The Study Of Pharmacokinetics Of Phellodendrine And Its Glucuronides In Rats

Objective1.To develop an accurate and reliable UHPLC-LTQ-Orbitrap XL qualitative method（LC-MSⁿ）for the analysis of phellodendrine glucuronides in rats urine.2.To prepare theⅡphase metabolites of phellodendrine glucuronides from the urine of rats after administration and identify their structure.3.To develop an accurate,reliable and sensitive liquid chromatography-tandem mass spectrometric（LC-MS/MS）method for the simultaneous determination of phellodendrine and its glucuronides in rats plasma samples.4.To study the pharmacokinetics of phellodendrine and its glucuronides in normal rats after single intragastric administration of phellodendrine in low,medium and high dose;in diabetic rats after single intragastric administration of phellodendrine in medium dose;in normal rats after single intragastric administration of Huangbai decoction freeze-dried powder and Zhimu-Huangbai decoction freeze-dried powder by means of the established LC-MS/MS method.Methods1.UHPLC-LTQ-Orbitrap XL qualitative method for the analysis of phellodendrine glucuronides in rats urine:The chromatographic separation was carried out on a Hypersil C18 column（4.6×250 mm,10μm）by isocratic elution with ultrapure water containing 0.1%formic acid-acetonitrile（65:35,V/V）at the flow rate of 400（?）L·min^-1.The column temperature was set at 30℃and the injection volumn was 10（?）L under the measure wavelength of 284 nm.The analytes was performed by mass spectrometry with Turbo Ionspray ionization（ESI）under CID mode.The first-class mass spectrometry was scanned by Fourier Transform Mass Spectrometry（FTMS）scanning mode,a resolution of 30000.The second-class and third-class mass spectrometry was scanned by data dependency scanning mode with resolution of 15000.The other operation parameters of mass spectrum were described as follows:spray voltage,4 k V;sheath gas,45arb;atomization temperature,350℃;auxiliary gas,10 arb;collision energy,35 e V.2.Gathering the urine of normal rats after oral administration of the effective part of phellodendrine.The glucuronic acid conjugates of phellodendrine were extracted and separated by means of AB-8 macroporous resin,ODS,Sephadex LH-20 chromatographic column and preparative liquid chromatograph.The structural formulas of the glucuronic acid conjugates of phellodendrine were appraised by means of UV,MS,¹H-NMR,¹³C-NMR,HSQC,HMBC.3.LC-MS/MS method for simultaneous determination of phellodendrine and its glucuronides:Aconitine was used as the internal standard（IS）.The plasma sample was extracted by protein precipitation with methanol-formic acid（9:1）.The chromatographic separation was carried out on a Agilent ZORBAX Eclipse XDB-C18 column（4.6×250 mm,10μm）.The mobile phase consisted of phase A（water containing 0.1%formic acid）and B（methanol）.The gradient program started as follows:0～1 min,5%B;1～6 min,5%～95%B;6～8 min,95%B;8～8.5 min,95%～5%B;8.5～10 min,5%B.The analysis time was 10 min and flow rate was 300μL·min^-1.The column temperature was set at 35℃and the sample injector temperature was set at 4℃.The injection volumn was 5（?）L.The quantification of the analytes was performed by mass spectrometry with Turbo Ionspray ionization（ESI）under positive ion multiple reaction monitoring（MRM）mode.The other operation parameters of mass spectrum were described as follows:Temperature,600℃;Ion Spray（IS）,5000 V;Collision gas,6 psi;Curtain gas（CUR）,25 psi;Ion source gas 1（GS1）,60 psi;Ion source gas2（GS2）,50 psi.4.Rats were randomly divided into 6 groups:single intragastric administration of monomer phellodendrine in the dose of 20,40,80 mg·kg^-1;type 2 diabetic rats single intragastric administration of monomer phellodendrine in dose of 40 mg·kg^-1;Huangbai decoction freeze-dried powder;Zhimu-Huangbai decoction freeze-dried powder.The blood was collected at different times from the suborbital venous plexus.The plasma samples were determined by the established LC-MS/MS method to calculate the concentration of