Simultaneous detection of ketamine, lorazepam, midazolam and sufentanil in human serum with liquid chromatography-tandem mass spectrometry for monitoring of analgosedation in critically ill patients.

Abstract

A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed and validated for the determination and quantification of four predominantly used analgosedatives in the intensive care unit: ketamine, lorazepam, midazolam and sufentanil in human serum. The extraction procedure consisted of protein precipitation of serum samples with acetonitrile and subsequent centrifugation. D5-fentanyl and D4-midazolam served as internal standards (ISTD). Separation of analytes was performed with a Hypersil C18 column and a mobile phase with acetonitrile and 0.1% formic acid (60/40, v/v) under isocratic conditions at a flow rate of 280μl/min. Analytes were simultaneously detected with a triple-stage quadrupole mass spectrometer (LC-MS/MS) in a selected reaction monitoring (SRM) mode with positive heated electrospray ionization (HESI) within a single 2-min run. Calibration curves were linear over a range of 50-2000 for ketamine, 10-1000 for lorazepam, 5-500 for midazolam and 1-100 for sufentanil (ng/ml). The limit of detection and the lower limit of quantification were 0.01 and 10.00 for ketamine, 0.005 and 10.00 for lorazepam, 0.018 and 5.00 for midazolam and 0.068 and 0.25 for sufentanil (ng/ml). Intra- and inter-day accuracies and precisions of all analytes were less than 15%. Bench stability with spiked serum samples was ensured after 12, 24 and 48h at room temperature, freeze- and thaw-stability after 3 cycles of thawing and freezing. The method was successfully established according to International Conference on Harmonization (ICH) guideline Q2 (R1) "Validation of Analytical Procedures" and applied in critically ill adult patients in the intensive care unit. We suggest its suitability for parallel quantification of the sedative analgesics ketamine, lorazepam, midazolam and sufentanil. The method serves as an instrumental tool for therapeutic drug monitoring (TDM) and pharmacokinetic studies [1].