AUTOMATED-DETERMINATION OF MIDAZOLAM IN HUMAN PLASMA BY HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY USING COLUMN-SWITCHING

An automated gradient high-performance liquid chromatographic method u sing a column-switching technique was developed in order to determine and quantify midazolam (separated from the metabolite alpha-hydroximid azolam) in human plasma. After dilution with an internal standard (flu razepam) solution, containing 20% acetonitrile, 400 mul of the plasma samples were injected onto a precolumn (17 x 4.6 mm I.D., C18 Corasil 37-53 mum) and retained. Proteins and polar plasma components were was hed out using a 0.1 M sodium hydroxide solution, followed by an equili bration with a phosphate buffer of pH 8.0. After column-switching mida zolam and flurazepam were eluted and transferred to the analytical col umn (RP-select B) in the backflush mode, separated by gradient elution and detected at 230 nm by ultraviolet detection. Precision of replica te analyses on the same day was 1.5% for midazolam and 0.7% for fluraz epam. Recovery of midazolam was in the range 80-89% and the detection limit was 2 ng/ml plasma.