SPDS EXPERT REVIEW PANEL

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

• (+) Acceptable chromatography • (-) Arcane mobile phase using unconventional modifiers (IPA)

• (-) Some justification for using formic acid – especially in the light of what is stated about its potential to facilitate conversion of trans- to cis-yangonin – would be welcome. • (-) The use of 25-cm columns to develop a new method cannot be faithfully applauded to; • (-) Absence of temperature control during separation should no longer treated as a testament of a robust method; it is beneficial to thermostat the column • (-) Preparation of standard and sample solutions is not described clearly, including dilution volumes, the need for repeated sonications • (-) Two basic easy-to-perform tests should be recommended in method development: (i) PTFE filter retention study and (ii) solution stability study. The effect of prolonged sonication should ideally be investigated as well. • (-) The specification of a 4-place balance is clearly insufficient considering the standard amounts weighed • (-) Dilutions of the standard solutions must be including micropipettes to deliver the specified sub-mL volumes. Given that the diluent is methanol, the use of micropipettes should be generally discouraged as it may result in a very substantial error. If absolutely necessary, positive displacement pipettes, or a calibrated syringe could be recommended. • (-) The inconsistent diluents employed in standard and sample solution preparation: methanol, acetonitrile, then 70% acetonitrile are puzzling. Ideally, the solvents should be the same for the samples and the standards. • (-) The injection solvent may be too strong, especially for the standard solutions. • (-) The decision to modify the separation to address cis-yangonin issue has led, arguably to a more substantial coelution of yangonin and desmethylyangonin. • (-) If indeed there is conversion of yangonin to a cis-form, more work should be expended: explore the kinetics of the process, better yet, find a solvent where such conversion is minimal

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

See above

G. Any general comments about the method?

See above and below

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.

No.

Method should benefit from additional development to address numerous points specified in IV (E), and then from a more rigorous validation that would address:  Proper precision, intermediate precision and accuracy/recovery studies  Matrices other than supercritical extract, as detailed in SMPR