柱前衍生化HPLC法测定藏药熏倒牛种子中15种氨基酸含量

Determination of15amino acids in Biebersteinia heterostemon Maxim seeds by precolumn derivation HPLC

目的:建立柱前衍生化HPLC法测定藏药熏倒牛种子中15种氨基酸的含量。方法:供试品在一定量6 moL·L^(-1)盐酸中110℃水解6 h,与异硫氰酸苯酯进行衍生化反应。采用CAPCELL PAK-C18(250 mm×46 mm,5μm)色谱柱,以80%乙腈溶液(A)-0056 moL·L^(-1)乙酸钠缓冲液(B)为流动相,梯度洗脱(0~15 min,5%A;15~20 min,5%A→20%A;20~35 min,20%A→36%A;35~45 min,36%A→70%A;45~50 min,70%→90%A;50~55 min,90%A;55~56 min,90%A→5%A;56~60 min,5%A),流速为10 mL·min^(-1),检测波长254 nm,色谱柱温度40℃。结果:15种氨基酸衍生物能够达到基线分离,在096~5856μg·mL^(-1)范围内线性关系良好,r≥09991,精密度、重复性、稳定性试验的RSD值分别小于273%、285%、289%,加样回收率介于952%~1044%之间,RSD值均小于246%。经测定,3批熏倒牛种子中氨基酸总含量分别为164%、155%和108%。结论:此法简便易行,可用于藏药熏倒牛种子中15种氨基酸的含量测定。

Objective To establish a precolumn derivation HPLC method for the determination of 15 amino acids in Biebersteinia heterostemon Maxim seeds.MethodsThe sample was hydrolyzed in 6 moL·L^(-1)hydrochloric acid at 110℃for 6 hours,and derivatized with phenyl isothiocyanate(PITC).The analysis was carried out on a CAPCELL PAK-C18 chromatographic column(250 mm×46 mm,5μm)with 80%acetonitrile(A)-0056 mol·L^(-1)sodium acetate buffer(B)as mobile phase by gradient elution(0~15 min,5%A;15~20 min,5%A→20%A;20~35 min,20%A→36%A;35~45min,36%A→70%A;45~50 min,70%→90%A;50~55 min,90%A;55~56 min,90%A→5%A;56~60 min,5%A),at a flow rate of 10 mL·min^(-1),the detection wavelength was 254 nm,the column temperature was set at 40℃.ResultsThe 15 amino acids were able to achieve baseline separation in a good linear range of 096~5856μg·mL^(-1)(r≥09991).The RSD values of precision,repeatability and stability tests were respectively less than 273%、285%、289%,and the recoveries of the 15 amino acids(n=6,RSD<246%)were between 952% and 1044%.The total contents of the amino acids in three batches of Biebersteinia heterostemon Maxim seeds were164%、155%和108%.ConclusionThis method is simple and easy to operate.It can be used for the determination of 15 amino acids in Tibetan medicine Biebersteinia heterostemon Maxim seeds.