Multiple Heart-Cutting Two-Dimensional HPLC-UV Achiral–Chiral Analysis of Branched-Chain Amino Acids in Food Supplements under Environmentally Friendly Conditions

Round 1

Reviewer 1 Report

In this manuscript the authors report a multiple heart-cutting two-dimensional HPLC-UV method for the direct analysis of branched-chain amino acids in food supplements under environmentally friendly conditions (using water and ethanol as solvents).

This research shows the development and validation of an achiral IP-RP-HPLC method that includes the scrutinous of different variables like the eluent flow rate, the concentration of the EtOH and the ion pair and the use of various perfluorinated carboxylic acids with different side chain length. For 1D achiral separation, this method has been optimized with UV detection. For the 2D chiral analysis, a CLEC system working with (S)-OBS and copper(II) nitrate was also optimised. This mLC-LC method has been successfully applied to the analysis of food supplements.

The manuscript is sufficiently introduced and explained, and the coverage of the relevant literature is exhaustive. Despite this, I think the following reference should be included: Sebastian K. Piendl, David Geissler, Laura Weigelt, and Detlev Belder; Multiple Heart-Cutting Two-Dimensional Chip-HPLC Combined with Deep-UV Fluorescence and Mass Spectrometric Detection. Anal. Chem. 2020.

Please note that in entries e, f and g of the table 1, it should be written NFPA instead of NPFA.

On the other hand, there are several format mistakes (especially spaces) that should be corrected, and the molecules settings and size should be homogenized.

I recommend publication in Separation once the authors have addressed the above comments and observations.

Author Response

We are very grateful to the eminent Reviewer for the precious comments and suggestions.

All of these have been addressed in the revised version of the manuscript.

Q1. The manuscript is sufficiently introduced and explained, and the coverage of the relevant literature is exhaustive. Despite this, I think the following reference should be included: Sebastian K. Piendl, David Geissler, Laura Weigelt, and Detlev Belder; Multiple Heart-Cutting Two-Dimensional Chip-HPLC Combined with Deep-UV Fluorescence and Mass Spectrometric Detection. Anal. Chem. 2020.

A1. The reference suggested by the Reviewer has been added in the revised version of the manuscript.

Q2. Please note that in entries e, f and g of the table 1, it should be written NFPA instead of NPFA.

A2. A correction of the manuscript has been done according to the Reviewer’s comment.

Q3. On the other hand, there are several format mistakes (especially spaces) that should be corrected, and the molecules settings and size should be homogenized.

A3. A correction of the manusxcript has been done according to the Reviewer’s comment.

Reviewer 2 Report

The manuscript "Multiple heart-cutting two-dimensional HPLC-UV achiral-chiral analysis of branched-chain amino acids in food supplements under environmentally friendly conditions" is an interesting work, however, it is necessary to include a statistical test to ensure that there is a significant difference between the results. It is necessary to organize the presentation and analysis of results, therefore, comments must be addressed to improve the structure of the work and make it fully understandable.

Comments for author File: Comments.pdf

Author Response

We are very grateful to the eminent Reviewer for the precious comments and suggestions.

All of these have been addressed in the revised version of the manuscript.

 

Q0. It is necessary to include a statistical test to ensure that there is a significant difference between the results.

 

A0. In the field of supplement control and, more in general, of nutritional labelling the compliance between the analytical result and the declared value (in label) is assessed using percentage tolerances as reported in Guidance Document for Competent Authorities for the Control of Compliance with EU Legislation ON: Regulation (EU) No 1169/2011 of the European Parliament and of the Council of 25 October 2011 on the provision of food information to consumers, amending Regulations (EC) No 1924/2006 and (EC) No 1925/2006 of the European Parliament and of the Council, and repealing Commission Directive 87/250/EEC, Council Directive 90/496/EEC, Commission Directive 1999/10/EC, Directive 2000/13/EC of the European Parliament and of the Council, Commission Directives 2002/67/EC and 2008/5/EC and Commission Regulation (EC) No 608/2004 And Council Directive 90/496/EEC of 24 September 1990 on nutrition labelling of foodstuffs and Directive 2002/46/EC of the European Parliament and of the Council of 10 June 2002 on the approximation of the laws of the Member States relating to food supplements with regard to the setting of tolerances for nutrient values declared on a label. December 2012. [https://food.ec.europa.eu/safety/labelling-and-nutrition/addition-vitamins-and-minerals_en].

Therefore, in our opinion, the application of a statistical approach (t-test) is not appropriate in this context since inherent problems due to manufacturing practices (e.g. encapsulation of tablets) must be taken into account when the compliance of labelled amounts must be judged.

This aspect has been better explained in the revised manuscript.

