Application of Normal-Phase Silica Column in Hydrophilic Interaction Liquid Chromatography Mode for Simultaneous Determination of Underivatized Amino Acids from Human Serum Samples via Liquid Chromatography–Tandem Mass Spectrometry

Round 1

Reviewer 1 Report

Dear Authors,

Please accept my apologies for the belated reply and review. Thank you for submitting your manuscript on the interesting topic of amino acids analysis.

The manuscript describes an interesting method for fast and accurate analysis of amino acids in patients' serum using a well-known HILIC separation. However, I have some questions and comments.

1. You described protein precipitation during the sample preparation. How about the removal of lipids? Did you check the influence of the presence and absence of lipids in the sample? Could you please comment on that?

2. Could you please prepare and attach, as Supplementary Information, figures showing the mass spectra of the MRM transitions and the base peak chromatograms of the standard sample and a selected biological/patient sample?

3. Could you please explain why the protein precipitation using acetonitrile led to better results than the precipitation with methanol?

4. Please rephrase the statement on "normal and reverse phase solvents". All eluents can be used in any chromatography mode.

5. Please rephrase the following sentence: "In the case of normal phase chromatography, however, the more polar solvent is the stronger one the concentration of which is increased during the gradient". 

6. How does gradient delay affect the elution of analytes within the gradient time? You claim that in this sentence "Due to the gradient delay of the chromatographic system (approximately 1 minute), every AA eluted until the end of the gradient part." 

7. The retention of polar analytes in HILIC happens when the analyte and the stationary phase undergo interactions helped by the solvent film on the surface of the stationary phase. In this manuscript, there is no use of salts, but just formic acid, a common ion-pairing agent in reversed-phase chromatography. Can you provide a description of amino acids' mechanism of interaction with the silica stationary phase when formic acid is used?

8.  Please explain why the sample dilution led to improved peak shapes.

9. Tables 3 and 4 provide a lot of information on calibration procedures. I suggest moving the table into the Supplementary part.

Thank you!

Please consult a native English speaker for corrective reading of the manuscript. 

Author Response

Thank you for your remarks and comments. We accepted all the suggestions and modified the manuscript.

Author Response File: Author Response.docx

Reviewer 2 Report

Manuscript titled “Application of Normal Phase Silica Column in Hilic Mode for Simultaneous Determination of Underivatized Amino Acids from Human Serum Samples by Liquid Chromatography–Tandem Mass Spectrometry” reports the development of an MS/MS method used to quantify amino acids in human serum, for the purposes of aiding in neonatal screening. Data reported is interesting and has significant potential applicability in clinical settings. There are some comments and suggestions for the authors:

1.       The introduction gives a clear and concise justification of the problem, which provides support for the experiments performed. The authors mention screening of metabolic diseases or inherited metabolic disorders; could you briefly mention in the introduction some specific examples of diseases whose diagnostic would benefit from your method?

2.       Please verify subsection numbering, since they are all “1.1” or “1.2”.

3.       Section “Biological samples” mentions “newborns and unhealthy children”. Could the authors rephrase to specify if it was a single group (unhealthy newborns) or two groups (a group of healthy newborns and a group of diagnosed children). This is not immediately clear as currently written.

4.       Related to the previous comment, what do you mean by “unhealthy”? Later in the manuscript it is mentioned that some values were outside of healthy range, however, it is also stated that these values “may be caused by different health statuses”, implying that the authors do not know with certainty if a sample was obtained from a healthy or diseased patient. If the authors do know the health status of the individuals, it could be briefly mentioned here, otherwise, this could be clarified.

5.       In section “Development of sample preparation”, 100 uL of sample are said to have been mixed with 300 uL of acetonitrile to precipitate proteins. Did you centrifuge the precipitated samples?

6.       Section “Method validation” references three works of the FDA, Eurachem and ICH, however, these are not included in the reference list, please add them.

7.       Table 1 is referenced in “Experimental” section. Since this table shows optimized conditions, would it be more appropriate to include it in results and discussion, section “MS optimization” instead?

8.       Section “Stability” mentions that samples were kept at -20°C until they arrived at the laboratory. Do you know how much time passed from sample collection to when you received them? If so, please state it here.

9.       Table 4 lists the results of 12 samples analyzed, while the main text mentions that “some samples are significantly out of the healthy values”. Could you add the normal/healthy ranges expected for each amino acid? This would facilitate interpreting the data shown in this table.

10.   Although the work is quite interesting, it does not mention any potential limitations. Please consider listing some limitations of your study or any aspects that could be improved in subsequent works.

Author Response

Thank you for your remarks and comments. We accepted all the suggestions and modified the manuscript.

Author Response File: Author Response.docx