直链淀粉-三[(S)-1-苯乙基氨基甲酸酯]手性固定相拆分布地奈德对映体及其制剂含量的测定

22R-布地奈德的药物活性比22S-布地奈德的强2~3倍,开发布地奈德对映体拆分和定量分析方法,可为其药物研发及质量控制提供重要依据。目前,主要以反相C₁₈固定相对布地奈德对映体进行拆分,而采用手性固定相对其进行拆分少有报道。通过考察固定相、流动相和柱温对布地奈德对映体拆分的影响,建立了基于直链淀粉-三(S)-1-苯乙基氨基甲酸酯]手性固定相快速拆分和检测布地奈德对映体的高效液相色谱方法,其色谱条件如下:色谱柱为Chiralpak AS-RH色谱柱(150 mm×4.6 mm, 5.0 μm),流动相为乙腈-水(45∶55, v/v),柱温40 ℃,流速1.0 mL/min,二极管阵列检测器(DAD),检测波长246 nm,进样量10 μL。在该色谱条件下,布地奈德的两个对映体得到较好拆分,22R-布地奈德和22S-布地奈德的保留时间分别6.40 min和7.77 min,分离度为4.64; 22R-布地奈德和22S-布地奈德分别在各自范围内线性关系良好,相关系数(R²)均为0.9999,检出限分别为0.05 μg/mL和0.07 μg/mL,定量限分别为0.16 μg/mL和0.20 μg/mL; 4个添加水平的样品加标回收率为102.63%~104.17%,相对标准偏差(RSD)为0.08%~0.57%(n=6)。将该方法应用于1批次4个吸入用布地奈德混悬液实际样品进行检测,22R-布地奈德和22S-布地奈德的含量分别为283.15~284.63 μg/mL和259.86~261.51 μg/mL。该方法操作简便,分析时间短,重复性好,准确度高,可用于布地奈德对映体的拆分及其制剂的质量控制。

Separation of budesonide enantiomers with amylose-tris-[(S)-1-phenylethyl carbamate]chiral stationary phase and determination of its contents in pharmaceutical preparations

The drug budesonide exists as 22R and 22S enantiomers.However,the drug activity of 22R-budesonide is 2-3 times stronger than that of 22S-budesonide.The development of enantiomeric separation and quantitative analysis methods for budesonide can provide an important basis for its drug development and quality control.At present,the enantiomers of budesonide are separated on a reversed C₁₈ solid phase column.However,chiral stationary phases are rarely reported for the separation of the enantiomers of budesonide.In this study,a high performance liquid chromatography(HPLC)method with a chiral stationary phase was developed for the rapid separation and determination of budesonide enantiomers.The effects of the type of chiral stationary phase,mobile phase additives,and column temperature on the resolution of the budesonide enantiomers were also investigated.The results showed that the chiral stationary phase amylose-tris-(S)-1-phenylethyl carbamate]was more suitable for the separation of budesonide enantiomers.The mobile phase additives used in the experiment had no significant effect on the chromatographic parameters(peak height,peak width,and resolution)of the budesonide enantiomers.However,with an increase in the column temperature,the peak width of the budesonide enantiomers decreased,while the peak height and resolution increased.The optimized HPLC conditions were as follows:column,Chiralpak AS-RH(150 mm×4.6 mm,5.0μm);mobile phase,acetonitrile-water(45∶55,v/v);column temperature,40℃;flow rate,1.0 mL/min;detector,diode array detector(DAD);detection wavelength,246 nm;injection volume,10μL.The external standard method was used to quantify the budesonide enantiomers.Under the optimized conditions,the enantiomers were well separated,and the retention times of 22R-budesonide and 22S-budesonide were 6.40 min and 7.77 min,respectively.The resolution of the enantiomers was 4.64.The linear ranges of 22R-budesonide and 22S-budesonide were 0.16-1000μg/mL and 0.20-1000μg/mL,respectively.The peak area of the enantiomers showed a good linear relationship with the corresponding concentration,and the correlation coefficients(R²)were 0.9999.The limits of detection(LODs)of 22R-budesonide and 22S-budesonide were 0.05μg/mL and 0.07μg/mL,respectively,based on a signal-to-noise ratio of 3.The limits of quantification(LOQs)were calculated to be 0.16μg/mL and 0.20μg/mL,respectively,based on a signal-to-noise ratio of 10.The recoveries at four spiked levels were in the range of 102.63%to 104.17%,with the relative standard deviations(RSDs)of 0.08%to 0.57%(n=6).The budesonide solution was stored in dark at 4℃for 24 h,and no obvious degradation was observed.Finally,the method was applied to determine four actual samples of budesonide suspension for inhalation in a batch.The samples were dissolved in methanol,filtered through a 0.45μm microporous membrane,and then analyzed.The amounts of 22R-budesonide and 22S-budesonide in the samples were in the ranges of 283.15-284.63μg/mL and 259.86-261.51μg/mL,respectively.This method is simple and rapid,in addition to having good repeatability and high accuracy.It can be used for the resolution of budesonide enantiomers and for quality control in budesonide preparations.