柱前衍生化HPLC法同时测定龟甲胶中14种水解氨基酸含量

目的: 建立一种利用HPLC法同时测定龟甲胶中14种氨基酸的含量。方法: 样品以6 mol·L^-1盐酸于110 ℃水解7 h,以6-氨基喹啉-N-羟基琥珀酰亚胺活化环氨基甲酸酯(AQC)进行衍生化反应,采用Kromasil C₁₈色谱柱(5 μm,4.6 mm×250 mm),柱温37 ℃,以0.14 mol·L^-1三水合乙酸钠(pH 4.85)为流动相A,60%乙腈为流动相B,梯度洗脱,流速1.4 mL·min^-1,检测波长248 nm。结果: 在一定的浓度范围内,各种氨基酸峰面积和浓度呈良好的线性关系,r值均大于0.999 0;加样回收率(n=9)为94.5%~103.6%;不同样品中14种氨基酸含量范围分别为羟脯氨酸7.08%~8.22%、谷氨酸5.94%~8.21%、丝氨酸2.48%~3.12%、组氨酸0.25%~0.44%、甘氨酸14.35%~16.86%、精氨酸4.78%~5.76%、苏氨酸1.24%~1.75%、丙氨酸4.65%~6.56%、脯氨酸6.78%~8.83%、酪氨酸0.55%~0.84%、缬氨酸0.95%~1.40%、异亮氨酸0.70%~0.95%、亮氨酸1.59%~2.06%、苯丙氨酸1.35%~1.57%。结论: 该方法准确、可靠,具有良好的重复性和稳定性,可用于龟甲胶中羟脯氨酸、谷氨酸、丝氨酸、组氨酸、甘氨酸、精氨酸、苏氨酸、丙氨酸、脯氨酸、酪氨酸、缬氨酸、异亮氨酸、亮氨酸、苯丙氨酸的检测。

Objective: To establish an HPLC method for simultaneous determination of 14 kinds of amino acids in tortoise plastron glue.Methods: The sample was hydrolyzed at 110 ℃ for 7 hours in the 6 mol·L^-1 hydrochloric acid, and derived with 6-aminoquinolyl-N-hydroxysuccinimidyl-carbamate(AQC).HPLC was performed on a Kromasil C₁₈ column(5 μm, 4.6 mm×250 mm)with gradient elution of 0.14 mol·L^-1 sodium salt trihydrate(pH 4.85, mobile phase A)and 60% acetonitrile(mobile phase B)at the flow rate of 1.4 mL·min^-1, detected at 248 nm.Results: In a certain concentration range, the peak area and the concentration of each amino acid showed a good linear relationship.Correlation coefficient r values were more than 0.999 0.The recoveries ranged in 94.5%-103.6%.The content ranges of hydroxyproline, glutamicacid, serine, histidine, glycine, arginine, threonine, alanine, proline, tyrosine, valine, isoleucine, leucine, phenylalanine were 7.08%-8.22%, 5.94%-8.21%, 2.48%-3.12%, 0.25%-0.44%, 14.35%-16.86%, 4.78%-5.76%, 1.24%-1.75%, 4.65%-6.56%, 6.78%-8.83%, 0.55%-0.84%, 0.95%-1.40%, 0.70%-0.95%, 1.59%-2.06% and 1.35%-1.57% respectively in different samples.Conclusion: The developed method is accurate with high repeatability, which is helpful for the determination of hydroxyproline, glutamicacid, serine, histidine, glycine, arginine, threonine, alanine, proline, tyrosine, valine, isoleucine, leucine, phenylalanine in tortoise plastron glue.