Screening of 258 Pesticide Residues in Silage Using Modified QuEChERS with Liquid- and Gas Chromatography-Quadrupole/Orbitrap Mass Spectrometry
, Xingqiang Wu 1, Yanling Song 2, Yini Sun 2, Kaixuan Tong 1, Xiaoxuan Yu 1, Chunlin Fan 1 and Hui Chen 1,*
Round 1
Reviewer 1 Report
Dear Authors,
my oppinion can be read in attached file.
Best regards
Comments for author File: 
Comments.pdf
Author Response
Reviewer's opinion about MS entitled “Screening of 258 pesticide residues in silage by Modified QuEChERS with Liquid- and Gas- Chromatography-Quadrupole/Orbitrap Mass Spectrometry”.
General comments:
Paper is a typical methodology work, which implies lots of practical advantages. The achieved results are handy for the simply screening and quantitive determination of pesticide residues in silage. Besides, I have to mention, that silage-based plant cultivation can be characterized by an extensive production technology, which means that pesticide-using in these plants is very rare.
Specific remarks.
INTRODUCTION
It would be lucky to mention the main Chinese silage-plants and its production technology in a short paragraph.
Reply: Based on the reviewers' comments, we have added relevant content in the first paragraph of the introduction by reviewing the literature. The content is" and its production process mainly includes the steps of harvesting, processing, transportation, filling, compaction, and capping [3-5]. As high-quality roughage, silage is rich in nutrients after 45 days of fermentation. It is an essential source of fermentable carbohydrates for ruminants and is known as the "king of feed"[4,6,7]. The silage of interest in this study mainly uses corn stover as raw material, which has high nutritional value and wide market demand. ".
What kind of pesticides and what against can be applied to silage crops? Please, mention the main ranges! I think so, that these thoughts are essential to the explanation of the later results
Reply: Thanks for your suggestion. We revised the manuscript. we have added relevant content in the second paragraph of the introduction by reviewing the literature. For details, the content is "The demand for silage is growing with the rise of the livestock industry in China. To increase silage yield, irrational application of pesticides may exist during maize cultivation [8]. Pesticide residues in silage are mainly derived from pesticide spraying during planting to prevent diseases and pests [9]. According to the literature, the main pesticides involved in maize cultivation are organophosphorus, organochlorine, and carbamate [10,11]. Besides incorrect use of pesticides, pollution in the environment can also cause silage to contain a small number of pesticide residues, such as water and soil contaminated by pesticides [12,13].".
Please, delete "to the best of our knowledge" in line 62, because the hypothesis needs to be well established (by citations). The assumption is only speculatory, which cannot be part of scientific work.
Reply: Yes, it has been rewritten.
METHODOLOGY
The instruments and reagents are well introduced but I think more empty lines need to be placed between the subsections in order the transparency and the easy reading.
Reply: Thanks for your suggestion. we revised the manuscript. For details, see " Section 2.1 ".
Unfortunately, lack of plant sample acquisition and origin presentation
Reply: Thanks for your suggestion. we revised the manuscript. We have added relevant content based on your suggestions, as detailed in section 2.2.
Important tasks, that: the numbers in chemical symbols must be subscripted
no place before °C
the units of measurement should be changed everywhere (if the criteria of the newspaper highlight it): etc. “μg/mL” to “μg mL-1”
Reply: Yes, it is improved.
RESULTS AND DISCUSSION
Some paragraphs have displayed the entire without the comparison with the related results. Please complement them, where they can be.
Reply: Thanks for your suggestion. we revised the manuscript. We have added relevant content based on your suggestions, as detailed in section 3.1.
Please check the Figure numbering style "Figure 4(a)"
Reply: Thanks for your suggestion. These have been rewritten and improved.
Latin originating abbreviations need to be written in italics (etc.: etc):)
Reply: Yes, it is improved.
Plentiful active ingredients can be read in Table 1. Many kinds of organochlorine and organophosphate are on the list, which belong to third-generation pesticides. I think the using area of the ingredients should be displayed in a separate column, such as herbicide, insecticide or fungicide
Reply: Thanks for your suggestion. We have added relevant content based on your suggestions, as detailed in Table 1.
Besides, it would increase the quality of the work, if the authors would give practical advice and conclusions from the Chinese pesticide use in silage crops
Reply: Thanks for your suggestion. We have added relevant content based on your suggestions, as detailed in section 3.5.
Overall, the manuscript is valuable and it contains lots of practical benefits. With the above-mentioned adjustments and the remarks, the manuscript can be released for publication after minor revision in Agriculture, which will be valuable for practical experts, who are employed in qualitative inspection of stored silage items.
