Unconjugated Morphine in Blood by Radioimmunoassay and Gas Chromatography/Mass Spectrometry

Abstract

Morphine, the active metabolite of heroin, is rapidly inactivated by glucuronidation at the 3 carbon. Unconjugated (pharmacologically active) morphine was measured in postmortem blood by radioimmunoassay using an antibody-coated tube kit. The kit shows less than 0.2% cross-reactivity with codeine and morphine-glucuronide. Unconjugated morphine concentrations were confirmed by gas chromatography/mass spectrometry (GC/MS) using deuterated morphine as the internal standard. The blood was precipitated with 10% trichloroacetic acid (TCA) and concentrated hydrochloric acid (HCl), centrifuged, and decanted. The supernatant was then either diluted (unhydrolyzed) or heated to 100 degrees C, 30 min (hydrolyzed), followed by a wash with 4:1 chloroform:isopropranol. The upper aqueous layer was then saturated with sodium bicarbonate (NaHCO3) and extracted with 4:1 chloroform:isopropranol. The organic layer was evaporated, derivatized with trifluoroacetic anhydride (TFA), and analyzed by selected ion monitoring (SIM) GC/MS. Comparison of the results for unconjugated morphine by radioimmunoassay and unhydrolyzed morphine by GC/MS gave a correlation coefficient of r = 0.98, n = 100. Unconjugated morphine ranged from 0 to 100% of total morphine with a mean of 42%, n = 200, for heroin or morphine involved deaths. Review of 56 putative rapid deaths gave a mean of 68% unconjugated morphine with a range of 26 to 100%. The ratio of unconjugated to total morphine was found to be stable in postmortem blood after more than a year of storage at room temperature, within the precision of the method.