Gas chromatography/chemical ionization triple quadrupole mass spectrometry analysis of anabolic steroids: ionization and collision-induced dissociation behavior.

RATIONALE: The detection of new anabolic steroid metabolites and new designer steroids is a challenging task in doping analysis. Switching from electron ionization gas chromatography triple quadrupole mass spectrometry (GC/EI-MS/MS) to chemical ionization (CI) has proven to be an efficient way to increase the sensitivity of GC/MS/MS analyses and facilitate the detection of anabolic steroids. CI also extends the possibilities of GC/MS/MS analyses as the molecular ion is retained in its protonated form due to the softer ionization. In EI it can be difficult to find previously unknown but expected metabolites due to the low abundance or absence of the molecular ion and the extensive (and to a large extent unpredictable) fragmentation. The main aim of this work was to study the CI and collision-induced dissociation (CID) behavior of a large number of anabolic androgenic steroids (AAS) as their trimethylsilyl derivatives in order to determine correlations between structures and CID fragmentation. Clarification of these correlations is needed for the elucidation of structures of unknown steroids and new metabolites. METHODS: The ionization and CID behavior of 65 AAS have been studied using GC/CI-MS/MS with ammonia as the reagent gas. Glucuronidated AAS reference standards were first hydrolyzed to obtain their free forms. Afterwards, all the standards were derivatized to their trimethylsilyl forms. Full scan and product ion scan analyses were used to examine the ionization and CID behavior. RESULTS: Full scan and product ion scan analyses revealed clear correlations between AAS structure and the obtained mass spectra. These correlations were confirmed by analysis of multiple hydroxylated, methylated, chlorinated and deuterated analogs. CONCLUSIONS: AAS have been divided into three groups according to their ionization behavior and into seven groups according to their CID behavior. Correlations between fragmentation and structure were revealed and fragmentation pathways were postulated.

GC-C-IRMS in routine doping control practice: 3 years of drug testing data, quality control and evolution of the method.

In order to detect the misuse of endogenous anabolic steroids, doping control laboratories require methods that allow differentiation between endogenous steroids and their synthetic copies. Gas chromatography combustion isotope ratio mass spectrometry (GC-C-IRMS) is capable of measuring the carbon isotope ratio of urinary steroids and this allows differentiation between both. GC-C-IRMS and its application to doping control has evolved a lot during the last decade and so have the World Anti-Doping Agency (WADA) technical documents that describe how GC-C-IRMS should be applied. Especially the WADA technical document of 2014 introduced a number of obligatory quality controls and a fixed methodology that should be used by all the doping control laboratories. This document imposed more uniform methods between the laboratories in order to decrease the interlaboratory standard deviation and acquire similar results for the analysis of the same urine samples. In this paper, 3 years of drug testing data of our GC-C-IRMS method in routine doping control practice is described, with an emphasis on the new WADA technical document and its implementation. Useful data for other doping control laboratories is presented focussing on general method setup, quality control and data collected from routine samples. The paper concentrates on how IRMS results shift or remain similar by switching to the 2014 WADA technical document and gives insight in a straightforward approach to calculate the measurement uncertainty.

Metabolism of methylstenbolone studied with human liver microsomes and the uPA⁺/⁺-SCID chimeric mouse model.

Anti-doping laboratories need to be aware of evolutions on the steroid market and elucidate steroid metabolism to identify markers of misuse. Owing to ethical considerations, in vivo and in vitro models are preferred to human excretion for nonpharmaceutical grade substances. In this study the chimeric mouse model and human liver microsomes (HLM) were used to elucidate the phase I metabolism of a new steroid product containing, according to the label, methylstenbolone. Analysis revealed the presence of both methylstenbolone and methasterone, a structurally closely related steroid. Via HPLC fraction collection, methylstenbolone was isolated and studied with both models. Using HLM, 10 mono-hydroxylated derivatives (U1-U10) and a still unidentified derivative of methylstenbolone (U13) were detected. In chimeric mouse urine only di-hydroxylated metabolites (U11-U12) were identified. Although closely related, neither methasterone nor its metabolites were detected after administration of isolated methylstenbolone. Administration of the steroid product resulted mainly in the detection of methasterone metabolites, which were similar to those already described in the literature. Methylstenbolone metabolites previously described were not detected. A GC-MS/MS multiple reaction monitoring method was developed to detect methylstenbolone misuse. In one out of three samples, previously tested positive for methasterone, methylstenbolone and U13 were additionally detected, indicating the applicability of the method.

