Optimization of Plant Extract Purification Procedure for Rapid Screening Analysis of Sixteen Phenolics by Liquid Chromatography
Round 1
Reviewer 1 Report
The manuscript "SPE optimization with follow-up HPLC–DAD method for determination of sixteen phenolics with wide range of polarity in various plant matrices" by Petra Ranušová et. al., describes the development of SPE-HPLC method for simultaneous monitoring of sixteen different phenolics in various plant matrices.
EVALUATION
In my opinion, this article does not fit in the aim and scope of Separations journal, being more focused on the extraction technique than on the chromatographic analysis.
Moreover, the present study does not present relevant developments in the analysis of polyphenols in plant matrices, where none optimization was performed in HPLC analysis or separation performance was evaluated.
In addition, the extractive technique used, SPE, has already been widely applied in similar analyzes, and currently there are miniaturized extractive techniques that present better performance and are more environmentally friendly due to less use of solvents, such as microextraction by packed sorbents (MEPS) and micro solid phase extraction (μ-SPEed).
Author Response
Dear editorial board and reviewers,
On behalf of all co-authors, we want to thank very much to all the reviewers for careful reading of our manuscript and their valuable comments. We carried out the corrections suggested and below we describe in detail our answers to the comments and raised questions:
In my opinion, this article does not fit in the aim and scope of Separations journal, being more focused on the extraction technique than on the chromatographic analysis.
Moreover, the present study does not present relevant developments in the analysis of polyphenols in plant matrices, where none optimization was performed in HPLC analysis or separation performance was evaluated.
We can see the point of the respected reviewer and agree that our work is not focused on chromatographic analysis in the narrow sense. Nevertheless a proper extraction technique, especially in plant matrices of different complexity, is a crucial prerequisite for successful chromatographic analysis; therefore our procedure can be helpful and beneficial for establishment and/or further development of separation methods for polyphenol screening. We also would like to stress our method offers simple sample pre-treatment and purification of complex (plant) matrices. Further, at least for routine screening with HPLC-DAD method, the number of simultaneously detectable phenolic with wide range of polarity is worth of attention.
In addition, the extractive technique used, SPE, has already been widely applied in similar analyzes, and currently there are miniaturized extractive techniques that present better performance and are more environmentally friendly due to less use of solvents, such as microextraction by packed sorbents (MEPS) and micro solid phase extraction (μ-SPEed).
The reviewer is certainly correct and we admit our approach cannot compete with the mentioned techniques. Still, as we argued in the manuscript, the robustness, cost efficiency and technical simplicity of the detailed method allows for a routine analysis of polyphenols in any commonly equipped laboratory and might be of potential interest for many researchers involved in polyphenol analysis in different plant tissues/matrices.
We believe that edits of our discussion will persuade the respected reviewer that our manuscript is worth of publishing. Thank you for reconsideration.
Reviewer 2 Report
This work does not introduce a significant novelty but it is a well designed and implemented study that accomplishes the simultaneous determination of a wide range of phenolic compounds in plant extracts. The authors have performed a detailed work and the final method shall be of wide interest.
Overall, I advise in favor of publication, not because of its novelty but because this method could be a useful tutorial for the determination of phenolic compounds.
Author Response
Dear editorial board and reviewers,
On behalf of all co-authors, we want to thank very much to all the reviewers for careful reading of our manuscript and their valuable comments. We carried out the corrections suggested and below we describe in detail our answers to the comments and raised questions:
This work does not introduce a significant novelty but it is a well designed and implemented study that accomplishes the simultaneous determination of a wide range of phenolic compounds in plant extracts. The authors have performed a detailed work and the final method shall be of wide interest.
Overall, I advise in favor of publication, not because of its novelty but because this method could be a useful tutorial for the determination of phenolic compounds.
We thank for the positive attitude and comments of the respected reviewer.
Reviewer 3 Report
The manuscript entitled “SPE optimization with follow-up HPLC–DAD method for determination of sixteen phenolics with wide range of polarity in various plant matrices” addresses a very interesting and little-publicized topic that is in line with the magazine's scopus.
I could suggest a change to the proposed title because I consider it quite redundant in addition to including abbreviations that are not normally recommended to be included in the title that appears as the first impact of the reader.
The manuscript is well written and structured and supported by current references. I believe, however, that the discussion should be revised because the results should be discussed with the bibliography in order to make the article more interesting and complete.
In addition, I request that certain points be checked, namely:
- In point 2.5, when referring to the name of the plant species that they used, they must present the scientific name of the species in full.
Still at this point, it is not clear the procedure of the extracts after evaporation. Is the extract evaporated to dryness or only methanol evaporated?
