Heroin is one of the most dangerous abused drugs in the world. Tramadol is an additive recently found at high concentration levels in street heroin seizures in Egypt. This substance could affect the usual analytical method for the detection of heroin and metabolites, as well as the pharmacokinetic and disposition of single analytes. One shortfall regarding this issue is present in the literature. This study describes a validated, simple, sensitive and selective method to determine tramadol, O-desmethyltramadol, 6-acetylmorphine and free morphine in the blood, brain, liver and kidney of Wistar rats, intraperitoneally treated with a combination of heroin and tramadol (10 and 70 mg/kg, respectively) using liquid–liquid extraction and gas chromatography–mass spectrometry detection. The calibration curves of tramadol, O-desmethyltramadol and 6-acetylmorphine in blood were linear in the concentration range from 25–5,000 ng/mL and morphine was found in the concentration range 50–5,000 ng/ mL. The analytes were detected in all tested matrices, except 6-acetylmorphine, which was not detected in liver. The highest concentrations of tramadol and O-desmethyltramadol were observed in kidney (22,9381 and 28,498 ng/g), while 6-acetylmorphine and morphine were found at the highest levels in brain (3,280 and 3,899 ng/g, respectively). The present method is simple, rapid and sensitive and can be used to study the pharmacokinetics, disposition and interaction of these drugs in several animal models.
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