肉豆蔻中赭曲霉毒素的测定及赭曲霉毒素产毒菌的分离鉴定

Determination of ochratoxins in nutmeg as well as isolation and identification of ochratoxin-producing fungus

建立了同时测定肉豆蔻中3种赭曲霉毒素的超高效液相色谱-串联质谱法(UPLC-MS/MS)。样品经70%甲醇超声提取,HLB柱净化,采用Agilent Proshell 120 EC C_(18)色谱柱,以乙腈(含0.1%甲酸)-水(含0.1%甲酸)为流动相进行梯度洗脱,于电喷雾离子源正负离子模式下多反应监测模式检测。结果表明,肉豆蔻基质中3种赭曲霉毒素基质效应为91.0%~112.0%,采用基质匹配标准曲线法定量时各浓度范围下线性关系良好(R^(2)>0.999)。样品在高、中、低3个浓度加标水平下,回收率为63.3%~88.4%,相对标准偏差(RSD)小于6.0%,检出限(LOD)和定量限(LOQ)分别为0.1~1.0μg/kg和0.3~3.0μg/kg。另从肉豆蔻中分离得到一株产毒菌,采用紫外荧光筛选、形态学鉴定、DNA测序结合液质联用检测的方法最终鉴定为菌核曲霉。

A simultaneous determination method for 3 ochratoxins in nutmeg was established by ultra-high performance liquid chromatography tandem mass spectrometry(UPLC-MS/MS).The ochratoxins were extracted from the sample through ultrasonic extraction using 70%methanol and purification using HLB column.The separation of the ochratoxins was performed by a Agilent Proshell 120 EC C_(18) column(150 mm×3.0 mm,2.7μm)with gradient elution using the mobile phase involving the acetonitrile containing 0.1%formic acid and the water containing 0.1%formic acid.Simultaneous detection was performed in multiple reaction monitoring(MRM)mode with electrospray ionization(ESI)source in both positive and negative ionization modes.The results showed that the matrix effect of the nutmeg was slight(91.0%-112.0%),and the established method provided a satisfied linearity for each of the 3 ochratoxins with the correlation coefficients above 0.999.The average recoveries ranged from 63.3%to 88.4%with the relative standard deviations(RSDs)lower than6.0%.The limits of detection(LODs)and quantitation(LOQs)ranged from 0.1 to 1.0μg/kg and 0.3 to 3.0μg/kg,respectively.In addition,a strain of mycotoxin-producing fungus was isolated from the nutmeg,which was identified as Aspergillus sclerotiorum by ultraviolet fluorescence screening,morphological identification,DNA sequencing and UPLC-MS/MS determination.