A Validated Method for Simultaneous Determination of Codeine, Codeine-6-Glucuronide, Norcodeine, Morphine, Morphine-3-Glucuronide and Morphine-6-Glucuronide in Post-Mortem Blood, Vitreous Fluid, Muscle, Fat and Brain Tissue by LC–MS

• Published in: Journal of analytical toxicology Vol. 39; no. 3; pp. 203 - 212
• Main Authors: Frost, Joachim, Løkken, Trine N., Brede, Wenche R., Hegstad, Solfrid, Nordrum, Ivar S., Slørdal, Lars
• Format: Journal Article
• Language: English
• Published: England Oxford University Press 01.04.2015
• Subjects: Adipose Tissue - chemistry; Autopsy; Brain; Calibration; Cause of Death; Chromatography, High Pressure Liquid - standards; Codeine - analogs & derivatives; Codeine - blood; Forensic Toxicology - methods; Humans; Limit of Detection; Mass Spectrometry - standards; Morphine Derivatives - blood; Muscle, Skeletal - chemistry; Opioid-Related Disorders - blood; Opioid-Related Disorders - diagnosis; Opioid-Related Disorders - mortality; Reference Standards; Reproducibility of Results; Solid Phase Extraction; Substance Abuse Detection - methods; Substance Abuse Detection - standards; Vitreous Body - chemistry
• ISSN: 0146-4760; 1945-2403
• DOI: 10.1093/jat/bku145

The toxicodynamics and, to a lesser degree, toxicokinetics of the widely used opiate codeine remain a matter of controversy. To address this issue, analytical methods capable of providing reliable quantification of codeine metabolites alongside codeine concentrations are required. This article presents a validated method for simultaneous determination of codeine, codeine metabolites codeine-6-glucuronide (C6G), norcodeine and morphine, and morphine metabolites morphine-3-glucuronide (M3G) and morphine-6-glucuronide (M6G) in post-mortem whole blood, vitreous fluid, muscle, fat and brain tissue by high-performance liquid chromatography mass spectrometry. Samples were prepared by solid-phase extraction. The validated ranges were 1.5–300 ng/mL for codeine, norcodeine and morphine, and 23–4,600 ng/mL for C6G, M3G and M6G, with exceptions for norcodeine in muscle (3–300 ng/mL), morphine in muscle, fat and brain (3–300 ng/mL) and M6G in fat (46–4,600 ng/mL). Within-run and between-run accuracy (88.1–114.1%) and precision (CV 0.6–12.7%), matrix effects (CV 0.3–13.5%) and recovery (57.8–94.1%) were validated at two concentration levels; 3 and 150 ng/mL for codeine, norcodeine and morphine, and 46 and 2,300 ng/mL for C6G, M3G and M6G. Freeze–thaw and long-term stability (6 months at −80°C) was assessed, showing no significant changes in analyte concentrations (−12 to +8%). The method was applied in two authentic forensic autopsy cases implicating codeine in both therapeutic and presumably lethal concentration levels.