AQC 柱前衍生高效液相色谱方法测定康复新液中游离氨基酸和总肽的含量

目的 应用 6-氨基喹啉基-N-羟基琥珀酰亚氨基氨基甲酸酯 （AQC） 柱前衍生化技术，建立康复新液中游离氨基酸和总肽的高效液相色谱 （HPLC） 含量测定方法，并以总肽含量为指标对市售样品进行检测。方法 以十八烷基硅烷键合硅胶为填充剂 （Kromasil 100-5 C18 色谱柱） ；60% 乙腈 （A） -0.14 mol·L-1三水合乙酸钠溶液，用磷酸调节 pH 值至 5.0 （B） 为流动相，梯度洗脱；柱温：39 ℃；流速：1.0 mL·min-1，检测波长：248 nm。结果 14 种氨基酸均能达到良好的分离效果；门冬氨酸、谷氨酸、丝氨酸、组氨酸、甘氨酸、精氨酸、苏氨酸、丙氨酸、脯氨酸、缬氨酸、赖氨酸、异亮氨酸、亮氨酸和苯丙氨酸分别在 2.0~99.7、3.4~168.5、2.8~139.6、4.0~201.4、 4.8~238.1、 6.7~336.9、 3.3~167.5、 9.7~487.3、 4.1~202.5、 4.4~221.6、 5.4~270.5、 3.3~166.8、4.8~240.8、4.7~236.6 μg·mL-1范围内呈现良好的线性关系 （r=0.999 5~1.000 0） ；游离氨基酸的平均加样回收率（n=6） 为 86.8%~108.1%，RSD 为 2.8%~4.4%，总肽酸水解氨基酸的平均加样回收率 （n=6） 为 83.2%~102.7%，RSD 为 0.1%~3.1%；仪器精密度、重复性、稳定性实验的 RSD 均小于 5.0%。结论 该方法与现行质量标准的紫外分光光度法比较，二者测定的总氨基酸含量结果基本一致，但前者的专属性、重现性更优；以具有生物活性的总肽为质控指标，针对性更强，可为康复新液的质量评价提供更科学、合理的方法。

Determination of Free Amino Acids and Total Peptides in Kangfuxin Liquid by HPLC Coupled with AQC Pre-column Derivatization

Objective To establish a method for determining the content of free amino acids and total peptides in Kangfuxin Liquid by HPLC coupled with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate （AQC） pre-column derivatization，and to evaluate the quality of commercial samples with total peptides as indexes. Methods HPLC separation was performed on Kromasil 100-5 C18 column with mobile phase comprising of 60% acetonitrile（A）and 0.14 mmol·L-1 sodium acetate trihydrate solution （adjusted to pH 5.0 with phosphoric acid）（B） in a gradient elution manner at the flow rate of 1.0 mL·min-1. The column temperature was 39 ℃. The detection wavelength was 248 nm. Results 14 amino acids （aspartate，glutamic acid，serine，histidine，glycine，arginine，threonine，alanine，proline，valine，lysine，isoleucine，leucine，and phenylalanine） could achieve good separation，and present good linear relationship （r=0.999 5~1.000 0） in the concentration range of 2.0~99.7，3.4~168.5，2.8~139.6， 4.0~201.4， 4.8~238.1， 6.7~336.9， 3.3~167.5， 9.7~487.3， 4.1~202.5， 4.4~221.6， 5.4~270.5， 3.3~166.8，4.8~240.8 and 4.7~236.6 μg·mL-1，respectively. The average recoveries of free amino acids and hydrolyzed amino acids from total peptides were 86.8%~108.1%（n=6）and 83.2%~102.7%（n=6），with RSDs at 2.8%~4.4% and 0.1%~3.1%，respectively. The RSDs of precision，repeatability and stability tests were all lower than 5.0%. Conclusion As compared to ultraviolet spectrophotometry method for the current quality standard，the total amino acids content determined by this method is in consistent to UV. However，this method showed better specificity and reproducibility than UV. When total peptides with biological activity were used as quality control indicators，the pertinence of this method was strengthened. Our study may provide a scientific and reasonable method for the quality control of Kangfuxin Liquid.