Development and validation of UHPLC-MS/MS method for analysis of sedative drugs and their metabolites in blood plasma

Abstract

In intensive care units, the precise administration of sedative drugs is crucial in order to avoid under- or over sedation – both of which can be very harmful. It is especially important in case of paediatric patients. A highly sensitive simultaneous UHPLC-MS/MS method was developed for the quantification of morphine, morphine-3-β-glucuronide, morphine-6-β-glucuronide, clonidine, midazolam and 1’-hydroximidazolam in human plasma samples. The lowest limit of quantification for all analytes was 50 pg/mL using only 100 μL of blood plasma. Analytes were separated chromatographically using C18 column with weak ion-pairing additive 1,1,1,3,3,3-hexafluoro-2-propanol with pH = 9 (adjusted using ammonium hydroxide) and methanol. Method was fully validated and method was using matrix matched calibration in the range of 0.05-250 ng/mL for all analytes. Within-day accuracy for all analytes remained 87-113 %, but within-day precision remained within 3-11 % for all analytes at all concentration levels over the calibration range. The method will be applied for real patients’ samples in the EU FP7 project CloSed – "Clonidine for Sedation of Paediatric Patients in the Intensive Care Unit”.