Toxicokinetics Of Dexrabeprazole Sodium For Injection And Its Metabolites In Beagles

Objective:1.To establish a high performance liquid chromatography-tandem mass spectrometry(HPLC-MS/MS)method for the simultaneously determination of(R)-and(S)-rabeprazole,(R)-and(S)-desmethylrabeprazole and Rabeprazole sulfone in beagle dogs plasma.2.To establish a high performance liquid chromatography-mass spectrometry(HPLC-MS)method for the determination of Rabeprazole thioether in beagle dogs plasma.3.To study the toxicokinetics of dexrabeprazole and its metabolites for injection in beagles,and then expoud the relationship between exposure and dose under venenous test condition.Methods:1.The(R)-and(S)-rabeprazole,(R)-and(S)-desmethylrabeprazole and rabeprazole sulfone were all determined by HPLC-MS/MS.Plasma samples were extracted with ethyl acetate and separated on CHIRAL-AGP chiral column(4.0×150 mm,5 μm)by the mobile phase of 10 mmol ammonium acetate-acetonitrile at a flow rate of 0.5 mL·min-1 with a gradient elution program.The column temperature was maintained at 25 ℃.Mass spectrometer was operated using ESI with positive ion mode at following parameters:capillary voltage 4000 V;drying gas flow 11 L·min-1;nebulizer gas 45 psi;source temperature 350 ℃.The following ion transitions(m/z):360.3→242.1 for(R)-and(S)-rabeprazole,346.2→228.2 for(R)-and(S)-desmethylrabeprazole,376.2→240.1 for Rabeprazole sulfone and 368.2→190.2 for omeprazole were selected for quantification in MRM mode.The fragment electric voltage for them were 110 V,135 V,100 V and 70 V,and energy of collision gas were 10 eV,22 eV,7 eV and 10 eV,respectively.2.The rabeprazole thioether were determined by HPLC-MS.Plasma samples were processed by protein precipitation with acetonitrile and separated on Inertsil(?)ODS-3 column(4.6×150 mm,5 μm)by the mobile phase of 5 mmol ammonium acetate-acetonitrile(65:35,V/V)at a flow rate of 0.7 mL·min-1.The column temperature was maintained at 25 ℃.Mass spectrometer was operated using ESI with positive ion mode at following parameters:capillary voltage 4000 V;drying gas flow 11 L·min-1;source temperature 350 ℃;nebulizer gas 50 psi.The ions for rabeprazole thioether and diazepam(internal standard)were m/z 344.2 and m/z 285.1,which were selected as the target ions for quantification in the selected ion monitoring(SIM)mode,and fragment electric voltage for them were 50 V and 90 V,respectively.3.40 beagles dogs were randomly divided into 4 groups,and received a single dose of 2.5 mg·kg-1(low)、10 mg·kg-1(middle)、40 mg·kg-1(high)dexrabeprazole and 40 mg·kg-1 rabeprazole(control),respectively.Plasma samples were collected on the first day and 30 days and stored at-80 ℃ for pending analysis.The(R)-and(S)-rabeprazole,(R)-and(S)-desmethylrabeprazole,rabeprazole sulfone and rabeprazole thioether levels in plasma were determined by established HPLC-MS/MS and HPLC-MS method,and the following pharmacokinetic parameters were calculated:Cmax,Tmax,t1/2,AUC0-t(from 0 to last measurable time)and AUC0-∞(from 0 to infinity),using a software DAS 2.1.1.Then the differences between single and multiple dose were observed by SPASS 17.0 software,and the relationship between exposure and dose were also analyzed.Results:1.The matrix effects and recovery of(R)-and(S)-rabeprazole,(R)-and(S)-desmethylrabeprazole,rabeprazole sulfone and omeprazole(I.S.)met the determination requirements,and matrix in plasma had no interference on the determination.Linearity of(R)-and(S)-rabeprazole were achieved over a concentration range from 10 to 2500 ng·mL-1;(R)-and(S)-desmethylrabeprazole were achieved over a concentration range from 5 to 1000ng·mL-1;rabeprazole sulfone was achieved over a concentration range from 10 to 1000ng·mL-1.Lower limit of quantification(LLOQ)for(S)-and(R)-rabeprazole and rabeprazole sulfone were 5 ng·mL-1,(S)-and(R)-desmethylrabeprazole were 10 ng·mL-1.Both intra-and inter-assay precisions were less than 15%.(R)-and(S)-rabeprazole,(R)-and(S)-desmethylrabeprazole,rabeprazole sulfone in plasma were stable after storage at room temperature for 15 h after extraction,two freeze-thaw cycles and storage at-80 ℃ for 60 days.2.The matrix effects and recovery of rabeprazole thioether and diazepam(I.S.)were meeting the determination requirements,matrix in plasma had no interference on the determination.Linearity of rabeprazole thieother was achieved over a concentration range from 2 to 800 ng·mL-1.Lower limit of quantification(LLOQ)was 2 ng·mL-1.Both intra and inter assay precision were less than 15%.Rabeprazole thioether in plasma was stable after storage at room temperature for 12 h after extraction,two freeze-thaw cycles and storage at-80 ℃ for 65 days.3.After single dose and multiple dose,the exposure of(R)-rabeprazole,(R)-desmethylrabeprazole,rabeprazole sulfone and rabeprazole thioether increaced with increacing dose.Conclusions:1.Simple,sensitive and accurate HPLC-MS/MS and HPLC-MS method were established in the study,which were applied for the determination of(R)-and(S)-rabeprazole,(R)-and(S)-desmethylrabeprazole,rabeprazole sulfone and rabeprazole thioether in beagle plasma.2.Of(R)-rabeprazole,(R)-desmethylrabeprazole,rabeprazole sulfone and rabeprazole thioether after injection(R)-rabeprazole sodium,the Cmax and AUC act as positive correlation with dose,but no linear correlation.Compared to single dose,the exposure of rabeprazole thioether significantly incresed,and clearance reduced.