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.