Simultaneous Determination of Pesticides and Veterinary Pharmaceuticals in Environmental Water Samples by UHPLC–Quadrupole-Orbitrap HRMS Combined with On-Line Solid-Phase Extraction

Round 1

Reviewer 1 Report

This manuscript develops and validates an analytical method using modern instrumental tools to the simultaneous determination of multiple pesticide and veterinary pharmaceutical residues in environmental water samples. The manuscript fits within the scope of the journal and the authors have knowledge and expertise in this field of study. The idea of this work is not particularly original and several major improvements are still needed in order to achieve a suitable manuscript: - I have observed that the authors are writing this manuscript using very long sentences. It would be better if they shorten their sentences to be clearer and simpler for the readers. - Introduction should be enriched by including some relevant reviews published in the last years focused on the trends in analysis of pesticide and veterinary pharmaceutical residues and the use of the instrumental analytical tools at legislative level, in order to provide additional information to the readers. - The approach to perform sample preparation/extraction of residues depends of the kind of matrix, i.e. food samples/QuEChERS (e.g. “J. AOAC Int. 2003, 86, 412-431”, etc.), soil/QuEChERS (e.g. “Molecules 2018, 23, 2009”, etc.), water/SPE (e.g. “Talanta 2010, 83, 410-424”, etc.). This fact should be highlighted in the text, including some relevant works, in order to contextualise the problem and to achieve a complete understanding of the concern for those non specialist readers. - All analytical standard materials used in an analytical method for monitoring purposes must be commonly available at a reasonable cost. In this sense, this reviewer doubts about the suitability of use in this work stable isotope labelled compounds as internal standards and worse to quantify non-equivalent compounds. This fact must be well argued or the work could be considered improperly planned from the beginning. - It should also be guaranteed that the analytical standard materials used as internal standard are the most suitable to determine those chemicals that do not have an equivalent stable isotope labelled compound, at least in comparison with those used ones. - The authors should clarify if the limits of quantification (LOQ) achieved in this study are suitable attending to the legislation requirements. For drinking water or groundwater the LOQ must meet 0.1 μg/L. For surface water the LOQ must comply with the lowest effect concentration, e.g., LC50, EC50, NOEC, etc. - The authors should clarify the difference between method detection limit (MDL) and limit of detection (LOD); and between method quantitation limit (MQL) and LOQ. - The way in which the samples are stored before extraction should be provided. How many days are stored the samples before extraction? Do the authors take into account the stability of residues before extraction? And the residues adsorption on polypropylene tubes? What percentage of pesticides is degraded during the whole storage? Has been the possibility of forming side products assessed? These issues should be clarified in the text, together with their influence on the results. Otherwise, this research work would be incomplete. This reviewer considers these issues key. - The authors should clarify the way in which the blank samples are obtained. - Lines 267-268: The reference “(EU, 2017)” should be included in the manuscript. - The current work does not assess the main factors and interactions among the variables, in sample preparation method and chromatographic analysis. A modelisation of the system, e.g., by response surface methodology, could solve this problem. The authors should solve or justify properly this lack. - It should be clearer described/correlated in the text/table 1/table 2 the internal standards used to determine each target agrochemical. - In the current work, variability represents a concern for this reviewer. The authors should explain in more detail how this concern was controlled/studied in sample preparation steps. - It should be provided the intra- and inter-day recoveries of the analytical method. - It should be provided a believable statement of the currently not filled necessities that could be covered by the suggested method. The discussion should be enriched with this issue. - Conclusions should include a proposal of future work. - References need to be updated. All references before 2009 should be removed unless they are essential for understanding the manuscript. Taking into account the comments to the authors, I consider that major revisions are still needed before the manuscript could be accepted in this journal. However, this reviewer encourages the authors to improve the manuscript and address the issues commented above, because the work is of interest.

Author Response

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Reviewer 2 Report

Dear Authors,

thank you for submitting your work on on-line sample preparation and separatopn of veterinary pharmaceuticals in environmental water.

Generally, I do not have objectiosn except for one matter:

Page 3/19, Line 202 ff.: It is known that the analytes you have analyzed tend to build stable Na-addukts, especially in environmental samples. Can you explain why did you not observe and did not use for analysis any Na-addukt, except for virginamycin?

