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Purpose: To develop a solid phase extraction (SPE) method that utilizes reverse-phase high performance liquid chromatography (RP-HPLC) to determine mitragynine (MG) in rat and human urine, and to investigate the influence of caffeine (CF) on urinary excretion of MG in rats. Methods: A two-dimensional wash-elute study was conducted using Oasis ® HLB cartridge. The optimized SPE procedures consisted of washing with 5 and 70 % methanol containing 2 % ammonium hydroxide and eluting with methanol containing 2 % acetic acid. The SPE-HPLC method was validated according to United States Food and Drug Administration guidelines. Two groups of rats were used for the study and received an oral administration of either alkaloid extract (AE) of kratom (100 mg/kg), or AE (100 mg/kg) combined with CF (25 mg/kg). The 24-h urine samples after administration were analyzed using the developed method for the content of MG excreted. Results: Validation indicate good linearity ( r > 0.9991) and high precision in rat (1.18 - 5.97) and human urine (0.67 - 3.41). Accuracy for rat and human urine ranged from -9.11 - 19.64 and -7.20 - 13.72 %, respectively. Recovery of MG ranged from 92.75 - 100.83 %. Co-administration of AE and CF significantly increased urinary excretion of MG. Conclusion: The developed SPE method is simple, fast and reliable, and can be suitably applied to pharmacokinetic studies Keywords: Mitragynine, Mitragyna speciosa , Solid phase extraction, Caffeine, Urine

Solid phase extraction method for determination of mitragynine in urine and its application to mitragynine excretion study in rats receiving caffeine