Establishment And Application Of A LC-MS/MS Analytical Method For The Determination Of Baicalein And Its Seven Metabolites

Baicalein is one of the active ingredients of Scutellaria baicalensis which is isolated from the Scutellaria baicalensis Georgi.Baicalein is a flavonoid and it has numerous pharmacological activities such as anti-inflammatory,anti-oxidant,neuroprotective,anti-allergic,antibacterial or viral infection,anticardiovascular diseases,anticancer.Baicalein is extensively metabolized in the body.And has many metabolits because of its extensive metabolism in vivo.Up to now,several reseaches have been reported for the determination and pharmacokinetic study of baicalin and baicalein,but the identification of its metabolites in human plasma and the simultaneous determination of baicalein and its metabolites has not been reported so far.The aim of this study was to identify the metabolites of bicalein in human plasma and develope a simple,selective,sensitive,and reproducible LC-MS/MS method of baicalein and its metaolites for the simultaneous determination and apply the method to a study.Method: Plasma samples before and after administration were measured with a high resolution mass spectrometer,respectively.Compare the two chromatograms and screen metabolites combined with metabolic software.According to the structure of baicalein,metabolism rule and ragmentation patterns of drugs,probable metabolites structures were speculated.In order to identify the metabolites,the probable metabolites standard was synthesized.Then the metabolites were chromatographed,the metaobolites including the isomers were identified according to the retention time.The method was based on two liquid chromatography systems.Liquid–liquid extraction was used for pretreatment of plasma for the determination of baicalein.The mobile phase consisted of acetonitrile–0.5% fomic acid chromatographic separation was performed using a SUPELCO Ascentis-C18(50 mm×4.6 mm I.D.,5 ?m particle size)chromatographic column with gradient elution.Protein precipitation method was used for pretreatment of plasma for the determination of seven metabolites.The mobile phase consisted of acetonitrile–0.1 % fomic acid and 10 mM ammonium acetate in water.the metabolites were separated on the Agilent HC-C18(150 mm×4.6 mm I.D.,5 ?m particle size).The flow rate was 1.2ml/min.Concentrations of baicalein and metabolites were measured in multiple reaction monitoring(MRM)mode via a positive electroSpray ionization source.The MRM transitions were m/z 271.1?m/z 123.1,m/z 255.0 ? m/z 153.0 for baicalein and chrys;m/z 447.1 ? m/z 271.1 for 6BG,7BG;m/z 351.0?m/z 271.0 for 6BS,7BS;m/z 623.2?m/z 271.0 for BGG;m/z 461.1?m/z 284.9 for 6-MeBG;m/z 609.1?m/z 271.1 for BGGlu;m/z 287.0?m/z 135.0 for luteolin.The calibration range in plasma samples was 1-500 ngm L for baicalein;1-300 ng/mL for 6BG;1-500 ng/m L for 7BG;10-500 ng/m L for 6BS;10-3000 ng/m L for 7BS;50-5000 ng/m L for BGG;1-200 ng/mL for 6-MeBG;1-300 ng/m L for BGGlu.LLOQ was 1 ng/mL for baicalein;1 ng/mL for 6BG;1 ng/m L for 7BG;10 ng/m L for 6BS;10 ng/m L for 7BS;50 ng/mL for BGG;1 ng/m L for 6-MeBG;1 ng/m L for BGGlu.The specificity,linearity,sensitivity,precision and accuracy,extraction recovery and matrix effect,Stability of the methode were tested for validate the method.The LC-MS/MS method was applyed to quantitation of baicalein and seven metabolites in human plasma.And we analyze the pharmacokinetic parameters included the Cmax,Tmax,t1/2,AUC0–t,AUC0-?,CLz/F,Vd/F,MRT.Result:The metabolites of baicalein in human plasma were identified in human plasma,totally 8 metabolites were identified as baicalein6,7-di-O-glucuronide(BGG,M1),baicalein6-O-glucoside-7-O-glucuronide(BGGlu,M2),baicalein7-O-glucur onide(7BG,M3),baicalein6-O-glucuronide(6BG,M4),6-O-glucoside-baicalein-7-O-sul fate(7BS,M5),7-O-glucoside-baicalein-6-O-sulfate(6BS,M6),6-methoxybaicalein7-Oglucuronide(6-MEBG,M7),7-methoxybaicalein6-O-glucuronide(7-MEBG,M8).A sufficient method to determin baicalein and its seven metabolites in human plasma was development.The method was proved to be accurate and specific,sensitive.recovery rates were achieved satisfactorily for each analyte and matrix effectwas negligible.the method was able and sufficiently for the determination and pharmacokineticanalysis of baicalein and metabolites in human plasma.A sufficient method to determin baicalein and its seven metabolites in human plasma was developed.The method was proved to be accurate and specific,sensitive.recovery rates were achieved satisfactorily for each analyte and matrix effect was negligible.The method was able and sufficiently for the determination and pharmacokineticanalysis of baicalein and metabolites in human plasma.The results of plasma analysis showed that the baicalein was widely and rapidly metabolized.The plasma concentration-time curve became a multi-peak phenomenon,and the levels of baicalein metabolites were higher.There were multiple peaks of baicalein and metabolites in the plasma drug concentration-time profile,this phenomenon was because they baicalein undergoes enterohepatic recirculation.In contrast to the concentration leves of baicalein in plasma,several metabolites show the relatively high systemic levels.And the t1/2 of baicalein and some metabolites was not consistent among all the sujects,it could be caused by the large difference among individuals and the extensive significant metabolism.