Reviewer 2 Report
In this manuscript, a modified QuEChERS method combined with LC and GC coupled to Q-orbitrap/MS was used to determine 258 multiclass pesticides in silage. The authors optimized the extraction and clean-up conditions, but only traditional reagents that have been used for years were tested. The optimized method was adequately validated at 3 different levels using matrix matched calibration, yielding acceptable recoveries, repeatability, and sensitivity. Although the methodology is not new or innovative, it works well. Furthermore, the manuscript is well organized, and the discussions of the results are good. All this together gives an appropriate manuscript for the journal, but the following points should be checked:
Point 1. The English of the text needs a thorough revision. There are many mistakes scattered all over the text that must be fixed before the manuscript can be considered suitable for publication. For example, in lines 2, 4, 11, 13, 24, 48, 50, 52, 53, 113, 114, and a very long etcetera.
Point 2. Line 46: There are several publications in which pesticide residues in silage were analyzed using the QuEChERS method. Even one of these works was developed by the father of the QuEChERS method. The authors must analyze them and argue the gap that this work is filling.
Point 3. Line 167: The authors added 1% acetic acid during sample extraction for all experiments. As is well known, acetic acid is added together with NaOAC in the AOAC version of the QuEChERS method to form an acetic acid/acetate buffer that prevents the degradation of alkali-sensitive pesticides (reference 15). However, in the end, the authors selected EN salts that include a citrate buffer for the same purpose. So, why did the authors add acetic acid in the EN version?
Point 4. Lines 169-293. In each section from 3.1.1 to 3.1.5, all other variables must be detailed, not just the one being optimized. How many replicates were performed for each set of experiments? In this sense, the repeatability results must be included both in the text and in figures 2-5. Were all the experiments performed at 100 µg/kg? This must be specified.
Point 5. Lines 169-293: The authors evaluated many variables, except the amount of sample. Why was 2 grams of sample selected?This should be explained in the text.
Point 6. Lines 220-223: For future work, I recommend using ammonium formate as the extraction salt, as it has been shown to be a better option than sodium and magnesium salts, particularly for MS: https://doi.org/10.1016/j.chroma.2014.06.104
Point 7. Line 237: Likewise, the effect of CarbonX could have been studied, as it has been shown to be an effective substitute for GCB to remove pigments without loss of planar pesticide recoveries: https://unitedscience.com/shop/carbonx/carbonx-plus-carbon-quechers/. In fact, this would have been very pertinent in view of most pesticides were affected by a medium and strong matrix effect. Another recommendation is to determine the amount of coextracted material from the matrix that is injected into the chromatographic system by gravimetry. In this way, in addition to the recoveries, it could be used to decide which combination of clean-up sorbents is the best. This is important to reduce the need for maintenance of the chromatographic system.
Point 8. “LOQs were determined as the lowest validated spike level based on the recovery results by spiking a series of mixed standard solutions in blank samples.”. Consequently, these are not LOQs (typically defined as 10 times the S/N ratio).
Point 9. Line 368: Can the authors provide more information about the real samples (geographical origin, composition, etc.)?
Point 10. Other comments:
- The keywords must be different from those of the title.
- Table 1: It could be included as complementary material due to its length. In addition, it requires revision regarding the format since many inconsistencies are observed. What do the numbers below the ions correspond to? What were the collision energies used?
Author Response
In this manuscript, a modified QuEChERS method combined with LC and GC coupled to Q-orbitrap/MS was used to determine 258 multiclass pesticides in silage. The authors optimized the extraction and clean-up conditions, but only traditional reagents that have been used for years were tested. The optimized method was adequately validated at 3 different levels using matrix matched calibration, yielding acceptable recoveries, repeatability, and sensitivity. Although the methodology is not new or innovative, it works well. Furthermore, the manuscript is well organized, and the discussions of the results are good. All this together gives an appropriate manuscript for the journal, but the following points should be checked:
Point 1. The English of the text needs a thorough revision. There are many mistakes scattered all over the text that must be fixed before the manuscript can be considered suitable for publication. For example, in lines 2, 4, 11, 13, 24, 48, 50, 52, 53, 113, 114, and a very long etcetera.
Reply: Thanks for your suggestion. These have been rewritten and improved.
Point 2. Line 46: There are several publications in which pesticide residues in silage were analyzed using the QuEChERS method. Even one of these works was developed by the father of the QuEChERS method. The authors must analyze them and argue the gap that this work is filling.
Reply: Thanks for your suggestion. These have been added and improved. For details, the content is “The literature research shows that the current published QuEChERS method has achieved satisfactory stability and recovery, but the number of pesticides detected and the sensitivity of the method need to be improved.”
Point 3. Line 167: The authors added 1% acetic acid during sample extraction for all experiments. As is well known, acetic acid is added together with NaOAC in the AOAC version of the QuEChERS method to form an acetic acid/acetate buffer that prevents the degradation of alkali-sensitive pesticides (reference 15). However, in the end, the authors selected EN salts that include a citrate buffer for the same purpose. So, why did the authors add acetic acid in the EN version?
