Rapid Detection of Aspergillus spp. and Quantitative Simultaneous Analysis of Aflatoxins (B1, B2, G1 and G2) in Kenyan Hybrid Maize Cultivars Using FT-IR and LC-ESI-MS/MS Spectro-analysis Techniques
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The present study combined Fourier-Transform Infrared Spectroscopy (FT-IR) analysis with high resolution liquid chromatography tandem mass spectroscopy equipped with an electrospray ionization source (LC-ESI-MS/MS) in the screening and quantification of four naturally occurring aflatoxins (AFs) AFB1, AFB2, AFG1 and AFG2 in milled hybrid maize cultivars (n = 171) from selected counties in the Rift-Valley region of Kenya. Samples were extracted in a single-step process using acetonitrile/water 80:20 v/v in 0.5% formic acid without further clean-up steps. Aflatoxins were analyzed in both positive and negative ionization modes using the multi-reaction monitoring (MRM) acquisition process that allowed confirmation and quantification of the aflatoxins in a single run. The MRM mode was utilized for quantitative analysis while qualitative analysis was done using the enhanced product ion mode. Aflatoxin presence was detected in the following ranges and frequencies: AFB1 (0-9.36μg/kg), AFB2 (0-6.62μg/kg), AFG1 (0-7.72μg/kg) and AFG2 (0-9.18μg/kg). Additional mycotoxins recovered included the following; sterigmatocystin (0-8.3μg/kg) and deoxynivalenol (0-7.3μg/kg). In overall, low amounts of aflatoxins were recovered from the milled maize samples with an additional low total aflatoxin content above the stipulated regulatory limit of 10μg/kg. Given that slow continued exposure to aflatoxins (especially in low amounts) has been proven to be a leading cause of various forms of cancer, urgent intervention measures and national surveillance programs ought to be introduced to protect consumers from gradual aflatoxin poisoning emanating from the consumption of contaminated Kenyan maize.
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Comments to Author
Dear Authors, I got the chance to evaluate your manuscript. First and foremost, I would like to commend you for addressing an important public health concern through your study. The work presents a significant contribution to food safety research, particularly in the African context, by combining spectroscopic and mass spectrometric methods to assess mycotoxin contamination in maize. The sample size, analytical strategy, and regional relevance of the study all add notable strength and applicability to the work. That said, I have a few concerns and suggestions I believe could further strengthen your manuscript before it is considered for publication. I have outlined these BELOW. As I usually find helpful the detailed response, the evaluation is presented in a detailed paragraph-based format. I began by …
Comments to Author
Dear Authors, I got the chance to evaluate your manuscript. First and foremost, I would like to commend you for addressing an important public health concern through your study. The work presents a significant contribution to food safety research, particularly in the African context, by combining spectroscopic and mass spectrometric methods to assess mycotoxin contamination in maize. The sample size, analytical strategy, and regional relevance of the study all add notable strength and applicability to the work. That said, I have a few concerns and suggestions I believe could further strengthen your manuscript before it is considered for publication. I have outlined these BELOW. As I usually find helpful the detailed response, the evaluation is presented in a detailed paragraph-based format. I began by carefully understanding your intentions, methodology, and the logical flow of your manuscript. I then correlated each of your major points, results, and interpretations with the overall structure and claims of your work. Based on this understanding, I raised rational, scientifically grounded questions and observations. My feedback is cited against your manuscript's sections and lines where possible and is supported by reasoned critique and commentary to assist you in refining the manuscript. That is why you might find my assessment in elaborative paragraphs formats. Please take my comments in the spirit of constructive peer review. I sincerely hope they are helpful to you in revising and improving the scientific clarity, reproducibility, and alignment of your work. Best wishes as you refine this valuable manuscript, and I wish you great success with this and your future scholarly endeavors. Warm regards, ===================================================================================== Review Report : Access Microbiology OVERALL ASSESSMENT BASED Questions: 1. What specific features in the FT-IR spectra were used to screen for Aspergillus spp., and how were these validated against known fungal presence or infected controls? Can authors explain should this FT-IR's contribution interpret as exploratory or diagnostic? Okay consider this way; How did the authors validate that FT-IR spectra from bulk maize samples reflect the presence of Aspergillus flavus or A. parasiticus, given the dominance of grain matrix over microbial content? 2. If FT-IR is presented as a rapid screening tool for mycotoxigenic fungi, why were no performance metrics (accuracy, specificity, sensitivity) provided to support this claim? 3. Were any fungal isolates obtained from the maize samples to confirm fungal species by culture, microscopy, or genetic methods? 4. Were any fungal isolations, culturing, or molecular identifications (e.g., ITS sequencing) performed to confirm FT-IR predictions or PCA cluster assignments? 