Midazolam (8-chloro-6-(2-fluorophenyl)-1-methyl-4H-imidazo[1,5-α][1,4] benzodiazepine), a short-acting benzodiazepine with hypnotic properties, is used for conscious sedation to perform short diagnostic and endoscopic procedures and for induction and maintenance of general anaesthesia [1]. Midazolam is metabolized by the cytochrome P-450 enzyme system to several metabolites including an active metabolite, α-hydroxymidazolam [2].

We have developed a simple and reproducible HPLC-MS/APCI method to determine the concentration of midazolam and its α-hydroxy metabolite in human plasma. The method consists of a solid-phase extraction procedure (SPE) as the sample preparation step.

The SPE operations were performed automatically by means of a sample processor equipped with a robotic arm (Gilson ASPEC XL). The ASPEC was programmed to condition each Bakerbond SPE C2 extraction column with 1 mL of methanol followed by 1 mL of water and 1 mL of 1% acetic acid just before use. A 0.5 mL of plasma sample containing the internal standard (flunitrazepam) was then applied on the cartridge. The washing step was performed with 2 x 1 mL of 0.01 M ammonium acetate. Finally, the analytes were eluted with 2 x 1 mL of 0.2 M ammonium acetate (pH 9.0) in methanol (10:90,v/v). The eluent was evaporated to dryness. The sample residue was dissolved in 200 μL of mobile phase, transferred to autosampler vial and 60 μL aliquot was injected onto a LC-MS system. The chromatography separation was achieved on a 100 x 4.6 mm Chromolit RP-18e column operated with a mobile phase of acetonitrile and 0.01 M ammonium acetate buffer pH 4.3 (52:48,v/v) at a flow-rate of 1 mL/min. The mass spectrometer equipped with atmospheric pressure chemical ionization (APCI) source was run in the positive ion mode and set with a selected ion monitoring method (SIM) of ion m/z = 325.75, 341.75 and 313.75 for midazolam, α-hydroxymidazolam and flunitrazepam, respectively

The recoveries from plasma samples for midazolam and α-hydroxymidazolam ranged from 84.8 to 97.7% and from 85.8 to 88.4%, respectively. The limit of detection was established as 0.1 ng/mL for both compounds. The linearity was assessed in the concentration range 1.5-100 ng/mL for midazolam and 1.5-60 ng/mL for α-hydroxymidazolam. The intra- and inter-run precision and accuracy values were less than 10% for both compounds. No matrix effect was observed.

The sensitive and reproducible procedure described offers possibility to measure plasma level of midazolam and α-hydroxymidazolam after oral administration during the pharmacokinetic studies in humans.

1. M.Bebin, T.P.Bleck, Drugs 1994, 48, 153.

2. A.Ghosal, H.Satoh, P.E.Thomas et al. Drug Metab.Dispos. 1996, 24, 940.

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