免疫磁珠富集净化-超高效液相色谱法同时测定陈皮中4种黄曲霉毒素

doi:10.3724/SP.J.1123.2015.06001

色谱 ›› 2015, Vol. 33 ›› Issue (12): 1320-1326.DOI: 10.3724/SP.J.1123.2015.06001

免疫磁珠富集净化-超高效液相色谱法同时测定陈皮中4种黄曲霉毒素

邢言言¹, 佟玲², 陈楠², 于治国¹, 赵云丽¹

1. 沈阳药科大学药学院, 辽宁沈阳 110016;
2. 天士力控股集团研究院药物分析所, 天津 300402

收稿日期:2015-06-01 出版日期:2015-12-08 发布日期:2012-06-21
通讯作者: 赵云丽
基金资助:
国家"重大新药创制"科技重大专项(2013ZX09402202).

Simultaneous determination of four aflatoxins in Citrus reticulatablanco by ultra performance liquid chromatography coupled with immunomagnetic beads for enrichment and purification

XING Yanyan¹, TONG Ling², CHEN Nan², YU Zhiguo¹, ZHAO Yunli¹

1. School of Pharmacy, Shenyang Pharmaceutical University, Shenyang 110016, China;
2. Pharmaceutical Analysis Institute of Tasly Holding Group, Tianjin 300402, China

Received:2015-06-01 Online:2015-12-08 Published:2012-06-21

摘要/Abstract

摘要：

将ProtElut NHS(N-羟基丁二酰亚胺)偶联磁珠与抗黄曲霉毒素总量单克隆抗体偶联得到黄曲霉毒素总量免疫磁珠,其具有较好的分散性,良好的磁性能和特异的选择性。本文建立了陈皮中4种黄曲霉毒素的免疫磁珠富集净化,超高效液相色谱(UPLC)检测方法。样品经甲醇-PBS缓冲溶液(2:8, v/v)提取后用免疫磁珠富集净化,1 mL甲醇洗脱;经ACQUITY UPLC HSS T3 C₁₈色谱柱(100 mm×2.1 mm, 1.8 μm)分离,以水和甲醇为流动相梯度洗脱,采用汞/氙灯荧光检测器测定。实验结果表明,4种黄曲霉毒素在各自的线性范围内峰面积与其质量浓度线性关系良好,相关系数(r²)大于0.999;检出限(信噪比为3)为0.013~0.038 μg/kg,定量限(信噪比为10)为0.044~1.2 μg/kg;平均回收率为63.9%~115.0%,相对标准偏差为0.4%~14.2%,均符合痕量分析的要求。该方法简单快速、准确可靠,可用于陈皮中4种黄曲霉毒素的测定。

关键词: 超高效液相色谱法, 陈皮, 黄曲霉毒素, 免疫磁珠

Abstract:

Immunomagnetic beads (IMBs) were formed by coupling the ProtElut NHS (N-hydroxysuccinimide) magnetic beads and anti-aflatoxins monoclonal antibody. The synthesized IMBs presented uniform size, good magnetic property and specific selectivity. In this work, a novel and facile pretreatment method of sample enrichment and purification based on the IMBs for the determination of aflatoxins (AFs) (AFB₁, AFB₂, AFG₁, AFG₂) in Citrus reticulatablanco samples by ultra performance liquid chromatography (UPLC) were developed. The sample was extracted with 20%(v/v) methanol-PBS buffer solution (pH 7.4), followed by a cleanup procedure with the IMBs. The target compounds were eluted using 1 mL methanol. The four afatoxins were separated on an ACQUITY UPLC HSS T3 C₁₈ column (100 mm×2.1 mm, 1.8 μm) adopting a gradient program within 12 min. The UPLC equipped with a fluorescence detector with a special mercury/xenon lamp was used to detect the aflatoxins. The satisfactory correlation coefficients (r²>0.999) of the four aflatoxins were obtained within their respective linear ranges. The limits of detection (S/N=3) were between 0.013 μg/kg and 0.038 μg/kg, and the limits of quantification (S/N=10) were between 0.044 μg/kg and 1.2 μg/kg. The recoveries were in the range of 63.9%-115.0% with the relative standard deviations (RSDs) of 0.4%-14.2%. The validation results meet the requirements of trace assay. It's indicated that the IMBs were good pretreatment alternatives and the developed method is simple, fast, accurate, and can be applied for the simultaneous determination of the four aflatoxins in Citrus reticulatablanco.

Key words: Citrus reticulatablanco, aflatoxins (AFs), immunomagnetic beads (IMBs), ultra performance liquid chromatography (UPLC)

中图分类号:

O658

邢言言, 佟玲, 陈楠, 于治国, 赵云丽. 免疫磁珠富集净化-超高效液相色谱法同时测定陈皮中4种黄曲霉毒素[J]. 色谱, 2015, 33(12): 1320-1326.

XING Yanyan, TONG Ling, CHEN Nan, YU Zhiguo, ZHAO Yunli. Simultaneous determination of four aflatoxins in Citrus reticulatablanco by ultra performance liquid chromatography coupled with immunomagnetic beads for enrichment and purification[J]. Chinese Journal of Chromatography, 2015, 33(12): 1320-1326.

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免疫磁珠富集净化-超高效液相色谱法同时测定陈皮中4种黄曲霉毒素

Simultaneous determination of four aflatoxins in Citrus reticulatablanco by ultra performance liquid chromatography coupled with immunomagnetic beads for enrichment and purification

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