SPDS EXPERT REVIEW PANEL

D. Based on the supporting information, is the method written clearly and concisely? If no, please specify the needed revisions.

Generally: The method is well described and substantively prepared. The project of the method is well integrated and includes a clear and concise description.

My remarks and recommendations to the method are as follows: 1. Identification of the components of the samples Authors presents results in the appendix as: - Chromatograms and UV-spectra (pp. 15-19/50, Appendix 10.1 and 10.5. Appendix 5 – Chromatograms); - by comparison of the spectra of the analytes with those in spectra libraries - to confirm their presence in the samples (pp. 21-23/50, Appendix 10.1). Could Authors add determination of the purity of peaks (If possible - examples)? 2. Measurement of Resolution The authors presented (figure 19) manual determination of the resolution between Methysticin and Dihydromethysticin (on page 24/50). The authors calculated the Rs values on the basis of chromatograms in a manual manner. It seems to me that there is a possibility to calculate the Rs value automatically (in the software depending on the type of report). The resolution of the peaks Methysticin and Dihydromethysticin is Rs=1.26. Baseline resolution corresponds to Rs > 1.5 for bands of similar size. 3. Graphs of the working range (pages 30-34/50, 10.4. Appendix 4): Could authors add standard deviation values of the "a" and "b" in equations of the regression line. 4. Matrix Some matrices have been omitted, or their suitable results are not presented in the described method.

E. Based on the supporting information, what are the pros/strengths of the method? F. Based on the supporting information, what are the cons/weaknesses of the method?

The developed method was evaluated following the definitions of AOAC SMPR 2018.005 with respect to values of LOD and LOQ, Linearity, Repeatability,

Reproducibility, Recovery. Analytical data was collected for all compounds/matrix combinations listed in AOAC SMPR 2018.005.

Authors may evaluate matrix effect and influence of them on the results.

G. Any general comments about the method?

I have no additional comments.

Recommendation for the Method

Do you recommend this method be adopted as a First Action and published in the Official Methods of Analysis of AOAC INTERNATIONAL? Please specify rationale.

In my opinion, the AOAC Candidate Method #KAV-004 entitled ‘HPLC analysis of flavokavins and kavapyrones from Piper methysticum’ described by Meissner and Haberlein (et al.: Mathias Schmidt, Steffen Radtke, Gabriele Adis, Eva Maute, Katherina Kardos, and Janina Schnabel) is applicable for the detection and quantitation of the content of kavapyrones and flavokavains in dried, ground, kava roots prepared as an ethanolic extract by HPLC-DAD. The AOAC Candidate Method #KAV-004 can be adopted (after revision) as a First Action Methods of Analysis of AOAC INTERNATIONAL.