采用液相色谱-电喷雾飞行时间质谱(LC-ESI-TOF-MS)法,选择阴离子电离模式,建立了一种同时检测虫生真菌菌丝中16种游离氨基酸的定性定量分析方法.研究结果表明:16种氨基酸的色谱特征和质谱离子特征明显,可用来对氨基酸进行准确定性分析.定量实验结果表明,16种氨基酸的线性范围广准确性高,在氨基酸浓度线性范围4.0 ～ 125 μmol/L,16种氨基酸的峰面积与浓度相关系数都大于0.998.在氨基酸浓度为50μmol/L时,16种氨基酸的相对标准偏差在0.84％ ～2.75％之间.用液质联用法对7种虫生真菌菌丝样品中16种游离氨基酸含量的测定结果显示,同一菌株中各种氨基酸的含量相差较大,但所有供菌株中含量最高的均为苯丙氨酸,它在不同供试菌株中的含量范围为0.718 ～2.173 mg/g,其中中国被毛孢的苯丙氨酸含量最高.供试菌株中含量较高的氨基酸还有脯氨酸、缬氨酸、亮氨酸、异亮氨酸、赖氨酸和谷氨酸,它们含量大部分都在0.3mg/g以上,较其他氨基酸含量明显较高,这些含量较高的氨基酸可作为判定虫生真菌菌丝中游离氨基酸的特征指标.不同菌株的游离氨基酸总量也存在较大差异,中国被毛孢中游离氨基酸总含量为7.537 mg/g,较蝉棒束孢(3.196 mg/g)的总含量高出1.36倍.%A method for simultaneous analysis of 16 free amino acids in entomogeneous fungi was developed using liquid chromatography-electrospray time-of-flight mass spectrometry ( LC-ESI-TOF-MS) under negative ionization mode. The results showed that chromatographies and ion features of the 16 amino acids had obvious characteristics which can be used for accurately qualitative analysis. Quantitative tests showed that the 16 amino acids had wide linear ranges and high accuracy. In the linear range from 4. 0 to 125μmol/L all the correlation coefficients between concentration and peak area of the 16 amino acid were above 0. 998. At the concentrations of 50μmol/L the relative standard deviations of the 16 amino acids were in the range from 0. 84% to 2. 75% . Results of LC-ESI-TOF-MS analysis of the 16 free amino acids in mycelia of 7 kinds of entomogenous fungi showed that the contents of the 16 amino acids were very different in the same strain. Of all the amino acids the content of phenylalanine was the highest one in all tested strains, and its range was from 0.718 to 2. 173 mg/g. Hirsutella sinensis contained the highest phenylalanine. Apart from phenylalanine the contents of proline, valine, leucine, isoleucine, lysine and glutamic acid were also obviously higher than the other amino acids, and most of them were higher than 0. 3mg/g. The amino acids with relative higher contents could be used as characteristic index for identification of free amino acids in entomogenous fungi. The total free amino acids contents in mycelia of different strains were also significantly different. The content of Hirsutella sinensis was 7. 537mg/g which was 1. 36 times higher than that of Isaria cicadae (3. 196mg/g).
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