Post-mortem analysis: development of an analytical strategy to determine a large panel of drugs on a small sample volume

AIMS: An 18 years old male died after a heavy struggle with four policemen. The body presented multiple but not severe blunt trauma injuries (contusions, abrasions and few lacerations to the head), not able to cause the death. The case was classified as sudden death after a violent struggle, with a probable induction of hypoxia. Besides, depositions of the deceased night-mates agreed that he claimed to have assumed different drugs of abuse. We were assigned the responsibility to determine whether a massive drug assumption could have generated such a major respiratory failure or rather the hypoxic condition, eventually responsible for the death, resulted from constriction in handcuffed, face down position, and increased oxygen need upon the scuffle. METHODS: Some months after the death we received tiny amounts of urine, blood and bile specimens. We were requested to screen for the presence of ketamine, norketamine, amphetamines, heroin, 6- MAM, 11-nor-9-carboxy-Δ9-THC and, possibly, anabolic steroids. Screening all these substances appeared problematic, since the blood volume available (0.5 mL) did not allow to perform different analytical procedures on separate aliquots. Furthermore, the specific gravity of the urine sample showed an anomalously low value, likely due to dilution during bladder washing, making this matrix unreliable for drug analysis. Both blood and bile specimens were processed using a specifically designed protocol of analysis. The samples were subjected to a preliminary enzymatic hydrolysis and a subsequent extraction step with tert-butyl methyl ether at pH 9. Then, the extracts were directly analyzed by GC-MS and HPLC-MS, using different selected ion monitoring (SIM) programs for ketamine, norketamine, amphetamines, morphine and 6-MAM. The extracts residues were subsequently derivatized with a mixture MSTFA-NH4IDTE and lastly re-analyzed by GC-MS, using different SIM methods to determine morphine, 6-MAM, THCmetabolites and various anabolic steroids. This protocol was utilized to build up 5-point calibration curves from spiked negative blood for all the analytes cited. Limits of detection, defined as the minimum concentration providing S/N > 3 ratio, were extrapolated from the lowest concentration level used for the calibration. All LODs resulted below 5 ng/mL. RESULTS AND CONCLUSIONS: Both blood and bile samples turned out positive for morphine. Using the HPLC-MS/MS calibration, the morphine concentration in blood was 26 ng/mL and in the bile sample was 4800 ng/mL. The GC-MS determination found a morphine concentration of 20 ng/mL in blood and 2100 ng/mL in bile. By considering that i) the concentration of morphine in blood was low, ii) the concentration of morphine in bile was high and iii) the metabolite 6-MAM was absent, we concluded that the presence of morphine had to be attributed to an earlier use of heroin or morphine. With a long elapsed time between drug use and death a link between these two events is unlikely. Therefore, the forensic pathologist together with other autopsy evidence determined that the sudden death was due to a respiratory and heart failure related to “excited delirium syndrome”.