High-performance liquid chromatography–tandem mass spectrometry for the determination of pidotimod in human plasma and its application to a pharmacokinetic study

A selective, rapid and sensitive high-performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) method was developed for the first time to determine pidotimod in human plasma and applied to a pharmacokinetic study. Diphenhydramine was used as the internal standard (I.S.). Sample pretreatment involved in one-step protein precipitation (PPT) with methanol of 0.1 mL plasma. The analysis was carried out on an Ultimate™ XB-C8 column with mobile phase of methanol–water containing 0.5% formic acid (65:35, v/v). The detection was performed on a triple quadrupole tandem mass spectrometer by multiple reaction monitoring (MRM) mode via electrospray ionization (ESI) source. Each plasma sample was chromatographed within 4.5 min. The linear calibration curves were obtained in the concentration range of 0.05–20.00 μg/mL (r2 ≥ 0.99) with the lower limit of quantification (LLOQ) of 0.0500 μg/mL. The intra- and inter-day precision (relative standard deviation, R.S.D.) values were below 15% and accuracy (relative error, R.E.) was from −5.1% to 3.9% at all quality control (QC) levels. The method was applicable to clinical pharmacokinetic study of pidotimod in healthy volunteers after oral administration.