Er (%) was ranging from -5.10% to 10.5%. Within- and between-run RSD (%) were lower 4.51% and 2.15%, respectively. Overall recovery of Bu was equal to 69.3±4.56% (RSD (%)). The present method is sensitive and specific, requiring a simple sample preparation procedure and short analysis time, advantages crucial for therapeutic drug monitoring of Bu in clinical practice and application in pharmacokinetic studies.