Automated determination of midazolam in human plasma by high-performance liquid chromatography using column switching

• Published in: Journal of chromatography. B, Biomedical applications Biomedical applications, 1994-03, Vol.654 (1), p.69-75
• Main Authors: Lauber, Rolf, Mosimann, Marcel, Bührer, Michael, Zbinden, Alex Martin
• Format: Article
• Language: English
• Subjects: Analysis; Autoanalysis; Biological and medical sciences; Chromatography, High Pressure Liquid - methods; Chromatography, High Pressure Liquid - statistics & numerical data; Drug Stability; General pharmacology; Humans; Hydrogen-Ion Concentration; Medical sciences; Midazolam - blood; Pharmacology. Drug treatments; Sensitivity and Specificity
• ISSN: 0378-4347; 1572-6495
• DOI: 10.1016/0378-4347(94)00017-4

An automated gradient high-performance liquid chromatographic method using a column-switching technique was developed in order to determine and quantify midazolam (separated from the metabolite α-hydroximidazolam) in human plasma. After dilution with an internal standard (flurazepam) solution, containing 20% acetonitrile, 400 μl of the plasma samples were injected onto a precolumn (17 × 4.6 mm I.D., C 18 Corasil 37–53 μm) and retained. Proteins and polar plasma components were washed out using a 0.1 M sodium hydroxide solution, followed by an equilibration with a phosphate buffer of pH 8.0. After column-switching midazolam and flurazepam were eluted and transferred to the analytical column (RP-select B) in the backflush mode, separated by gradient elution and detected at 230 nm by ultraviolet detection. Precision of replicate analyses on the same day was 1.5% for midazolam and 0.7% for flurazepam. Recovery of midazolam was in the range 80–89% and the detection limit was 2 ng/ml plasma.