产油丝状真菌高山被孢霉中蝶呤类化合物的提取及检测

doi:10.13386/j.issn1002-0306.2013.10.029

产油丝状真菌高山被孢霉中蝶呤类化合物的提取及检测

Extraction and determination of pteridines in the oleaginous fungus, Mortierella alpina

摘要

摘要: 建立了高效液相色谱-荧光检测法同时测定高山被孢霉体内新蝶呤、蝶呤和生物蝶呤的方法。高山被孢霉在液氮破壁、酸性条件下经碘-碘化钾溶液氧化1h、滴加抗坏血酸还原液后,体内的四氢生物蝶呤及其前体二氢蝶呤三磷酸盐和6-丙酮酰四氢生物蝶呤分别被氧化成相应的氧化产物新蝶呤、蝶呤、生物蝶呤。经离子交换树脂纯化后,利用高效液相色谱进一步分离得到了氧化产物蝶呤、新蝶呤和生物蝶呤,并采用电喷雾质谱对定性结果进行确认。色谱柱为Inertsil ODS-3C₁₈（5μm,150mm×4.6mm）,用10mmol/L的磷酸氢二钠（pH6.0）作为流动相,流速为1.2mL/min,荧光检测器激发波长为350nm,吸收波长为450nm。检测出的新蝶呤、生物蝶呤和蝶呤的检出限依次是:0.003、0.002、0.005μg/mL。本方法能快速并准确的检测各类蝶呤类化合物,为利用高山被孢霉发酵生产四氢生物蝶呤奠定一定基础。

Abstract: The BH4 and its precursors dihydroneopterin triphosphate and pyruvoyltetrahydropterin in the oleaginous fungus Mortierella alpina had been extracted and determined by high performance liquid chromatography (HPLC) with fluorescence detection.Mortierella alpina was triturated under liquid nitrogen and oxidized by I2KI under acidic condition for 1h.Then the sample was purified by ion exchange chromatography and analyzed by HPLC-fluorescence detection and electrospray ionization-mass spectrometry (ESI-MS) .HPLC was performed on an Inertsil ODS-3 C₍18_ column (5μm, 150mm×4.6mm) .Aliquots of reaction mixtures were injected into the column and eluted by 10mmol/L Na₂HPO₄ (pH6.0) at a flow rate of 1.2mL/min.The excitation and emission wavelengths were set at 350nm and 450nm, respectively.The detection limit of this method for neopterin, pterin and biopterin were 0.003, 0.002 and 0.005μg/mL, respectively.The method was accurate and rapid, which could lay the foundation for the production of BH4 by Mortierella alpina.