鹿角胶中4个主要氨基酸的测定研究

Research on determination of 4 main amino acids in deerhorn glue

目的:研究建立鹿角胶中L-羟脯氨酸、甘氨酸、丙氨酸、L-脯氨酸4个氨基酸的分析和测定方法。方法:样品以6 mol·L-1盐酸于沸水浴水解1 h,蒸干,甲醇溶解,以苯酚-0.5%硼砂溶液(4∶1)为展开剂,硅胶G为固定相,茚三酮试液为显色剂进行TLC分析。样品以6 mol·L-1盐酸于150℃水解1 h,以异硫氰酸苯酯(PITC)为衍生化试剂衍生后进行HPLC分析。采用Phenomenex C18色谱柱(4.6 mm×250 mm,5μm),柱温为43℃;流动相A为乙腈-0.1 mol·L-1醋酸钠(7∶93),流动相B为乙腈-水(4∶1),梯度洗脱,流速1.0 mL·min-1;检测波长为254 nm进行HPLC测定分析。结果:TLC能较好地鉴别出鹿角胶中的L-羟脯氨酸、甘氨酸、丙氨酸、L-脯氨酸。HPLC测定L-羟脯氨酸、甘氨酸、丙氨酸、L-脯氨酸的线性范围分别为0.02～0.34μg(r=1.000),0.04～0.71μg(r=1.000),0.02～0.30μg(r=0.9998),0.02～0.37μg(r=1.000);加样回收率(n=6)分别为99.5%(RSD=2.3%),97.8%(RSD=1.6%),100.8%(RSD=1.6%),97.9%(RSD=2.4%)。结论:该方法准确、可靠,具有良好的重复性和稳定性,可用于鹿角胶中L-羟脯氨酸、甘氨酸、丙氨酸、L-脯氨酸的检测。

Objective：To develop TLC and HPLC methods for simultaneous detection of 4 amion acids（L-hydroxyproline,glycine,alanine,L-proline） in deerhorn glue.Methods： The sample hydrolyzed for 1 hour was detected by TLC with phenol-0.5% borax solution（4∶1） as mobile phase,silica gel G as stationary phase,triketohydrindene as colour reagents.The sample hydrolyzed for 1 hour at 150 ℃,derived with phenyl siothiocyanate（PITC） was determined by HPLC.This HPLC determination was performed on a Phenomenex C18 column（4.6 mm×250 mm,5 μm）,with gradient elution of acetonitrile-0.1 mol·L-1 sodium acetate（7∶93）（A） and acetonitrile-water（4∶1）（B） at flow rate of 1.0 mL·min-1,detected at 254 nm.Results： L-hydroxyproline,glycine,alanine,L-proline had good linearity in the ranges of 0.02-0.34 μg（r=1.000）,0.04-0.71 μg（r=1.000）,0.02-0.30 μg（r=0.9998）,0.02-0.37 μg（r=1.000）,respectively.The average recoveries（n=6） were 99.5%（RSD=2.3%）,97.8%（RSD=1.6%）,100.8%（RSD=1.6%）,97.9%（RSD=2.4%）,respectively.Conclusion： The developed method is accurate with high repeatability,which is helpful for the determination of L-hydroxyproline,glycine,alanine,L-proline,4 amino acids in deerhorn glue.