鹿角胶中4个主要氨基酸的测定研究

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,5 μm),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0.02~0.34 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);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.