Analysis of midazolam and metabolites in plasma by high-performance liquid chromatography: probe of CYP3A.
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.
Carrillo
J A
JA
Department of Pharmacology and Psychiatry, Medical School, University of Extremadura, Badajoz, Spain.
Ramos
S I
SI
Agundez
J A
JA
Martinez
C
C
Benitez
J
J
D000328:Adult; D014151:Anti-Anxiety Agents / Q000097:blood*; D001189:Aryl Hydrocarbon Hydroxylases; D015415:Biomarkers / Q000097:blood; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000097:blood*; D016903:Drug Monitoring / Q000379:methods*; D006801:Humans; D008297:Male; D008874:Midazolam / Q000097:blood*; D010089:Oxidoreductases, N-Demethylating / Q000097:blood*; D012016:Reference Values; D012680:Sensitivity and Specificity
D014151:Anti-Anxiety Agents; D015415:Biomarkers; D003577:Cytochrome P-450 Enzyme System; D001189:Aryl Hydrocarbon Hydroxylases; D051544:Cytochrome P-450 CYP3A; D010089:Oxidoreductases, N-Demethylating; D008874:Midazolam