To Develop the Method for UHPLC-HRMS to Determine the Antibacterial Potential of a Central American Medicinal Plant
Round 1
Reviewer 1 Report
The purpose of this study was to develope a novel rapid sensitive UHPLC- HRMS method which could determine antibacterial activity directly by LC-MS using the mass difference due to the reduction of indicator resazurin to resorufin due to the bacterial growth. It is well known that resazurin is converted into resorufin in bacterial mitochondria. But, this paper has a title „Evaluation of Antibacterial Potential of a Central American Medicinal Plant using UHPLC-HRMS“ and in Results there are no data of antibacterial activity, except from prevoiusly published work from agar disk diffusion and agar well diffusion. Main results are absence of resazurin peak with the bacterial growth demonstrated by mass spectrometer or resazurin reduced to resorufin due to the bacterial growth. Results of antibacterial activity are missing and also correlations between UHPLC-HRMS results with previously determined one. Otherwise, the title should be changed, because, the main purpose is to develop direct method for determine antibacterial activity by LC-MS. So I don't recommend the paper for publication in the current status. Some minor issues are listed below.
1. Article needs an extensive English correction
2. Some notes should be clear. For examples,
3. Abstract should emphasize the purpose of this study
4. In Abstract line 13: plant belonging to the family Bromeleacea→Bromeliaceae
5. The sentence „The methanolic plant extract being dark, posed a hurdle in reading minimum inhibition concentration (MIC) values directly from the 96 well plate. But, the indicator resazurin added to the extract was reduced to resorufin with the bacterial growth and remained as is with bacterial inhibition“ needs to be improved for better understanding
6. The introduction should define the purpose of the work and its significance. The current state of the research field should be reviewed carefully and key publications cited.
7. In Line 46: „The purpose was to determine the MIC of the plant extract by reduction of resazurin to resorufin due to bacterial growth inhibition „- where are those results?
8. Instruments and reagents please add manufacturer, state
9. Line 98: A. magdalena plant source?
10. Line 101: Incucula →inoculum
11. Line 103: LB broth needs description and source
12. Line 112: The methanolic extract was roto-vaped at room temperature (Buchi, R-114) and then reconstituted in 20% DMSO. Explain why was methanol evaporated and then dissolved in DMSO?
13. Line 138: „Antimicrobial activity of A. magdalenae was determined using agar disc diffusion and agar well diffusion assays.“ It should be clear that You are reffering to your prevoiusly published work.
14. The writing seems incomplete, especially, in the Results and discussion section
15. Objective title based on results.
Author Response
Please see attachment.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
= General impression =
1/ the Authors have found a serious but simple problem and
2/ they solved it by the use of the overdone method.
Thus, before the publication, the Authors should answer the following questions and/or justify their strategy.
A/ What is the real advantage of using a high-tech UHPLC-HRMS to define concentrations of two easily detectable compounds only?
Consider 1: use of HRMS shot (direct injection of filtered medium liquid; with calibrating mixture) to define the ratio of resazurin/resorufin. No need to waste expensive solvents for UHPLC and time. If orbitrap is available, why not to do it in a highly specific MSn frag. ions monitoring mode? It should be still in the scope of interest of this Journal.
Consider 2: separation with defined (U)HPLC method but with the use of UV or FLD that are much cheaper detectors (equipment, its maintenance, and availability). Standard of resazurin is also cheap and easy-available.
Sensitivity of UHPLC-MS was announced but not proved. Is it betted than FLD?
B/
l.44
'we aimed to develop a highly rapid and sensitive UHPLC-HRMS method'
Is the analysis time concurrent to other popular assays for resazurin/resorufin determination?
Is the sensitivity of MS concurrent to other popular assays for resazurin/resorufin determination?
Was the reported 'sensitivity' analyzed at all? Supply the results.
Discuss the above questions with the available literature in the Results and Discussion chapter.
C/
Was it considered and checked if a solution of resazurin does not undergo reduction during ionization in experimental conditions?
D/
The proof is based on a statement that key redox reaction is possible only in the presence of living bacteria. Is it known whether the extract alone, incubated with resazurin for a long time of the experiment, does not influence this redox reaction?