High-temperature liquid chromatography-isotope ratio mass spectrometry methodology for carbon isotope ratio determination of anabolic steroids in urine.

BACKGROUND: Gas Chromatography Isotope Ratio Mass Spectrometry (GC-C-IRMS) has long been used in routine laboratories to determine the δ13C values of anabolic steroids in urine, differentiating between, e.g., endogenous and synthetic testosterone (T) in sports doping control. Until now, liquid chromatography (LC-IRMS) has not been used. The LC-IRMS setup doesn't allow organic solvents or modifiers in the mobile phase for δ13C determinations. Mid-to non-polar analytes such as steroids can be analysed in water heated to High Temperatures (HT, up to 200 °C) because at 200 °C has a similar polarity as 80/20 methanol/water at ambient temperature. In this work, we developed a method for steroids in urine, extending the application of the LC-IRMS to non-polar analytes in complex matrices. RESULT: An HT-LC-IRMS method capable of determining the δ13C values of four steroids (i.e., testosterone (T), 5α-androstane-3α,17ß-diol (ααß), 5ß-androstane-3α,17ß-diol (ßαß) and pregnanetriol (PT)) in urine was developed and validated. Accuracy ranged from 0.23 ‰ (ααß and ßαß) to 0.49 ‰ (T), and the detection limit was set at 10 ng mL-1 (T, ααß+ßαß). The validation data and a comparison of authentic urine samples analysed with HT-LC-IRMS and GC-C-IRMS indicated a comparable performance between HT-LC-IRMS and GC-C-IRMS. SIGNIFICANCE: HT-LC-IRMS can be used to determine δ13C values of anabolic steroids, extending the applicability of both HT-LC and LC-IRMS to non-polar substances determined in a complex matrix in routine laboratory practice.

Evaluation of analytical columns suitable for high-temperature liquid-chromatography-isotope-ratio-mass-spectrometry analysis of anabolic steroids.

Isotope ratio mass spectrometry (IRMS) can be used to determine the carbon isotope ratio of anabolic steroids. For example, in sports doping and food safety control, it enables determining an endogenous or synthetic origin of anabolic steroids. Generally, the steroids of interest are purified by liquid chromatography (LC) and analysed by gas chromatography combustion IRMS. LC-IRMS is not used since only mobile phases without carbon atoms can be used. For analysing mid-to apolar compounds, heated water can be used as an eluent as it has a similar polarity to a weak polar organic solvent. The silica-based columns are not robust enough at elevated temperatures in aqueous conditions. However, modified silica particles, metal oxides coated with polymers, and porous graphitic carbon are promising column materials for high-temperature LC (HT-LC) applications. Here, the stability of the stationary phase is crucial, and their chromatographic performance needs to be evaluated under the conditions mentioned above for anabolic steroid separations. Six columns using temperatures up to 200 °C were assessed, and only two were found to be appropriate. The ZirChrom-PBD column can be used for HT-LC-IRMS research purposes but is not recommended for routine laboratory practice applications due to the substantial loss of retention and resolution over time at elevated temperatures. Sachtopore-RP columns are the only suitable option for routine HT-LC-IRMS applications, even though they suffer from peak broadening over time when operating at elevated temperatures.

Prevalence of nandrolone preparations with endogenous carbon isotope ratios in Australia.

Nandrolone and its prohormones, including 19-norandrost-4-ene-3,17-dione and 19-norandrost-4-ene-3ß,17ß-diol, are anabolic steroids forbidden at all times in sports according to the World Anti-Doping Code Prohibited List and its metabolite 19-norandrosterone (19NA) is the preferred urinary target compound to identify their abuse. In recent years, an increasing number of 19NA isotope ratio mass spectrometry (IRMS) cases have arisen that, based on the initial testing procedure, were likely to result in an adverse analytical finding but were concluded negative after IRMS analysis. The current study was therefore set up to gain a better insight on the prevalence of nandrolone preparations with endogenous carbon isotope ratio values in Australia. Suitable workplace (non-athlete) urine samples that had previously been reported positive for 19NA were identified and analysed on IRMS. A total of 82% of the samples that were analysed were reported with enriched carbon isotope ratios of 19NA (i.e., 19NA greater than -26‰). This indicates that there is a high prevalence of nandrolone-containing anabolic androgenic steroid preparations in Australia that have 'endogenous' carbon isotope ratios which reduces the ability to identify exogenous nandrolone.

