title: Development of a rapid and sensitive high-performance liquid chromatographic method to determine CYP2D6 phenotype in human liver microsomes.
abstract: Dextromethorphan is a probe substrate to determine CYP2D6 phenotype. The conversion of dextromethorphan to dextrorphan by CYP2D6 accounts for approximately 60% of total metabolism. Most analytical methods utilize complicated labor- and time-intensive sample processing methods with several liquid-liquid extraction (LLE) steps. Our goal was to develop a non-LLE based rapid and sensitive HPLC method, to measure dextromethorphan metabolism in human liver microsomes. A solid-phase filtration based reverse-phase HPLC method with fluorescence detection was developed and validated. Human liver (n = 6) microsomal incubations were carried out with dextromethorphan, under optimum conditions. The analytes were separated by one-step centrifugal filtration with Nanosep separation units. The filtrate was injected ( 50 microL) into a Waters Alliance 2690 HPLC system. Metabolic incubations were also conducted to determine levels using LLE for comparisons. The Nanosep separation step reduced the extraction time from 3h to 40 min. The limit of quantitation was 23.8 nM (9.7 ng/mL), recovery was approximately 98%, the mean precision values were <10% RSD for the controls (80, 320 and 640 nM) and mean percentage error was <5%. Michaelis-Menten parameters were determined to distinguish CYP2D6 phenotypes. A rapid and sensitive HPLC method is reported, which may be suitable for automation and allows phenotyping of human liver microsomes.
authors.affiliation: VCU School of Pharmacy, MCV Campus, Richmond, VA 23298-0533, USA.
authors.forename: Bhaskar
authors.initials: B
authors.lastname: Rege
authors.forename: Clark
authors.initials: C
authors.lastname: March
authors.forename: Mohamadi A
authors.initials: MA
authors.lastname: Sarkar
mesh_terms: D000996:Antitussive Agents / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D019389:Cytochrome P-450 CYP2D6 / Q000235:genetics* / Q000378:metabolism; D003915:Dextromethorphan / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008862:Microsomes, Liver / Q000201:enzymology*; D010641:Phenotype; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013056:Spectrophotometry, Ultraviolet
chemical_list: D000996:Antitussive Agents; D003915:Dextromethorphan; D019389:Cytochrome P-450 CYP2D6