Accordingly, the following sentence has been added in the revised manuscript: “The results showed that BCAAs were present in amount very close (from 92% up to 99%) to those declared by the producer: 457 mg Leu/tablet, 239 mg Val/tablet, 248 mg Ile/tablet (mean of three replicates). The found amounts were compliant with those la-belled, i.e. 500 mg Leu/tablet, 250 mg Val/tablet and 250 mg Ile/tablet since in the offi-cial control a lower tolerance of 20 % is admitted (https://food.ec.europa.eu/safety/labelling-and-nutrition/addition-vitamins-and-minerals_en).”.

 

 

Q1.The abstract should start with the context of the problem, why it was decided to develop a multiple heart-cutting (mLC-LC) two-dimensional HPLC-UV achiral-chiral method for the direct analysis of branched-chain amino acids (BCAAs) in food supplements under environmentally friendly conditions?

A1. It is very well known that columns for enantioselective LC analysis suffer from limited chemiselectivity, especially for structurally related compounds, as it is the case for Leu and Ile. Two-dimensional analysis through the heart-cut approach are useful to cope with this limitation. According to the Reviewer’s comment, this aspect has been now cited in the revised version of the manuscript.

 

Q2. Line 38. The placement of citations 1-5 after the words "Green chromatography" is not logical. If the authors refer to what is an assigned term in those references, a more complete sentence should be generated.

A2. According to the Reviewer’s comment, the first sentence in the Introduction has been modified as follow in the revised version of the manuscript: Green chromatography [1-5] is a specific branch of Green Chemistry [6-8] originated from the very well-known “twelve principles” by Anastas, to minimize the impact of chromatographic processes on the environment and human health.”.

 

Q3. Line 135. Check spelling as there are two punctuation marks together.

A3. A correction has been done according to the Reviewer’s comment.

 

Q4. A comparison analysis of the means of the results should be included.

A4. According to the Reviewer’s comment, the following sentences have been added in the revised version of the maniscript; the first in the section related to the extraction process, the second in that describing the chromatographic analysis.

“In the context of extraction process, both plain water and other EtOH contents higher than 5% (v/v) were comparatively tested. The presence of a minor amount of EtOH re-sulted necessary to favor the extraction of the three rather hydrophobic AAs, while amount higher than 5%n (v/v) did not further improve the extraction extent, which was rather quantitative (> 98%).”.

“In the context of the optimization method for the achiral analysis, two additional while less performing volatile perfluoroalkyl acids were also comparatively tested trying to ameliorate the chemoselective extent between the structurally similar Ile and Leu: no-nafluoropentanoic acid (NFPA), and tridecafluoroheptanoic acid (TDFHA).”.

Still, the following sentences have been added to introduce the employed six-port switching valve: “For the mLC-LC applications, a manual two-position six-port switching valve assembled in our laboratory in the occasion of a previous study focused on the achiral-chiral analysis of an AA pool in a complex food matrix [27] was here efficiently used. This system, was very similar to that employed by other authors for similar applications [22,23] and included a 100 μL stainless steel loop.”.

 

A5. Lines 142.176. The authors begin by justifying the methods or procedures used, however, they must be presented in a way that helps justify the results. The organization of the results must be: 1. description of the results obtained, 2. justification of the results, 3. comparison with other similar investigations.

Q5. Following the Reviewer’s comment, section 3.1 has been re-organized in the revised version of the manuscript. Only the first few sentences have been maintained as before.

“In order to maximize the chemoselectivity of the IP-RP method representing the first dimension of the mLC-LC system, the effect of the following variables was iteratively scrutinized: (i) concentration of organic modifier (EtOH); (ii) type and concentration of IP agent; (iii) eluent flow rate. Based on some successful IP-RP-HPLC studies on the analysis of AA mixtures in food matrices [30,32,37,38], we decided to test a similar chromatographic method by simply replacing the commonly used acetonitrile (ACN) with the greener and less toxic EtOH.

Based on the satisfactory results recently obtained in our laboratory [33], a water/EtOH - 95/5 (v/v; 7mM of HFBA) mobile phase was initially tested. With a 7 mM HFBA concentration, the base-line separation of the three BCAAs was achieved, and the AAs were eluted in direct relation to their lipophilicity extent [37,38]. The elution order in the EtOH-based system was exactly the same obtained with water-ACN mobile phases [37,38], (Val < Ile < Leu), even though EtOH produced lower elution efficiencies than ACN, as a result of the slower mass transfer kinetics.

According to the IP-RP mechanism, the analysis time increases with increasing the concentration of the IP reagent [39]. This retention trend could be conveniently accompanied by improved selectivity. For this reason, the effect of HFBA concentration on the overall chromatographic performance was evaluated, trying to enhance the “critical chemoselectivity” of the system towards the pair Ile-Leu. As shown in Table 1 (entries a and b), by increasing the concentration of IP reagent up to 15 mM, the residence time of analytes in the columns is prolonged with only a marginal effect on critical selectivity and, accord-ingly, the lower tested HFBA concentration (7 mM) was preferred also to enhance the life span of LC column.

