RESUMEN
Pre-race dosing of horses with alkalinising agents to manipulate performance has been evident in racing worldwide for over 30 years. To regulate the use of alkalinising agents, racing authorities adopted thresholds for total plasma carbon dioxide (TCO2 ) in racehorses. Traditionally, racing laboratories have measured plasma TCO2 using ion selective electrode (ISE) technology, with the Association of Official Racing Chemists (AORC) approving the use of only three ISE instruments for measurement. Because of the manufacture and support of these instruments ceasing, racing laboratories have explored alternative techniques to measure plasma TCO2 . In this study, headspace gas chromatography mass spectrometry (HSGCMS) with fully automated sample preparation was investigated as an alternative technique to ISE. Sample preparation was carried out online on a Gerstel robot, where plasma was aspirated directly from sealed vacutainer tubes before further treatment and headspace injection into a GCMS. The method was successfully cross validated against a Beckman Unicel DxC®600, meeting all criteria stipulated in the AORC cross-validation protocol. The method achieved an accuracy of 99.8%, within-run relative standard deviation of 0.22% and interday reproducibility of 0.04 mM, all significant improvements on the authors ISE method. A population study was also conducted to ensure the plasma TCO2 threshold, established with ISE methodology, did not change with the developed HSGCMS method. The concentrations and standard deviations for the two methods were almost identical, HSGCMS mean 30.62 mM, standard deviation 1.65 mM, and ISE 30.65 and 1.55 mM. The results indicate that the fully automated HSGCMS method is suitable for measurement of equine plasma TCO2 for regulatory purposes.
Asunto(s)
Dióxido de Carbono , Plasma , Animales , Cromatografía de Gases y Espectrometría de Masas , Caballos , Reproducibilidad de los ResultadosRESUMEN
The doping of greyhound dogs with testosterone is done in an attempt to improve their athletic performance, but such doping cannot easily be confirmed, especially in male dogs owing to the natural presence of endogenous testosterone. As testosterone is usually administered as its esters, their direct detection in hair would provide confirmatory evidence of the administration of a pharmaceutical product. This article demonstrates that the use of a liquid chromatography-high resolution mass spectrometry method with heated electrospray ionisation (HESI) combined with the use of amino solid-phase extraction (SPE) cartridges for sample clean-up, is suitable for the sensitive determination of propionate, phenyl propionate, isocaproate, decanoate, and enanthate esters of testosterone in greyhound hair. The method is linear over the range, 0.1 µg/kg-10 µg/kg, for all the testosterone esters analysed. The limits of detection (LOD) are 0.05 µg/kg for testosterone phenyl propionate, isocaproate, and decanoate, 0.025 µg/kg for testosterone propionate, and 0.25 µg/kg for testosterone enanthate. This method was applied to hair samples collected from male greyhounds before and after a single administration of a product containing several testosterone esters, each of which could be detected up to 100 days post-administration. The study also demonstrates that tail hair is the specimen of choice for the analysis of testosterone in dog hair and that washing of dogs does not impact the analysis of testosterone esters in hair. This method may be useful in racing regulation for the detection of illegitimate use of testosterone in all species.
Asunto(s)
Anabolizantes/análisis , Pelaje de Animal/química , Perros , Detección de Abuso de Sustancias/métodos , Espectrometría de Masas en Tándem/métodos , Testosterona/análisis , Andrógenos/análisis , Animales , Cromatografía Liquida/métodos , Perros/metabolismo , Doping en los Deportes , Esterificación , Ésteres/análisis , Límite de Detección , Masculino , Extracción en Fase Sólida/métodosRESUMEN
Zolpidem is a non-benzodiazepine hypnotic that has been implicated in both drug-facilitated sexual assault and drink spiking. Detection of the drug in urine is extremely difficult because of its extensive metabolism. A method is presented for the detection and quantitation of zolpidem carboxylic acid (ZCA), the major urinary metabolite of zolpidem. The metabolite was extracted from urine at pH 4.5-5.0 with chloroform/isopropanol alkylated with ethyl iodide and identified by gas chromatography-mass spectrometry in the selected ion-monitoring mode. Following a single ingestion of 10 mg zopidem, ZCA is detected for up to 72 h. The limit of detection is 2 ng/mL, with an overall recovery of 80%. Using this procedure, zolpidem was identified in two cases of alleged drug-facilitated sexual assault.
Asunto(s)
Medicina Legal/métodos , Cromatografía de Gases y Espectrometría de Masas , Hipnóticos y Sedantes/orina , Piridinas/orina , Detección de Abuso de Sustancias/métodos , Biotransformación , Femenino , Humanos , Hipnóticos y Sedantes/farmacocinética , Piridinas/farmacocinética , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Delitos Sexuales , ZolpidemRESUMEN
Protopine was extracted from Fumaria officinalis and purified by column chromatography. Urine samples were collected from horses and a human volunteer that had been administered either F. officinalis or protopine free base. Plant and urine samples were acetylated and analysed by GCMS after solid-phase extraction (SPE). The urinary metabolites of protopine were identified as 4,6,7,13-tetrahydro-9,10-dihydroxy-5-methyl-benzo[e]-l,3-benzodioxolo [4,5-1][2] benzazecin-12(5H)-one, 4,6,7,13-tetrahydro-10-hydroxy-9-methoxy-5-methyl-benzo[e]-1,3-benzodioxolo[4,5-1][2] benzazecin-12(5H)-one and 4,6,7,13-tetrahydro-9-hydroxy-10-methoxy-5-methyl-benzo[e]-1,3-benzodioxolo[4,5-l][2] benzazecin-12(5H)-one, chelianthifoline, isochelianthifoline and 2-O-desmethylchelianthifoline. The metabolic formation of the tetrahydroprotoberberines by closure of the bridge across N5 and C13 is rate limited and protopine-like metabolites accumulate only when the route is overloaded. Metabolism was qualitatively similar in the horse and human.
Asunto(s)
Alcaloides de Berberina/orina , Animales , Benzofenantridinas , Cromatografía de Gases y Espectrometría de Masas , CaballosRESUMEN
The influence of sampling variables on the concentration of the dopamine metabolites 3-methoxytyramine (3MT), dihydroxyphenylacetic acid (DOPAC) and homovanilic acid (HVA) was examined in equine urine. A logarithmic transformation of the data for all horses gave distribution which approximated the normal distributions for each metabolite. The mean urinary concentration of 3 MT in horses was 214 ng/mL and the application of a threshold with a probability of 1 in 10,000 gave an actionable level of 4 microg/mL. Environmental variables were not forensically significant in determining the population distribution. HVA was not found to be a reliable indicator of dopamine or levodopa administration.