Reply: Thanks for your suggestion. The original unbuffered version evolved into two official methods using citrate buffering at a relatively low buffering capacity (CEN Standard Method EN 15662, developed by Anastassiades) or acetate buffering at higher concentration to give a greater buffering strength (AOAC Official Method 2007.01, developed by Lehotay). Both methods make the pH value of the extraction system about 5, which is conducive to the satisfactory recovery of sensitive pesticides under acidic or alkaline conditions. Acetonitrile solution with the addition of 1% acetic acid has less effect on the pH of the citrate buffer system, while this extraction solvent is widely used in plant-derived matrices. Further investigation revealed that for the development of multi-residue methods, 1% acetate acetonitrile exhibited better extraction effects and compatibility with different polar pesticides [27,28]. Therefore, in this study, 1% acetate acetonitrile was used as the extraction solvent, while EN salt was finally selected as the salting agent through experimental comparison.
Point 4. Lines 169-293. In each section from 3.1.1 to 3.1.5, all other variables must be detailed, not just the one being optimized. How many replicates were performed for each set of experiments? In this sense, the repeatability results must be included both in the text and in figures 2-5. Were all the experiments performed at 100 µg/kg? This must be specified.
Reply: All experiments involved in this work were carried out at 100 ug kg-1 and were done in 3 parallels. The article's related questions have been revised according to the reviewers' comments. At the same time, the reproducibility results are included in Figures 2-5 based on the experimental data.
Point 5. Lines 169-293: The authors evaluated many variables, except the amount of sample. Why was 2 grams of sample selected? This should be explained in the text.
Reply: Thanks for your suggestion. These have been added and improved. For details, the content is “Considering silage's high pigment and fiber-rich characteristics, the sample weight should be appropriately reduced when using QuEChERS for pre-treatment. Combined with the research of the previous group on forage matrix pre-treatment [32], the sample weight was fully considered for instrument detection sensitivity and matrix interference, so 2 g of substance was selected for further research in this work. The QuEChERS procedure was evaluated due to the potential for matrix interference, one of the most challenging situations in high-throughput screening and essential for validating quantitative determination. Therefore, different parameters based on the QuEChERS method have been evaluated.”.
Point 6. Lines 220-223: For future work, I recommend using ammonium formate as the extraction salt, as it has been shown to be a better option than sodium and magnesium salts, particularly for MS: https://doi.org/10.1016/j.chroma.2014.06.104
Reply: Thanks for your suggestion. Our team has carefully studied the material you provided, and we will focus on comparing this condition's effect on the follow-up study's experimental results. Thanks again to the reviewer for sharing.
Point 7. Line 237: Likewise, the effect of CarbonX could have been studied, as it has been shown to be an effective substitute for GCB to remove pigments without loss of planar pesticide recoveries: https://unitedscience.com/shop/carbonx/carbonx-plus-carbon-quechers/. In fact, this would have been very pertinent in view of most pesticides were affected by a medium and strong matrix effect. Another recommendation is to determine the amount of coextracted material from the matrix that is injected into the chromatographic system by gravimetry. In this way, in addition to the recoveries, it could be used to decide which combination of clean-up sorbents is the best. This is important to reduce the need for maintenance of the chromatographic system.
Reply: Thanks for your suggestion. Based on the above information, we have reviewed a large amount of literature and have some understanding of the purification material CarbonX. In the subsequent study, we will focus on testing, comparing, and optimizing this purification filler and applying it to the purification of highly pigmented substrates.
Point 8. “LOQs were determined as the lowest validated spike level based on the recovery results by spiking a series of mixed standard solutions in blank samples.”. Consequently, these are not LOQs (typically defined as 10 times the S/N ratio).
Reply: Thanks for your suggestion. The LOQ values were tested according to that specified in the SANTE/11312/2021 guidance document, i.e., the lowest spike level meeting the method performance criteria for trueness and precision. Meanwhile, the response S/N ratios of all pesticides at the limit of quantification concentration levels were greater than 10, which met the definition of LOQ.
Point 9. Line 368: Can the authors provide more information about the real samples (geographical origin, composition, etc.)?
Reply: Thanks for your suggestion. These have been added and improved. For details, see in section 2.2.
Point 10. Other comments:
- The keywords must be different from those of the title.
Reply: Thanks for your suggestion. we revised the manuscript.
Table 1: It could be included as complementary material due to its length. In addition, it requires revision regarding the format since many inconsistencies are observed. What do the numbers below the ions correspond to? What were the collision energies used?
Reply: Table 1 provides a wealth of information about the study, and considering the reader's ease of reading the article, we would like to continue to keep it in the original text. Previously Table 1 was due to our negligence in not adjusting the table properly to show that there were numbers underneath, thus leading to a misunderstanding by the reviewer. We have standardized the table and reduced the length significantly. The acquisition mode of LC-Q-Orbitrap/MS is Full MS/dd-MS2, so the collision energies are set to stepped normalized collision energies of 20, 40, and 60. The acquisition mode of GC-Q-Orbitrap/MS is Full MS, so the collision energies are set to standard collision energies of 70 eV, see in section 2.1.1 and 2.1.2.
Round 2
Reviewer 2 Report
The manuscript can be published.
