High-performance liquid chromatography–atmospheric pressure chemical ionization/tandem mass spectrometry for the determination of a thiol compound in plasma Original Research Article

A sensitive, reliable high-performance liquid chromatography (HPLC) coupled with tandem mass spectrometry (MS/MS) method was developed for the determination of a sulfhydryl-containing compound in mouse, rat, dog, monkey and human plasma samples in support of pharmacokinetic (PK) studies. The reversed-phase chromatographic system was interfaced with an atmospheric pressure chemical ionization (APCI) source. Methyl acrylate (MA) was employed to react with the sulfhydryl group of the analyte to form a derivative that was stable in dog, monkey and human plasma at room temperature. Phenylmethylsulfonyl fluoride (PMSF), a potent inhibitor of hydrolase, was used to stabilize the test thiol-MA compound in plasma samples. Sample extracts following protein precipitation were injected into HPLC–MS/MS system. The derivatized compound was detected by a triple quadrupole mass spectrometric detector in the positive ion multiple-reaction monitoring mode. The calibration curve was linear from 2.5 to 5000 ng/ml. This paper reports the development of a HPLC–APCI/MS/MS method for the quantitative determination of a thiol component in dog plasma after derivatization with methyl acrylate derivative. The procedure was used to quantitate plasma levels following oral administration of the thiol compound to beagle dogs.