phellodendrine and its glucuronides and draw the drug concentration-time curve.DAS 2.1.1 software was applied to calculate the pharmacokinetic parameters while SPSS 19.0 software was used for statistical analysis.Results1.The metabolite of m/z～694 was authenticated as double-glucuronides of phellodendrine while m/z～518 was monoglucuronide by developing qualitative method and the comparison of chromatogram and mass spectrometry information of blank urine with dosing phellodendrine.An accurate,reliable and rapid LC-MSⁿ method was established for the analysis of phellodendrine glucuronides.2.Three glucuronides of phellodendrine were prepared by means of AB-8 macroporous resin,ODS,Sephadex LH-20 and preparative liquid chromatograph.They were phellodendrine-2,11-di-O-β-D-glucuronide（purity>99%）,phellodendrine-2-O-glucuronide,phellodendrine-11-O-β-D-glucuronide（purity>98%）.3.The method for the determination of phellodendrine and its glucuronides in rat plasma:The linear ranges of phellodendrine,phellodendrine-11-O-β-D-glucuronide,and phellodamine-2,11-di-O-β-D-glucuronide were 1～100,1～1200,10～1000 ng·m L^-1,respectively.The lower limit of quantitation were 1,1,10 ng·m L^-1;the recovery was（71.93±3.87）%～（91.50±4.76）%;the matrix effect was（90.39±1.62）%～（127.69±4.84）%;accuracy at（96.08±1.88）%～（110.20±4.97）%;intraday and inter-day precision RSD≤10.53%;The plasma samples during 4 h in room temperature,24 h at 4℃,30 d at-30℃,freeze-thaw 3 times and other conditions were stable.4.Pharmacokinetics study of phellodendrine and its glucuronids:After intragastric administration,all three components were detected in plasma,and the concentration of phellodendrine-11-O-β-D-glucuronide in plasma was highest,phellodendrine-2,11-di-O-β-D-glucuronide followed,the phellodendrine minimum.After administration of 20,40 and 80 mg·kg^-1doses of phellodendrine,the C_max and AUC of the three components increased with increasing dose and showed a positive correlation.The AUC increased proportionally with the dose,while the increase in C_max was not proportional to the dose of single glucuronide of phellodendrine.The pharmacokinetics of phellodendrine and its glucuronides in diabetic rats and normal rats changed,but there was no statistical difference.The change trends of plasma concentrations of the three components under different compatibility conditions were consistent,except that the C_max of the diglucuronides in the Huangbai group was significantly higher than that of the Zhinu-Huangbai group,and there was no statistical difference between the other parameters.ConclusionThis article systematically studied the phellodendrine glucuronides for the first time.The established UHPLC-LTQ-Orbitrap XL（LC-MSⁿ）method was sensitive,accurate and rapid and could be used for the qualitative analysis of phellodendrine glucuronides.The technical route of preparing metabolites from urine was mature,reliable and rapid.Three new phellodendrine glucuronides were isolated and prepared from urine to provide a material basis for the quantitative study of phellodendrine and its glucuronides in rats.The established LC-MS/MS method was accurate,rapid and sensitive and could be used for the determination of plasma concentrations of phellodendrine and its glucuronides in rats.Phellodendrine was absorbed rapidly in rats and rapidly metabolized into glucuronides.The plasma concentration of phellodendrine-11-O-β-D-glucuronide was the highest,the phellodendrine-2,11-di-O-β-D-glucuronide was the second,and the phellodendrine was the least.The linear kinetic of phellodendrine diglucuronides within the dose range of20～80 mg·kg^-1 of phellodendrine,and the pharmacokinetics of phellodendrine and glucuronide conjugates at different doses varied.The pharmacokinetic characteristics of phellodendrine and its glucuronides in pathological and normal conditions of typeⅡdiabetes have changed.There was no significant change in the pharmacokinetics of phellodendrine and its glucuronides after administration of Huangbai and Zhimu-Huangbai.