Development and validation of a gas chromatography-mass spectrometry method for opiates and cocaine in human bone

A method based on gas chromatography-mass spectrometry (GC-MS) is described for the determination of opioids (6-monoacetylmorphine, morphine, methadone and tramadol) and cocaine and its major metabolite in human bone. After the addition of nalorphine as internal standard, pulverized samples were incubated in acetonitrile for 1 h under ultrasounds. After adjusting the pH of the samples to 6, they were subjected to solid phase extraction and the analytes were eluted using 2 ml of dichloromethane/isopropanol/ammonia (78:20:2). Chromatography was performed on a fused silica capillary column and analytes were determined in the selected-ion-monitoring (SIM) mode. The assay was validated in the range 0.3-1 ng/mg (depending on the drug) to 150 ng/mg, the mean absolute recoveries ranging from 66% to 110%, the matrix effect from 62% to 121% and process efficiency from 61% to 89% depending on the analyte. The intra- and inter-assay accuracy values were always better than 20%. The validated method was then successfully applied to real bone samples from forensic cases in which toxicological analysis for these drugs in blood was positive. Drugs were detected in bone in 12 of the 15 blood positive results. The approximate concentration range was 3-5 ng/g for 6-monoacetylmorphine, 3-7 ng/g for morphine, 14-28 ng/g for methadone and 6 ng/g and 11 ng/g for tramadol and benzoylecgonine.