Abstract: AIM: To develop and validate an HPLC-MS/MS for the determination of pidotimod in human serum. METHODS: Glipizide was added as internal standard.The sample preparation included a simple deproteinization step with methanol.Chromatographic separation was achieved on a Lichrospher C₁₈ column(4.6 mm×150 mm,5 μm)at 30 ℃ with the mobile phase consisted of methanol-2.5 mmol/L NH₄Ac (90∶10, V/V). The mobile phase was eluted at a flow rate of 0.5 mL/min.Tandem mass spectrometer equipped with electrospray ionization source was applied and operated in the negative ion mode. Multiple reaction monitoring using the precursor to product ion combinations of m/z 242.9/153.0 and m/z 444.0/169.9 was performed to quantify pidotimod and glipizide, respectively. RESULTS: The calibration curves were linear over the range of 0.1-10 mg/L(r=0.9995). RSD of intra-batch and inter-batch were less than 15%, extraction recovery of three level concentrations were 85.52%-98.30%. CONCLUSION: The method is shown to be convenient, accurate, sensitive, and suitable for clinical pharmacokinetic study of pidotimod.

Key words: Pidotimod, Glipizide, LC-MS/MS, Pharmacokinetics, Serum, Deproteinization