title: 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.
abstract: 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.
authors.affiliation: Department of Laboratory Medicine, Children's and Womens's Health, Norwegian University of Science and Technology (NTNU), Trondheim, Norway Department of Clinical Pharmacology, St. Olav University Hospital, NO-7006 Trondheim, Norway joachim.frost@stolav.no.
authors.forename: Joachim
authors.initials: J
authors.lastname: Frost
authors.affiliation: Department of Laboratory Medicine, Children's and Womens's Health, Norwegian University of Science and Technology (NTNU), Trondheim, Norway.
authors.forename: Trine N
authors.initials: TN
authors.lastname: Løkken
authors.affiliation: Department of Clinical Pharmacology, St. Olav University Hospital, NO-7006 Trondheim, Norway.
authors.forename: Wenche R
authors.initials: WR
authors.lastname: Brede
authors.affiliation: Department of Clinical Pharmacology, St. Olav University Hospital, NO-7006 Trondheim, Norway.
authors.forename: Solfrid
authors.initials: S
authors.lastname: Hegstad
authors.affiliation: Department of Laboratory Medicine, Children's and Womens's Health, Norwegian University of Science and Technology (NTNU), Trondheim, Norway Department of Pathology and Medical Genetics, St. Olav University Hospital, Trondheim, Norway.
authors.forename: Ivar S
authors.initials: IS
authors.lastname: Nordrum
authors.affiliation: Department of Laboratory Medicine, Children's and Womens's Health, Norwegian University of Science and Technology (NTNU), Trondheim, Norway Department of Clinical Pharmacology, St. Olav University Hospital, NO-7006 Trondheim, Norway.
authors.forename: Lars
authors.initials: L
authors.lastname: Slørdal
mesh_terms: D000273:Adipose Tissue / Q000737:chemistry*; D001344:Autopsy; D001921:Brain; D002138:Calibration; D002423:Cause of Death; D002851:Chromatography, High Pressure Liquid / Q000592:standards; D003061:Codeine / Q000031:analogs & derivatives* / Q000097:blood; D053593:Forensic Toxicology / Q000379:methods*; D006801:Humans; D057230:Limit of Detection; D013058:Mass Spectrometry / Q000592:standards; D009022:Morphine Derivatives / Q000097:blood*; D018482:Muscle, Skeletal / Q000737:chemistry*; D009293:Opioid-Related Disorders / Q000097:blood* / Q000175:diagnosis / Q000401:mortality; D012015:Reference Standards; D015203:Reproducibility of Results; D052616:Solid Phase Extraction; D015813:Substance Abuse Detection / Q000379:methods* / Q000592:standards; D014822:Vitreous Body / Q000737:chemistry*
chemical_list: D009022:Morphine Derivatives; C010414:norcodeine; C035349:morphine-6-glucuronide; C061773:codeine-6-glucuronide; C018108:morphine-3-glucuronide; D003061:Codeine