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.

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.

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.

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.

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.

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.

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.