Simultaneous determination of alfentanil and midazolam in human plasma using liquid chromatography and tandem mass spectrometry

A fast, sensitive and selective liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for the determination of alfentanil and midazolam in human plasma has been developed and validated. Alfentanil and midazolam were extracted from plasma using a mixed-mode cation exchange solid phase extraction method, with recoveries of both compounds greater than 80% at 3 different concentrations (1, 10 and 100 ng/ml). Compounds were analyzed on a C18 column with a water and methanol mobile phase gradient with acetic acid as an additive, at a flow rate of 0.3 ml/min. The working assay range was linear from 0.25 to 100 ng/ml for each compound. The signal to noise ratio was 80 and 40 for alfentanil and midazolam, respectively, at the lowest concentration calibration standard, with less than 10% matrix suppression by human plasma at this concentration. Alfentanil and midazolam were stable in human plasma during storage at −80 °C, processing, and analysis. The procedure was validated and applied to the analysis of plasma samples from healthy human subjects administered oral and intravenous alfentanil and midazolam.