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Taylor P.,HFL Sport Science A Quotient Bioresearch Ltd. Company | Scarth J.P.,HFL Sport Science A Quotient Bioresearch Ltd. Company | Hillyer L.L.,British Horseracing Authority
Bioanalysis | Year: 2010

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. © 2010 Future Science Ltd. Source


Scarth J.P.,HFL Sport Science A Quotient Bioresearch Ltd. Company | Spencer H.A.,HFL Sport Science A Quotient Bioresearch Ltd. Company | Hudson S.C.,HFL Sport Science A Quotient Bioresearch Ltd. Company | Teale P.,HFL Sport Science A Quotient Bioresearch Ltd. Company | And 2 more authors.
Steroids | Year: 2010

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. © 2009. Source


Scarth J.P.,HFL Sport Science A Quotient Bioresearch Ltd. Company | Spencer H.A.,HFL Sport Science A Quotient Bioresearch Ltd. Company | Timbers S.E.,HFL Sport Science A Quotient Bioresearch Ltd. Company | Hudson S.C.,HFL Sport Science A Quotient Bioresearch Ltd. Company | Hillyer L.L.,British Horseracing Authority
Drug Testing and Analysis | Year: 2010

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 referencematerial 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. Copyright © 2010 John Wiley & Sons, Ltd. Source


Scarth J.P.,HFL Sport Science A Quotient Bioresearch Ltd. Company | Clarke A.,HFL Sport Science A Quotient Bioresearch Ltd. Company | Hands J.,HFL Sport Science A Quotient Bioresearch Ltd. Company | Teale P.,HFL Sport Science A Quotient Bioresearch Ltd. Company | And 4 more authors.
Chromatographia | Year: 2010

The use of anabolic steroids as growth promoting agents in food production is prohibited under European Union legislation, but there is currently no internationally accepted method for detecting the abuse of the anabolic steroid nandrolone in the porcine. Therefore, an analytical biomarker approach based on gas chromatography-tandem mass spectrometry (GC-MS-MS) analysis of the major urinary free fraction nandrolone metabolite 19-noretiocholanolone was developed and validated. The lower and upper limits of quantification of the assay were 25 and 3,000 pg mL-1 respectively. The limit of detection was calculated as 13.2 pg mL-1, which is significantly lower than previously reported methods. When applied to a population of untreated animals, 19-noretiocholanolone distributions in boars and gilt were bimodal, with a small number of concentrations in each sex at around the 1,000 pg mL-1 region and the majority of concentrations closer to the lower endof the calibration range. Statistical analysis of the data was carried out in order to suggest screening and confirmatory threshold approaches for this steroid in the urine of boars and gilts. The adopting of particular screening thresholds would be at the discretion of the individual regulating authorities, but at a false non-compliance rate of 1 in 10,000 of the normal population, the suggested confirmatory thresholds (7,501.6 pg mL-1 for boars and 19,200.4 pg mL-1 in gilts) are able to detect the abuse of nandrolone for several weeks following administration of this steroid. © 2010 Vieweg+Teubner Verlag | Springer Fachmedien Wiesbaden GmbH. Source


Scarth J.P.,HFL Sport Science A Quotient Bioresearch Ltd. Company | Clarke A.D.,HFL Sport Science A Quotient Bioresearch Ltd. Company | Teale P.,HFL Sport Science A Quotient Bioresearch Ltd. Company | Pearce C.M.,HFL Sport Science A Quotient Bioresearch Ltd. Company
Steroids | Year: 2010

Effective detection of the abuse of androgenic-anabolic steroids in human and animal sports often requires knowledge of the drugs metabolism in order to target appropriate urinary metabolites. Designer steroids are problematic since it is difficult to obtain ethical approval for in vivo metabolism studies due to a lack of a toxicological profile. In this study, the in vitro metabolism of estra-4,9-diene-3,17-dione is reported for the first time. This is also the first study comparing the metabolism of a designer steroid in the three major species subject to sports doping control; namely the equine, canine and human. In order to allow the retrospective analysis of sample testing data, the use of a high-resolution (HR) accurate-mass Thermo LTQ-Orbitrap LCMS instrument was employed for metabolite identification of underivatised sample extracts. The full scan HRLCMS Orbitrap data was complimented by several further experiments targeted at elucidating more detailed structural information for the most abundant metabolites. These included; HRLCMS/MS of the underivatised metabolites, functional group selective chemical derivatisation followed by full scan HRLCMS, enzyme inhibition experiments and full scan electron ionization GCMS analysis of methoxyaminetrimethylsilyl derivatives. The major metabolite detected in all species, and therefore the most suitable candidate for screening of estra-4,9-diene-3,17-dione abuse, was proposed to be an isomer of 17-hydroxy-estra-4,9-dien-3-one. Less significant metabolic pathways in all species included hydroxylation and reduction followed by hydroxylation. Reductive metabolism in the canine was less significant than in the other two species, while the equine was unique in producing a di-reduced metabolite (proposed to be an isomer of estra-4,9- diene-3,17-diol) and also relatively large quantities of d-ring hydroxy and hydroxy-reduced metabolites. © 2010 Elsevier Inc. All rights reserved. Source

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