False Positive Identification of Pesticides in Food Using the European Standard Method and LC-MS/MS Determination: Examples and Solutions from Routine Applications
Round 1
Reviewer 1 Report
Manuscript title: False positive identification of pesticides in food using the European standard method and LC-MS/MS determination: examples and solutions from routine applications
General comments
The manuscript contains latest standard method for pesticides in food and feed. Their findings can be of interest for the Journal readership. However, the revision by an English native speaker is warranted. The manuscript is well conceived and conducted with adequate technical standards. The authors are encouraged to evaluate the carcinogen health risk assessment. The discussion is not well written and the findings should be discussed with the results of other published articles. There are some points to address before its further consideration.
Please find a list of needed corrections below:
Specific comments
Line 15: add “a” after make in “make a decision
Line 18: the sentence is incomplete. tebufenpyrad in dried ginger and… and what. Please correct it
Line 34: add “a” after proposes in “proposes maximum residue limit (MRL) in food”
Line 38: add “a” after such in “such high number of analytes”
Line 83: please change “compound” to “compounds” in “The adequate separation of 480 compound”
Line 140: correct “mixture of PSA” to “a mixture of PSA”
Line 202: please correct “later” in “The screening validation was performed latter than the confirmatory validation
Line 255: “showed up this trace.” Should be “showed this trace”. Correct it.
Line 276: “intensity ration” Should be “intensity ratio” Correct it.
Line 284: mycobutanil?? Please correct spell
Line 336: correct “distinguised” to “distinguished”
Line 361: correct “identifed” to “identified”
Line 382: please delete highlight mark
Author Response
Reviewer 1
General comments
The manuscript contains latest standard method for pesticides in food and feed. Their findings can be of interest for the Journal readership. However, the revision by an English native speaker is warranted. The manuscript is well conceived and conducted with adequate technical standards. The authors are encouraged to evaluate the carcinogen health risk assessment. The discussion is not well written and the findings should be discussed with the results of other published articles. There are some points to address before its further consideration.
Answer: thank you. These examples from routine applications and such investigations and solutions with the presented “exotic” matrices have not been published so far. This is the main novelty of the paper. Hence, a real comparison in the discussion section to earlier published papers cannot be done appropriately, however we added important thoughts in the conclusion section based on the results obtained. We tried to better discuss the results in the revised version. We believe this is appropriate now.
Please find a list of needed corrections below:
Specific comments
Line 15: add “a” after make in “make a decision
Answer: thank you, we corrected it.
Line 18: the sentence is incomplete. tebufenpyrad in dried ginger and… and what. Please correct it
Answer: thank you, we corrected it.
Line 34: add “a” after proposes in “proposes maximum residue limit (MRL) in food”
Answer: thank you, we corrected it.
Line 38: add “a” after such in “such high number of analytes”
Answer: thank you, we corrected it.
Line 83: please change “compound” to “compounds” in “The adequate separation of 480 compound”
Answer: thank you, we corrected it.
Line 140: correct “mixture of PSA” to “a mixture of PSA”
Answer: thank you, we corrected it.
Line 202: please correct “later” in “The screening validation was performed latter than the confirmatory validation
Answer: thank you, we corrected it.
Line 255: “showed up this trace.” Should be “showed this trace”. Correct it.
Answer: thank you, we corrected it.
Line 276: “intensity ration” Should be “intensity ratio” Correct it.
Answer: thank you, we corrected it.
Line 284: mycobutanil?? Please correct spell
Answer: thank you, we corrected it.
Line 336: correct “distinguised” to “distinguished”
Answer: thank you, we corrected it.
Line 361: correct “identifed” to “identified”
Answer: thank you, we corrected it.
Line 382: please delete highlight mark
Answer: thank you, we corrected it.
Reviewer 2 Report
I have some suggestions to improve manuscript quality.
Please write the scientific name as well with common name at first mentioning in manuscript.
Unit and number must not be attached. Please revise in whole manuscript.
