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.