- Regarding the chromatographic methodology, they used HPLC and DAD. Wouldn't it also have been convenient to use MS?
- Line 198 remove parentheses and add a comma before "respectively"
- Check and standardize references
Author Response
Dear editorial board and reviewers,
On behalf of all co-authors, we want to thank very much to all the reviewers for careful reading of our manuscript and their valuable comments. We carried out the corrections suggested and below we describe in detail our answers to the comments and raised questions:
The manuscript entitled “SPE optimization with follow-up HPLC–DAD method for determination of sixteen phenolics with wide range of polarity in various plant matrices” addresses a very interesting and little-publicized topic that is in line with the magazine's scopus.
I could suggest a change to the proposed title because I consider it quite redundant in addition to including abbreviations that are not normally recommended to be included in the title that appears as the first impact of the reader.
Thank you for the suggestion. In accordance to it we altered the title as: “Optimization of plant extract purification procedure for rapid screening analysis of sixteen phenolics by liquid chromatography”. Accordingly, we slightly adjusted the keywords.
The manuscript is well written and structured and supported by current references. I believe, however, that the discussion should be revised because the results should be discussed with the bibliography in order to make the article more interesting and complete.
Thank you for the valuable suggestion. We have edited our discussion by some examples mentioned in literature to point on the benefits and drawbacks of our approach. The edited sentences are indicated with red colour and we believe the edits will meet the expectation of the respected reviewer.
There is an option to extend the discussion by examples with respect to the individual steps of our procedure. However mostly these are not directly comparable due to different experimental setups, e.g. types of sorbent, samples and analytes. Therefore we prefer not to incorporate them to keep the straightforwardness and clarity of paper discussion. Nevertheless, if the respected reviewer insists, we are ready to add them.
In addition, I request that certain points be checked, namely:
- In point 2.5, when referring to the name of the plant species that they used, they must present the scientific name of the species in full.
We agree with the reviewer and added the full scientific names of the studied plant species when first mentioned.
Still at this point, it is not clear the procedure of the extracts after evaporation. Is the extract evaporated to dryness or only methanol evaporated?
We are thankful for pointing on this un-clarity. We edited the corresponding text (p. 6, lines 147-151) to clarify that only methanol was evaporated from extracts. We also added some more specific information.
- Regarding the chromatographic methodology, they used HPLC and DAD. Wouldn't it also have been convenient to use MS?
The reviewer is absolutely correct, nevertheless MS is much more costly technique and still cannot be considered as a common laboratory equipment. The comment, however, is justified and we addressed it in the text (p. 11, lines 256-264).
- Line 198 remove parentheses and add a comma before "respectively"
The suggestion was accepted and the text changed as requested.
- Check and standardize references
All the references were thoroughly checked again and formally adjusted into a uniform and standardized style.
Round 2
Reviewer 1 Report
The manuscript "SPE optimization with follow-up HPLC–DAD method for determination of sixteen phenolics with wide range of polarity in various plant matrices" by Petra Ranušová et. al., describes the development of SPE-HPLC method for simultaneous monitoring of sixteen different phenolics in various plant matrices.
EVALUATION
Once again, I reiterate that in my opinion this manuscript does not fit in the aim and scope of Separations journal. Although the preparation of samples and the extraction/purification of compounds are important for the separation of analytes, the chromatographic techniques are the step that effectively guarantees the separation between analytes.
Thus, in the Separations journal the focus should be on chromatographic separation and the respective identification technique (DAD, MS, etc.). The present study does not present relevant developments HPLC analysis.
Moreover, the unique reference in manuscript for separation performance was described in line 167 “The obtained data confirmed good resolution of peaks (Rij ≥ 1.5) within 65 minutes of analysis time. Retention times varied within range of ±2%.”, being an information given in a precarious and very summarized manner.
In addition, the “Validation characteristics of HPLC methodology” described in line 166 was performed with or without the optimized parameters of SPE? If yes, why was described before the optimization of SPE procedure? Also, the results of the quantification of polyphenols should be highlighted in the manuscript.
Despite recognizing some scientific merit of the study with regard to the extraction technique, the manuscript needs to focus on the results obtained during the HPLC analysis, mainly on the qualitative parameters of an analyte separation, such as peak resolution, symmetry of the peak, among others. In addition, the optimization of some parameters/conditions of the chromatographic analysis are indispensable, such as the type of column, column temperature, flow used, and even the eluents used, these being key elements to evaluate the chromatographic performance of a method analytical. Furthermore, no study is mentioned to support the choices of these parameters in the HPLC analysis. That is, why was this procedure used in the HPLC analysis? Are the best chromatographic conditions for these analytes under study? These are questions that have to be answered.
