Expand sample processing methodology, especially wrt blanks and BMIS

[wkumler]

Reviewer 2 | 04 Oct 2024 | 12:20

• [x] Blanks are mentioned but it is not shown how these were used for the data analysis.
• [x] In line 142 the method for the evaluation of the matrix effect is mentioned, but no results are given.
• [x] The experimental could be clearer in line 132 ff “The samples were then bead-beaten three times, followed by triplicate extraction of the aqueous layer...” Was the organic layer collected or was the aqueous layer collected (as suggested by the rest of the sentence?). Was phase separation occurring spontaneously or was a centrifugation required? Was separation done by pipetting?
• [x] Line 177: I would rather state “targeted analytical methods”. Metabolomics is by definition rather untargeted.
• [x] Line 185: The data are discussed quantitatively but for some compounds no standard was available. The claim that results were normalized “to the best match standard”. How was this decided? Are the different response-factors in the MS considered?

[wkumler]

Discussion of blanks being used to visually assess background was added with 497272a

[wkumler]

Rephrasing of organic/aqueous extraction added with 8563bec

- The samples were then bead-beaten three times, followed by triplicate extraction of the aqueous layer and addition with fresh methanol/water mixture with additional bead-beatings in between. Samples were then dried down under ultrapure nitrogen gas and warmed using a Fisher-Scientific Reacti-Therm module.
+ The samples were then bead-beaten three times, followed by triplicate extraction of the aqueous layer into a separate vial after spontaneous separation and replacement with fresh methanol/water mixture using a glass Pasteur pipette with additional bead-beatings in between. The aqueous fraction was then dried down under ultrapure nitrogen gas and warmed using a Fisher-Scientific Reacti-Therm module.