Wang et al., (2017) Please cite the references correctly as per journal guideline
Please justify the novelty and originality of this study by corroborating previous research gaps in the introduction section
Please describe that how this study helped to overcome these research gaps
Please improve the coherence in writeup
Please cite the references in text of not more than 5 years old
Error bars are missing
Please combine the results and discussion sections
Mechanistic details ae missing in results and discussion. Please add the relevant details.
Discussion is not up to the mark. Please improve the discussion by providing supportive or conflicting results from other published reports.
Use full form of acronym only at first usage. Abbreviate subsequently.
It has limited scientific novelty or generality.
Author Response
Reviewer 2
Please write the scientific name as well with common name at first mentioning in manuscript.
Answer: thank you, we carefully checked the abbreviations.
Unit and number must not be attached. Please revise in whole manuscript.
Answer: thank you, we carefully checked and detached the number and units.
Wang et al., (2017) Please cite the references correctly as per journal guideline
Answer: we have no reference from Wang et al. (2017).
Please justify the novelty and originality of this study by corroborating previous research gaps in the introduction section
Answer: thank you, we justified the novelty and originality in the introduction.
Please describe that how this study helped to overcome these research gaps
Answer: thank you. The presented examples from routine applications and such investigations can help other experts avoid false positive cases in the future as these solutions are presented for the first time.
Please improve the coherence in writeup
Answer: thank you, we enhanced the coherence in writeup.
Please cite the references in text of not more than 5 years old
Answer: thank you, we cited a number of paper from the last 5 years, but there are several important papers published earlier. Again, relevant studies on this topic has not been published recently.
Error bars are missing
Answer: thank you, we did not discussed values with uncertainties in the manuscript.
Please combine the results and discussion sections
Answer: thank you, they are combined.
Mechanistic details are missing in results and discussion. Please add the relevant details.
Answer: thank you, mechanistic details are in the Materials and Methods section where they shall be written. We cited the latest standard method that contains all relevant information on the mechanistic details. We would like to avoid to all the details that can easily be found in the standard not to lose the real message of the paper.
Discussion is not up to the mark. Please improve the discussion by providing supportive or conflicting results from other published reports.
Answer: thank you. As there are no other published papers on this topic, the discussion is limited and real comparison could not be done appropriately, but we tried to improve it. A real comparison in the discussion section to earlier published papers cannot be done appropriately, however we added important thoughts in the conclusion section based on the results obtained
Use full form of acronym only at first usage. Abbreviate subsequently.
Answer: thank you, the abbreviations were checked carefully.
It has limited scientific novelty or generality.
Answer: thank you. These examples from routine applications and such investigations have not been published so far. This is the main novelty of the paper.
Reviewer 3 Report
Detailed comments are included in the review file attached below.
Comments for author File: 
Comments.pdf
Author Response
Reviewer 3
A brief summary and general concept comments:
The aim of this study was to summarize the cases where false positive identification can
occur in pesticide analysis because of setting aside the relevant confirmatory analysis.
The reviewed work includes relevant information on the application of the standard
method on pesticide analysis in food. Firstly, analytical criteria for the analysis of pesticides,
which are laid down in SANTE/11312/2021 guideline was briefly characterized. In the research, it was stated that some food matrices, especially the novel and “exotic” matrices might have not been considered and validated according to SANTE guidelines and it is inevitable to deal with novel interferences on the MRM transitions of the hundreds of analysed pesticides.
Furthermore, it was noted that sometimes the common approach of using neat solvent-based
calibration and monitoring only two MRMs had been reported to fail. It was therefore
concluded that as some of the analytes possess low MRLs, the involvement of HRMS conditions might not offer the right solution for selective identification, and thus, the use of minimum three MRM transitions should be regarded as a general and usual policy, which should be often confirmed by the standard addition process. It was noticed that standard addition was useful not only for the background compensating in mass spectrometric detection and the identification process, when negligible retention time difference was observed between the analytes and the interfering matrix compounds.
In reviewer’s opinion, analysing overall merit and rating interest to the readers and
taking all the conclusions from the study into account, the results of this study may be
of high interest to readers. However, the results obtained and conclusions do not bring much
novelty to the state of knowledge in this field.
