Validation of a sensitive and specific LC-MS/MS method and application to evaluate the systemic exposure and...

A specific and sensitive LC-MS/MS method using d₃ -glycopyrrolate as the internal standard (IS) was developed for the quantitative determination of glycopyrrolate (GLY) in human plasma over a concentration range of 4.00-2000 pg/ml. The GLY and IS were extracted using a solid-phase extraction cartridge, then eluted with 0.5% formic acid in 70:30 acetonitrile-water. The eluate was directly injected into an Agilent Pursuit 5PFP column for analysis using an isocratic mobile phase of 50:50 A:B at a flow-rate of 0.500 ml/min (A, 10 mm ammonium acetate in 1% formic acid; B, methanol). The MS detection was in positive mode by monitoring m/z 318.3 → 116.1 (GLY) and 321.3 → 119.1 (IS). The method validation showed the linearity of r²  ≥ 0.9960, intra-/inter-run precisions of ≤11.1% coefficient of variation and accuracies ranging from -2.5 to 12.8% relative error for all levels of quality control samples. This method was successfully employed to support a clinical study to compare absorption and bioavailability of GLY administered by a Magnair^® eFlow nebulizer to a Seebri^® Breezhaler^® with/without a charcoal blockade of gastric absorption. By comparison with intravenous administration, respective bioavailabilities of ~15% for GLY/Magnair and ~22% for the Seebri Breezhaler were found. The bioanalytical reliability was also demonstrated by satisfactory incurred sample reanalysis performance.