title: Enantiomeric determination of tramadol and O-desmethyltramadol in human plasma by fast liquid chromatographic technique coupled with mass spectrometric detection.
abstract: A rapid and sensitive method using liquid chromatography-tandem mass spectrometry (LC-MS/MS) for enantiomeric determination of tramadol and its primary phase metabolite O-desmethyltramadol in human plasma has been developed. Tramadol hydrochloride-(13)C, d(3), was used as an isotopic labeled internal standard for quantification. The method involves a simple solid phase extraction. The analytes and internal standard were separated on Lux Cellulose-2 packed with cellulose tris(3-chloro-4-methylphenylcarbamate) using isocratic elution with hexane/isopropanol/diethylamine (90:10:0.1, v/v/v) at a flow rate of 1.3 mL/min. The APCI positive ionization mass spectrometry was used with multiple reaction monitoring of the transitions at m/z 264.2-->58.2 for tramadol, m/z 250.1-->58.2 for O-desmethyltramadol and m/z 268.2-->58.2 for internal standard. Linearity was achieved between 1-800 ng/mL and 1-400 ng/mL (R(2) > or = 0.999) for each enantiomer of tramadol and O-desmethyltramadol, respectively. Intra-day accuracies ranged among 98.2-102.8%, 97.1-109.1% and 97.4-102.9% at the lower, intermediate, and high concentration for all analytes, respectively. Inter-day accuracies ranged among 95.5-104.1%, 99.2-104.7%, and 94.2-105.6% at the lower, intermediate, and high concentration for all analytes, respectively. This assay was successfully used to determine the concentration of enantiomers of tramadol and O-desmethyltramadol in a pharmacogenetic study.
authors.affiliation: Institute of Forensic Medicine and Toxicology, 1st Faculty of Medicine, Charles University in Prague, 121 08 Prague 2, Czech Republic. lukas.chytil@lf1.cuni.cz
authors.forename: Lukás
authors.initials: L
authors.lastname: Chytil
authors.forename: Olga
authors.initials: O
authors.lastname: Matousková
authors.forename: Olga
authors.initials: O
authors.lastname: Cerná
authors.forename: Pavla
authors.initials: P
authors.lastname: Pokorná
authors.forename: Václav
authors.initials: V
authors.lastname: Vobruba
authors.forename: Frantisek
authors.initials: F
authors.lastname: Perlík
authors.forename: Ondrej
authors.initials: O
authors.lastname: Slanar
mesh_terms: D002138:Calibration; D002853:Chromatography, Liquid / Q000379:methods*; D019389:Cytochrome P-450 CYP2D6 / Q000235:genetics; D006801:Humans; D057230:Limit of Detection; D013058:Mass Spectrometry / Q000379:methods*; D010641:Phenotype; D015203:Reproducibility of Results; D052616:Solid Phase Extraction; D013237:Stereoisomerism; D013997:Time Factors; D014147:Tramadol / Q000031:analogs & derivatives* / Q000097:blood / Q000737:chemistry
chemical_list: C080580:O-demethyltramadol; D014147:Tramadol; D019389:Cytochrome P-450 CYP2D6