An ultrasensitive LC-MS/MS method and application to clinical dose-range finding for glycopyrrolate via an...

Aim: Glycopyrrolate (GLY) has been widely used to treat chronic obstructive pulmonary disease (COPD). Recent technical advancement significantly improves the drug delivery efficiency. In a clinical dose-range determination study via electronic nebulizer, the starting dose was as low as 3-μg, thus, a bioanalytical method capable of measuring down to sub-pg/ml or fg/ml level was required to characterize pharmacokinetics of GLY. Methods: A straightforward solid-phase extraction coupled with reversed-phase LC-MS/MS method was successfully developed to quantify plasma GLY concentrations of 0.500-250 pg/mL. MS/MS detection was at ESI+ by monitoring m/z 318.3→116.1 for GLY and 321.3→119.1 for the internal standard GLY-d₃. Results and Conclusion: The method was validated with adequate selectivity, intra- and inter-assay precision and accuracy, dilution linearity, and sufficient stability for measuring GLY in human plasma. This method has been used for sample analysis of a clinical dose-range finding study (3-50 µg twice daily doses via an electronic nebulizer). The assay exhibited good precision, accuracy, and robustness. Pharmacokinetic analysis showed good dose proportionality over the studied dose range, where C_max was found to be Y = 1.354*X + 1.968 (r² = 0.9984), and AUC_{0-12 h} was Y = 4.661*X + 23.32 (r² = 0.9976). The study clearly demonstrated the importance of an ultrasensitive (500 fg/ml LLOQ) method.