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    同位素稀释-超高效液相色谱-串联质谱法测定中药材中的17种真菌毒素

    Determination of Seventeen Mycotoxins in Chinese Herbal Medicines by Ultra-High Performance Liquid Chromatography-Mass Spectrometry with Isotope Dilution

    • 摘要: 称取2.000 g试样于50 mL离心管中,加入乙腈-水-甲酸(70+29+1)混合溶液20.0 mL,浸泡60 min,于振荡混匀器上提取30 min,超声提取30 min,离心后,取上清液2.0 mL,加入同位素内标混合溶液50 μL,用磷酸盐缓冲溶液稀释至20.0 mL得样品溶液。样品溶液以1~3 mL·min-1流量通过免疫亲和柱,用5 mL水淋洗免疫亲和柱,弃去全部流出液,再用2 mL甲醇-乙酸(98+2)溶液洗脱免疫亲和柱2次,收集全部洗脱液于试管中,于50℃下氮吹至近干,加入乙腈(1+9)溶液0.5 mL溶解残渣,离心后,上清液供超高效液相色谱-串联质谱法分析。为了校正样品净化过程和离子化过程的损失以及消除基质效应,选用稳定性同位素13C为内标物。17种真菌毒素的质量浓度在一定范围内与其峰面积与内标的峰面积的比值呈线性关系,检出限(3S/N)在0.1~2.0 μg·L-1之间。对空白中药材样品进行加标回收试验,回收率在84.3%~104%之间,测定值的相对标准偏差(n=6)在1.9%~13%之间。

       

      Abstract: The sample (2.000 g) was placed in 50 mL centrifuge tube, and 20.0 mL of the mixed solution of acetonitrile-water-formic acid (70+29+1) was aded. The mixture was soaked for 60 min. The mixture was extracted on an oscillating mixer for 30 min and ultrasonically extracted for 30 min. After centrifugation, 2.0 mL of the supernatant was taken and 50 μL of the mixed solution with internal standard of isotope was added, and the mixture was diluted with phosphate buffer solution to 20.0 mL to obtain the sample solution. The sample solution was passed through the immune affinity column at a flow rate of 1-3 mL·min-1. The immune affinity column was eluted with 5 mL of water, and all effluent was discarded. Then the immune affinity column was eluted with 2 mL of methanol-acetic acid (98+2) solution for 2 times, and all the eluent was collected in the test tube. The eluent was evaporated to near-dryness by N2-blowing at 50℃, and 0.5 mL of acetonitrile-water (1+9) solution was used to dissolved the residue. After centrifugation, the supernatant was analyzed by ultra-high performance liquid chromatography-tandem mass spectrometry. Stable isotopes 13C internal standards were used to correct the loss of sample purification and ionization process and eliminate matrix effect. Linear relationships between the ratios of peak areas of 17 mycotoxins to peak areas of the internal standards and the mass concentration of 17 mycotoxins were found in definite ranges, with detection limits (3S/N) in the range of 0.1-2.0 μg·L-1. Test for recovery was made by standard addition method on the base of blank Chinece herbal medicine sample, giving results in the range of 84.3%-104%, and RSDs (n=6) ranged from 1.9% to 13%.

       

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