Development of a Sensitive HILIC-MS/MS Method for Quantification of Melamine, Derivatives, and Potential Precursors in Various Water Matrices

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript analytica-3754451-1 reported HILIC-MS/MS analysis of melamine and the derivatives in various water samples. The experiment in the manuscript is well designed, carried out with caring quantitative and qualitative analysis of the targets, and well presented. The reviewer considered that it should be published after minor revisions. Followings are comments.

 

1. For the analysis of melamine, the use of the ZIC-HILIC column is recommended by FDA (Laboratory Information Bulletin (LIB) 4422: Melamine and Cyanuric Acid Residues In Foods, 2008). The authors also tried ZIC-HILIC column (line 119-120), but there is no description why they finally decided to Luna HILIC. Did authors find any problem in the case of the LC separation on ZIC-HILIC column? Please add some description.

 

2. Caption in Fig.2 claimed that the mobile phases were mixtures of acetonitrile and pure water (no buffer salts). In lines 178-179, authors described that “10 mM ammonium acetate and 0.1% formic acid” were added to the aqueous part, for the explanation of the chromatographic conditions used in Figure 2. Pure water, or ammonium acetate solution? Which is correct?

 

3. No chromatographic conditions for Figure 3 were given in the caption, so please add the information.

 

4. Isotope effect by ¹²C and ¹³C is so small in common HPLC separation, and there is no possibility to separate the isotopomers by such a short column, but the separation of the isotopologues based on ¹H and ²H (D) is sometimes possible. Did the internal standard elute even not showing peak top separation? In the case of HILIC, ¹H moiety elutes faster than the corresponding ²H moiety generally.

Author Response

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Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Dear Authors,

 

I appreciate the opportunity to review your manuscript and would like to offer some constructive feedback.

 

Firstly, the language used throughout the manuscript requires refinement. It may be beneficial to consult a language expert to enhance the overall writing style, which currently has a colloquial tone. Additionally, the introduction would greatly benefit from a more comprehensive literature review to provide a robust foundation for your research.

 

The abstract does not clearly articulate the novelty of your work or its outcomes. Emphasizing these aspects more explicitly would strengthen this section.

 

In the method optimization section, while you indicate that both isocratic and gradient elutions were tested alongside various injection volumes, the lack of discussion regarding the chromatograms from these experiments needs to be addressed. An explanation for this omission would add clarity.

 

It is noted that the methodology largely derives from previous literature. Therefore, it would be beneficial to create a comparative table that highlights the advantages of your approach in relation to existing methods in the field.

 

Furthermore, I recommend that the SPE protocols be included in the main article rather than the supplementary materials, as they are critical to understanding the methodology employed.

 

In the method validation section, it would enhance the discussion to include a comparison with an acceptable range of 75 - 125% for any regulatory benchmarks. Additionally, a detailed table including the limits of detection (LOD), limits of quantification (LOQ), linear equations, and correlation coefficients will provide further clarity and rigor to your validation process.

 

Finally, clarity regarding the conclusion of the developed analytical technique would be beneficial. A well-articulated statement in this regard would enhance the overall impact of your findings.

 

Thank you for considering these suggestions. I look forward to your revised manuscript.

Author Response

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Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The authors did a good job in presenting their work. The paper can be accepted in its present form

Author Response

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Author Response File: Author Response.pdf

Reviewer 4 Report

Comments and Suggestions for Authors

In the paper Analytica-3754451, the authors present a method for the determination of melamine and five of its derivatives in water samples using HILIC-mode separation coupled with MS/MS detection.

The manuscript is clearly written, and the principles, methodology, and results are sufficiently described and discussed. Overall, the study may be of interest to some readers, although it does not represent a major innovation in the field of melamine analysis. Both HILIC separation and MS/MS detection are well-established approaches commonly applied in this context.

1. The authors claim that SPE method is more time-consuming than evaporation method. Is that really true? How long does preconcentration by evaporation take? According to the schedule in table 1, it is 12 hours, if I understand the set program correctly.
2. A more in-depth comparison between the evaporation and SPE methods would enhance the overall quality and impact of the manuscript. For instance, the matrix effect observed for HMMM could be significantly reduced when using the SPE method, likely due to the more effective removal of interfering compounds.

Author Response

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Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

Dear Authors,

 

Thank you for your valuable time on this manuscript.