Enantiomeric Separation Of ?-blocker Enantioners By Supercritical Fluid Chromatography With Liquid-phase Microextraction

Chiral?-blockers have been widely used in cardiovascular disease,and have been playing a leading role in cardiovascular field.There exist huge differences between two enantiomers such as activity,toxicity and biodegradability.Therefore,it is significant to study the enantioseparation of?-blocker and its derivatives for the safety and rationality.Six chiral?-blockers?atenolol,carvedilol,metoprolol tartrate,acebutolol,indenolol and celiprolol?were separated by supercritical fluid chromatography?SFC?with CO₂ as main mobile phase.The optimal separation conditions were studied by changing the chiral stationary phase?CSP;Chiralcel OD-H?Chiralpak AS-H?Chiralpak AD-3?Chiralpak IB?Lux 3u Cellulose-1 and Sino-Chiral OJ?,the volume fraction of the additive diethylamine?0⁰.3%?,different type and percentage of modifiers?methanol,ethanol and isopropanol?,column temperature?29⁴1??and back pressure?13¹7 MPa?.Meanwhile,hollow fiber liquid phase microextraction?HF-LPME?was used as a pretreatment method for sample tablets,and SFC was used as a subsequent separation/detection method to determine the enantiomeric content of metoprolol tartrate tablets.Variables affecting the extraction efficiency of 3p-HF-LPME were studied and optimized,and methodological validation was carried out.Six chiral?-blockers were baseline separated on experimental contidions,considering the speed of elution and the degree of separation,Chiralcel OD-H column is suitable for atenolol,metoprolol tartrate,indenolol and celiprolol,while the Chiralpak AD-3 has better separation capacity for other two compounds.Adding DEA can effectively solve the problem of peak tailing.Considering the influence on separation parameters and baseline stability,0.1%DEA is selected.And the type and percentage of modifiers played a key role in enantioseparation.Adding the same type of modifier,the retention factor and resolution decreased gradually with increasing percentage of a given modifier.When using the different modifier of the same concentration,retention factor of?-blockers decreased in order of 2-propanol,ethanol,methanol.The column temperature has an important influence on the separation process and parameters of chiral compounds by SFC.Based on van't hoff equation,under the temperature range assayed,the chiral separation process of six chiral?-blockers were all controlled by enthalpy,the separation factor decreased when increasing the column temperature with limits.Changing in system back pressure can cause a change in the density of the mobile phase as a whole,thereby affecting the enantiomeric separation.As the back pressure was increased,a corresponding decreased in retention factor and resolution was observed.The separation factor changed within limits.Considering the high detection limit of SFC and the low concentration of analyte,enrichment pretreatment of analytes by hollow fiber liquid phase microextraction is necessary.The conditions of 3p-HF-LPME were selected and optimized,the conditions were as follows:n-octanol as organic phase,sodium chloride was not added into the sample solution,the pH value of the sample solution was adjusted to 10,the pH value of the acceptance phase was 2,and the extraction time was 60 minutes at the stirring rate of 40?and 800 rpm.The enrichment ratio of metoprolol tartrate was119 times,the recovery rate was between 90.3%and 101.3%,and the intra-and inter-day RSD was between 4.5%and 9.7%.The detection limit of the method was0.35?g·mL^-1.Finally,the concentration of the actual metoprolol tartrate tablet was determined by liquid phase microextraction-supercritical fluid chromatography,and the measured result was 97.9%of the theoretical value.This method is a powerful tool for quantitative analysis of chiral separation of?-blockers.It has many advantages,such as simplicity,environmental protection,no need to use a large number of organic reagents and good determination results.