High-throughput assay for simultaneous quantification of the plasma concentrations of morphine, fentanyl, midazolam and their major metabolites using automated SPE coupled to LC-MS/MS

摘要

rapid LC-MS/MS assay method for simultaneous quantification of morphine, fentanyl, midazolam and their major metabolites: morphine-3-β-d-glucuronide (M3G), morphine-6-β-d-glucuronide (M6G), norfentanyl, 1'-hydroxymidazolam (1-OH-MDZ) and 4-hydroxymidazolam (4-OH-MDZ) in samples of human plasma has been developed and validated. Robotic on-line solid phase extraction (SPE) instrumentation was used to elute the eight analytes of interest from polymeric SPE cartridges to which had been added aliquots (150μL) of human plasma and aliquots (150μL) of a mixture of two internal standards, viz. morphine-d3 (200ng/mL) and 1'-hydroxymidazolam-d5 (50ng/mL) in 50mM ammonium acetate buffer (pH 9.25). Cartridges were washed using 10% methanol in ammonium acetate buffer, pH 9.25 (1mL, 2mL/min) before elution with mobile phase comprising 0.1% formic acid in water (A) and acetonitrile (B) with a flow rate of 0.6mL/min using an 11.5min run time. The analytes were separated on a C18 X-Terra ~? analytical column. The linear concentration ranges were 0.5-100ng/mL for fentanyl, norfentanyl and midazolam; 1-200ng/mL for 4-hydroxymidazolam, 2.5-500ng/mL for 1'-hydroxymidazolam and 3.5-700ng/mL for morphine, M3G, and M6G. The method showed acceptable within-run and between-run precision (relative standard deviation (RSD) and accuracy <20%) for quality control (QC) samples spiked at concentrations of 80% and 50% of the ULOQ, 3 times higher than the LLOQ, and also at the LLOQ. Furthermore, analytes were stable in samples (after mixing with internal standard) for at least 48h in the autosampler (except for 4-hydroxymidazolam which decreased by 22% after 24h), 5h at room temperature and after three cycles of freeze and thaw. No autosampler carry-over was observed and the absolute recovery (the area ratio of analyte in plasma relative to that in ammonium acetate buffer 50mM, pH 9.25) was in the range 40% (midazolam) to 110% (morphine). The assay was applied successfully to the measurement of the analytes of interest in plasma samples from patients on extracorporeal membrane oxygenation (ECMO).