HPLC-PDA METHOD FOR THE QUANTIFICATION OF MITRAGYNINE IN FRESH KRATOM (<em>MITRAGYNA</em> <em>SPECIOSA</em>) LEAF

Abstract

Kratom (Mitragyna speciosa) is a psychotropic tropical plant that is used as a traditional remedy in Southeast Asia. It has many medicinal properties and has great potential for clinical applications. Mitragynine and 7-hydroxymitragynine (7-OH) are the important pharmacologically active compounds found in kratom with mitragynine as the major alkaloid found in the leaf. We report the development and validation of a high-performance liquid chromatography-photodiode array method for the determination and quantification of mitragynine from M. speciosa fresh leaf extract. Fresh leaf samples were cryogenically ground and freeze-dried before methanol extraction. Gradient elution was conducted using acetonitrile and ammonium bicarbonate buffer (pH 9.5) at a flow rate of 1 mL min^-1. Chromatogram separation was achieved using Kinetex EVO C18 column in a duration of 25 min with MG eluted at 15.75 min. The calibration curves were in a linear relationship with determination coefficients (R²) ≥ 0.99. Mitragynine quantification was calculated based on the calibration curve equation. The precision value (RSD) was less than 2% and accuracy range from 93%–100%. The values for limit of detection (LOD) and limit of quantification (LOQ) were 0.67 μg mL^-1 and 1.51 μg mL^-1, respectively. This method has been validated and found suitable for the quantification of mitragynine in fresh leaf. We found that freeze-drying during sample preparation from fresh leaf yielded more reliable mitragynine quantification, compared with that of conventional oven-drying method.