An HPLC-MS/MS method for the quantitative determination of 4-hydroxy-anethole trithione in human plasma and its application to a pharmacokinetic study

A selective, rapid and sensitive method for the quantitation of 4-hydroxy-anethole trithione (ATX) in human plasma based on high-performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) was developed and validated. Paracetamol was used as the internal standard (I.S.). After liquid–liquid extraction of 500 μL plasma with ethyl acetate, ATX and the I.S. were chromatographed on an Inertsil^® ODS-3 column. The mobile phase was consisted of methanol–water (75:25, v/v) with a flow rate of 0.25 mL/min. The detection was performed on a triple quadrupole tandem mass spectrometer by multiple reaction monitoring (MRM) mode via electrospray ionization (ESI) source. The calibration curve was linear over the range of 0.452–603 ng/mL (r² ≥ 0.99) with a lower limit of quantitation (LLOQ) of 0.452 ng/mL. The intra- and inter-day precision (relative standard deviation, R.S.D.) values were below 13% and the accuracy (relative error, R.E.) was from −2.7% to −7.5% at three quality control levels. The assay herein described was successfully applied to a pharmacokinetic study of anethole trithione (ATT) tablet in healthy volunteers after oral administration.