title: A novel incubation direct injection LC/MS/MS technique for in vitro drug metabolism screening studies involving the CYP 2D6 and the CYP 3A4 isozymes.
abstract: A direct injection LC/MS/MS method involving a novel incubation technique was developed for the inhibition screening of CYP 2D6 and CYP 3A4 isoenzymes using dextromethorphan and midazolam as probe substrates. Both assays were performed using an electrospray ionization source in the positive ion mode. Direct injection was possible by using a short C 18, LC column (2 mm x 20 mm) with large particle diameter packing (10 microm). Analytical characteristics of the direct injection technique were studied by examining matrix effects, which showed suppression of the ESI signal between 0.20 and 0.65 min. The retention times for analytes were adjusted to approximately 0.8 min (k'>3), resulting in no matrix effect. Column lifetime was evaluated and determined to be approximately 160 direct injections of the matrix. The precision and accuracy of the control samples for the quantitation of dextromethorphan was between -0.53 and -12.80, and 3.73 and 6.69% respectively. Unlike conventional incubation techniques, incubations were carried out in an autosampler equipped with a heating accessory. This novel incubation method, which involved no stirring of the incubation mixture, estimated the Cl(int in vitro) for dextromethorphan and midazolam in human liver microsomes to be 1.65+/-0.22 ml/(hmg) and 0.861 ml/(min mg) respectively. The autosampler tray maintained uniform temperature and was sensitive to changes in temperature between 33 and 41 degrees C. High-throughput screening was performed using known inhibitors of the CYP 2D6 isozyme, and the system was evaluated for its ability to differentiate between these inhibitors. The strong inhibitor quinidine resulted in a 25.6% increase in t(1/2), the medium potency inhibitor chlorpromazine resulted in an increase of 6.14% and the weak inhibitor primaquine had no significant effect on half-life. This technique involves no sample preparation, demonstrated run times of 2 min per injection and can be fully automated. The method should therefore prove to be a valuable tool in the drug discovery process.
authors.affiliation: Department of Pharmaceutics, Medical College of Virginia, Virginia Commonwealth University, P.O. Box 980533, Richmond, VA 23298-0533, USA.
authors.forename: S
authors.initials: S
authors.lastname: Bhoopathy
authors.forename: B
authors.initials: B
authors.lastname: Xin
authors.forename: S E
authors.initials: SE
authors.lastname: Unger
authors.forename: H T
authors.initials: HT
authors.lastname: Karnes
mesh_terms: D000465:Algorithms; D000818:Animals; D000996:Antitussive Agents / Q000378:metabolism; D002746:Chlorpromazine / Q000494:pharmacology; D002853:Chromatography, Liquid; D019389:Cytochrome P-450 CYP2D6 / Q000378:metabolism*; D065690:Cytochrome P-450 CYP2D6 Inhibitors; D051544:Cytochrome P-450 CYP3A; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D003915:Dextromethorphan / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006207:Half-Life; D006801:Humans; D006993:Hypnotics and Sedatives / Q000378:metabolism; D066298:In Vitro Techniques; D007202:Indicators and Reagents; D007527:Isoenzymes / Q000378:metabolism; D008862:Microsomes, Liver / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D008874:Midazolam / Q000378:metabolism; D004364:Pharmaceutical Preparations / Q000378:metabolism*; D011319:Primaquine / Q000494:pharmacology; D011802:Quinidine / Q000494:pharmacology; D051381:Rats; D015203:Reproducibility of Results; D021241:Spectrometry, Mass, Electrospray Ionization
chemical_list: D000996:Antitussive Agents; D065690:Cytochrome P-450 CYP2D6 Inhibitors; D065607:Cytochrome P-450 Enzyme Inhibitors; D004791:Enzyme Inhibitors; D006993:Hypnotics and Sedatives; D007202:Indicators and Reagents; D007527:Isoenzymes; D004364:Pharmaceutical Preparations; D003915:Dextromethorphan; D003577:Cytochrome P-450 Enzyme System; C104464:CYP3A protein, human; D019389:Cytochrome P-450 CYP2D6; D051544:Cytochrome P-450 CYP3A; D011802:Quinidine; D011319:Primaquine; D008874:Midazolam; D002746:Chlorpromazine