Expert Review Panel for Dietary Supplements

II. Review of the Method Only 1. Does the applicability of the method support the applicability of the SMPR? If not, please explain what is missing.

The SMPR applicability states, "Methods must be able to quantitate mitragynine, 7-hydroxymitragynine, and separate other relevant indol alkaloids of Mitragyna speciosa...". "Methods should be able to quantitate the analytes for which appropriate standards are available and account for interfering compounds." The rreeport's Results and Discussion section stated, "The resolution was >1.5 for 7-OH mitragynine and mitragynine to close eluting components (Figure 2)", however, no 7-OH mitragynine peak is seen in Figure 2 at the scales presented. This is followed with a description of requiring the use of spiked samples to monitor acceptable separation of the target analytes. If accepted, the official method should include this monitoring step and how to respond if the target analytes are not resolved from interference. Also, the protocol on how to evaluate if a separation is acceptable. SMPR lists a 7-OH mitragynine LOQ of 0.005% with the method determined LOQ being 0.011%. Slightly higher but likely acceptable since this is 10x below the analytical range. The method's LOQ for mitragynine is met. SMPR lists a 7-OH mitragynine LOD of 15ppm with the method determined LOD 40ppm. Slightly higher but still well below the analytical range. The method's LOD for mitragynine is met. The authors stated the procedure of determining MDL & LOQ used a neat sample diluted to low concentrations to "account for matrix effects". However, diluting the matrix alongside the targeted analytes reduces matrix effects by reducing potential interferences. Ideally having a sample of material with low target analytes or spiking a similar matrix with target analytes best assesses MDL/LOQ. I believe the used approach underestimates LOD/LOQ which is an issue with the reported LOD/LOQ for 7-OH mitragynine being higher than stipulated in the SMPR. The spike/recovery study results met SMPR requirements. Comfrey leaves were used as a matrix blank and spiked at 3 levels spanning the listed SMPR ranges. NOTE: this approach can be used to better assess LOD/LOQ. The analytical range was reported as 1-500 ug/mL (mitragynine) with the calibration range spanning 25-500 ug/mL. SMPR lists the needed range as 0.1 - 15% (wt/wt) and converting the calibration range wt/vol to wt/sample wt was not possible because the sample prep stated a "dried extract" was diluted 1:10 which was confusing. Can the authors supply the conversion factors. Also, the analytical range for 7-OH mitragynine was not listed but the calibration range was 2-25 ug/mL.

2. Does the analytical technique(s) used in the method meet the SMPR? If not, please specify how it differs from what is stated in the SMPR.

3. Are the definitions specified in the SMPR used and applied appropriately in the method? If no, please indicate how the terms are used.

Yes for the analytes and materials. Concerns with LOD, LOQ, %recovery are listed above.

The HORRAT portion needs to be adjusted. The Horwitz equation that was used predicts RSD(R) which is reproducibility between laboratories. These predicted values were compared to the author's within-lab data (repeatability). The calculated HORRAT values therefore need to be multiplied by 1.5 to 2.0. This will raise the reported HORRATs.