5. On what biological basis were the PCA clusters interpreted (e.g., county, contamination, composition)? Without fungal confirmation, how can these be confidently attributed to fungal infection? Why was only PCA used? If the intent was classification or detection, why was no supervised method (e.g., PLS-DA) used to assess FT-IR's predictive capability? 6. What does the phrase "peak area ratio of one toxin to another" refer to in your quantification? Was an internal standard used, or were toxins quantified independently? 7. Several figures and statements imply recovery of A. flavus/A. parasiticus from samples. How were these species confirmed? If not, should these claims be reframed as speculative? 8. Can FT-IR truly be called a "rapid detection" method in this context if no fungal identity, specificity, or accuracy metrics were demonstrated? 9. I would recommend that Table 1 (sample-by-sample toxin data) be moved to supplementary material. figure legends be revised to clearly explain "-/-", "f/g" notations, and peak labels? 10. Given the complex matrix, how were co-extracted components (lipids, pigments, etc.) accounted for in FT-IR and LC-MS/MS analyses? 11. How were aflatoxins quantified below the lowest calibration range (e.g., 50%) aflatoxins present alongside an additional mycotoxin" is unclear. Since only 55 samples had any aflatoxin, ">50%" of what? Perhaps it mean over half of the aflatoxin-positive samples had co-contamination. Given 28 samples had co-occurrence of all three toxins, that is 28/55 = 50.9%. If so, it should be explicitly ">50% of AF-positive samples had at least one additional mycotoxin". Otherwise, it misleadingly sounds like >50% of all samples were co-contaminated, which contradicts earlier figures. We recommend rewriting it for clarity. * Figure Spectra: Figures 2 and 3 show example chromatograms and spectra. The text does not refer to them except in captions. If these figures are intended to illustrate co-contamination or MRM peaks, the authors should describe at least one panel in the text (e.g. "Figure 2A shows the MS/MS chromatogram of sample UG/025 with peaks for AFB₁, sterigmatocystin (STE), and DON"). Otherwise, the figures remain unused by the narrative. Recommendation is about spectra discussion, although figures are stated and linked in text. * Table-text synchrony: Table 3 provides calibration slopes and LOD/LOQfile-48pir6fgl5melfehyarg1b. It is good to see these, but the text should mention R² or linearity (none is given). For instance, after describing LOD/LOQ, the authors could state "All four AF calibration curves were linear over the tested range (r² > 0.99, data not shown)." If such metrics are unavailable, at least include them in Table 3. Otherwise, the regression equations (with intercepts) are provided without context. * Correlation analysis: A correlation (ρ=0.19) between AF and STE is reported (p=0.0065). While statistically significant, ρ=0.19 is very weak. The text says, "positive correlation," but readers may assume a strong link. The authors should temper this, noting that although statistically non-zero, the correlation explains
Please rate the manuscript for methodological rigour
Good
Please rate the quality of the presentation and structure of the manuscript
Good
To what extent are the conclusions supported by the data?
Strongly support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
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The work presented is clear and the arguments well formed. This study would be a valuable contribution to the existing literature. This is a study that would be of interest to the field and community. The reviewers have highlighted major concerns with the work presented. Please ensure that you address their comments.
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Comments to Author
Extremely well written, highly informative, and well-evidenced. Very minor issues to address, such as: Line 42 - 'Overall, low amounts...' Line 100 - Quite confusing, could benefit from 'and' before 'optimization of protocols' Figure 2 - Some of the chromatograms are cropped at the margins, obscurring the axis, and the third is missing the X axis label. Line 415 - I think it is fairer to say 'advanced' rather than 'augmented' the findings of past studies. Line 511 - Are cancer rates higher in a manner that could be attributable to mycotoxins, such as with oesopheageal cancer? Saying 'cancer cases have been previously reported' is very vague. Otherwise, very robust, a pleasure to read, and makes a strong case for action by regulatory systems and for the improvement of mycotoxin screening.
…
Comments to Author
Extremely well written, highly informative, and well-evidenced. Very minor issues to address, such as: Line 42 - 'Overall, low amounts...' Line 100 - Quite confusing, could benefit from 'and' before 'optimization of protocols' Figure 2 - Some of the chromatograms are cropped at the margins, obscurring the axis, and the third is missing the X axis label. Line 415 - I think it is fairer to say 'advanced' rather than 'augmented' the findings of past studies. Line 511 - Are cancer rates higher in a manner that could be attributable to mycotoxins, such as with oesopheageal cancer? Saying 'cancer cases have been previously reported' is very vague. Otherwise, very robust, a pleasure to read, and makes a strong case for action by regulatory systems and for the improvement of mycotoxin screening.
Please rate the manuscript for methodological rigour
Very good
Please rate the quality of the presentation and structure of the manuscript
Good
To what extent are the conclusions supported by the data?
Strongly support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
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