RESEARCH ARTICLE Immunochromatographic assay for the detection of kwakhurin and its application for the identification of Pueraria candollei var. mirifica (Airy Shaw & Suvat.) Niyomdham Suppalak Phaisan 1 | Gorawit Yusakul 1 | Poomraphie Nuntawong 2 | Seiichi Sakamoto 2 | Waraporn Putalun 3,4 | Satoshi Morimoto 2 | Hiroyuki Tanaka 5 1 School of Pharmacy, Walailak University, Thaiburi, Thasala, Nakhon Si Thammarat, 80160, Thailand 2 Department of Pharmacognosy, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka, 812-8582, Japan 3 Faculty of Pharmaceutical Sciences, Khon Kaen University, Khon Kaen, 40002, Thailand 4 Research Group for Pharmaceutical Activities of Natural Products Using Pharmaceutical Biotechnology (PANPB), National Research University-Khon Kaen University, Khon Kaen, 40002, Thailand 5 Department of Pharmacognosy and Kampo, Faculty of Pharmaceutical Sciences, Sanyo- Onoda City University, 1-1-1 Daigaku-Dori, Sanyo-Onoda City, Yamaguchi, 756-0884, Japan Correspondence Asst.Prof. Gorawit Yusakul, Ph.D., School of Pharmacy, Walailak University, Thasala, Nakhon Si Thammarat 80160, Thailand. Email: gorawit.yu@mail.wu.ac.th Funding information Research on Food Safety, the Health and Labour Sciences Research Grants, the Ministry of Health, Labour and Welfare, Japan; Thailand Research Fund and Office of the Higher Education Commission, Thailand Abstract Introduction: The plant Pueraria candollei var. mirifica (Airy Shaw & Suvat.) Niyomdham (PM), known by its common Thai name as white Kwao Krua, is sometimes misidentified because it presents similar botanical characteristics to those of Butea superba (red Kwao Krua). The phytochemicals in PM are phytoestrogens in the class of isoflavonoids, but Butea superba contains flavonoids that exhibit androgenic and antiestrogen effects. Objectives: This research aims to develop a simple analytical method for identifica- tion and to differentiate PM from red Kwao Krua and other Pueraria species. Methods: A gold nanoparticle-based immunochromatographic assay (ICA) was developed for the detection of kwakhurin (Kwa), a unique compound found in PM. The parameters, including sensitivity, accuracy, precision, and specificity, were validated. All samples were analyzed using ICA and high-performance liquid chroma- tography with UV detector (HPLC-UV). The results of the two methods were com- pared for consistency checking. Results: The cutoff limit of Kwa detection was 160 ng/mL, which was lower than in the HPLC-UV method. The repeatability and reproducibility of the ICA preparation and assembly showed high precision. The cross-reactivity to related isoflavonoids was less than 0.32%, which implied high specificity of the ICA for Kwa. Moreover, false-positive and false-negative results from other plant extracts were not observed. Conclusion: The developed ICA is applicable for distinguishing PM from red Kwao Krua and other Pueraria species. This simple analytical method can be applied for the identification of raw PM materials in the industrial and agricultural sectors. KEYWORDS Butea superba, identification, immunochromatographic assay, kwakhurin, Pueraria mirifica, white Kwao Krua 1 | INTRODUCTION The plant Pueraria candollei var. mirifica (Airy Shaw & Suvat.) Niyomdham (PM) is known by its common Thai name as white Kwao Krua. PM benefits the health of menopausal women because of the estrogenic activity of its chemical compounds. Pueraria species such as PM, P. lobata, P. phaseoloides, and Glycine max. contain common iso- flavonoids, such as puerarin, daidzin, daidzein, genistin, and genistein. Received: 15 June 2020 Revised: 4 August 2020 Accepted: 1 September 2020 DOI: 10.1002/pca.2998 Phytochemical Analysis. 2020;1–9. wileyonlinelibrary.com/journal/pca © 2020 John Wiley & Sons, Ltd. 1