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 (R2 = 0.9995), y = −2.21 × 10−4 (±3.93 × 10−5) × x2 + 5.85 × 10−2 (±5.27 × 10−3) × x + 4.08 × 10−3 (±4.82 × 10−4). For the three QC concentrations (QC1 0.252, QC2 2.52, and QC3 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.