雌、雄地鳖中17种氨基酸的含量测定及指纹图谱研究

Study on HPLC Fingerprint and Determine Amino Acids in Female and Male Eupolyphaga sinensis

目的:建立地鳖17种游离氨基酸的HPLC-FLD指纹图谱,考察雌、雄地鳖中17种氨基酸成分的含量差异。方法:0.1 mol/L盐酸超声提取游离氨基酸,OPA为衍生试剂,Agilent ZOBAX SB-C18(250 mm×4.6 mm,5μm)色谱柱,流动相A为磷酸盐缓冲液(0.1 mol/L,pH=7.5),流动相B为甲醇-乙腈-水(45∶45∶10),梯度洗脱,FLD检测器激发波长340 nm,发射波长450 nm,流速1.0 m L/min,柱温30℃。结果:建立了地鳖中游离氨基酸HPLC指纹图谱,标定了21个共有峰,采用国家药典委员会"中药色谱指纹图谱相似度评价系统"进行相似度评价,15批雌地鳖药材相似度均不低于0.97;定量分析了地鳖中17种氨基酸成分,不同批次地鳖样品中各氨基酸含量差异较大,湖北产较江苏、河北产总游离氨基酸含量高;地鳖雌虫与雄虫总游离氨基酸含量比较,雌虫显著高于雄虫。结论:所建立的地鳖指纹图谱准确、重复性好,可为地鳖质量控制标准的建立提供数据依据;地鳖雌、雄虫中17种氨基酸含量差异较大,为传统以地鳖雌虫入药提供新的科学依据。

Objective： To establish the fingerprint of free amino acids in Eupolyphaga sinensis,and to study the difference of the content of various amino acids in female and male Eupolyphaga sinensis. Methods： The free amino acids were extracted by ultrasonic in0. 1 mol/L HCl. O-Phthalaldehyde（OPA） was used as the pre-column derivation reagent,and the content of amino acids was determined by HPLC method. Agilent ZOBAX SB-C18（250 mm × 4. 6 mm,5 μm） was used as chromatographic column,mobile phase A was sodium phosphate buffer（0. 1 mol/L,pH = 7. 5）,mobile phase B was methanol-acetonitrile-water（45∶45∶10）; the excitation wavelength was set at 340 nm and emission wavelength was set at 450 nm,the flow rate was 1. 0 m L/min in gradient elution,and the column temperature was 30 ℃. Results： The fingerprint of free amino acids in Eupolyphaga sinensis was established,21 common peaks were calibrated in the fingerprint and 17 amino acids contents were quantitatively analysed. The similarities of 15 batches of female Eupolyphaga sinensis were no less than 0. 97,which analyzed by＂Similarity Evaluation System for Chromatographic Fingerprint of TMC＂edited by China Pharmacopoeia Committee. The content of amino acids was large different in different batches of Eupolyphaga sinensis samples,and the content of amino acids of Eupolyphaga sinensis in Hubei province was higher than that in Jiangsu province and Hebei province,and the content of total free amino acids in female Eupolyphaga sinensis was higher than that in males Eupolyphaga sinensis. Conclusion： The method is accurate and simple,and thereby can be used specifically for the quality control of Eupolyphaga sinensis. There are differences content of the free amino acid between in female and male Eupolyphaga sinensis,which can provide new scientific basis for female Eupolyphaga sinensis for its medical use.