Application of DBS for the Quantitative Determination of Midazolam and Hydroxymidazolam using an AB Sciex QTrap 6500 LC-MS/MS System

摘要

A highly sensitive, accurate, precise, and robust UHPLC-MRM assay has been developed for the quantitative determination of MDZ and HDMZ in rat DBS samples, meeting all acceptance criteria for a validatable method. The five-fold gain in sensitivity of the QTrap 6500 over the QTrap 5500 allowed a less abundant but more selective MRM transition to be utilized for HMDZ, in addition to compensating for the relatively poor DBS recovery of both MDZ and HMDZ. Calibration curves for MDZ which exhibited quadratic behavior on the QTrap 5500 were linear on the QTrap 6500 due to the increased saturation point of the pulse-counting detector (108 cps). Linear regressions for MDZ and HMDZ at an LLOQ of 0.1 ng/mL (180 fg on-column) were > 0.9990 over three orders of magnitude. The precision and accuracy of the assay was shown to be immune to spotted blood volume, punch location, and punch size.