Owing to its advantageous low viscosity, full miscibility with water and low UV ab-sorption, ACN has been and is actually still being the most widespread organic modifier for IP- RP-HPLC applications. However, in spite of all the just mentioned favorable characteristics, ACN is well-recognized to have some toxic effect (mostly of dermal and oral character), beside to be highly flammable and volatile. All these characteristics well-justify its inclusion in the list of hazardous air pollutants [40]. On the other hand, compared to ACN, EtOH exhibits lower toxicity, reduced cost, it is easier to dispose, and it has demonstrated to provide comparable or even superior characteristics for separation of biomolecules under IP-RP conditions [9,41]. There is currently only one published paper that ap-plies the IP-RP-HPLC approach to analysis of underivatized proteinogenic AAs using EtOH as organic modifier of aqueous mobile phase [33].”.

 

Q6. Table 1. The title and footer of the table must be adjusted to the width of the table and not of the sheet. The spelling of the title should be checked (particularly the use of upper- and lower-case letters).

A6. Focused modifications have been done according to the Reviewer’s comment.

 

Q7. Table 2. The table is not well explained in the main text, if it is essential, it should be deleted and only "mentioned" in the text.

A7. According to the Reviewer’s comment, Table 2 has been removed in the revised version of the manuscript and the following sentence has been added in the text: “The tested linearity concentration ranges were 0.125-0.75 mg/mL for the three BCAAs, while the coefficient of determination (R2) resulted higher than 0.9991.”.

 

Q8. Table 3. The headers of the table have repeated information in the title, it must be synthesized. For example, section as intra-day and inter-day with their respective RSD (%) and Recovery (%), because RSD and recovery were defined in the title.

A8. According to the Reviewer’s comment, the following sentence has been added in the revised version of the manuscript: “Data in Table 2 are referred to three replicates of each external test solution within one day and for three consecutive days.”. Moreover, the Table’s heading has been shortened as follows: “Table 2. Intra-day and inter-day RSD% and Recovery% values.”. 

 

Q9. Lines 262-275. The results in the table are not described or discussed in depth in the main text. It is necessary to carry out the complete analysis with a comparison with results of other authors.

A9. Based on the Reviewer’s comment, a short comparison with recent literature data has been reported. Accordingly, the following sentence has been added in the revised version of the manuscript: “The results obtained in the validation study were in line with those reported by other authors [43-45] in the analysis of food matrices, thereby confirming the good quality of the developed method.”.

 

Q10. Figure 4. The title of the figure should fit the width of the figure (and not the main text).

A10. A modification has been done according to the Reviewer’s comment.

 

Q11. The authors present a summary of the activities that were carried out, which were previously addressed in the abstract and introduction. The conclusions are not a summary.

It should start with a short context (one or two lines) and go immediately to the relevant results to end with the "sale of the results", that is, explain why the results were important, their relevance, the contribution to society or to research etc.

A11. According to the Reviewer’s comment, a sensitive shortening of the Conclusion section has been done. In the current and more focused version, the following final sentence has been added: “This study clearly demonstrates that environmentally sustainable chromatographic methods can be conveniently applied for the overall characterization of AA-based food supplements, which have seen a widespread production and use in the last decade.”. 

 

Q12. Less than 20% of the references are from the last 5 years, it is necessary to update the information cited.

A12. According to the Reviewer’s comment, the following references have ben added in the revised version of the manuscript: Food Chem. 2018, 240, 196-203; Food Chem. 2022, 390, 133204; ACS Omega 2022, 7, 31106-31114; Anal. Chem. 2020, 92, 3795-3803.

 

 

 

Round 2

Reviewer 2 Report

Regarding the previous comment: "it is necessary to include a statistical test to ensure that there is a significant difference between the results".

I understand the point of the authors who based or supported their results on European Regulations, however, statistical analysis is ALWAYS necessary to establish a critical parameter on the results. That is, it helps to understand if the samples were representative, if they were executed correctly or even so that other authors can decide if they can repeat their method.

Author Response

Based on the Reviewer's comment, the standard deviation for each determined value has been added in the revised version of the manuscript. Accordingly, the revised sentence is as follows: “The results showed that BCAAs were present in amount very close (from 92% up to 99%) to those declared by the producer: 457.0 ± 2.6 mg Leu/tablet, 239 ± 4 mg Val/tablet, 248.0 ± 3.6 mg Ile/tablet (mean of three replicates).”.

Moreover, in section 2.2. (Extraction of the AA pool from the food supplement), the following sentence has been added to better explain the applied procedure: “This procedure was repeated thrice and each solution was submitted to chromato-graphic analysis.”.