高效液相色谱-质谱联用法分析微生物降解植物甾醇为雄甾烯酮

Preliminary Study of Bio-transformation of Phytosterol by HPLC- MS

摘要:

采用高效液相色谱-质谱联用法(HPLC/MS)分析测定植物甾醇侧链降解过程中产物雄甾-1,4-二烯-3,17-二酮(ADD)及雄甾4-烯-3,17-二酮(AD)。其中液相色谱的条件为色谱柱Alltima C18 ODS-2(5 μm, 250 mm ×4.6 mm); 流动相甲醇:水(V/V=7:3); 流速1 mL/min; 柱温室温; 紫外检测器的检测波长 244 nm。质谱为 ZMD Micromass 电喷雾质谱仪。结果测得ADD与AD标准样品的保留时间分别为9.70 min与11.13 min, 发酵样品的HPLC与MS图谱与ADD与AD标准样品的图谱一致。采用高效液相色谱法定量ADD与AD的线性范围在0.01 mg/mL ~ 0.09 mg/mL, 产物回收率分别为102.6%与105.90%, 日内精密度分别为3.02%与3.08%, 日间RSD分别为3.50%与3.24%。该方法灵敏度高、选择性好、操作简便、定量准确, 适用植物甾醇微生物侧链降解过程中产物的分析及产品质量控制。

Abstract:

The method for analysis and determination the cleavage of soybean sterol, in which the soybean sterol was degraded and the products androst-1,4-diene-,17-dione (ADD) and androst-4-ene-3,17-dion (AD) were developed by Liquid Chromatography-mass Spectrometry. The HPLC conditions adopted were: a Alltima ODS-2 column (250 mm×4.6 mm, 5 μm), a mobile phase consisted of menthanol - water (70:30), a flow rate of 1.0 mL/min, a room column temperature. and the detective wavelength was 244 nm.The ZMD Micromass electrospray ionization (ESI)-mass spectrometer was employed. In such conditions the corresponding HPLC chromatogram and MS spectrum were obtained. The method has a linear ranger of 0.01 mg/mL ~ 0.09 mg/mL, R2 =0.9999, the recoveries of ADD and AD were 102.6% and 105.90%, the RSD of ADD and AD were 3.02%, 3.5% and 3.08%, 3.24%. This method showed high sensitivity, accuracy and easy to perform. It is suitable to analysis the process cleavage of soybean sterol as well as quality control of product.