Determination of helicidum and its metabolites in dog plasma by LC/UV/MS/MS and its application to pharmacokinetic studies

A simple, rapid and reliable method was developed for the identification and quantification of helicidum and its metabolites in beagle dog plasma by liquid chromatography/ultra-violet/electrospray ionization-ion trap mass spectrometry (LC/UV/ESI-ITMS). Two metabolites were identified by MS: formylphenyl-O-β-d-pyranosyl alloside (I) and hydroxylmethylphenyl-O-β-d-pyranosyl alloside (II). UV was used for concentration determination with the wavelength of 270 nm. Liquid–liquid extraction was used and the extraction recovery exceeded 90%. Kromacil C18 column (5 μm, 4.6 mm i.d. × 250 mm) was used as the analytical column. Linear detection responses were obtained for helicidum concentration ranging from 1.76 × 10−4 to 70.4 × 10−4 μmol/mL (0.050–2.00 μg/mL). The precision and accuracy data, based on intra- and inter-day variations over 3 days, were less than 5%. The limit of determination and quantitation (LOD, LOQ) for helicidum was 0.010 and 0.030 μg/mL, respectively. Pharmacokinetic data of helicidum and the two metabolites were obtained with this method after administration of intravenous injection and a single oral dose of tablets to six beagle dogs, respectively.