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目的:建立板蓝根中4个主要氨基酸苏氨酸、脯氨酸、精氨酸、缬氨酸的RP-HPLC柱前衍生化法,研究板蓝根氨基酸含量变化。方法:采用2,4-二硝基氟苯柱前衍生化,Eclipse XDB C18反相色谱柱(250 mm×4.6 mm,5μm)色谱柱分离,以0.014 mol·L-1磷酸盐缓冲液(pH8.2)与乙腈水溶液[乙腈-水=(1∶1)]为流动相梯度洗脱,测定板蓝根水提液、人工胃液、人工肠液和酸水解液中的氨基酸含量。结果:苏氨酸、脯氨酸、精氨酸、缬氨酸的线性范围分别为0.117~1.170(r=0.999 9)、0.067~0.670(r=0.999 1)、2.95~29.50(r=0.999 0)、0.044~0.440μg(r=0.999 9),线性关系良好,平均回收率分别为99.90%(RSD=3.14%)、100.82%(RSD=2.03%)、97.83%(RSD=2.18%)、100.98%(RSD=3.84%)。结论:板蓝根多肽经人工胃液和人工肠液消化后,未水解为游离氨基酸,该法操作简单,重复性好,准确可靠。
OBJECTIVE: To establish a RP-HPLC pre-column derivatization method for the determination of four main amino acids, threonine, proline, arginine and valine, in Radix isatidis. Methods: The compounds were separated on a Eclipse XDB C18 reversed-phase column (250 mm × 4.6 mm, 5 μm) using 2,4-dinitrofluorobenzene pre-column derivatization. The column was eluted with 0.014 mol·L-1 phosphate buffer .2) and acetonitrile aqueous solution [acetonitrile-water = (1: 1)] were used as the mobile phase to determine the content of amino acids in the aqueous extracts of Radix isatidis, artificial gastric juice, artificial intestinal juice and acid hydrolyzate. RESULTS: The linear ranges of threonine, proline, arginine and valine were 0.117-1.170 (r = 0.999 9), 0.067-0.670 (r = 0.999 1), 2.95-29.50 (r = 0.999 0 ) And 0.044-0.440 μg (r = 0.999 9) respectively. The average recoveries were 99.90% (RSD = 3.14%), 100.82% (RSD = 2.03%), 97.83% % (RSD = 3.84%). Conclusion: The Banlangen polypeptide is not hydrolyzed to free amino acids after being digested by artificial gastric juice and artificial intestinal juice. The method is simple, reproducible, accurate and reliable.