柱前衍生高效液相色谱法测定灵芝孢子粉中氨基酸的含量

Assay of Amino Acids in Ganoderma Lucidum Spore Powder by Pre-column Derivatization HPLC

目的:建立用于测定灵芝孢子粉中氨基酸含量的柱前衍生化高效液相色谱方法。方法:采用邻苯二甲醛(OPA)和9-氯甲酸芴甲酯(FMOC)联用对灵芝孢子粉中的氨基酸进行衍生,色谱条件为:Advance Bio AAA(100 mm×4.6 mm,2.7μm)色谱柱,流动相A为0.01 mol/L磷酸氢二钠和0.01 mol/L硼酸钠溶液,pH值为8.2±0.1,流动相B为甲醇︰乙腈︰水(45︰45︰10)。流速:0.8 mL/min,柱温:45℃,进样量:10μL,检测波长:338 nm和262 nm。结果:灵芝孢子粉中检测的17种氨基酸在60 min内分离良好;在一定范围内线性关系良好(r≥0.999,n=6);加样回收率为95.42%~99.38%,实验方法的精密度、重复性和稳定性的相对标准偏差(RSD)均小于3.0%。结论:经方法学验证显示,本研究所建立的实验方法适用于灵芝孢子粉中17种氨基酸的含量测定。

Objective To establish a pre-column derivatization-high performance liquid chromatography method for the determination of amino acids in ganoderma lucidum spore powder.Methods The amino acids in ganoderma lucidum spore powder were derivatized with o-phthalaldehyde(OPA)and 9-fluorenylmethoxycarbonyl chloride(FMOC).The chromatographic conditions were Advance Bio AAA(100 mm×4.6 mm,2.7μm)column,and Mobile phase A was 0.01 mol/L disodium hydrogen phosphate and 0.01 mol/L sodium borate solution(pH:8.2±0.1).Mobile phase B was methanol:acetonitrile:water(45:45:10),flow rate was 0.8 mL/min,the column temperature was 45℃,the injection volume was 10μL and the detection wavelength was fixed at 338 nm and 262 nm.Results 17 amino acids in Ganoderma lucidum spore powder were separated satisfactorily in 60 minutes.All the components exhibited good linear correlation in the given concentration range(r≥0.999,n=6).The recovery rate of the sample was between 95.42%and 99.38%.The RSD of the precision,repeatability and stability of the experimental method were all less than 3.0%.Conclusion The methodological verification shows that the established experimental method is suitable for the determination of 17 amino acids in ganoderma spore powder.