Development Of An Immunoassay For Mitragynine

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Date
2016-05
Authors
Lee, Mei Jin
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Abstract
Kratom is a tropical plant used in traditional medicine for pain relief and to treat diarrhea. However, in Malaysia kratom is commonly misused as a recreational drug due to its central nervous system stimulatory effect and its frequency of abuse has been on the rise since 2005. Mitragynine (MG) an active ingredient in kratom has been proven to be addictive. Thus, possession of kratom is now illegal and controlled under the Poisons Act 1952. Therefore, a rapid screening urine test needs to be developed. For effective enforcement to monitor the kratom abuse, the main objective of this project was to develop an immunoassay for the detection of mitragynine residues and its metabolites in human urine. To raise anti-mitragynine antibodies, mitragynine was conjugated to the carrier protein, cationized-bovine serum albumin (cBSA) directly using the Mannich reaction. The synthesized antigen was injected into two rabbits to raise polyclonal antibodies against mitragynine. The antibodies were harvested a week post the (2nd) booster immunization. Horseradish peroxidasemitragynine (HRP-MG) conjugate was synthesized via Mannich reaction and used as a tracer. These antibodies and enzyme-conjugate were optimized for antibody characterization. The antibody assay using HRP-MG produced an IC50 of 8.38 ng/mL. The antibody cross-reacted with 7α-hydroxy-7H-mitragynine (82.65%), speciociliatine (63.20%), and paynantheine (54.35%) using HRP-MG. The immunoassay developed showed a limit of detection (LoD) of 15.05 ng/mL (ppb) and a limit of quantification (LoQ) of 27.23 ng/mL (ppb) in urine whereby the urine samples were diluted 10 times with dilution buffer. The variation of intra-day and inter-day assay results ranged from 1.50 – 8.05%. A liquid chromatography tandem mass spectrometry (LC-ESI-MS/MS) method was developed and validated for the detection of mitragynine and its metabolites in human urine. Therefore, the developed immunoassay was validated and its results correlated with the LC-ESI-MS/MS data. Ten positive human urine samples were screened and quantified using the developed immunoassay method and their results compared with that of the LC-ESI-MS/MS method showed good correlation of R2 = 0.7426. Data collected from the immunoassay showed positive urine samples with total concentration ranging from 6.62 – 81.51 μg/mL (ppm). These positive samples analysed with the LC-ESI-MS/MS showed 0.45 – 9.81 μg/mL (ppm) of mitragynine, 5.52 – 24.23 μg/mL (ppm) of 9-O-DM-MG, and 4.01 – 14.85 μg/mL (ppm) of 16-COOH-MG thus, giving a total concentration range of 11.00 – 44.35 μg/mL (ppm). Therefore, it was concluded that an immunoassay was successfully developed and validated for the detection of mitragynine and its metabolites in human urine. The validated LC-ESI-MS/MS method was suitable to be used as a confirmation method.
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To develop an immunoassay for the detection of mitragynine residues , and its metabolites in human urine.
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