M. Koupparis

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.

Structural characteristics of anabolic androgenic steroids contributing to binding to the androgen receptor and to their anabolic and androgenic activities. Applied modifications in the steroidal structure.

Anabolic androgenic steroids (AAS) are synthetic derivatives of testosterone introduced for therapeutic purposes providing enhanced anabolic potency with reduced androgenic effects. Androgens mediate their action through their binding to the androgen receptor (AR) which is mainly expressed in androgen target tissues, such as the prostate, skeletal muscle, liver and central nervous system. This paper reviews some of the wide spectrum of testosterone and synthetic AAS structure modifications related to the intended enhancement in anabolic activity. The structural features of steroids necessary for effective binding to the AR and those which contribute to the stipulation of the androgenic and anabolic activities are also presented.

Schemes of metabolic patterns of anabolic androgenic steroids for the estimation of metabolites of designer steroids in human urine

Unified metabolism schemes of anabolic androgenic steroids (AAS) in human urine based on structure classification of parent molecules are presented in this paper. Principal components analysis (PCA) was applied to AAS molecules referred in the World Anti-Doping Agency (WADA) list of prohibited substances, resulting to their classification into six distinct groups related to structure features where metabolic alterations usually occur. The metabolites of the steroids participating to these six groups were treated using the Excel(c) classification filters showing that common metabolism routes are derived for each of the above PCA classes, leading to the proposed metabolism schemes of the present study. This rule-based approach is proposed for the prediction of the metabolism of unknown, chemically modified steroids, otherwise named as designer steroids. The metabolites of three known, in the literature, AAS are estimated using the proposed metabolism schemes, confirming that their use could be a useful tool for the prediction of metabolic pathways of unknown AAS.

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.

Direct injection LC/ESI-MS horse urine analysis for the quantification and identification of threshold substances for doping control. I. Determination of hydrocortisone

Two simple and rapid LC/MS methods with direct injection analysis were developed and validated for the quantification and identification of hydrocortisone in equine urine using the same sample preparation but different mass spectrometric systems: ion trap mass spectrometry (IT-MS) and time-of-flight mass spectrometry (TOF-MS). The main advantage of the proposed methodology is the minimal sample preparation procedure, as particle-free diluted urine samples were directly injected into both LC/MS systems. Desonide was used as internal standard (IS). The linear range was 0.25-2.5 microg ml(-1) for both methods. Matrix effects were evaluated by preparing and analyzing calibration curves in water solutions and different horse urine samples. A great variation of the signal both for hydrocortisone and the internal standard was observed in different matrices. To overcome matrix effects, the unavailability of blank matrix and the excessive cost of the isotopically labeled internal standard, standard additions calibration method was applied. This work is an exploration of the performance of the standard additions approach in a method where neither nonisotopic internal standards nor extensive sample preparation is utilized and no blank matrix is available. The relative standard deviations of intra and interday analysis of hydrocortisone in horse urine were lower than 10.2 and 5.4%, respectively, for the LC/IT-MS method and lower than 8.4 and 4.4%, respectively, for the LC/TOF-MS method. Accuracy (bias percentage) was less than 9.7% for both methods.

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.

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.

Direct injection liquid chromatography/electrospray ionization mass spectrometric horse urine analysis for the quantification and confirmation of threshold substances for doping control. II. Determination of theobromine

In equine sport, theobromine is prohibited with a threshold level of 2 microg mL(-1) in urine, hence doping control laboratories have to establish quantitative and qualitative methods for its determination. Two simple liquid chromatography/mass spectrometry (LC/MS) methods for the identification and quantification of theobromine were developed and validated using the same sample preparation procedure but different mass spectrometric systems: ion trap mass spectrometry (ITMS) and time-of-flight mass spectrometry (TOFMS). Particle-free diluted urine samples were directly injected into the LC/MS systems, avoiding the time-consuming extraction step. 3-Propylxanthine was used as the internal standard. The tested linear range was 0.75-15 microg mL(-1). Matrix effects were evaluated analyzing calibration curves in water and different fortified horse urine samples. A great variation in the signal of theobromine and the internal standard was observed in different matrices. To overcome matrix effects, a standard additions calibration method was applied. The relative standard deviations of intra- and inter-day analysis were lower than 8.6 and 7.2%, respectively, for the LC/ITMS method and lower than 5.7 and 5.8%, respectively, for the LC/TOFMS method. The bias was less than 8.7% for both methods. The methods were applied to two case samples, demonstrating simplicity, accuracy and selectivity.

The effect of ion pairing reagents in the retention profile of zwitterionic cephalosporins

Abstract The retention of three zwitterionic cephalosporins, Cefepime, Cefpirome, and Ceftazidime, in a broad pH range was studied by means of reversed phase high performance liquid chromatography (RP‐HPLC) and reversed phase ion‐pair liquid chromatography (RP‐IPC), using an octadecylsilane stationary phase and acetonitrile as organic modifier. Sodium hexane‐, heptane‐, and octane‐sulphonate and tetrabutylammonium hydroxide were used as sources of counter ions in ion pair chromatography. The presence of the permanently charged quaternary nitrogen in the cephalosporin molecules leads to zwitterionic species over a broad pH range, masking the carboxylic acid function and stabilizing the retention. The effect of the counter ion depends on the ionization state of the compounds. Formation of ion pairs with the opposite charged center of the molecules led to significant increase in retention. At pH values favoring the zwitterionic species a decrease in retention was observed as a result of the disruption of the intramolecular interactions and the deliberation of the free charge. The extent of ion pair formation and the competition with the zwitterionic species, depended on the counter ion concentration in the mobile phase and was reflected in retention. The effect of the size of the counter anion alkyl chain on retention was investigated at different pH as well.

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.