Development of a sensitive GC-C-IRMS method for the analysis of androgens.

The administration of anabolic steroids is one of the most important issues in doping control and is detectable through a change in the carbon isotopic composition of testosterone and/or its metabolites. Gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS), however, remains a very laborious and expensive technique and substantial amounts of urine are needed to meet the sensitivity requirements of the IRMS. This can be problematic because only a limited amount of urine is available for anti-doping analysis on a broad spectrum of substances. In this work we introduce a new type of injection that increases the sensitivity of GC-C-IRMS by a factor of 13 and reduces the limit of detection, simply by using solvent vent injections instead of splitless injection. This drastically reduces the amount of urine required. On top of that, by only changing the injection technique, the detection parameters of the IRMS are not affected and there is no loss in linearity.

Evaluation of alternative gas chromatographic and mass spectrometric behaviour of trimethylsilyl-derivatives of non-hydrolysed sulfated anabolic steroids.

Sulfated metabolites have shown to have potential as long-term markers (LTMs) of anabolic-androgenic steroid (AAS) abuse. The compatibility of gas chromatography-mass spectrometry (GC-MS) with trimethylsilyl (TMS)-derivatives of non-hydrolysed sulfated steroids has been demonstrated, where, after derivatisation, generally, two closely eluting isomers are formed that both have the same molecular ion [M-H2 SO4 ]•+ . Sulfated reference standards are in limited commercial availability, and therefore, the current knowledge of the GC-MS behaviour of these compounds is mainly based on sulfating and analysing the available standard reference material. This procedure can unfortunately not cover all of the current known LTMs as these are often not available as pure substance. Therefore, in theory, some metabolites could be missed as they exhibit alternative behaviour. To investigate the matter, in-house sulfated reference materials that bear resemblance to known sulfated LTMs were analysed on GC-MS in their TMS-derivatised non-hydrolysed state. The (alternative) gas chromatographic and mass spectrometric behaviour was mapped, evaluated and linked to the corresponding steroid structures. Afterwards, using fraction collection, known sulfated LTMs were isolated from excretion urine to confirm the observed findings. The categories that were selected were mono-hydroxy-diones, 17-methyl-3,17-diols and 17-keto-3,16-diols as these are commonly encountered AAS conformations. The ability to predict the GC-MS behaviour of non-hydrolysed sulfated AAS metabolites is the corner stone of finding new metabolites. This knowledge is also essential, for example, for understanding AAS detection analyses, for the mass spectrometric characterization of metabolites of new designer steroids or when one needs to characterize an unknown steroid structure.

Detection time comparison of non-hydrolysed sulphated metabolites of metenolone, mesterolone and 17α-methyltestosterone analysed by four different mass spectrometric techniques.

The frequent detection of anabolic androgenic steroids (AAS) indicates their popularity among rule-breaking athletes. The so called long-term metabolites play a crucial role in their detection, and non-hydrolysed sulphated metabolites have gained renewed interest, as research has demonstrated their extended detection time compared to the more conventional markers (e.g., for metenolone and mesterolone). Their potential has been investigated using liquid and gas chromatography-mass spectrometry (LC- and GC-MS). However, due to their complementary nature, chances are that the most promising metabolite on one technique does not necessarily exhibit the same behaviour on the other and vice versa. Therefore, a comparison was carried out where as a trial model, metenolone, mesterolone and 17α-methyltestosterone were selected and the most likely long-term sulphated metabolites identified on four mass spectrometric instruments. Additionally, using a modified sample preparation procedure, comparison between conventional and non-hydrolysed sulphated metabolites between different GC-MS instruments was also included. When focusing on each individual marker, no cases were observed where a single metabolite provided a superior detection time on all instruments. Furthermore, for each AAS, there were incidences where a metabolite provided the best detection time on one instrument but could only be detected for a shorter period or not at all on other instruments. This demonstrates that metabolite detection windows and hence their added-value as target substance are unique and dependent on the analytical technique and not only on their pharmacokinetic behaviour. Consequently, in each case, a metabolite versus instrument evaluation is needed to maximise the probabilities of detecting doping offences.

An antibody-free, ultrafiltration-based assay for the detection of growth hormone-releasing hormones in urine at low pg/mL concentrations using nanoLC-HRMS/MS.

