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

Abstract

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 100ng/ml). Compounds were analyzed on a C(18) column with a water and methanol mobile phase gradient with acetic acid as an additive, at a flow rate of 0.3ml/min. The working assay range was linear from 0.25 to 100ng/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.

Figure 1

Representative chromatograms of the lowest calibration standard (0.25 ng/ml) of (A) alfentanil and (B) midazolam in human plasma.

Figure 2

Representative chromatograms of blank human plasma to assess potential interfering peaks at the retention times and MRM transitions for (A) midazolam, (B) midazolam d₄, (C) alfentanil, and (D) alfentanil d₅.

Figure 3

Representative LC-MS/MS chromatograms of human plasma from a subject administered alfentanil and midazolam. Shown are (A) midazolam, (B) midazolam d₄, (C) alfentanil, and (D) alfentanil-d₅.

Figure 4

Plasma concentration vs time profiles of alfentanil and midazolam after administering (A) intravenous midazolam, (B) intravenous alfentanil, (C) oral midazolam, and (D) oral alfentanil.