What is the influence of DMSO, that is known itself as an oxidizing factor under certain conditions?
E/
What was the acceptable absence level of resazurin that was treated as a positive result? 0% of the area when compared with a standard solution? 1%, 5%, other?
= Several parts of manuscripts are futile =
l.22-24
'The sample preparation for LC-MS assay included centrifugation of the samples taken from 96 well plate followed by filtration of the supernatant through a 0.45um PTFE syringe filter, before exposing them to C-18 column'
What is the reason to put such obvious details to the Abstract?
l.39-40
'The antibacterial potential of acetic acid was confirmed in our laboratory using agar disc diffusion assay performed on gram negative bacteria.'
Did you really need to do it? That is why acetic acid is used as a 'natural' conserving agent for gherkins, mushrooms, ketchup or tabasco.
l.62-63, chapter 2.1
'The XYZ arm mechanism picks the software assigned sample from the vial in X-Y plane with the help of fine needle' and so on.
Separations Journal readers are familiar with autosampler idea, and also with the idea of quadrupole and so on described below. Why pay a fee for printing this?
The abstracts and manuscript should be rewritten. Leave only the necessary elements.
= other =
l.35
'[...] use of A. magdalenae as caustic for wounds [3]. Therefore, it was hypothesized that plant A. magdalenae has antibacterial properties.'
OK, we know several caustic substances, e.g. solutions of NaOH, HCl and similar. Also in medicine (e.g. AgNO3). 'Caustic' means: able to destroy/dissolve other substances and also: able to generate blisters on the (over)exposed skin. It is hard to conclude that caustics are APPLICABLE antimicrobials. If you mean the general property of Bromeliaceae plants (accumulation of peptide-digestive enzymes, like bromelain, that can be btw. hazardous for microbes) - clear your intentions, please.
l.38
'the most abundant (>90%)'
Do you mean 'the relatively most abundant among both volatile and EI-ionizable compounds'? The rhizome of this plant consists mainly of water, cellulose and other polysaccharides. Was it fresh enough and uninfected? Was it analyzed by other methods?
l.47-48
'MIC of A. magdalenae'
Consider 'MIC of dry methanol extract of A. magdalenae (DER ...)'. Define DER.
= typos & omissions =
'Bromeleacea', repeated in text
Correct to 'Bromeliaceae'
l.11-12
'Ultra High-Performance Liquid Chromatography – High Resolution Mass Spectrometry (UHPLC-HRMS)'
If 'high-performance' than 'high-resolution'.
l. 55 fig.1
Bonds correction in a structure of the resazurin is needed.
l.81
'KV' -> 'kV'
2.4 and 2.5.1
The extraction was conducted in a ratio 6g/60mL, this was concentrated to ~dryness and redissolved in an unknown volume of 20%DMSO.
What was the final concentration of stock extract? And -> how the MIC was calculated, to repeat your experiment.
l.110
'1mg/mL gentmicin' in water? in medium?
l.114
'0.1% resasurin' in water? in medium?
How was the MS detector calibrated? What was the m/z range of detection?
Finishing, readers do not know if the method works in practice. Was the MIC of your extract finally defined?
Author Response
Please see attached
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
As the purpose was to develop the method to detect the masses of resazurin and resorufin to determine the antibacterial potential of the plant extract by reduction of resazurin to resorufin, now the results and the manuscript title are adequate.
Authors addressed every comment but need some English improvement.
Author Response
Dear Reviewer,
Thank you for your suggestions.
We have revised the paper for grammar, style, and other English language improvements.
Regards,
Authors
Reviewer 2 Report
= General impression =
Corrected but still insufficient.
0
Reread the work and remove REPETITIONS and BASICS. Materials and Methods are Materials and Methods while Discussion is Discussion. What is generally known is known. The work needs to be a show, not mumbling.
Correct 'u' to 'μ' character when needed (numerous places).
l.11-12
Just decide to use or not to use '-' connecting the same types of words.
l.12-13, 33
Names of botanists after plant name -> without italics.
l.17-19
'extract was dark [...] To overcome this problem, a blue indicator [...] was added [...] got reduced to pink [...] while remained blue [...]'