This work presents an ultrafiltration-based, validated method for the screening and confirmation of prohibited growth hormone-releasing hormone (GHRH) analogues (sermorelin/CJC-1293, sermorelin metabolite, CJC-1295 and tesamorelin) in urine by nanoLC-HRMS/MS. Sample preparation avoids the use of laborious antibody-based extraction approaches and consists solely of preconcentration by ultrafiltration. Even in the absence of immuno-affinity purification steps, high sensitivity was still ensured as limits of detection between 5 and 25 pg/mL and limits of identification between 25 and 50 pg/mL were established. The robustness of the miniaturized chromatographic setup was evaluated through the injection of 200 + preconcentrated urinary extracts. In a comparison with immuno-affinity purification, enhanced recoveries (59 - 115%) and similar sensitivity were achieved, yet at lower operational costs. Stability experiments showed the importance of the proper handling of urine samples to avoid degradation of these peptide hormones, especially for sermorelin and its metabolite which were found to rapidly degrade at temperatures > 4 °C and pH values < 7 in accordance with earlier studies. Without the need for specific antibodies, this method may be expanded to cover emerging peptide drugs (≥ ~3 kDa), as well as their metabolites in the future to facilitate coverage for this class of prohibited substances.

A uniform sample preparation procedure for gas chromatography combustion isotope ratio mass spectrometry for all human doping control relevant anabolic steroids using online 2/3-dimensional liquid chromatography fraction collection.

Androgenic anabolic steroids are the most misused substances in sports because of their performance-enhancing effects. Often synthetic analogues of endogenously present steroids are administered. To determine their endogenous or exogenous origin, Gas Chromatography Combustion Isotope Ratio Mass Spectrometry (GC-C-IRMS) is used in the field of doping control. Compounds subjected to IRMS analysis must be interference-free, with liquid chromatography fraction collection (HPLC-FC) being the crucial clean-up step. However, this clean-up is challenging, particularly for compounds present at low concentrations in samples with pronounced matrix effects. The compounds of interests for IRMS analyses in doping control are testosterone (T) and its main metabolites (androsterone, etiocholanolone, 5α-androstane-3α,17ß-diol, 5ß-androstane-3α,17ß-diol), epitestosterone, 19-norandrosterone (19-NA), boldenone (B) and its main metabolite (BM), formestane (F) and 6αOH-androstenedione (6aOHADION). Currently, the available methods only deal with a selection of the above-mentioned compounds. Some of these compounds (e.g., 19-NA, B, BM, 6aOHADION) are present in very low concentrations, requiring an extensive and dedicated sample clean-up, and this makes it challenging to develop a universal clean-up procedure. Many of these methods require different and multiple offline HPLC-FC setups, which are labour-intensive and time-consuming. That is problematic during, e.g., large sports events, where reporting time is limited (e.g., 72 h). Therefore, in the current work, we developed a uniform online 2D/3D HPLC-FC method, capable of purifying all relevant target compounds in a single run, leading to the fastest clean-up procedure so far (i.e., 31 min for T and its main metabolites; 46 min for 19-NA, F and 6aOHADION; 48 min for B and BM).

Enabling the inclusion of non-hydrolysed sulfated long term anabolic steroid metabolites in a screening for doping substances by means of gas chromatography quadrupole time-of-flight mass spectrometry.

The World Anti-Doping Agency (WADA) publishes yearly their prohibited list, and sets a minimum required performance limit for each substance. To comply with these stringent requirements, the anti-doping laboratories have at least two complementary methods for their initial testing procedure (ITP), one using gas chromatography - mass spectrometry (GC-MS) and the other using liquid chromatography-MS (LC-MS). Anabolic androgenic steroids (AAS) have in previous years consistently been listed as the most frequently detected class of compounds. Over the last decade, evidence has emerged where a longer detection time is attained by focusing on sulfated metabolites of AAS instead of the conventional gluco-conjugated metabolites. Despite a decade of research on sulphated AAS using LC-MS, no LC-MS ITP has been developed that combines this class of compounds with the other mandatory targets. Such combination is essential for economical purposes. Recently, it was demonstrated that the direct injection of non-hydrolysed sulfates is compatible with GC-MS. Using this approach and by taking full use of the open screening capabilities of the quadrupole time of flight MS (QTOF-MS), this work describes for the first time a validated ITP that allows the detection of non-hydrolysed sulfated metabolites of AAS while, simultaneously, remaining capable of detecting a vast range of other classes of compounds, as well as the quantification of endogenous steroids, as required for an ITP compliant with the applicable WADA regulations. The method contains 263 compounds from 9 categories, including stimulants, narcotics, anabolic androgenic steroids and beta-blockers. Additionally, the advantages of the new method were illustrated by analysing excretion samples of drostanolone, mesterolone and metenolone. No negative effects were observed for the conventional markers and the detection time for mesterolone and metenolone increased by up to 150% and 144%, respectively compared to conventional markers.

