Analytical method validation with development for the detection and quantification of kratom alkaloids using...

In this investigation, a highly sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay was designed both to develop and to detect kratom alkaloids that are naturally occurring organic compounds present in the foliage and flowers of the Mitragyna speciosa tree.

We utilize a deuterated analog of mitragynine (mitragynine-D3) as an internal standard (IS) that is spiked into and extracted from kratom extracts using liquid-liquid extraction with ethanol. Water and acetonitrile with 0.1 % formic acid constituted the mobile phase operated at a flow-rate of 0.75 mL/min through a Luna 5 μm C18(2) 100 Å, LC Column 150 × 4.6 mm analytical column and kept at 40 °C. The gradient program started at 0 min with 20 % B, then at 2 min increased to 43.7 % at 12.50 min. We used a period of 12.50 to 14.50 min, increased to 90 % B and held for 19 min, used for washing. Then, an equilibration period of the mobile phase B was maintained at 20 % for 25 min. The LC-MS/MS detection was carried out by electrospray positive ionization in the multiple-reaction monitoring (MRM) mode.

We established an LC-MS/MS method to simultaneously quantify multiple kratom alkaloids. The optimized MRM transitions are mitragynine, speciogynine, speciociliatine, and mitracillatine (m/z 399.20 → 174.00); corynantheidine (369.20 → 144.05); paynantheine (397.20 → 174.00); 7-OH mitragynine (415.20 → 190.00); ajmalicine (353.00 → 144.05); mitraphylline (369.00 → 159.95); and the IS (402.20 → 177.10). Retention times ranged from 6.15 to 11.98 min. The assay (total run time 25 min) had excellent linearity over the concentration range of 5-100 ng/mL (R² > 0.99), with a Lower Limit of Quantification of 5 ng/mL and Limit of Detection 1.5 ng/mL with a two μL injection. Analytes were well-resolved with the method, which exhibited acceptable intra- and inter-day accuracy and precision, along with confirmed stability under various conditions. Application of kratom extracts yielded reproducible and reliable quantification of alkaloid content.

The findings provide a standard analytical technique for testing kratom-based drug release, dissolution, comparison of different kratom products, and development of formulation. This method is also applicable for regulatory compliance and for producing validated results in fields like kratom adulteration that are unregulated and non-standardized.