Determination of Sildenafil in Health-care Foods by Ultrasensitive Chemiluminescence Enzyme Immunoassay

CHENG Zhi; ZHANG Yong-yi; YANG Jin-yi; XU Zhen-lin; WANG Hong; LEI Hong-tao; SUN Yuan-ming; SHEN Yu-dong

doi:10.19969/j.fxcsxb.21022602

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Determination of Sildenafil in Health-care Foods by Ultrasensitive Chemiluminescence Enzyme Immunoassay

Experimental Techniques and Methods | 更新时间：2023-02-13

- Determination of Sildenafil in Health-care Foods by Ultrasensitive Chemiluminescence Enzyme Immunoassay
- Journal of Instrumental Analysis Vol. 40, Issue 10, Pages: 1503-1508(2021)
- 作者机构：
  
  华南农业大学食品学院广东省食品质量安全重点实验室，广东广州 510642
- 作者简介：
- 基金信息：
- DOI：10.19969/j.fxcsxb.21022602
  CLC：
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程智,张咏仪,杨金易等.保健食品中西地那非化学发光免疫分析方法研究[J].分析测试学报,2021,40(10):1503-1508.

CHENG Zhi,ZHANG Yong-yi,YANG Jin-yi,et al.Determination of Sildenafil in Health-care Foods by Ultrasensitive Chemiluminescence Enzyme Immunoassay[J].Journal of Instrumental Analysis,2021,40(10):1503-1508.
程智,张咏仪,杨金易等.保健食品中西地那非化学发光免疫分析方法研究[J].分析测试学报,2021,40(10):1503-1508. DOI： 10.19969/j.fxcsxb.21022602.

CHENG Zhi,ZHANG Yong-yi,YANG Jin-yi,et al.Determination of Sildenafil in Health-care Foods by Ultrasensitive Chemiluminescence Enzyme Immunoassay[J].Journal of Instrumental Analysis,2021,40(10):1503-1508. DOI： 10.19969/j.fxcsxb.21022602.

摘要

针对保健食品中西地那非药物的非法添加问题，该研究采用辣根过氧化物酶（HRP）与鲁米诺为信号输出系统，结合直接竞争模式探索了保健食品中西地那非的直接竞争化学发光酶免疫分析方法。基于特异性多克隆抗体，通过逐步优化策略，确定最佳免疫分析条件为：包被原质量浓度为41.67 ng/mL，酶标抗体质量浓度为1.25 μg/mL，封闭液和洗涤液的吐温－20含量为0.05%，稀释液的甲醇添加量为5%，竞争反应时间为40 min。在该条件下，建立了西地那非的直接竞争化学发光免疫分析方法，该方法对西地那非的半抑制浓度（IC,50,）为0.17 ng/mL，线性检测范围（IC,20,～IC,80,）为0.024 ~ 1.21 ng/mL，检出限（IC,10,，LOD）为0.008 ng/mL。与他达那非等功能类似物无显著交叉；西地那非样品的加标回收率为82.0%～114%，相对标准偏差均小于15%。盲样检测结果与HPLC－MS/MS确证方法具有良好一致性，说明该方法准确可靠，适用于样品中西地那非的快速筛查。

Abstract

To address the issues of sildenafil adulteration in health-care foods，a direct competition chemiluminescence enzyme immunoassay method（dcCLEIA） was developed for the detection of sildenafil in so-called health-care foods in this work．Horseradish peroxidase（HRP） and luminol were used as signal output system．Based on specific polyclonal antibody，the optimal assay conditions were finalized as follows： the concentration of coating antigen was 41.67 ng/mL，the concentration of antibody was 1.25 μg/mL，the content of Tween－20 in blocking stock solution and washing buffer was 0.05%，the content of methanol in sildenafil dilution solution was 5% and the competitive reaction time was 40 min．Under the optimal conditions，the dcCLEIA method for sildenafil detection was established．The assay showed a half-inhibition concentration（IC,50,） of 0.17 ng/mL，with a linear detection range（IC,20,－IC,80,） of 0.024－1.21 ng/mL，and the limit of detection （LOD，IC,10,） was 0.008 ng/mL．Meanwhile，the cross-reactivity of dcCLEIA towards tadalafil analogues was less than 0.1%．The recovery test results showed that the average recoveries of the developed dcCLEIA for sildenafil in samples ranged from 82.0% to 114%，with relative standard deviation（RSD） less than 15%．The detection results for random blind samples by dcCLEIA had a good correlation with those of high performance liquid chromatography tandem mass spectrometry（HPLC－MS/MS）．The developed dcCLEIA method was accurate and reliable，and it was suitable for the rapid detection of sildenafil in real samples.

关键词

西地那非直接竞争化学发光酶联免疫分析（dcCLEIA）保健食品辣根过氧化物酶（HRP）

Keywords

sildenafildirect competitive chemiluminescence enzyme immunoassay（dcCLEIA）health-care foodhorseradish peroxidase （HRP）

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