Analytical studies of morphine and related substances using LC-MS/MS

Sammanfattning: Morphine is considered to be metabolized in three distinct metabolic pathways; glucuronidation, sulfation and N-demetylation. However, identification of morphine-3-sulfate (M3S) and morphine-6-sulfate (M6S) as morphine metabolites has not been convincing according to previous literature due to lack of reliable reference material and identification based on thin layer chromatography. In this thesis reference material for M3S and M6S was developed, and a sensitive analytical method for quantification of M3S and M6S in urine and plasma with mass spectrometry was also developed. Urine and plasma were analysed from different study groups; newborns, heroin addicts and terminal cancer patients. M3S was present in both urine and plasma from all study groups. The plasma ratio M3S/morphine-3-glucuronide was found to be 30 times higher in newborns than in adults. There was weak evidence that M6S actually forms in-vivo since only two samples contained detectable concentrations of M6S. It was demonstrated that both M3S and M6S was formed in-vitro by human liver homogenate but in small amounts. Nevertheless, we have demonstrated that both M3S and M6S are morphine metabolites in humans. Heroin is a highly addictive morphine derivative that is present on the illicit drug market. One of the primary interests in clinical and forensic drug testing is determination/identification of heroin intake. In this thesis a new validated routine LC-MS/MS method for urine drug testing of opiates has been evaluated leading to increased selectivity and separation power compared to earlier GC- MS methods. The evaluation displayed that the 6-AM biomarker is a good and dependable criterion for a heroin intake. In addition, we have also demonstrated that this method can be reduced regarding number of analytes. In 11.5 % of 6-AM positive urine samples (n=693) an atypical metabolic pattern of morphine and 6-AM was observed after a heroin intake. The atypical pattern seemed not to be related to a genetic polymorphism in the enzymes involved since the same individual can produce both “normal” and atypical pattern. In- vitro study using liver homogenates revealed that a strong inhibition of 6-AM formation was seen for a rearrangement product of thebaine (compound 3). Indeed, this compound could also be identified in all patient samples showing the atypical pattern.