Investigation of the urinary excretion of prednisolone and metabolites after nasal administration: Relevance to doping control.

Glucocorticosteroid use in sport is restricted to non-systemic (nasal/ophtamological/dermatological/intra-articular) use. Systemic use is prohibited because of strong inflammatory suppressing effects. Prednisolone is a GC proven to be very effective in the treatment of nasal congestions and allergic rhinitis and its therapeutic use is allowed. To establish normal urinary concentration ranges for nasally administered prednisolone, an excretion study was performed with Sofrasolone® (nasal-inhaler). Six volunteers were administered a high dose (4.5 mg prednisolone in four gifts over a 9-h period). Samples were analysed using a validated LC-MS/MS method monitoring prednisolone (PRED) and the metabolites prednisone (PREDON), 20ß-dihydroprednisolone (20ßPRED) and 20α-dihydroprednisolone (20αPRED) in the total fraction (glucuroconjugated and free). Maximum concentrations were 266, 500, 350 and 140 ng/ml for PRED, PREDON, 20ßPRED and 20αPRED, respectively. These results show that the current reporting limit of 30 ng/ml in urine can be easily exceeded after therapeutic use. Hence, to avoid false-positive findings related to nasal application, this limit should be increased. To investigate the degree of glucuronidation of PRED and its metabolites also the free fraction was investigated. This shows that PREDON has the highest glucuroconjugation (50%). PRED, 20ßPRED and 20αPRED only show less than 20% conjugation.

Development and validation of a liquid chromatography high-resolution mass spectrometry orbitrap method for the sensitive quantification of amoxicillin, piperacillin, tazobactam and meropenem in human faeces.

Beta-lactam antibiotics are of vital importance for the treatment of infections in a broad range of patients. Although most systemically administered antibiotics will be excreted renally, a fraction will reach the gastro-intestinal tract, affecting the intestinal microbiome by eradicating a wide range of bacterial species while facilitating the growth of antimicrobial-resistant species. A better understanding of the kinetics of beta-lactam antibiotics in the gastro-intestinal tract is essential to study their role in the development of antibiotic resistance in bacteria and to help develop future therapies to prevent damage to, or restore, the intestinal microbiome. Analysis of beta-lactam antibiotics in faeces is particularly challenging due to the heterogeneous nature of the matrix, rapid degradation of some beta-lactam antibiotics in faeces and very strong ion suppression when using mass spectrometry. Sample preparation was optimized using a sequential strategy of experimental designs. It resulted in lyophilization, a MOPS buffer system and the addition of the beta-lactamase inhibitor avibactam to minimize degradation of antibiotics allowing sensitive quantification. The developed liquid chromatography method with high-resolution mass spectrometric detection was successfully validated according to bioanalytical EMA guidelines and had a linear range of 1-200 µg g-1 lyophilized faeces for amoxicillin, piperacillin and meropenem; and 0.5-100 µg g-1 lyophilized faeces for tazobactam. Despite the highly complex and heterogeneous composition of faeces, the accuracy (0.1-15%) and precision (1.7-12.1%) were in line with those obtained for quantification methods of beta-lactam antibiotics in plasma, the golden standard matrix for therapeutic drug monitoring. The applicability of the method was illustrated by successful quantification of piperacillin and tazobactam in faeces from an intensive care unit patient receiving piperacillin/tazobactam in a continuous intravenous infusion. Both piperacillin and tazobactam were still present six days after discontinuation of the therapy.

Development and validation of a fast gas chromatography combustion isotope ratio mass spectrometry method for the detection of epiandrosterone sulfate in urine.

