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.

Mice convert melatonin to 6-sulphatoxymelatonin.

The principal objective of this study was to establish whether mice can convert melatonin to 6-sulphatoxymelatonin (aMT6s). Precision-cut liver slices from C3H/He, C57BL/6, and BALB/c mice were incubated with melatonin, and the concentration of aMT6s in the culture media was determined using a sensitive and specific radioimmunoassay procedure. All three strains of mice generated aMT6s in a time-dependent manner; no significant strain differences were observed. When samples of the media were treated with sulphatase prior to analysis, aMT6s was not detectable. In contrast, similar treatment with beta-glucuronidase had no effect. 6-Sulphatoxymelatonin was present in the urine of both control and melatonin-treated C3H/He and C57BL6 mice. Treatment with melatonin led to a dramatic rise in the urinary levels of aMT6s in both mouse strains. Pre-treatment of the urines with sulphatase, but not beta-glucuronidase, markedly decreased the levels of aMT6s. Finally, in both strains urinary excretion of aMT6s displayed diurnal rhythmicity, peak excretion occurring during the dark hours. It may be inferred that: (a) mice can convert melatonin to aMT6s, both in vivo and in vitro, and (b) mice generate aMT6s in a rhythmic manner. Finally, the present studies confirm that determination of aMT6s rhythms in mice could provide an alternative, non-invasive, approach for assessing circadian clock function.