In the manuscript titled “Chemical Composition Analysis of Qingyan Dropping Pills by UPLC-Q-TOF-MS”, the author deployed an LC-MS method through MRM to determine 87 compounds in Qingyan Dropping Pills (QDP), including various flavonoids, terpenoids, organic acids and esters, tannins, etc.. Fragmentation was utilized to verify the structure of those compounds, despite some isomers with similar structures. Overall, this study addresses a real analytical need, and the reported results can be applied to improve people’s understanding on a traditional Chinese medicine. However, the literature review is not sufficient, and the design of experiment is sloppy, which must be improved. Therefore, this manuscript should be under MAJOR REVISION before its publication. The points to be inproved are listed below:

1.In the introduction part, a citation number “0” was listed. Should be “1”.
2.Literature review on analytical methods are not sufficient. The authors only provided one reference regarding the application of LC/MS in study of traditional Chinese Medicine, which cannot provide the readers a comprehensive understanding of LC, LC/MS and q-TOF’s application to the pharmaceutical study. I recommend the authors read, including but not limited to, the papers list below, and rewrite the second paragraph in the introduction part, to introduce the wide usage of LC/MS techniques in pharmaceutical area: (1) Raman N, Prasad A, Reddy K R. Journal of pharmaceutical and biomedical analysis, 2011, 55(4): 662-667.(2) Guo, T.; Shi, Y.; Zheng, L.; Feng, F.; Zheng, F.; Liu, W., J. Chromatogr. A 2014, 1355, 73 (3) Feng, E.; Ma, X.; Kenttämaa, H. I., Anal. Chem. 2021, 93, 7851. (4) Ma X. Molecules, 2022, 27(19): 6466. (5) Niyonsaba E, Easton M K W, Feng E, et al. Analytical chemistry, 2019, 91(17): 11388-11396. (6) Feng E, Ma X, Kenttämaa H I. . Analytical Chemistry, 2021, 93(22): 7851-7859.
3.In the part of UPLC and MS conditions, the authors listed the flow rate and temperature used to separate the compounds in QDP. However, the selection of these critical parameters has not been justified. As the compounds to be separated are small molecules, their performance in UHPLC can be completely different if flow rate and temperature are changed. An optimization experiment must be conducted to guarantee a best condition is deployed.
4.Same part, the authors used acetonitrile as mobile phase A and 0.1% FA in water as mobile phase B. It is very weird to apply dominant amount of acetonitrile at the beginning of sequence, and increase the percentage of aqueous mobile phase thereafter, as a reverse phase LC was used. The authors must explain the reason to do that, or re-develop the method. Also, the authors need to justify the usage of 0.1% FA with any method optimization evidence, or the pKa of some typical compounds in QDP.
5.In the gradient, dead volume was not considered. Generally, a gradient need to have 100% or a relative high percentage of aqueous mobile phase at the beginning 3-5min and the ending 3-5 min to guarantee the dead volume does not affect the separation, and no potential mixing issue at the time of injection.
6.The result in Figure one shoed poor baseline and peak shape. This is as expected considering point 4 above, because the gradient and LC condition is so abnormal. The authors need to re-develop the method to fix all the issues.
7.LOD or LOQ has not been determined in this paper. This should be done for a complete LC-MS method development.
8.In Page 18, m/z 121 and 147 have not been ID’ed. They are over 20% ion abundance, hence need to be identified.

The author revised the manuscript and responded to the comment. I partially agreed the authors’ response to comment 1, 2, 7 and 8. However, I disagree with the authors’ response to comment 3, 4, 5, and 6.

To comment 3, the author mentioned the flow rate and temperature were adapted to detection instrument. Actually there is nothing to do with the detection instrument. The flow rate and temperature should only be relative to the performance of separation, and adjusted base on that too. The author need either (1) show their experiments that the flow rate and temperature they have chosen are better than flow rate of temperatures that are higher and lower; or (2) find conclusive reference that the temperature and flow rate they have chosen have been used for Qingyan or similar drug substances.

To comment 4, the author mentioned using 95% organic mobile phase is suggested based on HPLC literature. However, this is against the basic LC principle. As the authors are using reverse-phase column, using non-polar solvent (organic) at the beginning of separation will elute all compounds together at early retention time. This is nothing to do with methanol or acetonitrile, but the aqueous mobile phase (authors’ mobile phase B) should be dominant at the beginning of separation. The authors can do things below: (1) Looking for the literature and see if the authors are misunderstanding the mobile phase usage. Generally, mobile phase A should be 0.1% formic acid, and mobile phase B should be acetonitrile. Please make sure there is no mis-writing. (2) If the literature really uses 95% ACN to start the sequence, the author should not just follow it. THERE IS NO NEED TO REPEAT ERRORS. The authors need to try an isocratic method, using 95% ACN/5% formic acid solution as the condition during the whole separation, to see if it is better. If yes, please use the isocratic method. If not, then your method can be justified at only this circumstance.

To comment 5, the author need to list the previous literature to prove the dead volume does not matter.

To comment 6, same to above comment to #4, a comparative experiment using isocratic method needs to be conducted. Otherwise I cannot believe this is the best method for separation of Qingyan.