The Validation of Mitragynine And 7-Hydroxymitragynine Determination in Postmortem Blood by Chloroform Extraction

Kratom (Mitragyna speciosa) is traditionally used for medicinal purposes and acceptance as a medicinal plant with recently legal changes. Therefore, Kratom abuse has emerged as a significant public health concern in Thailand, particularly there is linked to traffic accidents. Mitragynine, and 7-hydroxymitragynine are effectively measured in human samples by using LC-MS/MS. The aims of this study were conducted on validations of chloroform use instead of methanol on blood mitragynine, and 7-hydroxymitragynine extraction prior analyzed by on LC-MS/MS.  The simulated postmortem blood was the pooling of ten expired blood units and added with different concentrations of mitagynine and 7-hydroxymitragynine (1.0-100 ppb), were extracted by chloroform and analyzed by LC-MS/MS. The results showed that the running time was about 10 min and the retention time (RT) of 7-hydroxymitragynine and mitragynine were 3.631 and 4.703 min, and each m/z profiles were correlated. The linearity of standard curves for mitragynine and 7-hydroxymitragynine were preferable. The LOD and LOQ of both metabolites were within 1.0 ppb as lowest point of standard concentration, whereas the standard concentrations at 10.0, 50.0 and 80.0 ppb were high linearity and preferable for the analytical range. The %CV of precision tests included within a day and between five days at all concentrations were acceptable by within ±20%. The peak area of mitragynine and 7-hydroxymitragynine within postmortem blood (n = 4) by different extractions, were statistically different (p = 0.012), which were implied that the yield of mitragynine and 7-hydroxymitragynine within postmortem blood extracted by chloroform was higher than by methanol.