羚羊角水解液的氨基酸薄层鉴别与含量测定

TLC identification and content determination of amino acids in the hydrolysate of antelope horn

目的建立羚羊角水解液中氨基酸类成分的薄层鉴别方法并测定其中游离氨基酸的含量,为羚羊角药材的质量控制提供依据。方法采用硅胶G薄层板,以十二烷基硫酸钠—正丁醇—正己烷—水按质量比(1.5∶7.0∶1.0∶1.7)组成的微乳液为展开剂,分别定性鉴别羚羊角水解液中的5种氨基酸,并对建立的薄层方法进行耐用性考察;采用异硫氰酸苯酯柱前衍生结合高效液相色谱法对水解液中16种游离氨基酸进行含量测定,并进行方法学考察。结果改变温度(4℃~30℃)、相对湿度(32%~88%)、微乳液含水量(10%~20%)等条件均能分离鉴别出羚羊角水解液中的5种氨基酸;16种氨基酸能完全分离,供试品溶液在24小时内稳定,平均回收率(n=6)在96.70%~103.02%,相对标准偏差均<3%,其中亮氨酸含量最高,组氨酸含量最低。结论建立的微乳薄层方法稳定、耐用性好,高效液相色谱法经方法学验证亦适用于羚羊角水解液中16种游离氨基酸的含量测定,可用于羚羊角药材的质量控制。

Objective To establish a thin layer chromatography(TLC)method for identifying amino acids in antelope horn hydrolysate and determine the content of free amino acids,so as to provide basis for the quality control of antelope horn.Methods Using silica gel-G plate,SDS-n-butanol-hexane-water at mass ratio(1.5∶7.0∶1.0∶1.7)as the developing agent,5 amino acids in antelope horn hydrolysate were qualitatively identified.The durability of the established TLC method was investigated.The content of 16 free amino acids was determined by pre-column derivatization of phenyl isothiocyanate and high performance liquid chromatography,and its methodology was investigated.Results When the conditions such as temperature(4℃~30℃),the relative humidity(32%~88%)and water content(10%~20%)of the microemulsion were changed,5 amino acids in antelope horn hydrolysate could be separated and identified.The 16 amino acids could be completely separated.The test solution was stable within 24 hours.The average recoveries were 96.70%~103.02%,and the RSDs were less than 3%.The content of leucine was the highest and the content of histidine was the lowest.Conclusion The established microemulsion TLC method is stable and durable.The high performance liquid chromatography method verified by methodology is also suitable for the determination of 16 free amino acids in antelope horn hydrolysate,and can be used for the quality control of antelope horn.