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This reviewer is in no position to comment on the biological significance of the specific *** studied by the authors but there is no question that the work presented here was conducted with considerable rigor. From an analytical perspective, even though much of the chromatography has relied on the use of previously described procedures, the authors went the extra step to further fine tune the methodology in order to improve the quality of the chromatography and the separation of the analytes. The calibration curves show excellent reproducibility, and the authors have taken care to produce a fully validated method (determined matrix effects, freeze/thaw cycles to establish analyte stability, etc.) that can be adapted by other users as well. The paper is very well written and easy to read.

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