Determination of opiates in whole blood and vitreous humor: A study of the matrix effect and an experimental design to optimize conditions for the enzymatic hydrolysis of glucuronides.

Undoubtedly, whole blood and vitreous humor have been biological samples of great importance in forensic toxicology. The determination of opiates and their metabolites has been essential for better interpretation of toxicological findings. This report describes the application of experimental design and response surface methodology to optimize conditions for enzymatic hydrolysis of morphine-3-glucuronide and morphine-6-glucuronide. The analytes (free morphine, 6-acetylmorphine and codeine) were extracted from the samples using solid-phase extraction on mixed-mode cartridges, followed by derivatization to their trimethylsilyl derivatives. The extracts were analysed by gas chromatography-mass spectrometry with electron ionization and full scan mode. The method was validated for both specimens (whole blood and vitreous humor). A significant matrix effect was found by applying the F-test. Different recovery values were also found (82% on average for whole blood and 100% on average for vitreous humor). The calibration curves were linear for all analytes in the concentration range of 10-1,500 ng/mL. The limits of detection ranged from 2.0 to 5.0 ng/mL. The method was applied to a case in which a victim presented with a previous history of opiate use.