Philip Teale

Presence and metabolism of endogenous androgenic-anabolic steroid hormones in meat-producing animals: a review

The presence and metabolism of endogenous steroid hormones in meat-producing animals has been the subject of much research over the past 40 years. While significant data are available, no comprehensive review has yet been performed. Species considered in this review are bovine, porcine, ovine, equine, caprine and cervine, while steroid hormones include the androgenic–anabolic steroids testosterone, nandrolone and boldenone, as well as their precursors and metabolites. Information on endogenous steroid hormone concentrations is primarily useful in two ways: (1) in relation to pathological versus ‘normal’ physiology and (2) in relation to the detection of the illegal abuse of these hormones in residue surveillance programmes. Since the major focus of this review is on the detection of steroids abuse in animal production, the information gathered to date is used to guide future research. A major deficiency in much of the existing published literature is the lack of standardization and formal validation of experimental approach. Key articles are cited that highlight the huge variation in reported steroid concentrations that can result when samples are analysed by different laboratories under different conditions. These deficiencies are in most cases so fundamental that it is difficult to make reliable comparisons between data sets and hence it is currently impossible to recommend definitive detection strategies. Standardization of the experimental approach would need to involve common experimental protocols and collaboratively validated analytical methods. In particular, standardization would need to cover everything from the demographic of the animal population studied, the method of sample collection and storage (especially the need to sample live versus slaughter sampling since the two methods of surveillance have very different requirements, particularly temporally), sample preparation technique (including mode of extraction, hydrolysis and derivatization), the end-point analytical detection technique, validation protocols, and the statistical methods applied to the resulting data. Although efforts are already underway (at HFL and LABERCA) to produce more definitive data and promote communication among the scientific community on this issue, the convening of a formal European Union working party is recommended.

High‐throughput ultra‐high‐performance liquid chromatography/tandem mass spectrometry quantitation of insulin‐like growth factor‐I and leucine‐rich α‐2‐glycoprotein in serum as biomarkers of recombinant human growth hormone administration

Insulin-like growth factor-I (IGF-I) is a known biomarker of recombinant human growth hormone (rhGH) abuse, and is also used clinically to confirm acromegaly. The protein leucine-rich alpha-2-glycoprotein (LRG) was recently identified as a putative biomarker of rhGH administration. The combination of an ACN depletion method and a 5-min ultra-high-performance liquid chromatography/tandem mass spectrometry (uHPLC/MS/MS)-based selected reaction monitoring (SRM) assay detected both IGF-I and LRG at endogenous concentrations. Four eight-point standard addition curves of IGF-I (16-2000 ng/mL) demonstrated good linearity (r(2) = 0.9991 and coefficients of variance (CVs) <13%). Serum samples from two rhGH administrations were extracted and their uHPLC/MS/MS-derived IGF-I concentrations correlated well against immunochemistry-derived values. Combining IGF-I and LRG data improved the separation of treated and placebo states compared with IGF-I alone, further strengthening the hypothesis that LRG is a biomarker of rhGH administration. Artificial neural networks (ANNs) analysis of the LRG and IGF-I data demonstrated an improved model over that developed using IGF-I alone, with a predictive accuracy of 97%, specificity of 96% and sensitivity of 100%. Receiver operator characteristic (ROC) analysis gave an AUC value of 0.98. This study demonstrates the first large scale and high throughput uHPLC/MS/MS-based quantitation of a medium abundance protein (IGF-I) in human serum. Furthermore, the data we have presented for the quantitative analysis of IGF-I suggest that, in this case, monitoring a single SRM transition to a trypsin peptide surrogate is a valid approach to protein quantitation by LC/MS/MS.

The application of in vitro technologies to study the metabolism of the androgenic/anabolic steroid stanozolol in the equine.

In this study, the use of equine liver/lung microsomes and S9 tissue fractions were used to study the metabolism of the androgenic/anabolic steroid stanozolol as an example of the potential of in vitro technologies in sports drug surveillance. In vitro incubates were analysed qualitatively alongside urine samples originating from in vivo stanozolol administrations using LC-MS on a high-resolution accurate mass Thermo Orbitrap Discovery instrument, by LC-MS/MS on an Applied Biosystems Sciex 5500 Q Trap and by GC-MS/MS on an Agilent 7000A. Using high-resolution accurate mass full scan analysis on the Orbitrap, equine liver microsome and S9 in vitro fractions were found to generate all the major phase-1 metabolites observed following in vivo administrations. Additionally, analysis of the liver microsomal incubates using a shallower HPLC gradient combined with various MS/MS functions on the 5500 Q trap allowed the identification of a number of phase 1 metabolites previously unreported in the equine or any other species. Comparison between liver and lung S9 metabolism showed that the liver was the major site of metabolic activity in the equine. Furthermore, using chemical enzyme inhibitors that are known to be selective for particular isoforms in other species suggested that an enzyme related to CYP2C8 may be responsible the production of 16-hydroxy-stanozolol metabolites in the equine. In summary, the in vitro and in vivo phase 1 metabolism results reported herein compare well and demonstrate the potential of in vitro studies to compliment the existing in vivo paradigm and to benefit animal welfare through a reduction and refinement of animal experimentation.

