Optimization of morphine extraction method for the assay of its urinary 3-glucuronideconjuguate by gas...

In the field of doping, a great interest is carried for the analysis of morphine, a powerful narcotic analgesic opiate which use is prohibited during competitions. In order to confirm the abnormal analytical result in our anti-doping laboratory, a sensitive and selective gas chromatography-mass spectrometry (GC-MS) method was performed for the quantification of urinary morphine. As sample preparation is a key step for the determination of drugs in biological samples, the aim of this work consists of the optimization of the urinary human sample pretreatment conditions before quantification by GC/MS. Enzymatic hydrolysis associated with liquid-liquid extraction constitute the major pre-treatment steps. Our study has first focused on the optimization of the extraction solvents then to enzymatic hydrolysis which morphine is released from its glucuronide conjugated form. Onboard premiums, a study involving the effect of "amount of enzyme", "incubation temperature" and "duration of hydrolysis" was conducted. This univariate study has enabled us to evaluate the influence of each of these operating variables on the area ratio of morphine to the internal standard (A_morphine/A_IS) response and to set the experimental fields for each one of them. Based on these results, an experimental design was established using the Box-Behnken model to determine, by multivariate analysis, the optimal operating conditions maximizing the "A_mophine/A_IS" response. After validation, the analysis of response surface makes it possible to set the optimum operating conditions, which the ratio "A_morphine/A_IS" is maximized. The retained conditions for enzymatic hydrolysis are 160μl of Escherichia coli glucuronidase enzyme during 6hours of incubation at a temperature of 36°C. The solvent mixture Methyl-t-Butyl Ether/isopropanol (4:1, v/v) was selected since it has improved morphine extraction from the urinary matrix allowing a gain of 50% when compared to that used in our routine laboratory. Our developed extraction method can be successfully applied for our forensic anti-doping analysis of morphin in human sample urine.