The authors proposed a simple, rapid and sensitive liquid chromatography-tandem mass spectrometric (LC-MS/MS) assay method for the simultaneous determination of saxagliptin and its active metabolite 5-hydroxy saxagliptin in human plasma. The developed method was fully validated as per the US FDA guidelines. The method utilized stable labeled isotopes saxagliptin-15 N d2 (IS1) and 5-hydroxy saxagliptin-15 N-d2 (IS2) as internal standards for the quantification of saxagliptin and 5-hydroxy saxagliptin, respectively. Analytes and the internal standards were extracted from human plasma by a single step solid-phase extraction technique without drying, evaporation and reconstitution steps. The optimized mobile phase was composed of 0.1% acetic acid in 5 mM ammonium acetate and acetonitrile (30:70, v/v) and delivered at a flow rate of 0.85 mL/min. The method exhibits the linear calibration range of 0.05-100 ng/mL for both the analytes. The precision and accuracy results for both the analytes were well within the acceptance limits. The different stability experiments conducted in aqueous samples and in matrix samples are meeting the acceptance criteria. The chromatographic run time was set at 1.8 min; hence more than 400 samples can be analyzed in a single day.

A simple, rapid and sensitive analytical method using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) detection with positive ion electrospray ionization was developed for the determination of dienogest in human K2 EDTA plasma using levonorgestrel d6 as an internal standard (IS). Dienogest and IS were extracted from human plasma using simple liquid-liquid extraction. Chromatographic separation was achieved on a Zorbax XDB-Phenyl column (4.6 × 75 mm, 3.5 µm) under isocratic conditions using acetonitrile-5 mm ammonium acetate (70:30, v/v) at a flow rate of 0.60 mL/min. The protonated precursor to product ion transitions monitored for dienogest and IS were at m/z 312.30 → 135.30 and 319.00 → 251.30, respectively. The method was validated with a linearity range of 1.003-200.896 ng/mL having a total analysis time for each chromatograph of 3.0 min. The method has shown tremendous reproducibility with intra- and inter-day precision (coefficient of variation) <3.97 and 6.10%, respectively, and accuracy within ±4.0% of nominal values. The validated method was applied to a pharmacokinetic study in human plasma samples generated after administration of a single oral dose of 2.0 mg dienogest tablets to healthy female volunteers and was proved to be highly reliable for the analysis of clinical samples.

A simple, rapid and sensitive ultra flow liquid chromatography-tandem mass spectrometry (UFLC-MS/MS) assay for the determination of norgestrel in human plasma was developed using levonorgestrel D6 as an internal standard (IS). Norgestrel and IS were extracted from human plasma via liquid-liquid extraction. Chromatographic separation was achieved on a Zorbax XDB-Phenyl column under isocratic conditions. Detection was done by tandem mass spectrometry, operating in positive ion mode. The protonated precursor to product ion transitions monitored for norgestrel and IS were at m/z 313.30→245.40 and 319.00→251.30 respectively. The method was fully validated as per current regulatory guidelines. Anticoagulant counter ion effect was also assessed with K2EDTA and K3EDTA. The method was validated with a linearity range of 304.356-50 807.337 pg/mL having run time of 2.0 min per sample. The method has shown tremendous reproducibility with intra- and inter-day precision (%CV) less than 11.0% and intra- and inter-day accuracy less than 9.0% of nominal values. The validated method was applied to a pharmacokinetic study in human plasma samples generated after administrating a single oral dose of 0.3 mg norgestrel tablets to healthy female volunteers and has proved to be highly reliable for the analysis of clinical samples.

A simple, rapid and sensitive liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) assay method has been developed and validated for the determination of anagrelide in human plasma samples.