AOAC SPIFAN Chlorate-02 Reviewer Form (July 2022)

AOAC Stakeholder Program on Infant Formula and Adult Nutritionals

CHLORATES/PERCHLORATES REVIEWER FORMS (CHLORATE-02)

Tuesday, July 26, 2022

AOAC INTERNATIONAL 2275 Research Blvd., Suite 300 Rockville, MD, 20850 USA

dboyd@aoac.org 301.924.7077 x126

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Expert Review Panel for Infant Formula and Adult Nutrition Evaluation of Method Chlorate-2

Title: A LC-MS/MS method for the sensitive and accurate quantification of Chlorate and Perchlorate in a broad range of food commodities, including baby food, nutritional formulas, and ingredients thereof Author: Huertas-Perez, JF, Mottier, P, Konings, E, et. Al. Reviewer Name: Reviewer 1 Summary of Method: Analytes are extracted with a mixture of water, acidified methanol, and dichloromethane. For dairy products and byproducts, extraction can be performed with water, acidified methanol, and EDTA, followed by a two-step clean-up (freezing and dispersive SPE with C18 in acetonitrile. Quantitation is by isotopic dilution liquid chromatography tandem mass spectrometry. Method Scope/Applicability: The method was validated for the following categories: Baby food, finished products; Baby food ingredients; Infant/adult formulas; Infant/adult formulas ingredients; and other human food or ingredients. The matrix categories were covered in the method as outlined in the SMPR. General comments about the method: The method is fairly straight forward and easy to do. The reproducibility, repeatability, accuracy, etc., look really good. The low-level spikes of 2 and 10 ppb are tough. There was only one question I had about the aliquots that get mixed with acetonitrile before filtering. Why such a low amount, i.e., 80 μ l, and 500 μ l, why not larger quantities. Method Clarity: The method is straight forward to use. Everything is easy to understand. The calculations, tables, etc., are well done. The only question I would have is the dilution of the samples using µl quantities. Pros/Strengths: • It can be used for multiple sample types. • Multiple samples can be extracted for a large run. • Data analysis is straight forward and can be done either with a spreadsheet or through the instrument software. • The use of 18 O 3 internal standards makes identification and quantitation easier. Cons/Weaknesses • The need of a mid-level analyst as a minimum. • Cost of the instrument (since this is the only one of three that could do the work, is it really a con?) • Cost of internal standards (this is the only way to do the two compounds reliably) Supporting Data • General Comment: The supporting data is easy to understand and to use. Tables 1-4 are well done, and easier to use. The use of another MS/MS would require infusing to determine the conditions for the

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transitions. The transitions should be the same as in the table. The remaining tables are easy to follow and contain the appropriate information to make necessary decisions about the method. Method Optimization: The method appears to be fully optimized with the appropriate matrices.

• Performance Characteristics:

Analytical Range: There is no analytical range specified in the SMPR. LOQ: The LOQ’s were met but should have been reported as consumed products. The “correction factors” should have been applied and the correct LOQ’s been reported. The SMPR concentrations apply to as consumed products. I am not sure how this would apply to the dried ingredients. Accuracy/Recovery: All baby food matrices analyzed met the requirement in the SMPR.

All baby food Ingredients analyzed met the requirement in the SMPR. All infant/adult formulas and ingredients analyzed met the requirement in the SMPR.

Precision (RSD r ): All baby food matrices analyzed met the requirement in the SMPR. All baby food ingredients analyzed met the requirement in the SMPR. All infant/adult formulas and ingredients analyzed met the requirement in the SMPR. Reproducibility (RSD R ): All baby food matrices analyzed met the requirement in the SMPR.

All baby food ingredients analyzed met the requirement in the SMPR. All infant/adult formulas and ingredients analyzed met the requirement in the SMPR.

• System suitability: I could find no indication of a system suitability check for the LC-MS/MS. The only thing is an instrument check test. This is just equilibration of MP, pressure check, and leak check. There was no indication of a standard injected multiple times to ensure the resolution is adequate, as well as the transitions are fine. 1. Is the Validation Study Report in a format acceptable to AOAC? It will need some work before publication. 2. Is the method described in sufficient detail so that it is relatively easy to understand, including equations and procedures for calculation of results (are all terms explained)? Yes. 3. Are the figures and tables sufficiently explanatory without the need to refer to the text? There are some issues with the LOQ’s where they are high when the “correction factor” is not applied. There should have been no need to do additional calculations. 4. Are all the figures and tables pertinent? Yes. It would have been nice to exclude the non SMPR matrices, and include more chromatograms.

