**Objective:** The aim of this study was to develop and validate a sensitive and specific liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantification of medroxyprogesterone acetate in human plasma.
**Methods:** Plasma samples were spiked with an internal standard, megestrol acetate, and extracted using n-hexane. The organic layer was evaporated under a nitrogen stream at 50°C and reconstituted in the mobile phase prior to LC-MS/MS analysis. Chromatographic separation was performed on an Alltech Alltima-C18 column (2.1 mm × 100 mm, 3 μm). The mobile phase consisted of methanol and 0.1% formic acid (72:28, v/v), delivered at a flow rate of 0.2 mL/min. The column temperature was maintained at 40°C. Electrospray ionization in positive mode was used, with selective reaction monitoring (SRM) for quantification. The precursor-to-product ion transitions were m/z 327.4 → 311.3 for medroxyprogesterone acetate and m/z 325.4 → 309.3 for the internal standard.
**Results:** The method showed a linear calibration range from 0.1 to 8.0 μg/L, with a lower limit of quantification (LLOQ) of 0.1 μg/L. The intra-day and inter-day precision (RSD) values were below 9.0%, indicating good reproducibility. The extraction recovery was 76.1%, demonstrating the method’s efficiency.
**Conclusion:** This developed LC-MS/MS method is highly specific, sensitive, and simple to perform, making it well-suited for clinical applications such as therapeutic drug monitoring or pharmacokinetic studies. The method provides reliable results and can be applied in routine laboratory settings.
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