柱后衍生阳离子交换色谱法同时测定新疆产贝母中17种氨基酸的含量

Simultaneous Determination of Contents of 17 Amino Acids in Fritillariae Pallidiflorae Bulbus Produced in Xinjiang by Post-column Derivatization Cation-exchange Chromatography

目的:建立同时测定新疆产贝母中17种氨基酸含量的方法。方法:采用阳离子交换色谱柱分离17种氨基酸;采用柱后衍生阳离子交换色谱法测定药材中17种氨基酸的含量:色谱柱为LCAK 06/Na柱,流动相A为柠檬酸三钠11.9 g、柠檬酸6 g、苯酚1g,加水溶解,再加65 m L甲醇和6 m L浓盐酸,加水定容(调p H至3.4),流动相B为柠檬酸三钠11.9 g、氢氧化钠2.8 g、苯酚1 g、硼酸5.0 g,加水溶解并定容(调p H至10.8)(梯度洗脱),流速为0.45 m L/min(A泵)、0.25 m L/min(M泵),检测波长为440 nm(脯氨酸)、570 nm(其余氨基酸),进样量为50μL。结果:天冬氨酸、苏氨酸、丝氨酸、谷氨酸、甘氨酸、丙氨酸、缬氨酸、蛋氨酸、异亮氨酸、亮氨酸、酪氨酸、苯丙氨酸、组氨酸、赖氨酸、精氨酸和脯氨酸检测浓度线性范围均为20~400 nmol/m L(除胱氨酸10~200 nmol/m L)(r均大于0.989 0);精密度、重复性、稳定性试验的RSD<2.0%;检测限除胱氨酸外(0.08 nmol/m L)其余氨基酸均为0.16 nmol/m L;加样回收率为98.5%~99.5%(RSD为0.06%~0.21%,n=6)。结论:该方法操作简便,精密度、稳定性、重复性好,可用于新疆产贝母中氨基酸含量的同时测定。

OBJECTIVE： To establish a method of contents determination of 17 amino acids in Fritillariae Pallidiflorae Bulbus produced in Xinjiang. METHODS： Cation-exchange column was used to separate 17 kinds of amino acids; post-column derivatization liquid chromatography was used to determine the contents of amino acids： the column was strong sulfonic acid cation exchange resin LCAK06/Na with mobile phase A （weighing trisodium citrate 11.9 g, citric acid 6 g, phenol 1 g, dissolving by water, then adding 65 mL of methanol and 6 mL of concentrated hydrochloric acid, bringing the volume with tap water, adjusting pH to 3.4）, mobile phase B （weighing trisodium citrate 11.9 g, NaOH 2.8 g, phenol 1 g, boric acid 5.0 g, adding water to dissolve, adjusting pH to 10.8）, gradient elution,flow rate was 0.45 mL/min for A pump and 0.25 mL/min for M pump,the detection wavelength was 440 nm for proline and 570 nm for the remaining amino acids, the injection volume was 50 -tL. RESULTS： The linear range were 20-400 nmol/mL of aspartic acid,threonine, serine, glutamic acid, glycine, alanine, valine, methionine, isoleucine, leucine, ty- rosine, phenylalanine, histidine, lysine, arginine, proline, 10-200 nmol/rnL of cystine（r were higher than 0.989 0） ; RSDs of precision, stability and reproducibility tests were lower than 2.0% ; limits of detection were 0.16 nmol/mL except for cystine（0.08 nmol/ mL） ; recovery was 98.5%-99.5% （RSD was 0.06%-0.21%, n=6）. CONCLUSIONS： The method is simple with good precision, stability and reproducibility, and can be used for the simultaneous determination of amino acids in Fritillariae Pallidiflorae Bulbus produced in Xinjiang.