SPDS EXPERT REVIEW PANEL

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

• Chromatography looks good, but it would be desirable to see actual sample chromatograms. It would be ultimately beneficial to transfer the method to a core-shell column and to achieve more reliable separation between Rg1 and Re. • The extraction procedure for the root powder omits the final 1-mL rinse of the sample that is used in reference [1]. This is a minor point, but worth addressing. • Hesitations arise from deriving the total post-hydrolysis volume from a combination of 70% methanol, and two aqueous solutions. The magnitude of error may be small, but this is a source of uncertainty that could be eliminated. E.g., procedure could be conducted in a 10-mL volumetric by combining 8 mL sample solution with 800 µL each of base hydrolysis solution and neutralizing solution, and subsequent adjustment to volume. • Another instance of small-volume jitters: filtration of the 1 mL of post- derivatization solution through a syringe filter. It would be ideal to confirm non- retention of analytes on the filter; the common practice is to filter a larger volume and discard the first couple of mLs of the filtrate, but it would be beneficial to check. • Are samples thermo-labile? Can sitting on the bench during the hydrolysis step and repeated sonication affect recovery? A short study can answer this. • Analytical column conundrum: 1. The reference [1] lists the column as C18(2) – presumably, Phenomenex Luna (?); 2. In reference [2] it is listed as Phenomenex Luna C18; 3. In the method draft the column is alternatively referred to as Poroshell 120 Bonus-RP (p. 47) or C18(2) (p. 48). It is likely that the former is a typo, although it could be a chromatographer's slip, and it would be desirable to try implementing the method on a superficially porous column. • Not certain why the method would instruct to make single injections of each test solution; most labs have internal SOPs and most analysts are comfortable with at least duplicate injections.

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

None substantial, but minor tweaks are proposed above

G. Any general comments about the method?

Despite the fact that the publication in JAOAC presumes rigorous peer review, it would be desirable to have access to raw analytical data, with weights, peak areas and concentrations, not just the summary tables

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.

Yes