James P. Scarth

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.

The use of in vitro technologies coupled with high resolution accurate mass LC-MS for studying drug metabolism in equine drug surveillance

The detection of drug abuse in horseracing often requires knowledge of drug metabolism, especially if urine is the matrix of choice. In this study, equine liver/lung microsomes/S9 tissue fractions were used to study the phase I metabolism of eight drugs of relevance to equine drug surveillance (acepromazine, azaperone, celecoxib, fentanyl, fluphenazine, mepivacaine, methylphenidate and tripelennamine). In vitro samples were analyzed qualitatively alongside samples originating from in vivo administrations using LC-MS on a high resolution accurate mass Thermo Orbitrap Discovery instrument and by LC-MS/MS on an Applied Biosystems Sciex 5500 Q Trap.Using high resolution accurate mass full-scan analysis on the Orbitrap, the in vitro systems were found to generate at least the two most abundant phase I metabolites observed in vitro for all eight drugs studied. In the majority of cases, in vitro experiments were also able to generate the minor in vivo metabolites and sometimes metabolites that were only observed in vitro. More detailed analyses of fentanyl incubates using LC-MS/MS showed that it was possible to generate good quality spectra from the metabolites generated in vitro. These data support the suggestion of using in vitro incubates as metabolite reference material in place of in vivo post-administration samples in accordance with new qualitative identification guidelines in the 2009 International Laboratory Accreditation Cooperation-G7 (ILAC-G7) document.In summary, the in vitro and in vivo phase I metabolism results reported herein compare well and demonstrate the potential of in vitro studies to compliment, refine and reduce the existing equine in vivo paradigm.

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.

Determination of fluoxetine and its major active metabolite norfluoxetine in human plasma by liquid chromatography-tandem mass spectrometry

SummaryA sensitive and specific bioanalytical method for the determination of fluoxetine and norfluoxetine in human plasma has been developed. Automated solid phase extraction on Oasis HLB cartridges was used to extract the analytes from human plasma. Analysis was by reverse phase liquid chromatography on a Xterra MS C18 column using a fast gradient. Fluoxetine, norfluoxetine and fluvoxamine (internal standard) were ionised using the Turbolonspray interface operating in positive ion mode. Detection was via multiple reaction monitoring (MRM) of the characteristic ion dissociation transitionsm/z 310.3→44.1, 296.2→134.3 and 319.2→71.1 for fluoxetine, norfluoxetine and fluvoxamine respectively. The method is linear over the range 0.5–50 ng mL−1 (using a sample volume of 0.5 mL). The method is accurate and precise with intra-batch and inter-batch precision (%CV) of<15% and accuracy (%RE) of <+-15% for both analytes. A run time of 4 minutes means a high throughput of samples can be achieved. The method has been be used to support a clinical study.

Impact of the emergence of designer drugs upon sports doping testing

Historically, dope-testing methods have been developed to target specific and known threats to the integrity of sport. Traditionally, the source of new analytical targets for which testing was required were derived almost exclusively from the pharmaceutical industry. More recently, the emergence of designer drugs, such as tetrahydrogestrinone that are specifically intended to evade detection, or novel chemicals intended to circumvent laws controlling the sale and distribution of recreational drugs, such as anabolic steroids, stimulants and cannabinoids, have become a significant issue. In this review, we shall consider the emergence of designer drugs and the response of dope-testing laboratories to these new threats, in particular developments in analytical methods, instrumentation and research intended to detect their abuse, and we consider the likely future impact of these approaches.

Determination of cetirizine in human plasma by liquid chromatography-tandem mass spectrometry

SummaryA liquid chromatography-tandem mass spectrometry (LC-MS-MS) method was developed for the quantitation of cetirizine in human plasma. Cetirizine and hydroxyzine (internal standard) were extracted from human plasma using a solid phase extraction porocedure with Oasis HLB cartridges. Elution from a reverse phase Xterra MS C18 high-performance liquid chromatography (HPLC) column using a fast gradient was followed by MS-MS-multiple reaction monitoring (MRM). Cetirizine and the internal standard were ionised using the TurbolonsprayTM interface operating in positive ion mode. The characteristic ion dissociation transitionsm/z 389.3→201.1 andm/z 375.3→201.1 were monitored for cetirizine and internal standard respectively. The limit of quantitation was 5 ng·mL−1 using 250 μl of plasma. Inter and Intra batch precision expressed by relative standard deviation was less than 9%. The assay was robust, sensitive, and highly specific and there was no interference from human plasma observed. With a total run-time of 6 minutes, the method was suitable for supporting clinical studies and applied to the analysis of samples from a bioequivalence study.