How can the addition of a blue indicator to dark extract solve the problem?
If the standard colorimetric reading of blue/pink change was impossible due to the color of extract and thus your method was developed - there is no need to describe the mechanism in Abstract and to start the mess. -> Short. Clear.
Btw. Was the colored extract a problem in [4]? MICs were determined in [4] without the help of HPLC. Why?
l.24-26
'The results obtained from [...] LC-MS spectrum [...] demonstrated the presence of resorufin from wells with bacterial growth and resazurin from wells with inhibition through peaks of relevant masses.'
You need to be clear everywhere and particularly in Abstract:
Did you demonstrate the presence of appropriate redox pigments in wells with known growth or lack of growth (that was stated impossible few lines above)?
OR
Did you demonstrate bacterial growth or lack of growth / MIC in unreadable wells/fractions by your method: by checking the redox pigments ratio?
l.38
The [4] was checked before sending the previous review, be sure.
The % of Probability (that analyzed peak is due to acetic acid -> see the caption of the table in your answer) is not % of peak Relative Area (of acetic acid among all volatiles) or particularly % of Content (of acetic acid in the plant). Do you even know how to interpret the GCMS results?
Another problem is the generalization. How can you say with a clear conscience that 'a plant has >90% acetic acid'? It is stupid. No cell can survive this. -> Refer [4] with sense.
l.59
'Fischer', correct
l.59-60
-> '[...] was -> UHPLC-ESI-LTQ Orbitrap [...] with Accela autosampler -> equipped with holder for 96 well plates.'
Add the calibration info from your answer.
END
l.60-72
Basic for this journal, useless, remove.
l.77
'lμ' -> 'μl'
l.84-85
simple but complicated
'The different masses [...] created due to the reduction [...] were used to determine antibacterial activity [...]'
-> e.g. 'Resazurin/resorufin ratio (based on m/z 230/214 measurement) was used to determine wells/fractions with antibacterial activity.' END
l.85-90
repetition of repetition, useless, remove.
l.92
The 'below' table?
Correct 'u' to 'μ' character.
Supply time with the units.
figures 2-4 can be cleverly merged
l.174
What is the conclusion for results from lines 162-174?
Discussion is overdone with antimicrobial topics that are not the essence of this work.
No reference on the use of HPLC in similar problematic situations.
Discussion is not 'what I have done' but 'what others have done or not have done and why my work is competitive'.
= Short comments to a previous revision, and what to do. =
answer A1
Like no answer... considered and what?
answer A2
It's obvious that MS can let you detect cpds in very low concentrations.
The problem of the work is that you don't define this level anywhere in the context of your cpds (-> LOD, LOQ) but instead repeat truism. If you want to say 'my method is highly sensitive' - measure it, prove, and report the results.
l.44
ANS1
We know this from the manuscript.
-> Introduce the primary HPLC articles concerned on this problem next declare the advantage of your method,
else -> clearly declare that there is no one similar work and you are first with a short and sensitive method.
ANS2
It is not the answer. No results on sensitivity. Convince me with your result.
C
If not - is this doubt added to discussion?
D
If no, -> this type of blank experiment should be done to ensure the specificity of your method.
Author Response
Please see the attachment.
Author Response File: Author Response.pdf
Round 3
Reviewer 2 Report
Thanks for any meaningful changes.
Have a good luck with novel projects. And go higher.
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LAST REV: Names of botanists after plant name -> without italics.
LAST ANS: The plant names have used in italics. Please check the following Pub Med links for reference.
THIS REV: Irrelevant links. If you don't understand what the problem is - try to learn, not to be rude / stubborn.
Plant name (italics): Aechmea magdalenae
Botanists (without italics) - person/s responsible for the classification and name: (Andre) for 1st=older accesion (-> Chevaliera magdalenae André); André ex Baker for 2nd=current naming (-> Aechmea magdalenae (André) André ex Baker)
It is not very hard to find botanical code.
https://www.iapt-taxon.org/nomen/main.php
