Quantitative determination of glycopyrrolate in human plasma by liquid chromatography-electrospray ionization mass spectrometry: The use of a volatile ion-pairing agent during both liquid-liquid extraction and liquid chromatography

The work presented here deals with the development of a quantitative tool for the determination of the quaternary ammonium anticholinergic glycopyrrolate in human plasma samples. Mepenzolate was used as an internal standard. The plasma samples were subjected to a suitable sample clean-up consisting of a simple and relatively fast, two step liquid-liquid ion-pair extraction procedure. The chromatography. using the same volatile ion-pair reagent heptafluorobutyric acid (HFBA), takes only 10 min. Relative standard deviation of retention times was never above 2.26% (n=36). The method was fully validated based on the US FDA Bioanalytical Method Validation Guidance for Industry. As such, a quantitative ESI-LC-MS(/MS) (TOF mass spectrometry) method was optimized for the absolute quantification of glycopyrrolate in human plasma in a concentration range from 0.101 to 101 ng/mL using a quadratic calibration function (R-2 = 0.9995), y = -2.21 x 10(-4) (+/- 3.93 x 10(-5)) x chi(2) + 5.85 x 10(-2) (+/- 5.27 x 10(-3)) x x + 4.08 x 10(-3) (+/- 4.82 x 10(-4)). For the three QC concentrations (QC(1) 0.252, QC(2) 2.52, and QC(3) 25.2 ng/mL) and the LLOQ (0.101 ng/mL), total precision was under 20% (18.0% (n = 6) at the LLOQ) and maximum accuracy was 112% (88.9% for the LLOQ, n = 6). Absolute matrix effect (maximum 133% +/- 9.59, n = 3), absolute recovery (better than 41.8% +/- 2.22, n = 3), relative (inter-subject) matrix effect (maximum 10.9% +/- 1.45, n = 4) and process efficiency (better than 45.2% +/- 5.74, n = 3) too were assessed at the 3 QC concentrations. (C) 2008 Elsevier B.V. All rights reserved.