柱前衍生RP-HPLC法测定狗尾草种子氨基酸含量
Determination of amino acids in seeds of Setaria viridis (L.) Beauv. by pre-column derivatization RP-HPLC

【目的】采用柱前衍生反相高效液相色谱（RP-HPLC）法检测狗尾草种子中游离氨基酸和水解氨基酸含量，为阐明其应用价值提供支撑。【方法】制备狗尾草种子的水解氨基酸样品溶液和游离氨基酸样品溶液，以异硫氰酸苯酯和三乙胺进行柱前衍生，建立RP-HPLC法，检测方法的精密性、稳定性、重复性。利用RP-HPLC法，采用梯度洗脱方法，检测狗尾草种子中游离氨基酸和水解氨基酸含量。【结果】21种氨基酸的检测浓度线性范围为0.001 9～2.700 0 μmol/mL,相关系数R2均≥0.999 0。21种游离氨基酸的平均加样回收率在87.50％～100.20％，相对标准偏差（RSD）在0.82％～2.46％；水解氨基酸的平均加样回收率在91.10％～100.21％，RSD 在0.52％～2.08％。必需氨基酸和半必需氨基酸在狗尾草21种游离氨基酸和水解氨基酸中均含有，但水解后色氨酸、天冬酰胺、组胺酰胺遭到了破坏，含量较低。【结论】建立的氨基酸测定方法稳定、快速、精密度高、线性范围宽，适合狗尾草种子中游离氨基酸和水解氨基酸的含量测定。狗尾草种子中含有丰富的游离和水解氨基酸。
【Objective】This study aimed to establish a method for determinating the contents of free and hydrolyzed amino acids from seeds of Setaria viridis (L.) Beauv.by pre-column derivatization RP HPLC.【Method】The sample solution including free and hydrolyzed amino acids from the seeds of Setaria viridis (L.) Beauv was prepared before being derived with phenyl isothiocyanate and triethylamine,and detected by HPLC.The precision,stability,and repeatability were detected before gradient elution chromatography was applied to determine the contents of free and hydrolyzed amino acids.【Result】The 21 amino acids had good linearity in the range of 0.001 9-2.700 0 μmol/mL with correlation coefficients all over 0.999 0.The average recovery of free amino acids was 87.50％-100.20％ (RSD=0.82％-2.46％).The average recovery of total amino acids was 91.10％-100.21％（RSD=0.52％-2.08％).There were essential amino acids and semi essential amino acids for human in seeds of Setaria viridis (L.) Beauv.But tryptophan,asparagine,and histamine amide were compromised after hydrolysis.【Conclusion】The established method was sensitive,accurate,reproducible,and reliable for determination of amino acids.Both free and hydrolyzed amino acids were rich in seeds of Setaria viridis (L.) Beauv