The role of banned substance residue analysis in the control of dietary supplement contamination

The potential for contaminated dietary supplements to result in a failed doping test remains a concern for athletes, trainers, and sporting authorities despite improvements to regulatory guidelines. Previous surveys of readily available supplements confirm that many are contaminated with steroids and stimulants prohibited for use in elite sport. Suggested responses to this issue include the complete avoidance of all supplements. Many athletes, however, use nutritional supplements to achieve effective training and also to ensure that daily nutritional requirements are met (e.g. recommended levels of vitamins and minerals). This ensures that the use of supplements is and will remain the norm for a range of sports. As a result, an alternative approach of rigorous testing of materials destined for use by elite athletes has been introduced in several countries. While the testing of final product for banned substances may help mitigate the problem, it will not help to remove the underlying issue of contamination. In this article we describe an alternative approach that uses appropriate quality assurance procedures backed up by testing to remove sources of contamination. The decrease in the incidence of contamination amongst supplement companies adopting such a system is explained, and contrasted with the relatively high incidences of contamination found in products that are not part of a quality system. These findings are of key importance to both supplement manufacturers and those involved in advising athletes about supplement use.

Development and validation of a potentiometric biosensor assay for tylosin with demonstrated applicability for the detection of two other antimicrobial growth-promoter compounds in feedstuffs

A potentiometric biosensor assay based on a commercially available polyclonal antibody was developed to detect tylosin residues in animal feed. The method can be used as a rapid (less than 45 min) laboratory-based procedure or as a portable field-test for the simultaneous measurement of up to 12 different samples. For both procedures the qualitative detection capability (CCβ) for tylosin was determined as 0.2 mg kg−1 in a range of animal feeds with a measurement repeatability at concentrations between 0.2 and 4 mg kg−1 of ≤13% coefficient of variation (%CV). The field-test format was capable of detecting tylosin residues at operating (external air) temperatures ranging between +4 and 37°C, although some reduction in signal was observed at the lower temperatures. The laboratory-based tylosin assay was evaluated using 16 medicated and 22 non-medicated feeds and was found to give comparable data with a confirmatory method based upon liquid chromatography-tandem mass spectrometry (LC-MS/MS). The potential to develop a multi-probe format assay for the simultaneous detection of tylosin, spiramycin and virginiamycin was also demonstrated. Cross-validation in a second laboratory showed the assay to be transferable, reliable and robust.

Identification of etamiphylline and metabolites in equine plasma and urine by accurate mass and liquid chromatography/tandem mass spectrometry

Etamiphylline camsylate (Millophylline V) was administered intravenously to two horses at a dose of 2.8 mg/kg. Urine and blood samples were taken up to 32 h post administration. Unhydrolyzed plasma and urine was extracted using solid phase extraction (SPE). The identity of the parent drug and metabolites was confirmed using a linear ion trap mass spectrometer and accurate mass analysis on an orbitrap mass spectrometer. Desethyletamiphylline (molecular weight 251) was the main metabolite observed in the urine and plasma samples and resulted from the N-deethylation of etamiphylline. The second metabolite detected in urine and plasma resulted from the demethylation of etamiphylline (molecular weight 265). The third minor metabolite detected in urine was proposed to have resulted from a simultaneous N-deethylation and demethylation of etamiphylline (molecular weight 238).

Development and validation of a high-throughput assay for the quantification of multiple green tea-derived catechins in human plasma

A rapid, accurate and robust method for the determination of catechin (C), epicatechin (EC), gallocatechin (GC), epigallocatechin (EGC), catechin gallate (Cg), epicatechin gallate (ECg), gallocatechin gallate (GCg) and epigallocatechin gallate (EGCg) concentrations in human plasma has been developed. The method utilizes protein precipitation following enzyme hydrolysis, with chromatographic separation and detection using reversed-phase liquid chromatography-tandem mass spectrometry (LC-MS/MS). Traditional issues such as lengthy chromatographic runtimes, sample and extract stability, and lack of suitable internal standards have been addressed. The method has been evaluated using a comprehensive validation procedure, confirming linearity over appropriate concentration ranges, and inter/intra-batch precision and accuracies within suitable thresholds (precisions within 13.8% and accuracies within 12.4%). Recoveries of analytes were found to be consistent between different matrix samples, compensated for using suitable internal markers and within the performance of the instrumentation used. Similarly, chromatographic interferences have been corrected using the internal markers selected. Stability of all analytes in matrix is demonstrated over 32 days and throughout extraction conditions. This method is suitable for high-throughput sample analysis studies.