Detection of endogenous steroid abuse in cattle: results from population studies in the UK

The use of steroids as growth-promoting agents in food production is banned under European Union legislation. Detecting the abuse of testosterone, nandrolone, boldenone, oestradiol and progesterone is complicated by the fact that these steroids are known to be endogenous in certain situations. In this study, the concentrations of characteristic metabolites of each of these steroids were quantified in populations of untreated steers and heifers. Steroid concentration population data were then used by a statistical model (the Chebyshev inequality) to produce threshold concentrations for screening and confirming the abuse of these steroids in steer and non-pregnant heifer urine. In addition to thresholds based on testing one animal (a ‘1 out of 1’ approach), new methods based on testing multiple animals from a herd (a ‘y out of n’ approach) allowed threshold concentrations to be significantly reduced and hence false compliances to be minimised. In the majority of cases, the suggested thresholds were found to be capable of confirming the abuse of endogenous steroids in steers and heifers. In the case of oestradiol abuse in the female, however, confirmation based on a threshold is not possible and alternative methods such as gas chromatography-combustion-isotope ratio mass spectrometry are required. In addition to the steer and heifer populations, a small number of pregnant animals were also tested, yielding insights into the biosynthetic pathways of some of the steroids.

Use of in vitro technologies to study phase II conjugation in equine sports drug surveillance.

BACKGROUND Within equine drug surveillance, there is significant interest in analyzing intact phase II conjugates of drugs in urine, but progress has been limited by a lack of reference material. METHOD In this study, in vitro techniques using equine liver fractions were employed to produce glucuronide and sulfate conjugates of stanozolol, 16β-hydroxystanozolol and nandrolone, the glucuronide conjugate of morphine and the glutathione metabolite of chlordinitrobenzene for the first time in equine sports drug surveillance. RESULTS The glucuronide conjugate of the synthetic progestagen altrenogest was also produced in vitro, removing the requirement for sample hydrolysis during routine urinalyses. CONCLUSION These results highlight the potential of in vitro studies for the production of phase II reference material, allowing the development of assays based on intact conjugates.

In vitro metabolism of tiletamine, zolazepam and nonbenzodiazepine sedatives: Identification of target metabolites for equine doping control

Within horseracing, the detection of prohibited substance doping often requires urine analysis; hence, it is necessary to understand the metabolism of the drugs in question. Here, the previously unknown equine metabolism of eight sedatives is reported in order to provide information on target metabolites for use in doping control. Phase I metabolite information was provided by incubation with equine liver S9 fraction. In vitro techniques were chosen in order to reduce the ethical and financial issues surrounding the study of so many compounds, none of which are licensed for use in horses in the UK. Several metabolites of each drug were identified using liquid chromatography-high resolution mass spectrometric (LC-HRMS) analysis on an LTQ-Orbitrap. Further structural information was obtained by tandem mass spectrometry (MS/MS) analysis; allowing postulation of the structure of some of the most abundant in vitro metabolites. The most abundant metabolites of alpidem, etifoxine, indiplon, tiletamine, zaleplon, zolazepam, zolpidem, and zopiclone related to hydroxylation/N-oxidation, deethylation, demethylation, deethylation, hydroxylation/N-oxidation, demethylation, hydroxylation/N-oxidation and hydroxylation/N-oxidation, respectively. In many cases, further work would be required to fully elucidate the precise positioning of the functional groups involved. The results of this study provide metabolite information that can be used to enhance equine anti-doping screening methods. However, the in vitro metabolites identified are at present only a prediction of those that may occur in vivo. In the future, any positive findings of these drugs and/or their metabolites in horse urine samples could help validate these findings and/or refine the choice of target metabolites.

Application of testosterone to epitestosterone ratio to horse urine - a complementary approach to detect the administrations of testosterone and its pro-drugs in Thoroughbred geldings

Detection of testosterone and/or its pro-drugs in the gelding is currently regulated by the application of an international threshold for urinary testosterone of 20 ng/mL. The use of steroid ratios may provide a useful supplementary approach to aid in differentiating between the administration of these steroids and unusual physiological conditions that may result in atypically high testosterone concentrations. In the current study, an ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method was developed to quantify testosterone (T) and epitestosterone (E). The method was used to analyze 200 post-race urine samples from geldings in order to generate the ratios for the reference population. Following statistical analysis of the data, an upper limit of 5 for T:E ratio in geldings is proposed. Samples collected from 15 geldings with atypical urinary testosterone concentrations (>15 ng/mL) but otherwise normal steroid profile, had T:E ratios within those observed for the reference population. The applicability of an upper T:E ratio to detect an administration was demonstrated by the analysis of a selection of incurred samples from testosterone propionate, dehydroepiandrosterone (DHEA), and a mixture of DHEA and pregnenolone (Equi-Bolic®) administrations. These produced testosterone concentrations above the threshold of 20 ng/mL, but also T:E ratios above the proposed limit of 5. In conclusion, consideration of the T:E ratio appears to be a valuable complementary aid to evaluate whether an atypical testosterone concentration could be caused by a natural biological outlier as opposed to the administration of these steroids. Copyright