Objective: This study aimed to 1) develop and validate a simple, accurate and reproducible high-performance liquid chromatography–tandem mass spectrometry method for determination and quantification of naltrexone and its reduced metabolite (6-beta naltrexol); 2) assess the effect of uremic toxins on metabolic reduction. Method: Sample preparation was conducted through liquid-liquid extraction of analytes spiked in 10 mM Tris-HCl incubation buffer (pH 7.4) using methyl tert-butyl ether. The chromatographic separation of analytes was achieved using Inert Sustain C18 (2.1 x 50 mm, 5 μm) analytical column under isocratic elution of solvent A (water containing 0.1% formic acid) and solvent B (methanol containing 0.1% formic acid) over a run time of 5 min. The analytes were detected by multiple reaction monitoring with electrospray ionization in the positive mode. Results: Excellent linearity was observed for all analytes over the concentration ranges of 100-8,000 ng/mL for naltrexone and 10-800 ng/mL for 6-beta naltrexol. The intra- and inter-day accuracy and precision for analytes were within ±15.0%. The method is accurate and precise and novel in its application to in vitro experiments assessing the effect of uremic toxins on metabolic reduction of naltrexone, which showed that indoxyl sulfate (100 μM) is a significant inhibitor of naltrexone reduction. Conclusion: The developed method is reproducible and accurate and well suited for conducting enzyme kinetic studies assessing metabolic reduction.
Key words: Naltrexone, 6-beta naltrexol, Reduction, LC-MS, Enzyme kinetics, Uremic toxins.