Quantitative Analysis Of Isosteviol And Metabolite Identification In Rat And Beagle Dog

Isosteviol is a chemical monomer isolated from stevioside. Research has found that it performs various physiological effects including preventing cardiovascular and cerebrovascular ischemia reperfusion injury and antidiabetic effect, with high medicinal value. But at present, there is lack of efficient analysis method for pharmacokinetic study of isosteviol. In this study, we applied ultra-high performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS) technique, established a quick and sensitive method, which was better than the existed, for quantitative analysis of isosteviol; investigated preliminary pharmacokinetics and metabolite identification of isosteviol in rat and beagle dog. The details were as follows:1) After optimization, the UPLC-MS/MS method was established like this: with medroxyprogesterone acetate worked as the internal standard, isosteviol was separated from biological samples processed by protein precipitation or liquid-liquid extraction on a UPLC C18 column by gradient elution of mobile phase consisted of water and acetonitrile both containing 0.1% formic acid(v/v). Ions were detected in the positive-ion mode and multiple reaction monitor scan was used with the precursor-product ion combination m/z 319.25→255.24 for acquiring signal of isosteviol.2) The result of method validation indicated that the method performed good selectivity and linearity between 2.000 ~ 1000 ng/m L in both rat and dog plasma samples. The lower limit of quantification is 2.000 ng/m L. The accuracy, precision, and stability are in line with requirement, with precision and reproducible recovery and matrix effect of isosteviol and internal standard suffered from plasma sample. Appling this method to investigate pharmacokinetics of isosteviol in rat and beagle dog, results showed that the isosteviol concentrations decline in a bi-exponential manner after intravenous injection, while the initial concentration C0, half life of distribution t1/2α, terminal half life t1/2z, and area under the plasma concentration-time curve AUC0~∞ were 84.59±12.75 μg/m L, 0.27±0.12 h, 10.13±5.61 h, 239.9±161.9 mg/L.h in rat after intravenous injection of 20 mg/kg isosteviol sodium(STVNa) and 14.20±8.220 μg/m L, 0.14±0.02 h, 6.92±2.65 h, 3.495±1.523 mg/L.h in dog after intravenous injection of 6 mg/kg STVNa, respectively. Besides, applying the method described above, preliminary analysis was carried out for tissue distribution research of isosteviol in rat.3) Metabolite identification of isosteviol was carried out by combining liver microsome incubate test in vitro and intravenous injection of STVNa in vivo, utilizing UPLC-MS/MS technique with MSE scan, fragment ion scan and multiple reaction monitor scan, and data analysis through MetabolynxXS software. It was found at the first time that 4 major metabolites were produced in rat, including 3 new discoveries of hydroxy group adductive metabolites from phase I metabolism and a known glucuronide conjugate from phase II metabolism, while glucuronide conjugate was the major metabolite of isosteviol found in dog.