Heroin-Related Compounds and Metabolic Ratios in Postmortem Samples Using LC-MS-MS

Analysis of postmortem samples with the presence of morphine can sometimes be challenging to interpret. Tolerance complicates interpretation of intoxications and causes of death due to overlap in therapeutic and fatal concentrations. Determination of metabolites and metabolic ratios can potentially differentiate between abstinence, continuous administration, and perhaps time of administration. The purpose of this study was to (a) develop and validate a method for quantitation of morphine-3β-D-glucuronide, morphine-6β-D-glucuronide, normorphine, codeine-6β-D-glucuronide, norcodeine, codeine, 6-acetylmorphine, and ethylmorphine in urine using liquid chromatography-tandem mass spectrometry; (b) apply the method to opiate related deaths; (c) compare metabolic ratios in urine in different causes of death (CoD) and after different drug intakes and (d) compare heroin intoxications in rapid and delayed deaths. Validation parameters such as precision, bias, matrix effects, stability, process efficiency, and dilution integrity were assessed and deemed acceptable. Lower limits of quantitation ranged from 0.01-0.2 μg/mL for all analytes. Autopsy cases (n=135) with paired blood and urine samples were analyzed. Cases were divided into three groups based on CoD; opiate intoxication, intoxication with other drugs than opiates, and other CoD. The cases were classified by intake: codeine (n=42), heroin (n=36), morphine (n=49), and ethylmorphine (n=3). Five cases were classified as mixed intakes and excluded. Heroin intoxications (n=35) were divided into rapid (n=15) or delayed (n=20) deaths. Parent drug groups were compared using metabolic ratio morphine-3β-D-glucuronide/morphine and significant differences were observed between codeine vs morphine (p=0.005) and codeine vs heroin (p≤0.0001). Urine and blood concentrations, and metabolic ratios in rapid and delayed heroin intoxications were compared and determined a significant difference for morphine (p=0.001), codeine (p=0.009), 6-acetylmorphine (p=0.02) in urine, and morphine (p=0.02) in blood, but there was no significant difference (p=0.9) between metabolic ratios. Morphine-3β-D-glucuronide results suggested a period of abstinence prior to death in 25% of the heroin intoxications.