Simultaneous Analysis of Seven Neonicotinoids in Commercial Milk Samples Using an UHPLC-MS/MS Method
Round 1
Reviewer 1 Report
In this work the authors present a method for the simultaneous quantification of 7 neonicotinoids in the UHPLC-MS/MS method. The authors present on the one hand the validation of the proposed method and on the other hand a possible application of this method to the analysis of commercial milk samples. The work is well written and straightforward; below are listed some comments which I hope could improve the quality of the paper.
Material and Methods:
On line 62, the authors state that they used a milk sample that contained no target analyte as a blank. I suggest to specify if this blank sample has been purchased or if the absence of the NEOs analytes has been verified by the authors themselves and if so, how?
The section “Materials and Methods” should be divided into subsections for ease of reading for examples (Reagents and Calibrators preparation; NEOs extraction; UHPLC-MS/MS analysis).
Results and Discussion
To improve the understanding of the results it would be better to divide this section into two separate paragraphs: methodological evaluation and application of the method.
Carrying out this subdivision, a critical point would emerge: the application part is lacking in information and this makes the paper unattractive.
I therefore suggest reformulating this last part by placing a more precise focus on the strengths of the method and its possible applications.
Conclusion:
The last sentence of the conclusion touches on an extremely interesting point on human breast milk screening, but no discussion on this topic has been addressed in the paper, can the authors explain this aspect better?
Author Response
Response to Reviewer 1 Comments
In this work the authors present a method for the simultaneous quantification of 7 neonicotinoids in the UHPLC-MS/MS method. The authors present on the one hand the validation of the proposed method and on the other hand a possible application of this method to the analysis of commercial milk samples. The work is well written and straightforward; below are listed some comments which I hope could improve the quality of the paper.
Point 1: Material and Methods: On line 62, the authors state that they used a milk sample that contained no target analyte as a blank. I suggest to specify if this blank sample has been purchased or if the absence of the NEOs analytes has been verified by the authors themselves and if so, how?
Response 1: We thank the reviewer for this comment. A blank milk sample was previously analyzed using UHPLC−MS/MS and selected in order to ensure the absence of analytes. Please see lines 50-51.
Point 2: The section “Materials and Methods” should be divided into subsections for ease of reading for examples (Reagents and Calibrators preparation; NEOs extraction; UHPLC-MS/MS analysis).
Response 2: We thank the reviewer for this constructive comment. We have divided into three subsections. Please see pages 2-3.
Point 3: Results and Discussion. To improve the understanding of the results it would be better to divide this section into two separate paragraphs: methodological evaluation and application of the method. Carrying out this subdivision, a critical point would emerge: the application part is lacking in information and this makes the paper unattractive. I therefore suggest reformulating this last part by placing a more precise focus on the strengths of the method and its possible applications.
Response 3: We thank the reviewer for this constructive comment. We have divided into two separate paragraphs and added the strengths of the method and its possible applications in lines 121-131.
“Compared with the aforementioned techniques, this study established a simple, fast, efficient and low cost liquid-liquid extraction–UHPLC–MS/MS method with satisfactory results for MRL in milk. The method developed shows satisfactory validation parameters in linearity, low LOD, accuracy and precision and fits for the simultaneous determination of seven NEOs in milk samples.
The presence of NEOs in breast milk has received increasing interest since human milk is the main nutrition for infants and reveals neonatal exposure. As our knowledge, limited data are available on the NEO residues in breast-milk [11]. Exposure of the neonates to NEOs appears to be important, because NEOs are neurotoxicants and may cause neurotoxicity caused by ACE and IMI [12,13]. Future studies are needed to monitor milk levels in breastfeeding women and to study potential risk for NEOs of newborns through milk consumption.”
Point 4:
Conclusion: The last sentence of the conclusion touches on an extremely interesting point on human breast milk screening, but no discussion on this topic has been addressed in the paper, can the authors explain this aspect better?
Response 4: We thank the reviewer for this constructive comment. We have discussed the importance of biomonitoring exposure to NEOs in breast milk. Please see lines 125-131.
“The presence of NEOs in breast milk has received increasing interest since human milk is the main nutrition for infants and reveals neonatal exposure. As our knowledge, limited data are available on the NEO residues in breast-milk [11]. Exposure of the neonates to NEOs appears to be important, because NEOs are neurotoxicants and may cause neurotoxicity caused by ACE and IMI [12,13]. Future studies are needed to monitor milk levels in breastfeeding women and to study potential risk for NEOs of newborns through milk consumption.”
Author Response File: 
Author Response.doc
Reviewer 2 Report
In my opinion the manuscript can be accepted if the authors explain my following two doubts:
- The authors claim that they use isotope dilution method for the analysis of seven compounds, however they have obtained isotope labeled standards of four compounds.
- The used transitions (Table 1) should be justified by respective literature data or by respective mass spectra (the latter could be shown in supplementary material).
(it is not necessary to use the compounds abbreviation in the abstract)
Author Response
Response to Reviewer 2 Comments
In my opinion the manuscript can be accepted if the authors explain my following two doubts:
Point 1: The authors claim that they use isotope dilution method for the analysis of seven compounds, however they have obtained isotope labeled standards of four compounds.
Response 1: We agreed with the reviewer’s comment. We have deleted isotope dilution method throughout the manuscript.
Point 2: The used transitions (Table 1) should be justified by respective literature data or by respective mass spectra (the latter could be shown in supplementary material).
(it is not necessary to use the compounds abbreviation in the abstract)
Response 2: We thank the reviewer for the comment. We have added respective mass spectra in the supplementary data (Figure S1). We have deleted the compounds abbreviation in the abstract.
Please see the attachment.
Author Response File: Author Response.doc
Round 2
Reviewer 2 Report
I still have doubts concerning the use of the isotope labeled standards for the determination of some of the compounds. It should be clearly described in the manuscript. The authors should briefly explain why the linearity, precision and accuracy are always similar, independently of whether the authors used isotope labeled standards or not.
There is no mass spectrum of thiacloprid.
Author Response
Response 1: We thank the reviewer for this comment. We have added explanation of the use of the isotope labeled standards in the section 3.1. Methodological evaluation. “Although quantitation without the use of isotope labeled standards considerably yielded similar results of the linearity, precision and accuracy, this may attribute to the stable method performance, the relatively small personal error, and no obvious matrix effect. Further studies with the isotope labeled standards are essential and encouraged for very accurate quantification in order to compensate matrix effects and mitigate measurement uncertainty [12].” Please see lines 104-109.
The mass spectrum of thiacloprid was shown in supplementary data (p18).
Please see the attachment.
Author Response File: Author Response.docx