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5. Could some be omitted and covered by a simple statement? There could have been a general summary statement comparing the two extraction methods. The table could have been put as supplementary data. 6. Are the references complete and correctly annotated? Yes. 7. Does the method contain adequate safety precaution reference and/or statements? Yes Recommendation: There just needs to be some modifications to the manuscript. There needs to be a system suitability section. It does meet the SMPR criteria. I would need to review the system suitability. I do not recommend this for 1 st action at this time.

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Expert Review Panel for Infant Formula and Adult Nutrition Evaluation of Method Chlorate 02

Title: A LC-MS/MS method for the sensitive and accurate quantification of Chlorate and Perchlorate in a broad range of food commodities, including baby food, nutritional formulas, and ingredients thereof. Author: José Fernando Huertas-Pérez, Pascal Mottier, Erik Konings, Quentin Baslé , Shi Ying Tan , Monika Kopeć -Durska , Patrycja Zawada , Ashley Griffin , María Guadalupe Sánchez-Calderón , Juan Silva , Yanira Gutiérrez Reviewer Name: Reviewer 2 Summary of Method: Chlorate and perchlorate are extracted with a mixture of water, acidified methanol, and dichloromethane by using a cell disruption instrument. The solution is then centrifuged, and an aliquot of the supernatant is diluted before injection on the LC-MS/MS. An optional sample preparation for dairy products and by-products commodities is included. This optional extraction is performed with a mixture of water, acidified methanol and EDTA, followed by a cleanup based on freezing out and dispersive solid phase extraction with C18 in acetonitrile. The purpose of this optional sample preparation is preserving the LC-MS-MS. Quantitative determination is carried out by isotopic LC-MS/MS.

Method Scope/Applicability: Baby food, infant/adult formulas, baby food ingredients, infant-adult formulas ingredients and other food commodities.

General comments about the method:

Method Clarity: The method is clearly written. Pros/Strengths:

• The method is straightforward. • Fast, good method for high throughput analysis • Uses common reagents present in all laboratories. • The method has an optional sample preparation for dairy products and by-products to preserve the LC MS/MS.

Cons/Weaknesses • No weaknesses.

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Supporting Data:

• General Comment:

Method Optimization: The method is well optimized. It has a general sample extraction and an optional sample preparation for dairy products and by-products to preserve the LC-MS/MS.

• Performance Characteristics:

Analytical Range: Wide analytical range. The method meets the SMPR requirement for LOQ and it analyzes a wide number of samples at different concentrations.

LOQ: General extraction (concentration as consumed products): Perchlorate: 0,002 mg/kg in baby food and infant/adult formula.

0,010 mg/kg or less in ingredients for baby food or infant/adult formula.

Chlorate: 0,005mg/kg or less in baby food and infant/adult formula.

0,010mg/kg or less in ingredients for baby food or infant/adult formula.

Optional extraction (concentration as consumed products): Perchlorate: 0,005 mg/kg or less in infant/adult formula.

0,010 mg/kg or less in infant/adult formula ingredients. Chlorate: less than 0,005mg/kg in infant/adult formula (except adult formula ready to drink that was analyzed at 0,01 mg/kg level). 0,010 mg/kg or less in infant/adult formula ingredients. Accuracy/Recovery: The results are generally in agreement with the SMPR. Some matrices are not fully validated, but matrix commodity checked for good results. A small percentage of results (recovery or repeatability) are slightly under SMPR requirements, but they are only a very small percentage of the total amount of samples analyzed. Precision (RSD r ): The results are generally in agreement with the SMPR. Some matrices are not fully validated, but matrix commodity checked for good results. A small percentage of results (recovery or repeatability) are slightly under SMPR requirements, but they are only a very small percentage of the total amount of samples analyzed.

Reproducibility (RSD R ): MLT was not performed.

