Development of Methyl Ester Antibody-Based Competitive Indirect ELISA for Quantitative Detection of Mitragynine in Human Urine

Radhiahtul Raehan Mustafa; Rashidah Sukor; Siti Mariam Mohd Nor; Nazamid Saari; Aliah Zannierah Mohsin

doi:10.1021/acsomega.3c02734

Development of Methyl Ester Antibody-Based Competitive Indirect ELISA for Quantitative Detection of Mitragynine in Human Urine

ACS Omega. 2023 Nov 17;8(50):47412-47426. doi: 10.1021/acsomega.3c02734. eCollection 2023 Dec 19.

Authors

Radhiahtul Raehan Mustafa¹, Rashidah Sukor^{2

3}, Siti Mariam Mohd Nor⁴, Nazamid Saari², Aliah Zannierah Mohsin²

Affiliations

¹ Academy of Islamic Civilisation, Faculty of Social Sciences and Humanities, Universiti Teknologi Malaysia, 81310 Skudai, Johor, Malaysia.
² Department of Food Science, Faculty of Food Science and Technology, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia.
³ Laboratory of Food Safety and Food Integrity, Institute of Tropical Agriculture and Food Security, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia.
⁴ Department of Chemistry, Faculty of Science, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia.

Abstract

Mitragynine is the main psychoactive compound of Mitragyna speciosa Korth. (kratom). This alkaloid could render psychotropic effects and is often misused as a substitute for commercial drugs. Nowadays, the increasing popularity of kratom has led to the development of a rapid and effective detection method. The detection of mitragynine in a biological sample such as urine requires a highly sensitive and specific method due to the complex nature of mitragynine in urine. Enzyme-linked immunosorbent assay (ELISA) is well known as a rapid screening method for biological samples. In this study, a competitive indirect ELISA was successfully developed using MG-22-OCH₃ IgG as a detection antibody for mitragynine in human urine. The mitragynine immunoassay showed a limit of detection and a limit of quantification of 0.412 and 1.25 μg/mL, respectively. The measurement range was between 0.01 and 100.0 μg/mL^, with a minimal inhibition (IC₅₀) value of 0.152 μg/mL. The developed ELISA was validated using a gold method such as high-performance liquid chromatography-mass spectrometry (HPLC-MS). The percentage of recovery and the coefficient of variation (CV) for the ELISA and LCMS/MS analyses were 84.0-95.70%, 99.20-112.0%, 7.69-9.78%, and 2.86-6.62%, respectively. This indicates that the developed ELISA is a reliable method that can be used as a rapid approach for quantifying mitragynine content in biological samples.