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目的:建立高效液相色谱法测定菌体中L-5-甲基四氢叶酸(L-5-MTHF)的方法。方法:加热法破碎细胞,强碱性阴离子交换树脂纯化,高效液相色谱柱为C18反向柱,流动相为K2HPO4(0.02 mol.L-1,pH7.2)-甲醇(84∶16)溶液,流速为1.0 mL.min-1,柱温为40℃,检测波长为290 nm。结果:L-5-MTHF在0.5～50μg.mL-1范围内线性良好,r=0.999 5,RSD为1.11%。结论:色谱法灵敏、准确、重现性好,可用于菌体中L-5-甲基四氢叶酸的定性与定量。
Objective: To establish a method for the determination of L-5-methyltetrahydrofolic acid (L-5-MTHF) in mycelia by high performance liquid chromatography. METHODS: Cells were disrupted by heating and strongly basic anion exchange resin. The high performance liquid chromatography column was C18 reversed-phase column. The mobile phase consisted of K2HPO4 (0.02 mol·L-1, pH 7.2) -methanol (84:16) , The flow rate was 1.0 mL.min-1, the column temperature was 40 ℃ and the detection wavelength was 290 nm. Results: L-5-MTHF showed a good linearity in the range of 0.5-50 μg.mL-1 with r = 0.999 5 and RSD 1.11%. Conclusion: Chromatography is sensitive, accurate and reproducible. It can be used for the qualitative and quantitative determination of L-5-methyltetrahydrofolic acid in mycelium.