When it comes to the presentation of results, in the reviewer’s opinion the manuscript’s
results are well presented but the discussion of the results should be more extensive.
Answer: thank you. The discussion is quite difficult here because similar examples has not been published so far. However, we have improved it in the revised manuscript. A real comparison in the discussion section to earlier published papers cannot be done appropriately, however we added important thoughts in the conclusion section based on the results obtained.
The specific comments to the manuscript:
Line 21 – 24 – the confirmatory analysis is almost the same as identification process in LCMS/MS, therefore this sentence should be written in a different way.
Answer: thank you. The screening and the confirmation are done on different ways (e.g. calibration, number of ion transitions). The standard addition is done only for those compounds that were identified under screening analysis that is performed using a simplifier calibration.
The aim of the study wasn’t presented in the Abstract. It should have been clearly written in
the Abstract.
Answer: thank you, we presented it.
Line 33-34 - Authors wrote: ‘…the European Food Safety Authority (EFSA) …. proposes
maximum residue limit (MRL) in food’ – these limits should be written in the plural form –
maximum residue limits (MRLs) , because there is no single limit for all pesticides.
Answer: thank you, we corrected it.
Line 68-70 - it is written incomprehensibly, what GC-MS 6 analysis/ or analyzes are about?
What here 6 means?
Answer: thank you, we deleted 6 here, it was a typo.
In the Introduction false identifications should have been reported, but all concerned
tandem mass spectrometry detection- especially LC-MS/MS, but also GC-MS/MS . The cases
of false identification from single MS detection shouldn’t have been presented (they should
have been omitted).
Answer: thank you, it is right, however a few similar papers can be found in the literature, so we would keep this technique (MS single stage) since it is allowed by the 15662:2018 standard.
Line 76 -77 – Authors wrote: ‘ Ghosh et al. (2020) detected delta-hexachlorocyclohexane in
tobacco…’ - it is advisable to present only examples with regard to the food matrix and not
to tobacco.
Answer: thank you, it is right, however a few similar papers can be found in the literature, so we would keep this matrix to highlight that such false identification can occur in complex matrices.
Line 87 -88 – Authors wrote ‘… setting aside the relevant confirmatory analysis’ – What did
the authors mean? These relevant confirmatory analysis should have been briefly listed here.
Answer: We meant to express the necessity of confirmatory analysis. If only screening is done, the false identification cannot be avoided. We rephrased the sentence. The confirmatory analysis is written in section 2.
Line 134-135 - The QuEChERS extraction salts and their amounts should have been listed and written here precisely (instead of in the subsection 2.1).
Answer: thank you. The method description shall be in section “Materials and Methods”. As the details are given in the 15662:2018 standard, we do not write the settings here. The aim of the study was not to present the method, it is well-written in the standard. The details would increase the long of the manuscript and make it boring for the readers. Moreover, the real message of the paper would lose.
Line 139 -142 - The amounts of added sorbents should be given here for selected examples
of samples. Furthermore, what followed the addition of sorbents to the previously obtained
extracts before the LC-MS/MS analysis? It should be written here precisely and clearly.
Answer: thank you. Again, as the details are given in the 15662:2018 standard, we do not write the settings here. The aim of the study was not to present the method, it is well-written in the standard. The details would increase the long of the manuscript and make it boring for the readers. Moreover, the real message of the paper would lose.
New extracts were done for confirmatory analysis as the standard addition is done with new samples. Previous extracts, which were used for screening, were disposed.
What do ‘the screening detection limits’ mean? It should be more precisely described.
Answer: thank you, we clarified it according to the SANTE guideline.
Line 164 - The MRM table should have been presented in the Supplementary Materials
Answer: thank you, the MRM setting are published in our previous work, so we would like to avoid the duplication, hence we only cited the corresponding paper.
Line 169-171 – ‘samples…. were quantified using an at least three-point standard addition
process at spiking levels that are proportional to the residue levels estimated with the help
of the screening method’ – these spiking levels should be revealed and briefly presented
here in the manuscript.
Answer: thank you. The levels are very different. They depend on the screened concentration. We wrote here a general approach we used.
