超高效合相色谱法拆分和测定克伦特罗对映体

Separation and determination of clenbuterol enantiomers by ultra-performance convergence chromatography

建立了基于超高效合相色谱技术(UPC^(2))的克伦特罗对映体拆分方法,并对所建立的方法进行了方法学考察及应用。实验考察了两种克伦特罗对映体标准溶液的稳定性,并对手性色谱柱、助溶剂、系统背压、色谱柱温度等色谱分离条件进行了优化。采用Acquity Trefoil AMY1(150 mm×3.0 mm,2.5μm)手性色谱柱进行分离,以超临界CO_(2)-含0.5%(v/v)10 mol/L醋酸铵的甲醇溶液为流动相,以流速2.0 mL/min梯度洗脱,检测波长为241 nm,进样体积为10μL,系统背压为13.8 MPa,柱温为40℃时,两种克伦特罗对映体分离效果最好。两种克伦特罗对映体的线性范围为1.0~20.0 mg/L,相关系数均大于0.9997,仪器检出限(S/N=3)均为0.5 mg/L。10.0 mg/L混合标准工作溶液重复进样6次,(+)、(-)克伦特罗对映体峰面积的相对标准偏差(RSD,n=6)分别为0.65%和0.76%。应用该方法对市售克伦特罗外消旋体标准品进行拆分,采用外标定量法计算克伦特罗外消旋体标准中间溶液10.0 mg/L中两种克伦特罗对映体含量,其中(+)-克伦特罗的含量为5.6 mg/L,(-)-克伦特罗的含量为5.5 mg/L。该计算结果与文献报道的工业品克伦特罗外消旋体中(+)-克伦特罗与(-)-克伦特罗的比例为1.02∶1.00基本相符。该方法具有分析速度快、分离效果好、有机溶剂消耗少等特点,适用于克伦特罗对映体的拆分,为其他手性化合物的拆分、药效精细分析和产品质量评定提供了可靠的技术支持。

Clenbuterol enantiomers differ greatly in their bioactivities.By optimizing the conditions for chromatographic separation and method validation,ultra-performance convergence chromatography(UPC^(2))was adopted to separate the enantiomers of clenbuterol.Standard solutions of(+)-clenbuterol and(-)-clenbuterol were stored at-18℃for 1,3,5,7,14,30,and 60 d,and then,their stability was monitored.The impacts of different chromatographic columns,cosolvents,system backpressure,and chromatographic column temperature on the separation of the two enantiomers were investigated.Acquity Trefoil AMY1(150 mm×3.0 mm,2.5μm)was used for separation,and CO_(2)-0.5%(v/v)ammonium acetate was used as the mobile phase.Gradient elution at a flow rate of 2.0 mL/min was adopted.The detection wavelength was set to 241 nm,and the injection volume was set to 10μL.The backpressure was set to 13.8 MPa,and the column temperature was maintained at 40℃.The two enantiomers showed good linear relationships in the range of 1.0 to 20.0 mg/L with correlation coefficients greater than 0.9997.The limits of detection(LODs,S/N=3)of(+)-clenbuterol and(-)-clenbuterol were both 0.5 mg/L.The relative standard deviation(RSD,n=6)for the peak area of the 10.0 mg/L mixed standard working solution with six replicate injections ranged from 0.65%to 0.76%.The effectiveness and practicability of this method were demonstrated by using it to detect standard clenbuterol racemate.The(+)-clenbuterol and(-)-clenbuterol contents were 5.6 mg/L and 5.5 mg/L,respectively,in the standard clenbuterol racemates,as determined by the external standard method of quantification.The detection results suggested that the content ratio of(+)-clenbuterol and(-)-clenbuterol was close to 1.02∶1.00,which is consistent with the literature data.The established method has the advantages of rapid analysis,good separation effect,and low consumption of organic solvents,and it is suitable for the separation of clenbuterol enantiomers.This method can also provide technical support for the separation of other chiral drugs,analysis of the effects of chiral drugs,and assessment of product quality.