A validated hybrid quadrupole linear ion-trap LC–MS method for the analysis of morphine and morphine glucuronides applied to opiate deaths

Abstract

A hybrid quadrupole linear ion-trap mass spectrometer using an electrospray ionisation ion source coupled to a HPLC system has been used to develop a method which can accurately measure morphine, morphine-3-glucuronide (M3G) and morphine-6-glucuronide (M6G) in plasma, whole blood and post-mortem blood following solid-phase extraction. The method can also qualitatively detect various other opioids and related compounds including: codeine, dihydrocodeine (and metabolites), noscapine, papaverine and 6-acetylmorphine (6-AM). The method has been favourably compared to an existing laboratory method using a now discontinued radio-immunoassay technique. The advantage of measuring the glucuronides directly rather than following deconjugation by β-glucuronidase has also been shown. Detection and quantification of compounds was achieved using multiple reaction monitoring (MRM) incorporating the use of deuterated morphine and M3G as internal standards. Precision and accuracy was determined to be less than 10% at both high and low levels for all analytes and the calibration curve was deemed linear over an acceptable range. Recovery in blood was greater than 90% and ion suppression/enhancement was shown to be less than 15%. This method was applied to over 130 post-mortem cases involving the use of heroin, prescribed morphine and codeine. The range of concentrations of morphine, M3G and M6G was large (particularly in heroin and prescribed morphine cases), reflecting the many different factors involved with therapeutic use or fatal opiate poisonings, including tolerance associated with regular use, variable dose regimens and co-administration of other drugs. Detection of other constituents of the opium poppy such as noscapine and papaverine and metabolites of diacetylmorphine in the blood (6-AM) was useful in determining the source of the morphine (i.e. illicit heroin) and the rapidity of death after administration.