Analysis of Midazolam and Metabolites in Plasma by High-Performance Liquid Chromatography: Probe of CYP3A

Abstract

Hydroxylation of midazolam (MDZ) is mediated almost exclusively by CYP3A isoforms. The authors describe a high-performance liquid chromatography assay involving MDZ, 1'-hydroxymidazolam, and 4-hydroxymidazolam in plasma. The compounds were eluted on an Ultrasphere ODS, 3-microm particle size, 7.5 cm x 4.6 mm reversed-phase column and monitored by ultraviolet absorbance at 254 nm. The composition of the mobile phase was 35.2% acetonitrile:4.8% methanol:60% buffer acetate (vol/vol/vol), 0.1 M, pH 4.7; the flow rate was 1 ml/minute. Calibration curves were linear (coefficients of correlation > 0.99) within the range of concentrations established (20 to 640 nM). Within- and between-day coefficients of variation were consistently better than 8%. The overall recovery was >90% and the lowest detectable concentration was 8 nM. This approach provides a simple, rapid, and sensitive assessment of MDZ and metabolites in plasma, with a very good accuracy and precision, which enables it as an in vivo marker of CYP3A activity in humans.

O-Dealkylation of Fluoxetine in Relation toCYP2C19Gene Dose and Involvement of CYP3A4 in Human Liver Microsomes

“…The activities of fluoxetine O-dealkylation were correlated with the activities of 250 M S-mephenytoin 4Ј-hydroxylation and 100 M midazolam 1Ј-hydroxylation. The rate of formation of 4Ј-hydroxymephenytoin and 1Ј-hydroxymidazolam was determined using highperformance liquid chromatography as described by Xie et al (1995) and by Carrillo et al (1998), respectively. Data Analysis.…”

“…The microsomal activities of fluoxetine O-dealkylation at low (5 M), medial (25 M), and high (100 M) substrate concentrations were measured for 11 liver microsomes from EMs of CYP2C19. The rate of formation of 4Ј-hydroxymephenytoin and 1Ј-hydroxymidazolam in the these liver microsomes was also determined by previously described methods of Xie et al (1995) and Carrillo et al (1998), which were reflected with the CYP2C19 activity and CYP3A4 activity. At a low substrate concentration of 5 M, a good correlation (r ϭ 0.740, P Ͻ 0.01) was found between fluoxetine O-dealkylation and S-mephenytoin 4Ј-hydroxylation (Fig.…”

Simultaneous determination of midazolam and 1′‐hydroxymidazolam in human plasma by liquid chromatography with tandem mass spectrometry

“…The drug is extensively metabolized to its oxidative analogs, 1′-hydroxymidazolam and 4-hydroxymidazolam, as a result of first-pass gastrointestinal and hepatic metabolism (Thummel et al, 1996), with the former being primary and pharmacologically active, and the latter minor and generally not detectable from human matrices by HPLC-UV (Carrillo et al, 1998;Thummel and Wilkinson, 1998). Such an oxidative metabolism process is catalyzed by substrate-specific and selective cytochrome P450, a super family of hemo-proteins that catalyze the metabolism of a large number of therapeutically important drugs and xenobiotics (Guengerich, 1999;Thummel and Wilkinson, 1998).…”

Effect of Multiple Dosing of Ketoconazole on Pharmacokinetics of Midazolam, a Cytochrome P-450 3A Substrate in Beagle Dogs

“…MDZ concentrations were analyzed by HPLC with UV detector as previously described (Carrillo et al, 1998) using diazepam as an internal standard. For plasma concentrations, thawed plasma (1 ml) was placed in 10-ml test tubes basified with glycine buffer (0.75 M, pH ϭ 9).…”