Anneleen Decloedt

A novel approach to the quantitative detection of anabolic steroids in bovine muscle tissue by means of a hybrid quadrupole time-of-flight-mass spectrometry instrument☆

In recent years, the analysis of veterinary drugs and growth-promoting agents has shifted from target-oriented procedures, mainly based on liquid chromatography coupled to triple-quadrupole mass spectrometry (LC-QqQ-MS), towards accurate mass full scan MS (such as Time-of-Flight (ToF) and Fourier Transform (FT)-MS). In this study, the performance of a hybrid analysis instrument (i.e. UHPLC-QuadrupoleTime-of-Flight-MS (QqToF-MS)), able to exploit both full scan HR and MS/MS capabilities within a single analytical platform, was evaluated for confirmatory analysis of anabolic steroids (gestagens, estrogens including stilbenes and androgens) in meat. The validation data was compared to previously obtained results (CD 2002/657/EC) for QqQ-MS and single stage Orbitrap-MS. Additionally, a fractional factorial design was used to shorten and optimize the sample extraction. Validation according to CD 2002/657/EC demonstrated that steroid analysis using QqToF has a higher competing value towards QqQ-MS in terms of selectivity/specificity, compared to single stage Orbitrap-MS. While providing excellent linearity, based on lack-of-fit calculations (F-test, α=0.05 for all steroids except 17β-ethinylestradiol: α=0.01), the sensitivity of QqToF-MS proved for 61.8% and 85.3% of the compounds more sensitive compared to QqQ-MS and Orbitrap-MS, respectively. Indeed, the CCα values, obtained upon ToF-MS/MS detection, ranged from 0.02 to 1.74μgkg(-1) for the 34 anabolic steroids, while for QqQ-MS and Orbitrap-MS values ranged from 0.04 to 0.88μgkg(-1) and from 0.07 to 2.50μgkg(-1), respectively. Using QqToF-MS and QqQ-MS, adequate precision was obtained as relative standard deviations for repeatability and within-laboratory reproducibility, were below 20%. In case of Orbitrap-MS, some compounds (i.e. some estrogens) displayed poor precision, which was possibly caused by some lack of sensitivity at lower concentrations and the absence of MRM-like experiments. Overall, it can be concluded that QqToF-MS offers good quantitative and confirmatory performance using the ToF-MS/MS mode whereas the full scan HR-ToF-MS allows screening for potential new designer drugs.

Mouldy feed: A possible explanation for the excretion of anabolic-androgenic steroids in horses

To ensure fair competition and to protect the horses welfare, horses have to compete on their own merits, without any unfair advantage that might follow the use of drugs. Therefore, regulatory authorities list all substances that are not allowed in competition, including most anabolic-androgenic steroids. As zero-tolerance is retained, the question arose whether the consumption of mouldy feed could lead to the excretion of steroids, due to the biotransformation of plant phytosterols to steroids. A rapid ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) analytical method, previously validated according to AORC (Association of Official Racing Chemists) and EC (European Commission) guidelines, was used to measure steroids in different sample types. Multiple mouldy feed samples were tested for the presence of steroids. The effect of digestion was tested by in vitro simulation of the horses hindgut in batch incubations. In most feed samples no steroids were detected, even when the products were mouldy. Mouldy corn however showed to contain up to 3.0 ± 0.4 µg/kg AED (4-androstenedione), the main testosterone precursor. This concentration increased when mouldy corn (with added phytosterols) was digested in vitro. An herbal phytosupplement also showed to contain α-testosterone. These results demonstrate that it is important to caution against the consumption of any feed or (herbal) supplement of which the detailed ingredients and quantitative analysis are unknown. The consumption of mouldy corn should especially be avoided, not only from a horse health and welfare point of view, but also to avoid possible inadvertent positive doping results. Copyright

Revealing the influence of glucocorticoid treatment on the excretion of anabolic-androgenic steroids in horses through in vitro digestive simulations and an in vivo case study

Anabolic-androgenic steroids (AAS) are strictly forbidden in equine sports because of their stimulating effect on muscle growth and performance. Nevertheless, low levels of AAS have been found in some horses, untreated with AAS. Glucocorticoids (GC), used as an anti-inflammatory therapy and structurally related to AAS, might play a role in this phenomenon. In order to unravel this possible correlation the influence of glucocorticoid treatment on the excretion of AAS was studied both in vivo and in vitro. In vivo effects were investigated by analysing urine samples collected from a gelding treated with betamethasone. Additionally, multiple in vitro digestion simulations were set up, according to a previously validated protocol, to study the possibility of a direct biotransformation of glucocorticoids to AAS, by the microbiota of the equine hindgut. Urine and in vitro digestion samples were extracted and analysed with UHPLC-MS/MS and UHPLC-Orbitrap-HRMS analytical methods. A significant influence on the urinary excretion of α-testosterone (αT), β-testosterone (βT) and androsta-1,4-diene-3,17-dione (ADD) was seen. αT-concentrations up to 20ng/mL were detected. ADD was not found before treatment but could be detected post-treatment. Cortisone and cortisol also peaked (>30ng/mL) between day 37 and 48 post-treatment. The in vitro digestion results however revealed no direct biotransformation of glucocorticoids to AAS by the microbiota of the equine hindgut. This study shows that a glucocorticoid treatment can disrupt the synthesis and excretion of AAS, not by direct biotransformation upon gastrointestinal digestion, but more likely by influencing the hypothalamic-pituitary-adrenal axis.

Plant-Based Beverages as Good Sources of Free and Glycosidic Plant Sterols

To address the ever-growing group of health-conscious consumers, more and more nutritional and health claims are being used on food products. Nevertheless, only very few food constituents, including plant sterols, have been appointed an approved health claim (European Commission and Food and Drugs Administration). Plant sterols are part of those limited lists of approved compounds for their cholesterol-lowering properties but have been praised for their anti-inflammatory and anti-carcinogenic properties as well. Despite this indisputable reputation, direct quantitative data is still lacking for naturally present (conjugated) plant sterols in beverages. This study aimed to fill this gap by applying a validated extraction and UPLC-MS/MS detection method to a diverse range of everyday plant-based beverages. β-sitosterol-β-d-glucoside (BSSG) showed to be by far the most abundant sterol in all beverages studied, with concentrations up to 60–90 mg per 100 mL in plant-based milk alternatives and fresh fruit juices. Ergosterol (provitamin D2) could be found in beers (0.8–6.1 µg per 100 mL, from the yeast) and occasionally in juices (17–29 µg per 100 mL). Overall, the results demonstrated that the concentrations of water-soluble sterol conjugates have been underestimated significantly and that specific plant-based beverages can be good, low-fat sources of these plant sterols.