Quantitation of seven sedative and analgesic drugs in whole blood from intensive care patients using liquid chromatography mass spectrometry

Summary We present the development and validation of a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for quantification of clonidine, dexmedetomidine, fentanyl, ketamine, ketobemidone, midazolam and morphine in whole blood. These are drugs predominately used in intensive care units (ICUs) but they are also encountered in forensic investigations. The analytes were recovered from 0.25 g of blood by protein precipitation with a mixture of acetonitrile and ethanol. Separation was performed on a BEH phenyl column. Mobile phases consisted of 0.05% formic acid in 10 mM ammonium formate and 0.05% formic acid in methanol, respectively, and the flow rate was 600 μL/min. The mass spectrometer was operated in positive electrospray ionization mode with multiple reaction monitoring. Validation included selectivity, qualitative matrix effects, calibration model, limit of detection, lower limit of quantification, within- and between-day accuracy and precision, process efficiency, dilution integrity, carry over and stability. Selectivity was high and no ion suppression or enhancement was observed in the areas were the analytes eluted. Calibration curves were linear over a range of 0.25–50 ng/g for dexmedetomidine, 0.05–50 ng/g for fentanyl and 5.0–500 ng/g for morphine and quadratic over a range of 0.5–50 ng/g for clonidine, 50–5000 ng/g for ketamine, 5.0–500 ng/g for ketobemidone and midazolam. The method showed acceptable within- and between-day accuracies and precisions. All analytes were stable in whole blood for three weeks at 4 °C. Concentrations in patient samples ranged between 42–760 ng/g for midazolam (n = 15), 0.3–1.5 ng/g for dexmedetomidine (n = 13), 0.6–6.4 ng/g for clonidine (n = 13), 8–62 ng/g for morphine (n = 16), 5–19 ng/g for ketobemidone (n = 5), 0.07–3.1 ng/g for fentanyl (n = 43), and 56–2000 ng/g for ketamine (n = 10). We conclude that the method was successfully validated and applied to ante-mortem and post-mortem blood samples from critically ill adult patients in a general ICU.