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

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 Cmax was found to be Y = 1.354*X + 1.968 (r2 = 0.9984), and AUC0-12 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.