Enantioseparation Of Five Proton Pump Inhibitors And Pharmacokinetic Study In Rats

Bioactivity of chiral drugs is closely related to the stereostructures. Nowadays development and production of optically pure drugs is the trend of new drugs research. The study on chiral drugs has become one of the important directions of new drugs research and development internationally. Analysis of chiral drugs, with increasing in importance in pharmaceutical research, is now a hot and difficult topic in analysis science. In this thesis, capillary electrophoresis (CE) and high performance liquid chromatography (HPLC) methods were developed for the chiral separation of proton pump inhibitors (PPIs) including tenatoprazole, pantoprazole, lansoprazole, rabeprazole and omeprazole. The optical determination method for (-)-pantoprazole and (-)-tenatoprazole and the seteroselective pharmacokinetics of tenatoprazole enantiomers in rats provided a reference for the development of optically pure PPIs.1. Chiral separation of four PPIs with capillary zone electrophoresisThe chiral separation of tenatoprazole, pantoprazole, lansoprazole and rabeprazole was studied with capillary zone electrophoresis (CZE) using negatively charged sulfobutyl ether-β-CD (SBE-β-CD) as chiral selector. The influence of type and concentration of CDs, running buffer pH, the type and concentration of organic modifier on the chiral separation was investigated. The separation mechanism was discussed by comparing the chiral separation results and the determination of the complexation binding constant. The chiral recognition mechanism was proposed on the difference of the complexation binding constant between two enantiomers. Under the condition of 50 mmol/L borox-150 mmol/L phosphate buffer (pH6.5) containing 40 mg/mL SBE-P-CD, applied voltage of 10 kV, the enantiomers of all PPIs were baseline separated. The optimized method for determination of tenatoprazole enantiomers was validated. The limits of detection (LOD) and quantification (LOQ) were 0.2 and 0.65μg/mL for both the enatiomers, respectively. The linearity of the method was in the range of 0.65～100μg/mL with r2≥0.9994. The inter-and tra-day assay precision was less than 3.0%(RSD) and recoveries were in the range 91.6～100%. The method was applied to the enantiomeric excess (ee) determination of (-)-tenatoprazole bulk samples.2. Chiral separation of pantoprazole enantiomers by HPLC using SBE-β-CD as chiral mobile phase additiveThe chiral separation of pantoprazole enantiomers was studied by HPLC using SBE-β-CD as chiral mobile phase additive (CMPA). The factors affecting the resolution such as concentration of SBE-β-CD, buffer pH, type and concentration of buffer solution, type and concentration of organic modifier were investigated. A baseline resolution of 2.1 was achieved within 15 min on a Spherigel C18 column (150mm×4.6mm,5μm) using acetonitrile and 10 mmol/L phosphate buffer (pH2.5) containing 20 mg/mL SBE-P-CD (15:85, v/v) as mobile phase with a flow rate of 0.9 mL/min at room temprature. The detection wavelength was 290 nm. The optimized method was extensively validated in terms of accuracy, precision and linearity according to the ICH guidelines and proved to be robust. The LOD and LOQ for (+)-pantoprazole were 0.2 and 0.5μg/mL, respectively. The linearity of the method was in the range of 0.5～6.0μg/mL with r2=0.9991 for (+)-pantoprazole. The precision of the method was less than 3.6%(RSD) and recoveries were in the range 92.1～101% for (+)-pantoprazole. The method was applied to the enantiomeric impurity determination of (-)-pantoprazole bulk samples.3. Chiral separation of five PPIs with chiral ligand-exchange chromatographyThe chiral separation of tenatoprazole, pantoprazole, lansoprazole, rabeprazole and omeprazole was studied with chiral ligand-exchange chromatography (CLEC). The type of chiral ligand, type and concentration of organic modifier, the concentration ratio of chiral ligand to metal ion and pH were studied for the optimization of separation condition. Under the optimized conditions,1 mmol/L L-His,0.5 mmol/L copper acetate in water (pH7.0)-acetonitrile (75:25, v/v), at flow rate of 1.0 mL/min, the enantiomers of omeprazole, pantoprazole, rabeprazole and lansoprzole were baseline separated, and the enantiomers of tenatoprazole could be resovled with the content of acetonitrile decreased to 18%. The minimal energy configurations were obtained by simulated annealing, and based on that, quantum chemical calculation was performed to discuss the mechanism of chiral recognition. The chiral recognition mechanism was proposed to be on the difference of the stability of ternary complexes between two enantiomers.4. Chiral separation of four PPIs by HPLC on chiral stationary phasesDirect enantioseparation of PPIs including tenatoprazole, pantoprazole, lansoprazole and omeprazole was studied using Amysole (Chiralpak AS-H) and Vancomycin (Chirobiotic V) as chiral stationary phases (CSPs). The effects of the concentration of mobile phase additive, composition of mobile phase and column temperature were investigated with Chiralpak AS-H as CSP. The enantiomers of all PPIs were resolved (Rs>3.3) within 15 min using n-hexane-ethanol-TEA (80:20:0.1%, v/v/v) as mobile phase with a flow rate of 0.4 mL/min at 40℃. The thermodynamic parameters of four PPIs in this mode were investigated, the enantioselectivety was proved to be enthalpocally controlled. The chiral recognition mechanism was discussed, inclusion interaction between the CSP and the analytes was confirmed to be the dramatic interaction responsible for the chiral discrimination. The separation mode, mobile phase and column temperature were also investigated when Chirobiotic V was adopted. Under the optimized conditions,20 mmol/mL ammonium acetate-tetrahydrofuran (93:7, v/v), at a flow rate of 0.5 mL/min and 20℃, the enantiomers of tenatoprazole got baseline separation, and the enantiomers of pantoprazole, lansoprazole and omeprazole got partial separation. The thermodynamic parameters of tenatoprazole in this mode was investigated, the enantioselectively was proved to be enthalpocally controlled. The results of enantiosparation on the two CSPs were compared with each other with Chiralpak AS-H CSP giving better results.5. Determination of tenatoprazole enantiomers and their stereoselective pharmacokinetics in ratsAn achiral-chiral sequential HPLC method was developed for the determination of tenatoprazole enantiomers in rat plasma. The method was used for the stereoselective pharmacokinetic study of tenatoprazole in rats after intra gastric administration of tenatoprazole racemate. The AUC0-∞value of (+)-tenatoprazole was significantly greater than that of (-)-tenatoprazole (p<0.001). There are also significant differences in t1/2 and CL/F (p<0.01 and p<0.001, respectively) values between enantiomers. The mean Cmax and AUC0-∞values for (+)-tenatoprazole were 2.5 and 7.6 times of those of (-)-tenatoprazole, which indicated that the pharmacokinetics of tenatoprazole in rats was stereoselective.