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• System suitability:

1. Is the Validation Study Report in a format acceptable to AOAC? Yes

2. Is the method described in sufficient detail so that it is relatively easy to understand, including equations and procedures for calculation of results (are all terms explained)? Yes

3. Are the figures and tables sufficiently explanatory without the need to refer to the text? Yes

4. Are all the figures and tables pertinent? Yes

5. Could some be omitted and covered by a simple statement? No

6. Are the references complete and correctly annotated? Yes

7. Does the method contain adequate safety precaution reference and/or statements? Yes

Recommendation: Good method, easy and fast to perform. The authors analyzed with very good results, a lot of matrices: matrices included in the SMPR, and other matrices not included, showing that the method has a broad applicability. Validation involved the two extraction procedures. Spiking samples, reference samples (not certified reference materials analyzed) and PT rounds were used for validation. The results are generally in agreement with the SMPR. Some matrices are not fully validated, but matrix commodity checked for good results. A small percentage of values (recovery or repeatability) are slightly under SMPR requirements, but they are only a very small percentage of the total amount of samples analyzed. Although SMPR does not require intermediate precision and uncertainty to be evaluated, their results from the method are very good, in agreement with SANTE guide.

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Looking at the chromatograms presented in the manuscript, the peaks have a very good resolution, and the symmetry of them is ok. As requested for the ERP, all the chromatograms at the LOQ levels in each matrix, corresponding matrix blanks and all calibration solution should be presented. Due to both analytes are present in the environment from both natural and anthropogenic source, another requirement of the ERP is to provide concentration in blank samples used for validation. Plant based infant formula was not validated. The authors mentioned that blank sample to validate the LOQ level was not available, but any level was validated for this matrix. The good performance of the method for this matrix was shown obtaining good results in a PT from NIST, but plant based infant formula is one of the matrices required for the SMPR to be validated. No validation data presented for liquid milk from a different species other than bovine. The authors mentioned this matrix was not available for validation at the LOQ level, but they did not present any data for this matrix. Before recommending the method for First Action, I would like the authors present all the chromatograms required and the concentration in blank samples used for validation.

I do not recommend at this moment the method for First Action status due to my comments above.

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Expert Review Panel for Infant Formula and Adult Nutrition Evaluation of Method CHLORATE-02

Title: 1 A LC-MS/MS method for the sensitive and accurate quantification of Chlorate and Perchlorate in a broad range of food commodities, including baby food, nutritional formulas and ingredients thereof Author: José Fernando Huertas-Pérez: Nestlé Research, Vers-chez-les-Blanc, 1000 Lausanne 26, Switzerland Reviewer Name: Reviewer 3 Summary of Method: LC-MS/MS method for the determination of chlorate and perchlorate in food commodities. Isotopic dilution used for quantitative analysis. Analytes are extracted with a mixture of water, acidified methanol, and dichloromethane.

Method Scope/Applicability: Method was validated in a wide range of food matrices in accordance with AOAC SMPR® 2021.001 Standard Method Performance Requirements (SMPRs®) for Determination of Chlorate and Perchlorate in Baby Foods, Infant/Adult Formulas, and Their Ingredients.

General comments about the method: Method is very well written and easy to follow. The details of the method and subsequent validation are thorough and meet the SMPR requirements fully.

Method Clarity: As stated above. The method is very well written and covers all the details necessary to be considered for First Action Status. The validation went above and beyond.

Pros/Strengths: • Simple Sample Preparation

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• Peak shape and separation. • Validation is thorough.

Cons/Weaknesses • Minor confusion about 2 different sample preparations. Results seem equivalent.

Supporting Data • General Comment: Significant data provided in easy-to-follow format.

Method Optimization:

• Performance Characteristics: Performance Characteristics meet SMPR requirements.

Analytical Range:

LOQ:

Accuracy/Recovery:

Precision (RSD r ):

Reproducibility (RSD R ):

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• System suitability: Covered under Operating Procedures section of the method document.

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Is the Validation Study Report in a format acceptable to AOAC? Yes, format is acceptable. 1. Is the method described in sufficient detail so that it is relatively easy to understand, including equations and procedures for calculation of results (are all terms explained)? Yes, method detail is sufficient.

2. Are the figures and tables sufficiently explanatory without the need to refer to the text? Yes, the figures and tables provide explanations that allow them to stand alone from the method document/manuscript.

3. Are all the figures and tables pertinent? Yes.

4. Could some be omitted and covered by a simple statement? No, All data tables and figures are necessary.

5. Are the references complete and correctly annotated? Yes.

6. Does the method contain adequate safety precaution reference and/or statements? Yes.

Recommendation: I recommend that this method is accepted as a First Action Method. The authors have covered all details and the method is well written. The exceptions to the SMPR are minor and have been explained with detail in the results/discussion section of the manuscript.

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