Kratom cocktail or the fatal 4 × 100 formula is defined as a mixture of boiled kratom leaves, cola drink, and cough syrup. In the present study, we focused on application of the indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) using the anti-mitragynine (MG) monoclonal antibody (anti-MG mAb) to the kratom cocktail. The ic-ELISA is a rapid method for quantification of the major kratom alkaloids including MG, paynantheine, and speciogynine in kratom cocktails. Because some matrices or additives may influence the binding affinity between the alkaloids and the anti-MG mAb, a liquid–liquid extraction method using chloroform was used to clean-up samples and minimize any cross-reactivity with anti-MG mAb. The anti-MG mAb showed slight cross-reactivity to caffeine, codeine, morphine, tramadol, and dextromethorphan (<0.5 %), which are also commonly added to a kratom cocktail. When applied to eight different kratom cocktail samples, the ic-ELISA using the anti-MG mAb allowed the determination of the combined kratom alkaloid content in the range of 0.083–576 mg/L, and these values were in agreement with the results of the high-performance liquid chromatography method (R2 = 0.9689). To our knowledge, this is the first report for the quantification of total amounts of kratom alkaloids including MG in kratom cocktail by a simple immunoassay. Because of the sharp rise in kratom cocktail abuse in the world, this method will be a useful tool for detection of kratom cocktail consumption.
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