Determination of levonorgestrel in human plasma by liquid chromatography–tandem mass spectrometry method: application to a bioequivalence study of two formulations in healthy volunteers

A rapid and sensitive liquid chromatography–tandem mass spectrometry (LC/MS/MS) method to determine levonorgestrel in human plasma was developed and fully validated. After hexane–ethyl acetate (70:30, v/v) induced extraction from the plasma samples, levonorgestrel was subjected to LC/MS/MS analysis using electro‐spray ionization. The MS system was operated in the selected reaction monitoring mode. Chromatographic separation was performed on a Hypersil BDS C 18 column (i.d. 2.1 × 50 mm, particle size 3 µm). The method had a chromatographic running time of 2.0 min and linear calibration curves over the concentration ranges of 0.25–90 ng/mL for levonorgestrel. The lower limit of quantification of the method was 0.25 ng/mL for levonorgestrel. The intra‐ and inter‐batch precision was 3.7–10.2 and 5.1–12.9%, respectively, for all quality control samples at concentrations of 0.5, 6.0 and 45.0 ng/mL. These results indicate that the method was efficient with a simple preparation procedure and a very short running time (2.0 min) for levonorgestrel compared with those methods reported in the literature and had high selectivity, acceptable accuracy, precision and sensitivity. The validated LC/MS/MS method was successfully used for a bioequivalence study of two tablet formulations of levonorgestrel in healthy volunteers. Copyright © 2008 John Wiley & Sons, Ltd.