Development and validation of a liquid chromatography coupled to tandem mass spectrometry method for the simultaneous quantification of five analgesics and sedatives, and six of their active metabolites in human plasma: Application to a clinical study on the determination of neurological death in the intensive care unit.

A sensitive and selective high-performance liquid chromatographic method coupled to tandem mass spectrometry was developed and validated for the quantification of morphine, hydromorphone, fentanyl, midazolam and propofol and their metabolites morphine-3-β-d-glucuronide, morphine-6-β-d-glucuronide, hydromorphone-3-β-d-glucuronide, 1'-hydroxymidazolam-β-d-glucuronide, α-hydroxymidazolam and 4-hydroxymidazolam in human plasma using potassium oxalate/sodium fluoride mixture as anticoagulant. Human plasma samples (0.4 mL) to which were added a mixture of eleven deuterated internal standards were subjected to solid phase extraction using a mixed-mode polymeric Oasis PRiME MCX in 96-well format. Propofol was selectively eluted and further derivatized using 2-Fluoro-1-methylpyridinium p-toluenesulfonate, whereas the remaining 10 analytes were eluted separately and further concentrated. The derivatized propofol was analyzed separately in a second injection. The analytes were chromatographically separated on a Kinetex phenyl-hexyl analytical column in gradient elution mode, using a mobile phase consisting of aqueous ammonium formate/formic acid buffer and methanol. The overall run time was 8 min. Detection was performed using an AB/SCIEX 4000 QTRAP instrument with positive electrospray ionization employing scheduled multiple reaction monitoring mode. The lower limits of quantification ranged from 0.02 to 5 ng/mL depending on the analyte. Calibration curves covered a concentration range of 1000× in all cases but 1'-hydroxymidazolam-β-d-glucuronide where it covered a range of 500 × . The validated method was accurate and precise, the intra-day accuracy and precision of quality control samples (4 concentration levels, n = 6 each) being within 91.5-112 % and 1.3-13.2 % (coefficient of variation), respectively, and inter-day (n = 24; 4 days) accuracy and precision of quality control samples (3 concentration levels) being within 94.8-103.5 % and 3.2-11.2 % (coefficient of variation). Mean absolute extraction recoveries were above 60 % for all compounds, except for hydromorphone-3-β-d-glucuronide (44 %) and for 1'-hydroxymidazolam-β-d-glucuronide (33 %). Internal standard corrected matrix effect ranged from -4.8 to 3.8 % in normal plasma and in plasma containing 1 % hemolyzed blood. Analytes were stable (above 90 %) in plasma and blood for 19 h at 22 °C, in blood for 90 h at 5 °C, in plasma for 60 days at -20 °C, for 4 months at -70 °C and after three freeze-thaw cycles, and in the injection solvent for at least 3 days in the autosampler. The present method is successfully being applied in a multicenter clinical study for the analysis of plasma samples from patients in intensive care units from a number of Canadian hospitals.
Competing Interests: Declaration of Competing Interest The authors declare no other competing interests. No writing assistance was utilized in the production of this manuscript.
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