Determination of kratom (Mitragyna speciosa) alkaloids in kratom cocktail using anti-mitragynine monoclonal antibody-based ELISA

Mitragynine is an exclusive indole alkaloid which found in leaves of kratom or Mitragyna speciosa (Roxb.) Korth. (Rubiaceae). This plant has been traditionally used to combat fatigue by chewing the fresh leaves or brewing tea. In the recent years, the kratom cocktail has been abuse in the teenagers and become a major concern in Thailand and Malaysia. It typically comprised of kratom leaves boiling in water and mixed with other substances such as cola drink, cough syrup, tranquilizer and even mosquito coil. Therefore, a simple and reliable analytical method is needed for the qualitative and/or quantitative analysis of the active compound, mitragynine, in the cocktail. We previously reported the establishment of monoclonal antibody (mAb) against mitragynine and developed an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) for determination of mitragynine in kratom leaves samples. In the present study, we applied the developed ic-ELISA using anti-mitragynine mAb to quantify mitragynine in kratom cocktail. The compounds which might be adulterated in cocktail were tested for the cross-reactivity. The benzodiazepines including alprazolam, diazepam and lorazepam and the mosquito coil showed the moderate cross-reactivity as 44%, 40%, 15% and 31%, respectively. In order to reduce the matrix interference, the cocktails were pretreated by liquid extraction using chloroform under basis condition. The recovery of mitragynine was 73% when compare with untreated cocktail. However, the ic-ELISA showed the results of total alkaloids contents (mitragynine, paynantheine and speciogynine) in kratom cocktail when compare with HPLC method.