title: Rapid and simultaneous measurement of midazolam, 1'-hydroxymidazolam and digoxin by liquid chromatography/tandem mass spectrometry: application to an in vivo study to simultaneously measure P-glycoprotein and cytochrome P450 3A activity.
abstract: In order to simultaneously determine in vivo P-glycoprotein (P-gp) and Cytochrome P450 3A (CYP3A) activity, a new, rapid and sensitive liquid chromatography/tandem mass spectrometry (LC-MS/MS) method has been developed and fully validated to simultaneously determine midazolam (MDZ, as CYP3A substrate), 1'-hydroxymidazolam (1'-OHMDZ) and digoxin (DG, as P-gp substrate) in rat plasma using digitoxin as the internal standard (IS). After a single step liquid-liquid extraction with tert-butyl methyl ether/dichloromethane (75:25, v/v), analytes were subjected to LC-MS/MS analysis using positive electro-spray ionization (ESI(+)) under selected reaction monitoring mode (SRM). Chromatographic separation was performed on an XTerra MS C18 column (50mm×2.1mm, i.d. 3.5μm). The MS/MS detection was conducted by monitoring the fragmentation of 326.05 → 244.00 (m/z) for MDZ, 342.02 →168.01 (m/z) for 1'-OHMDZ, 798.33 → 651.36(m/z) for DG and 782.67 → 635.24 (m/z) for IS. The method had a chromatographic running time of 3min and linear calibration curves over the concentrations of 2-400ng/mL for MDZ and 1'-OHMDZ and 0.5-100ng/mL for DG. The recoveries of the method were 86.8-96.3% for MDZ, 84.6-86.4% for 1'-OH MDZ, and 81.7-85.1% for DG. The lower limit of quantification (LLOQ) of the method was 2ng/mL for MDZ and 1'-OHMDZ and 0.5ng/mL for DG. The intra- and inter-batch precision were less than 15% for all quality control samples at concentrations of 5, 50 and 320ng/mL for MDZ and 1'-OHMDZ and 1, 10 and 80ng/mL for DG. The validated LC-MS/MS method has been successfully used to analyze the concentrations of MDZ, 1'-OH MDZ and DG in rat plasma for simultaneous measurement of in vivo P-gp and CYP 3A activity.
authors.affiliation: School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006, People's Republic of China.
authors.forename: Xinping
authors.initials: X
authors.lastname: Xue
authors.forename: Min
authors.initials: M
authors.lastname: Huang
authors.forename: Huanyu
authors.initials: H
authors.lastname: Xiao
authors.forename: Xiaoling
authors.initials: X
authors.lastname: Qin
authors.forename: Ling
authors.initials: L
authors.lastname: Huang
authors.forename: Guoping
authors.initials: G
authors.lastname: Zhong
authors.forename: Huichang
authors.initials: H
authors.lastname: Bi
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000378:metabolism*; D000818:Animals; D001711:Biotransformation; D002138:Calibration; D002316:Cardiotonic Agents / Q000097:blood* / Q000737:chemistry / Q000493:pharmacokinetics; D002851:Chromatography, High Pressure Liquid; D051544:Cytochrome P-450 CYP3A / Q000378:metabolism*; D004077:Digoxin / Q000097:blood* / Q000737:chemistry / Q000493:pharmacokinetics; D004347:Drug Interactions; D004355:Drug Stability; D057230:Limit of Detection; D008297:Male; D008874:Midazolam / Q000031:analogs & derivatives* / Q000097:blood* / Q000737:chemistry / Q000493:pharmacokinetics; D011897:Random Allocation; D051381:Rats; D017207:Rats, Sprague-Dawley; D015203:Reproducibility of Results; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D002316:Cardiotonic Agents; D004077:Digoxin; C040081:1-hydroxymethylmidazolam; D051544:Cytochrome P-450 CYP3A; D008874:Midazolam