In doping control, to confirm the exogenous origin of exogenously administered anabolic androgenic steroids (AAS), a gas chromatography combustion isotope ratio mass spectrometry (GC-C-IRMS) analysis is performed. Recently published work suggests that epiandrosterone sulfate (EpiAS) is a promising IRMS target compound for the detection of AAS, capable of prolonging the detection window. However, EpiAS is only excreted in urine in its sulfoconjugated form, while all other IRMS target compounds are excreted glucuronidated, meaning that EpiAS cannot be incorporated in the existing IRMS methods. A separate extensive sample preparation needs to be performed on this compound with a different hydrolysis and extraction procedure and a different liquid chromatography (LC) clean-up. The current work presents a new, fast, and easy to implement EpiAS IRMS method. The approach was based on the direct GC analysis of non-hydrolyzed EpiAS, making the solid phase extraction, hydrolysis, and acetylation step redundant. Sample preparation consisted of a simple liquid-liquid extraction, followed by LC fraction collection. A population study was performed to check compliance with the criteria drafted by the World Anti-Doping Agency (WADA). To verify the applicability of the developed approach, the method was applied to the samples of four administration studies (i.e. dehydroepiandrosterone (DHEA), testosterone gel (T gel), androstenedione (ADION), and intramuscular testosterone undecanoate. In contrast to previously published data, the strength of EpiAS as the target compound and the prolongation of the detection window in comparison with the conventional IRMS target compounds was less pronounced.

Searching for new long-term urinary metabolites of metenolone and drostanolone using gas chromatography-mass spectrometry with a focus on non-hydrolysed sulfates.

Sulfated metabolites have been shown to have potential as long-term markers of anabolic-androgenic steroid (AAS) abuse. In 2019, the compatibility of gas chromatography-mass spectrometry (GC-MS) with non-hydrolysed sulfated steroids was demonstrated, and this approach allowed the incorporation of these compounds in a broad GC-MS initial testing procedure at a later stage. However, research is needed to identify which are beneficial. In this study, a search for new long-term metabolites of two popular AAS, metenolone and drostanolone, was undertaken through two excretion studies each. The excretion samples were analysed using GC-chemical ionization-triple quadrupole MS (GC-CI-MS/MS) after the application of three separate sample preparation methodologies (i.e. hydrolysis with Escherichia coli-derived ß-glucuronidase, Helix pomatia-derived ß-glucuronidase/arylsulfatase and non-hydrolysed sulfated steroids). For metenolone, a non-hydrolysed sulfated metabolite, 1ß-methyl-5α-androstan-17-one-3ζ-sulfate, was documented for the first time to provide the longest detection time of up to 17 days. This metabolite increased the detection time by nearly a factor of 2 in comparison with the currently monitored markers for metenolone in a routine doping control initial testing procedure. In the second excretion study, it prolonged the detection window by 25%. In the case of drostanolone, the non-hydrolysed sulfated metabolite with the longest detection time was the sulfated analogue of the main drostanolone metabolite (3α-hydroxy-2α-methyl-5α-androstan-17-one) with a detection time of up to 24 days. However, the currently monitored main drostanolone metabolite in routine doping control, after hydrolysis of the glucuronide with E.coli, remained superior in detection time (i.e. up to 29 days).

Validation of an ultra-sensitive detection method for steroid esters in plasma for doping analysis using positive chemical ionization GC-MS/MS.

The standard approach to detect misuse with testosterone in sport is based on the determination and evaluation of the urinary steroid profile followed by the confirmation of atypical profiles using isotope ratio mass spectrometry. The detection capacity of these methods can be attenuated by confounding factors or testosterone preparations with endogenous isotopic fingerprints. An alternative detection method for misuse of an endogenous steroid in sports is the direct detection of the administered steroid ester present in most preparations. Thus unambiguous proof for doping misuse can be delivered. In this work, the sensitivity of gas chromatography coupled to a triple quadrupole with chemical ionization (GC-CI-MS/MS) is applied to detect trace levels of 10 testosterone and 2 nandrolone esters in plasma for in human doping analysis. The detection method was developed employing a liquid-liquid extraction and HPLC cleanup step before analysis on the GC-CI-MS/MS. The quantitative method was validated in a linear range of 100-2000 pg/ml and proved to be selective, reproducible and very sensitive with limits of detection as low as to 10 pg/ml. A clinical study with the administration of testosterone undecanoate in 3 volunteers was carried out and the compound was detectable up to 86 days after administration.

Peptide enrichment by ion-pair solid-phase extraction.

