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Quantitation of the enantiomers of tramadol and its three main metabolites in human whole blood using LC-MS/MS.
Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Medicine and Health Sciences. National Board of Forensic Medicine, Department of Forensic Genetics and Forensic Toxicology, Linköping Sweden.
Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Medicine and Health Sciences. National Board of Forensic Medicine, Department of Forensic Genetics and Forensic Toxicology, Linköping Sweden.
Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Region Östergötland, Center for Diagnostics, Department of Clinical Pharmacology. Linköping University, Faculty of Medicine and Health Sciences.
Linköping University, Department of Physics, Chemistry and Biology. Linköping University, Faculty of Science & Engineering. National Board of Forensic Medicine, Department of Forensic Genetics and Forensic Toxicology, Linköping Sweden.
2016 (English)In: Journal of Pharmaceutical and Biomedical Analysis, ISSN 0731-7085, E-ISSN 1873-264X, Vol. 119, p. 1-9Article in journal (Refereed) Published
Abstract [en]

The analgesic drug tramadol and its metabolites are chiral compounds, with the (+)- and (-)-enantiomers showing different pharmacological and toxicological effects. This novel enantioselective method, based on LC-MS/MS in reversed phase mode, enabled measurement of the parent compound and its three main metabolites O-desmethyltramadol, N-desmethyltramadol and N,O-didesmethyltramadol simultaneously. Whole blood samples of 0.5g were fortified with internal standards (tramadol-(13)C-D3 and O-desmethyl-cis-tramadol-D6) and extracted under basic conditions (pH 11) by liquid-liquid extraction. Chromatography was performed on a chiral alpha-1-acid glycoprotein (AGP) column preceded by an AGP guard column. The mobile phase consisted of 0.8% acetonitrile and 99.2% ammonium acetate (20mM, pH 7.2). A post-column infusion with 0.05% formic acid in acetonitrile was used to enhance sensitivity. Quantitation as well as enantiomeric ratio measurements were covered by quality controls. Validation parameters for all eight enantiomers included selectivity (high), matrix effects (no ion suppression/enhancement), calibration model (linear, weight 1/X(2), in the range of 0.25-250ng/g), limit of quantitation (0.125-0.50ng/g), repeatability (2-6%) and intermediate precision (2-7%), accuracy (83-114%), dilution integrity (98-115%), carry over (not exceeding 0.07%) and stability (stable in blood and extract). The method was applied to blood samples from a healthy volunteer administrated a single 100mg dose and to a case sample concerning an impaired driver, which confirmed its applicability in human pharmacokinetic studies as well as in toxicological and forensic investigations.

Place, publisher, year, edition, pages
2016. Vol. 119, p. 1-9
Keywords [en]
Enantiomer; LC–MS/MS; N, O-didesmethyltramadol; N-desmethyltramadol; O-desmethyltramadol; Tramadol
National Category
Analytical Chemistry
Identifiers
URN: urn:nbn:se:liu:diva-125284DOI: 10.1016/j.jpba.2015.11.012ISI: 000370211900001PubMedID: 26625281OAI: oai:DiVA.org:liu-125284DiVA, id: diva2:904613
Note

Funding agencies:The National Board of Forensic Medicine in Sweden funded this work.

Available from: 2016-02-19 Created: 2016-02-19 Last updated: 2018-11-09
In thesis
1. Forensic Toxicological Aspects of Tramadol: Focus on Enantioselective Drug Disposition and Pharmacogenetics
Open this publication in new window or tab >>Forensic Toxicological Aspects of Tramadol: Focus on Enantioselective Drug Disposition and Pharmacogenetics
2018 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

One of the most difficult parts in forensic toxicology is to interpret obtained drug concentrations. Was it therapeutic, toxic or even lethal to the particular individual that the blood sample was drawn from? Concentrations of opioid drugs are especially difficult to interpret, because of large interindividual differences in innate and acquired tolerance.

Tramadol is a complex drug. Not only is it an opioid, it is also a racemic drug with the (+)- and (-)-enantiomers of the parent compound and metabolites showing different pharmacological effects. Further, it is metabolized by polymorphic enzymes, which may affect the amounts of metabolites formed and possibly the enantiomer ratios of the parent compound and its metabolites. It has been speculated that particularly the (+)/(-)-enantiomer ratio of O-desmethyltramadol is related to the risk of adverse effects, and it has been shown that the ratio is affected by CYP2D6 genotype.

The overall aim of the thesis was to evaluate if forensic interpretations of tramadol, regarding toxicity and time since drug administration, may be improved by the use of genotyping and enantioselective concentration determination of tramadol and its three main metabolites.

To simultaneously quantify the enantiomer concentrations of tramadol, Odesmethyltramadol, N-desmethyltramadol and N,O-didesmethyltramadol in whole blood, a liquid chromatography tandem mass spectrometry (LCMS/MS) method was developed and validated. Genetic variation in CYP2D6, CYP2B6, CYP3A4 (encoding the tramadol metabolizing enzymes), ABCB1 (encoding a transport protein) and OPRM1 (encoding the μ-opioid receptor) was investigated, using pyrosequencing, xTAG, and TaqMan analysis. The methods were applied to the blood samples of two study populations; 19 healthy volunteers administered a therapeutic, single tramadol dose, and 159 tramadol positive autopsy cases.

The most important finding was the positive correlations between all four enantiomer ratios and time since tramadol administration in the healthy volunteers. All enantiomer ratios except the one of tramadol was also affected by the CYP2D6 genotype, which was apparent among the autopsy cases as well. Genetic variation in CYP2D6 and possibly CYP2B6 was shown to have an impact on tramadol pharmacokinetics, although no association to neither drug related symptoms nor tramadol related causes of death was found. Tramadol intoxications were predominantly characterized by low age (median 26 years) and male sex, often with a history of substance abuse and with other drugs (at fairly low concentrations) detected in blood.

In conclusion, enantiomer concentration determination combined with genotyping seems promising regarding estimations of time since drug administration, although is of low value concerning interpretations of toxicity in autopsy cases.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2018. p. 104
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 1647
National Category
Pharmaceutical Sciences Forensic Science
Identifiers
urn:nbn:se:liu:diva-152626 (URN)10.3384/diss.diva-152626 (DOI)9789176851968 (ISBN)
Public defence
2018-11-30, Berzeliussalen, Campus US, Linköping, 13:00 (English)
Opponent
Supervisors
Available from: 2018-11-09 Created: 2018-11-09 Last updated: 2018-11-09Bibliographically approved

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Haage, PernillaKronstrand, RobertCarlsson, BjörnJosefsson, Martin

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