RP-HPLC法测定珍珠明目滴眼液中16种氨基酸的含量

目的:柱前衍生HPLC法测定珍珠明目滴眼液中16种氨基酸的含量。方法: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 Gemini C₁₈ (250 mm×4.6 mm,5μm)色谱柱,流动相A为0.1 mol·L^-1醋酸钠缓冲液(pH 6.5)-乙腈(93:7),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254 nm,柱温43℃。结果:16种氨基酸质量浓度在一定范围内呈良好的线性关系(r=0.9990~0.9999);16种氨基酸平均回收率在82.7%~108.4%之间,RSD除天冬氨酸为4.5%、谷氨酸为4.3%,丝氨酸、酪氨酸和亮氨酸(Leu)为3.5%~4.0%外,其他均低于3.3%。不同厂家样品的氨基酸色谱指纹图谱差异较大,7个厂家(A~G)不同批次样品16种氨基酸总量平均值分别为44.6~50.0、20.9~29.4、47.9~55.5、154.2~164.3、13.3~15.0、8.9~13.4、181.3~192.7 μg·mL^-1。D和G厂家样品中谷氨酸(Glu)、甘氨酸(Gly)、精氨酸(Arg)、丙氨酸(Ala)和脯氨酸(Pro)5种氨基酸的含量高于其他厂家3~50倍。结论:本方法灵敏、准确,具有良好的重复性和稳定性,可用于珍珠明目滴眼液中氨基酸的质量控制。

Objective:To determine the contents of 16 amino acids in Zhenzhu Mingmu eye drops through RP-HPLC determination after precolumn derivatization.Methods:The sample was hydrolyzed with 6 mol·L^-1 hydrochloric acid for 1 hour at 150℃,and derivatized with phenyl isothiocyanate(PITC).HPLC was performed on a Phenomenex Gemini C₁₈ (250 mm×4.6 mm,5 μm)column with gradient elution of 0.1 mol·L^-1 sodium acetate buffer solution(adjusted to pH 6.5 with acetic acid)-acetonitrile(93:7)(A)and acetonitrile-water(4:1)(B)at the flow rate of 1.0 mL·min^-1,and the detection wavelength was at 254 nm.The column temperature was 43℃.Results:The mass concentrations of 16 amino acids had good linearity in the range of 0.525-1 375.0 μg·mL^-1 (r=0.999 0-0.999 9).The average recoveries were within 82.7%-108.4%; RSDs were less than 3.3%,except for Ser,Tyr and Leu whose RSDs were within 3.5%-4.0%,Asp 4.8% and Glu 4.3%.The chromatographic fingerprints of amino acids in the samples from different factories were obviously different.The average values for the total contents of 16 amino acids in different batches of samples from 7 factories were 44.6-55.0 μg·mL^-1,20.9-29.4 μg·mL^-1,47.9-55.5 μg·mL^-1,154.2-164.3 μg·mL^-1,13.3-15.0 μg·mL^-1,8.9-13.4 μg·mL^-1 and 181.3-192.7 μg·mL^-1,respectively.The contents of 5 amino acids,including Glu,Gly,Arg,Ala and Pro,in the samples from D and G factories,were 3-50 times higher than those from other factories.Conclusion:The method is sensitive and accurate with good stability and repeatability,which can be used to control the quality of amino acids in Zhenzhu Mingmu eye drops.