Development and Validation of an LC–MS/MS Method for the Quantification of HY‐078020 in Human Plasma Using Liquid–Liquid Extraction and Its Application to Human Pharmacokinetic Study

A sensitive and practical liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed and validated for the quantification of HY‐078020 in human plasma. Plasma samples (100 μL) were processed by liquid–liquid extraction with methyl tert ‐butyl ether using HY‐078020‐d6 as the internal standard. Chromatographic separation was achieved on a Phenomenex Luna C8(2) column with gradient elution, and detection was performed in positive electrospray ionization mode using multiple reaction monitoring. The monitored transitions were m/z 515.2 → 282.3 for HY‐078020 and m/z 521.1 → 282.3 for the internal standard. The assay was validated over the concentration range of 4.0–2400 ng/mL with a lower limit of quantification of 4.0 ng/mL. The method showed acceptable selectivity, accuracy, precision, matrix effect, extraction recovery, carryover, and stability in accordance with current bioanalytical validation criteria. Intra‐ and interbatch precision was within 9.03% and 5.46%, respectively, and the analyte remained stable under benchtop, freeze–thaw, processed‐sample, reinjection, and long‐term storage conditions. With its small plasma volume requirement, broad calibration range, low carryover, and robust stability, this method is suitable for routine quantification of HY‐078020 in human plasma and may support future pharmacokinetic studies.