Studies On Enantiomeric Separation Of ?-adrenergic Blocking Agents And Pharmacokinetics Of Arotinolol

?-adrenergic blocking agents were wildly used in the treatment of essential hypertension,the studies of enantiomeric separation,in vivo stereoselective pharmacokinetics and drug-drug interaction provides evidence for quality control,drug safety and clinical reaseach of this class of agents.In this paper,enantiomeric separation condition and enantioseparation mechanism using amylase derivative chiral stationary phase of Propanolol,Metoprolol,Arotinolol and Carvedilol was studied;on this basis,an LC-MS/MS analysis method combination with chiral stationary phase of arotinolol enantiormers in rat plasma was developed,and applied to a steroselective pharmacokinetic study in Wistar rats;a LC-MS/MS method for simultaneous determination of arotinolol and amlodipine was developed,and applied to pharmacokinetic studies of arotinolol and amlodipine.The details were presented as follows:1.Studies on the chiral separation using amylase derivative chiral stationary phase for four?-adrenergic blocking agentsThe enantiomeric separation of Propanolol,Metoprolol,Arotinolol and Carvedilol was achieved on amylase derivative chiral stationary phase under normal phase mode.The influences of different CSP,mobile phase modifier and its proportion,the concentration of mobile phase additive,column temperature and flow rate were evaluated,and then the separation condition was optimized.A baseline resolution of four?-adrenergic blocking agents was carried out on a chiral column using the mobile phase of n-hexane and ethanol in 0.03%diethylamine?20:80,v/v?at a flow rate of 0.550 mL/min and a colume temperature of 40°C.The resolution of Propanolol,Metoprolol,Arotinolol and Carvedilol was 1.37,1.80,2.09 and 4.70,respective.On the basis,the separation mechanism was discussed,and was used for the theoretical basis of pharmacology,pharmacokinetics and toxicology of?-adrenergic blocking agents.2.Studies on stereoselective pharmacokinetics of arotinolol in rat plasmaAn LC-MS/MS method combination with chiral stationary phase was developed for quantification of arotinolol enantiomers,and applied to determine the concentrations of arotinolol enantiomers in Wistar rat after oral administration of 10 mg/kg of arotinolol.After solid phase extraction of 50?L rat plasma,a baseline resolution of arotinolol enantiomers was achieved on a CHIRALPAK AD-H chiral column using the mobile phase of n-hexane and ethanol in 0.02%diethylamine?20:80,v/v?at a flow rate of 0.550 mL/min within 11.0 min.Haloperidol was selected as the internal standard.Acquisition of mass spectrometric data was performed on a triple-quadrupole massspectrometer in positive mode,multiple-reaction-monitoring using the transition m/z 372.1?316.1 for?±?-arotinolol and m/z 376.1?165.1 for haloperidol.The linear range for arotinolol enantiomers was 1.00ng/mL²00ng/mL,and linear relationship was good.The intra-and inter-day accuracy values were within±15%,precision values were below 15%.The extract recovery was between 87.2%and 96.2%.IS-normalized matrix effect was between0.88 and 1.17.The method was applied to determine the concentrations of arotinolol enantiomers in rat plasma,and then the main pharmacokinetics parameters were calculated.The C_max,T_max and AUC_0-?values were 121±64.2ng/mL,1.3±0.5hr and 474.4±100.2h*ng/mL for R-?-?-arotinolol;118±60.4ng/mL,1.2±0.4hr and 471.7±89.5h*ng/mL for S-?+?-arotinolol.The results showed that no significant difference of pharmacokinetic parameters of aortinolol enantiomers was observed,and there existed no stereoslectivity in pharmacokineitcs in Wistar rat.3.Studies on pharmacokinetics of a combination of arotinolol and amlodipineAn LC-MS/MS method for simultaneous determination of arotinolol and amlodipine was developed.The plasma was precipitated,and no endogenous or other substance was observed to disturb the determination of artinolol and amiodipine.Reverse phase separation was accomplished on Venusil XBP C18 column,and the run time was shortened to 5.0min under the gradient elute.The LLOQ of arotinolol and amlodipine was0.200ng/mL and 0.500ng/mL,the linear range was 0.200⁴0.0ng/mL and 0.500¹00ng/mL,the overall extraction recovery was 93.0%and 90.5%,and the overall IS normalized matrix effect was 0.96 and 0.97.The results satisfy the requirement of bioanalysis.This method was successfully applied to a combination of arotinolol and amlodipine pharmacokinetics study.The Wistar rats were divided into 3 groups randomly?six rats each group?,and were given a single oral dose of arotinolol,amlodipine and a combination of arotinolol and amlodipine,respectively.The interaction of drug combination was evaluated by pharmacokinetic parameters.The C_max and AUC_0-?values of single drug group and combined drugs group for arotinolol were 38.3±20.8ng/mL,105.8±44.8h*ng/mL and20.0±3.96ng/mL,86.1±16.9h*ng/mL;the C_max and AUC_0-?values of single drug group and combined drugs group for amlodipine were 41.3±16.5ng/mL,366.3±120.8h*ng/mL and 36.8±10.4ng/mL,325.5±95.5h*ng/mL.The results herein showed no significant difference appears in the pharmacokinetic parameters of Amlodipine between two groups;the C_maxvalue of combined drugs group for arotinolol was decreased,and it might be caused by individual difference.Further confirmatory analysis could be conducted by adding sample size to reduce individual differences.