Rapid determination of six metabolites from multiple cytochrome P450 probe substrates in human liver microsome by liquid chromatography/mass spectrometry: application to high-throughput inhibition screening of terpenoids.
A rapid liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed for the determination of six cytochrome P450 (CYP) probe substrate metabolites including paracetamol (PAR) for CYP1A2, 4-hydroxytolbutamide (OHTOL) for CYP2C9, 5-hydroxyomeprazole (OHOMe) for CYP2C19, dextrorphan (DEXM) for CYP2D6, 6-hydroxychlorzoxazone (OHCHL) for CYP2E1 and dehydronifedipine (DNIF) for CYP3A4. The triple-quadrupole mass spectrometer was operated in both positive and negative modes, and selective reaction monitoring was used for quantification. The method was validated over the concentration ranges (0.075/0.04/0.05/0.02/0.1/0.0625 microM to 4.8/2.56/3.2/1.28/6.4/4.0 microM) for PAR/OHTOL/OHOME/DEXP/OHCHL/DNIF analytes with acceptable accuracy and precision. The inhibitory effect on the six CYP enzymes has been verified with their known specific inhibitors. This high-throughput inhibition screening approach has been successfully applied to study the inhibitory effects of 18 terpenoids on CYP enzymes. Among them, tanshinone IIA and cryptotanshinone are found to be potent inhibitors to CYP1A2, while artemisinin is a marginal inhibitor to CYP1A2 and glycyrrhetic acid is a weak inhibitor to CYP2C9.
He
Fan
F
School of Pharmaceutical Sciences, Sun Yat-sen University, 74 Zhongshan Road II, Guangzhou 510080, China.
Bi
Hui-Chang
HC
Xie
Zhi-Yong
ZY
Zuo
Zhong
Z
Li
Jian-Kang
JK
Li
Xin
X
Zhao
Li-Zi
LZ
Chen
Xiao
X
Huang
Min
M
D002851:Chromatography, High Pressure Liquid; D003577:Cytochrome P-450 Enzyme System / Q000032:analysis / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000032:analysis* / Q000494:pharmacology; D006801:Humans; D066298:In Vitro Techniques; D020128:Inhibitory Concentration 50; D008862:Microsomes, Liver / Q000187:drug effects / Q000201:enzymology*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D013379:Substrate Specificity; D053719:Tandem Mass Spectrometry / Q000379:methods; D013729:Terpenes / Q000037:antagonists & inhibitors / Q000378:metabolism*
D004791:Enzyme Inhibitors; D013729:Terpenes; D003577:Cytochrome P-450 Enzyme System