Line 174-176- What do ‘blank samples’ mean? Were they analysed before fortification for
pesticides levels?
Answer: thank you, yes, they were and contained pesticides under LOD. We clarified it.
Line 190-191 – ‘Five samples were prepared at each level’ – which samples at which levels –
it’s unclear
Answer: thank you. Five replicates were done at each level for all sample matrices. We clarified it.
Line 192-194 – ‘Recovery and within-laboratory precision were calculated at 0.010 mg/kg
and 0.100 mg/kg spiking levels for the selected compounds’. What were these selected
substances?
Answer: thank you. The selected compounds are in Table S2.
Line 199 - What does ‘PT result’ means? Not everyone will guess, reading it. The abbreviation should be explained the first time it is introduced into the text. Moreover, it should be used in the plural form - PT results.
Answer: thank you, we clarified it.
Line 214 (and Tables 1-5) – How z-score was calculated should have been clearly described.
Answer: thank you, we cited here the ISO 17043 standard that describes the calculation of Z-score.
In the figures ‘neat solvent calibrant’ could be renamed into more simple name.
Answer: thank you. In the LC-MS/MS practice, it is very important to clarify whether the calibration was matrix-matched or not. So, the calibration in “neat solvent” means that matrix-matched calibration was not performed. The “neat solvent calibration” is commonly used in LC-MS practice since 2003 (Matuszewski et al, Strategies for the assessment of matrix effect in quantitative bioanalytical methods based on HPLC-MS/MS).
Line 276 – Authors wrote: ‘intensity ration of m/z 125 to m/z 70’ . Whole paths of ion
transitions should be written.
Answer: thank you, we corrected it.
In the Figure 3. b. - no named first chromatogram.
Answer: thank you, we corrected it.
Line 332 and 379 - Really ‘neat solvent’ as written in the text?
Answer: thank you, yes, please see it above.
Line 382 -383- Why 'additional ion transitions' are colored?
Answer: thank you, they are coloured for the better presentation.
Line 404 – Authors wrote: ‘As some of the analytes possess low ng/g MRLs…’ – it would be better to write: low MRLs (ng/g levels)
Answer: thank you, we corrected it.
In Supplementary Table S1: Screening detection Limits (SDLs) established for 415 pesticides
in olive oil under screening analysis were only shown, whereas MRM transitions for them
should have been also presented for them, although Ion transitions for 480 compounds were
listed in the study of Tóth et al.
Answer: thank you, we clarified it. “The method contains 480 compounds now, however only 415 pesticides were involved in the method when the screening analysis was done in the past.”
In Supplementary Table S2: What is the level of content (level of sample fortification) for
which the recovery (%) and RSD (%) were tested and presented? It is crucial to reveal this
information there, even below the table. Was these recoveries and RSDs calculated from 3
or more spiking levels?
Answer: thank you, we clarified it. “The first and second data correspond to 0.010 mg/kg and 0.100 mg/kg spiking levels, respectively.”
The question of formatting – in References: position no. 5 - there is hanging text instead of
indentation.
Answer: thank you, we corrected it.
English level:
is good and understandable, however the manuscript contains some linguistic errors.
Among them, the following can be mentioned e.g.:
Line 22 - Word ‘during’ used twice, reconsider changing it.
Answer: thank you, we corrected it.
Line 79 -81, 205, 227, 229 – Research papers should be written in a more impersonal way
Answer: thank you, we corrected them.
Line 223, 241, 271, 298, 326 - Change the colloquial style (ruling out) in the sentences /
reformulate them.
Answer: thank you, we corrected them.
Why word ‘neat‘ is used ? neat standard solution or simply: standard solution.
Answer: thank you, we please see it above.
Line 235, 239, and 391 – ‘the present’ or the presence?
Answer: thank you, we corrected it.
Line 276 – ‘intensity ration’? ratio without n
Answer: thank you, we corrected it.
The manuscript requires minor revision according to all the suggested comments.
Answer: thank you.
Round 2
Reviewer 2 Report
Acceptable in current form
