Sensitive assay for midazolam and its metabolite 1'-hydroxymidazolam in human plasma by capillary high-performance liquid chromatography

J Chromatogr B Biomed Sci Appl. 1998 Jun 12;710(1-2):165-71. doi: 10.1016/s0378-4347(98)00137-6.


A sensitive high-performance liquid chromatographic method is described for the quantification of midazolam and 1'-hydroxymidazolam in human plasma. Sample (1 ml plasma) preparation involved a simple solvent extraction step with a recovery of approximately 90% for both compounds. An aliquot of the dissolved residue was injected onto a 3 microm capillary C18 column (150 mm x 0.8 mm I.D.). A gradient elution was used. The initial mobile phase composition (phosphate buffer-acetonitrile, 65:35) was maintained during 16 min and was then changed linearly during a 1-min period to phosphate buffer-acetonitrile, 40:60. The flow-rate of the mobile phase was 16 microl/min and the eluate was monitored by UV detection. The limits of quantification for midazolam and 1'-hydroxymidazolam were 1 ng/ml and 0.5 ng/ml, respectively. The applicability of the method was demonstrated by studying the pharmacokinetics of midazolam, and its major metabolite 1'-hydroxymidazolam, in human volunteers following i.v. bolus administration of a subtherapeutic midazolam dose (40 microg/kg).

MeSH terms

  • Aryl Hydrocarbon Hydroxylases*
  • Chromatography, High Pressure Liquid / methods*
  • Cytochrome P-450 CYP3A
  • Cytochrome P-450 Enzyme Inhibitors
  • Cytochrome P-450 Enzyme System / metabolism
  • Enzyme Inhibitors / administration & dosage
  • Evaluation Studies as Topic
  • Humans
  • Midazolam / analogs & derivatives*
  • Midazolam / blood*
  • Midazolam / pharmacokinetics
  • Oxidoreductases, N-Demethylating / antagonists & inhibitors
  • Oxidoreductases, N-Demethylating / metabolism
  • Rifampin / administration & dosage
  • Sensitivity and Specificity


  • Cytochrome P-450 Enzyme Inhibitors
  • Enzyme Inhibitors
  • Cytochrome P-450 Enzyme System
  • 1-hydroxymethylmidazolam
  • Aryl Hydrocarbon Hydroxylases
  • Cytochrome P-450 CYP3A
  • Oxidoreductases, N-Demethylating
  • Midazolam
  • Rifampin