The technique of Solid-Phase Extraction (SPE) is widely used in various fields to concentrate samples and the search for tools to improve recoveries remains of outmost importance. The use of polymer based cartridges has become prevailing in a broad range of fields to enrich peptides from biological matrices. However, the existing SPE protocols are characterized by disparity. Ion-pairing (IP) reagents are commonly used in chromatographic applications, but their combination with SPE is less known. The aim of this study was to evaluate various SPE loading conditions, including the use of IP reagents, to improve the recoveries of nine selected peptide molecules. Control of pH and the use of IP reagents were found to be crucial to improve the enrichment of the peptides, especially cationic peptides, for which an up to ten-fold increase was observed. The practical potential of the presented theoretical findings were verified by employing IP-SPE for the development of an efficient extraction method for the doping relevant peptide Synacthen. The general proof of principle was obtained by analysis of excretion study urine samples and validation was performed with focus on the limit of detection (20 pg/ml) and recovery (37%).

Gas chromatography-mass spectrometry analysis of non-hydrolyzed sulfated steroids by degradation product formation.

Steroid detection and identification remain key issues in toxicology, drug testing, medical diagnostics, food safety control, and doping control. In this study, we evaluate the capabilities and usefulness of analyzing non-hydrolyzed sulfated steroids with gas chromatography-mass spectrometry (GC-MS) instead of the conventionally applied liquid chromatography-mass spectrometry (LC-MS) approach. Sulfates of 31 steroids were synthesized and their MS and chromatographic behavior studied by chemical ionization-GC-triple quadrupole MS (CI-GC-TQMS) and low energy-electron ionization-GC-quadrupole time-of-flight-MS (LE-EI-GC-QTOF-MS). The collected data shows that the sulfate group is cleaved off in the injection port of the GC-MS, forming two isomers. In CI, the dominant species (ie, [MH - H2 SO4 ]+ or [MH - H4 S2 O8 ]+ for bis-sulfates) is very abundant due to the limited amount of fragmentation, making it an ideal precursor ion for MS/MS. In LE-EI, [M - H2 SO4 ].+ and/or [M - H2 SO4 - CH3 ].+ are the dominant species in most cases. Based on the common GC-MS behavior of non-hydrolyzed sulfated steroids, two applications were evaluated and compared with the conventionally applied LC-MS approach; (a) discovery of (new) sulfated steroid metabolites of mesterolone and (b) expanding anabolic androgenic steroid abuse detection windows. GC-MS and LC-MS analysis of non-hydrolyzed sulfated steroids offered comparable sensitivities, superseding these of GC-MS after hydrolysis. For non-hydrolyzed sulfated steroids, GC-MS offers a higher structural elucidating power and a more straightforward inclusion in screening methods than LC-MS.

Development and validation of an open screening method for doping substances in urine by gas chromatography quadrupole time-of-flight mass spectrometry.

In anti-doping, a high number of classes of substances are prohibited and laboratories need to detect these at low urinary concentrations. Traditionally, testing is done using complimentary liquid chromatography mass spectrometry and gas chromatography mass spectrometry. High resolution mass spectrometric acquisition has some important advantages over triple quadrupole instruments (e.g., open screening due to full scan high resolution data acquisition with retrospectivity, compatibility with libraries and a straightforward and effortless addition and validation of new compounds in the future). Doping samples can be stored for 10 years and retrospective data analysis can be used to re-evaluate previously acquired data (e.g., searching for prohibited (designer) substances that were unknown at the initial moment of analysis). During the past decade, these advantages have led to the wide-scale transfer of liquid chromatography triple quadrupole mass spectrometry screening to liquid chromatography high resolution mass spectrometry screening for doping control purposes. Up to now, for gas chromatography a similar switch to high resolution screening has not yet occurred, because so far no method has been developed that combines sufficient sensitivity with wide-scale drug detection. In this work, the current gas chromatography triple quadrupole mass spectrometry screening method for human doping control purposes was successfully converted into an equivalent and complete gas chromatography high resolution acquisition screening method. This new screening method on a gas chromatography quadrupole time-of-flight mass spectrometer has been developed and validated. The method is compliant with the World Anti-Doping Agency requirements and allows the detection of 294 target compounds (and 14 internal standards), including diuretics, stimulants, narcotics, beta-2-agonists, beta-blockers, hormone modulators, anabolic agents and the quantification of 14 endogenous steroids in a single fast run (14.1 min).