Quantitative determination of levonorgestrel in beagle dog plasma after vaginal administration of intravaginal ring by high-performance liquid chromatography-tandem mass spectrometry.

A specific and sensitive high-performance liquid chromatography-tandem mass spectrometry method that could be used to determine the concentration of levonorgestrel (LNG) in beagle dog plasma was developed. Specifically, terfenadine was used as the internal standard (IS). The separation was achieved on a Kinetex-C18 110A column (3 × 30 mm i.d., 2.6 um, Phenomenex) and a gradient mobile phase consists of methanol (0.1% formic acid) and water (0.1% formic acid) was used. The flow rate was 0.8 mL/min and the injection volume was 10 μL. The detection was performed on a triple-quadruple tandem mass spectrometer by multiple reaction monitoring mode via electrospray ionization. Quantitative analysis was carried out at m/z 313.0 → 108.9 and m/z 472.6 → 436.2 for LNG and IS respectively. This method demonstrated the linearity of LNG over a concentration range of 0.5-50 ng/mL with a coefficient correlation (r) of 0.9973. The lower limit of quantification was 0.5 ng/mL. The intra- and inter-assay precisions were 2.9 and 11.2%, with an accuracy range from 94.8 to 108.4%. The stability data indicated that sample preparation and storage process had little effect on the concentration of LNG QC sample. The validated method was successfully applied to study the pharmacokinetics of LNG in beagle dog after vaginal administration of intravaginal ring.