Determination of midazolam and its major hydroxy metabolite in plasma by gas liquid chromatography. Application to a pharmacokinetic study

A sensitive gas liquid chromatographic (GLC) assay was developed for plasma determinations of 8‐chloro‐6‐(2′ ‐fluorophenyl)‐1‐methyl‐4 H ‐imidazo[1,5 α][1,4]benzodiazepine (compound I) and its hydroxymethylimidazo metabolite (compound II). The internal standards used were 8‐chloro‐6‐(2′ ‐chlorophenyl)‐1 ‐methyl‐4 H ‐imidazo[1,5 α][1,4]benzodiazepine (compound VI) and 7‐chloro‐5‐(2′ ‐fluorophenyl)‐1, 3‐dihydro‐1‐(hydroxyethyl)‐2 H ‐1,4‐benzodiazepin‐2‐one (compound VII) for compounds I and II, respectively. Following extraction, and silylation for compound II, compounds I and II were analyzed by GLC using a glass column packed with 5% OV‐101 on Gas‐Chrom Q, and a 63 Ni electron‐capture detector. The GLC method was validated by a CI‐GC/MS technique. The detection limit of the assay is approximately 4–5 ng/ml for compound I and 3 ng/ml for compound II. The method was used in comparative pharmacokinetic studies of the distribution of the two compounds in arterial and venous blood.