Developing an ultrasensitive immunochromatographic assay for authentication of an emergent fraud aminopyrine in herbal tea
Introduction
Herbal tea, defined as aqueous extracts from different parts of plants (roots, stems, leaves, flowers, fruits, and seeds), is consumed medicinally for disease prevention and nutrition improvement (Jin et al., 2016). As one of the first group of Chinese intangible cultural heritage projects, herbal tea has a very long history of cultural traditions (Zhao et al., 2013). With the development of living standards, the growing public awareness of health has boosted the rapid prosperity of the herbal tea market both in China and around the world (Fu et al., 2018). In recent years, some herbal teas claiming to have “exclusive efficacy” in China's Guangdong and Guangxi provinces have been inspected by the Administration of Market Regulation and found to contain prohibited western drugs such as aminopyrine (AMI) (Food China, 2021). However, aminopyrine, a nonsteroidal anti-inflammatory drug, belongs to the pyrazolone class, which is recently found illegally used for adulteration of traditional Chinese herbal teas because of the significant effects on clinical treatment of fever, headache, rheumatoid arthritis, and other symptoms (Dong et al., 2021). And aminopyrine is also classified as the first echelon of illegal added drugs detected in herbal tea by Guangdong Institute of Food Inspection because of its high detection rate, toxicity and potential safety hazards. Any herbal tea spiked with aminopyrine is illegal. This fraud is motivated by high commercial profits, which claims that such products can exhibit strong activity in treating cold and fever (Zhang et al., 2019). Long-term exposure to this compound will cause adverse side effects including allergic reactions, strong spasmolytic effect on smooth muscle of peripheral blood vessels, irritability, liver and kidney damage even gastrointestinal bleeding. (Karinen et al., 2015). Based on the drug safety concerns, the Food and Drug Administration (FDA) officially withdrew aminopyrine and its related drugs from the market in 2000. Production and use of aminopyrine have been banned in most countries (Huang et al., 2020). Considering the risk to public health, it is necessary to establish an analytical method for the detection of aminopyrine in herbal teas.
Currently, the detection methods for illegal addition in herbal tea mainly include thin layer chromatography (TLC) (Sherma & Rabel, 2018), liquid chromatography-tandem mass spectrometry (LC-MS/MS) (Ma et al., 2018), high performance liquid chromatography (HPLC) (Wang et al., 2021), ultrahigh performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) (Zhang et al., 2019), etc., which has the advantages of high accuracy, specificity, and reproducibility. However, the prolonged run time, high cost, sophisticated equipment, and skilled operators limit their application in rapid, on-site detection (Liu et al., 2022). Gold nanoparticles based immunochromatographic assay (AuNPs-ICA) is an alternative and mature technique for the detection of food contaminants due to its exemplar simplicity, rapidity, and low-cost (Li et al., 2021a). However, there is not yet any immunoassay for aminopyrine reported so far, it is urgent to develop a simple and rapid AuNPs-ICA.
In this study, a specific antibody against aminopyrine was obtained for the first time through a rational hapten design strategy. This was followed by the development of an ultrasensitive AuNPs-ICA for the authentication of aminopyrine in herbal teas, which was applied to authenticate blind real samples collected from the retail market and exhibited good reliability with the confirmatory method (LC-MS/MS).
Section snippets
Reagents and instruments
Aminopyrine, 1,5-dimethyl-4-(methylamino)-2-phenyl-1,2-dihydro-3H-pyrazol-3-one (DIM), 4-(bromomethyl) phenylacetic acid phenacyl ester, ethyl bromoacetate, ethyl 4-bromobutyrate, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), N,N-dimethylformamide (DMF), N-hydroxysuccinimide (NHS), ovalbumin (OVA), bovine serum albumin (BSA), and lactoferrin (LF) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Chloroauric acid, trisodium citrate, and polyvinyl pyrrolidone (PVP) were
Hapten design strategy
The structure of hapten plays a decisive role in the sensitivity and specificity of the antibody (Liang et al., 2022). In general, an appropriate hapten should mimic the molecular structure, stereochemistry, and electron distribution of the target analyte as far as possible (Cao et al., 2019). For small molecules like aminopyrine, a rational spacer arm is usually introduced to facilitate specific epitopes away from the carrier protein and maximize its exposure to the immune system (Wang et al.,
Conclusions
In this work, a specific antibody against aminopyrine was obtained by rational hapten design strategy, and an ultrasensitive AuNPs-ICA was established, which enabled the authentication of illegal aminopyrine addition in herbal tea within 10 min. The cut-off value of developed AuNPs-ICA for herbal tea samples was 100.00 ng/mL, the LOD and LOQ were 4.80 and 5.71 ng/mL, respectively. The sensitivity was about 20 times higher than that of the LC-MS/MS for herbal tea, which provided the possibility
CRediT authorship contribution statement
Yalin Fang: Conceptualization, Data curation, Formal analysis, Investigation, Writing – original draft. Zian Wang: Methodology, Validation. Qiqi Quan: Data curation, Software. Zhaodong Li: Conceptualization, Investigation. Kangliang Pan: Methodology, Validation. Yi Lei: Writing – review & editing, Validation. Xiaojun Yao: Investigation, Writing – review & editing. Xiangmei Li: Conceptualization, Writing – review & editing. Xing Shen: Conceptualization, Writing – review & editing. Anastasios
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
This work was financially supported by the National Scientific Foundation of China (31871883), HeYuan Planned Program in Science and Technology (2019041), Generic Technique Innovation Team Construction of Modern Agriculture of Guangdong Province (2021KJ130, 2022KJ130), National Key Research and Development Program of Thirteenth Five-Year Plan (2017YFC1601700).
We are grateful for their revision help by professor Boris B. Dzantieve and Dr. Anatoly V. Zherdev from A.N. Bach Institute of
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