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Reviewer 3 Report

The paper describes a method to determine 41 analytes belonging to veterinary drug and pesticide groups in water samples by LC-Q-orbitrap. The method is very fast thanks to the use of an on-line SPE purification system and its performance characteristics satisfactory (probably), but in fact the manuscript reports only the application of a commercial apparatus. The results of analysis of real water samples (groundwater and river water) do not furnish any information since the samples were not identified (number, type?). There is no novelty and also the English language is poor. Therefore I suggest to reject the paper.

Some specific comments:

2.2. Sampling and sample preparation

Lines 120-121. The collected groundwater and river water samples are not identified. How many samples have been analysed? Where were the sampling sites located with respect to the possible contamination sources?

2.4. Method validation

The validation study is very poor.

Some examples:

Lines 172-175 - The validation data are insufficient with only 3 replicates at 2 concentration levels. Which rule or guideline has been followed by the authors in order to validate the proposed method?

Lines 170-172. The approach to estimate MDLs and MQLs is not suitable since a standard solution furnish a too optimistic evaluation of standard deviation. For this reason also the discussion reported in Section 3.2.2. is questionable. The validation parameters must be always evaluated in real samples. Please consult the more important international guidelines or rules about method validation such as Commission Decision 2002/657/CE (veterinary drugs), SANTE/11813/2017 (pesticides) or the EURACHEM Guide “The Fitness for Purpose of Analytical Methods” (2014).

3.1 Operating conditions of the on-line SPE and UHPLC-Q-Orbitrap HRMS system

Lines 199-201 –“The CORTECS C18 column produced cleaner baselines and peak shapes compared to the 199 XBridge C18 column; hence, we concluded that the CORTECS C18 column was suitable for further studies.” No experimental data are shown to support this conclusion (e.g. comparative figures)

Lines 202-204. The authors probably do not know the specific terminology of mass spectrometry. [M+H]+ and [M+Na]+ are adducts.

3.2.1. Linearity

What is the criterion applied to decide if a curve is linear or not?

3.2.2. MDLs and MQLs

Lines 235-246. What is the interest on this discussion? The comparison about MDLs (or LOD/LOQs) demonstrates only that the proposed method has similar limits with respect to other procedures reported in literature. The authors cannot affirm that the MDLs are lower than those of Panditi et al. (2013) since, by the way, the differences could not be significant.

Table 1. Antibiotics, fungicides, insecticides etc … are not chemical classes

Table 2 should be unified with Table 1. The choice of the labelled ISs is questionable since five compounds belong to sulfonamide family, whereas other classes are not represented (e.g. tetracyclines)

Table 4 –Precision (n=7), accuracy (n=7)? In section 2.4 the authors report that 3 replicates/level have been carried out. The footnote “a” is not pertinent for precision

Figure 1 is wrong and also redundant since the LC gradient is reported in the text (section 2.3).

Author Response

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Reviewer 4 Report

This paper addresses a major problem for environmental pollution.

The analytical method presented make use of a very expensive tool (high resolution mass spectrometry detection) that cannot be used as routine tool. This should be commented in the conclusions.

However it is very good to have this results as a measure of the contamination level of the riverwater.

table 1 lists a series of numbers in the internal standards column but it is not clear what is the meaning of the numbers

Author Response

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Round 2

Reviewer 1 Report

In my judgment, after review carefully the manuscript, the changes are adequate and the authors have improved it properly. The confusing issues are also clearer. Currently, the article includes all the necessary information for a proper understanding of the work and results are of interest.

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Reviewer 3 Report

The paper remains unsatisfactory, since the lack of novelty is still evident. The English language is frequently redundant and the technical terminology is scarce.

As an example (lines 166-170):

“The exact masses of precursor ions (mass error < 5 ppm) and the retention times of target chemicals were confirmed to identify compounds. In order to determine the pesticides and veterinary pharmaceuticals in the target Q-orbitrap MS mode, all precursor ions were fragmented to ascertain the exact masses of the product ions for each precursor ion. The m/z values of the precursor ion and product ion for each target chemicals are listed in Table 1.”

could be shortly rewritten as follows:

“In order to confirm the identity of each analyte, the m/z of monitored adduct (mass error < 5 ppm), two fragment ions and the relevant retention time were verified (Table 1).”

In addition, “exact mass” is the calculated mass (m/z) and therefore the authors can only measure “accurate mass”.

Author Response

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