AOAC SPIFAN Chlorates Reviewer Form (March 2022)

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AOAC Stakeholder Program on Infant Formula and Adult Nutritionals

CHLORATES/PERCHLORATES REVIEWER FORMS (CHLORATE-01)

Wednesday, March 30, 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 01______ (Resubmission)

Title: Determination of Chlorate and Perchlorate in Baby Food, Baby Food Ingredients, Infant Formula and Infant Formula Ingredients by Liquid Chromatography – Tandem Mass Spectrometry

Author: ADELLE K. NANCEKIVELL, ELSA M. LOTZ, LISA A. GRAHAM Reviewer Name: Reviewer 1

Please Provide a Brief Summary of the Resubmitted Method: Samples are spiked with isotopically labelled internal standard, followed by extraction with acetonitrile and 2% formic acid in water. After centrifugation, the supernatant is subjected to a weak anion exchange solid phase extraction, eluted with 5% ammonia in acetonitrile then concentrated. The extract is analyzed by LC-MS/MS in multiple reaction monitoring (MRM) by electrospray ionization (ESI) in negative ion mode. Analytes are reported in terms of the free anion.

Was Method Scope/Applicability Updated? Yes. The method was improved to reach a reporting limit of 0,005 mg/kg.

Has the resubmitted method met the requirements outlined as per the ERP recommendation/comment(s)? Most of ERP requirements were included but not all of them.

Please provide documentation: Many samples were analyzed but not all minimum representative matrices were validated. Some matrices were analyzed in a low number of replicates (n=1) or only in one level of spiking. Regarding updates of references, the manuscript still has SANCO/12771/2013 as reference, but this version was replaced with other versions. Recovery and precision guidelines of last version are the same as 2013 version. The retention time acceptance criteria used for the lab (±2.5%) is not the value mentioned in 2013 version nor in newer version. The ±2.5% was used in older versions than 2013. The same happens with MRM ratio that is 30% in 2013 version and in newer version and not ±25%.

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Chromatograms: not all the required chromatograms were included. For calibration curve CS1 is included. The other chromatograms are registered as “samples” but is not clear what kind of samples are they. For one sample (maybe a blank matrix), the chlorate peak is not well resolved from an interference.

Table 2 indicates 10 µl injection but the chromatogram reports indicate 5 µl. Chlorate: 3rd transition in reports is 84.9-----69.1 but table 4 sets 82.95------69.1

Provide General comments: Chromatography was improved by increasing the aqueous component of mobile phase from 50% to 85%. The authors mentioned that no reference materials were available, but 1869A is available now, so they can use it when they analyze the samples they could not prepare until this moment . The authors mentioned that they are waiting for consumables to continue working with more matrices. A study of filter inertness was included but is not clear which filter must be used. In item Apparatus a nylon filter is mentioned but during Extraction Procedure a PVDF filter is recommended. Table 11: Regarding Whey Protein Hydrolysate I cannot see the difference between the first 3 rows and the last 3 rows. The incurred concentration is the same, the spiked concentration is the same, but they are listed in different rows. Final Recommendation: I recommend before moving to First Action, that the authors send the data of the samples they could not analyze due to shipment problems. More chromatograms are required with clear information about kind of sample and spiking level.

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Expert Review Panel for Infant Formula and Adult Nutrition Evaluation of Method Chlorate-01 (Resubmission)

Title: Method for the Determination of Chlorate and Perchlorate in Baby Food, Baby Food Ingredients, Infant Formula and Infant Formula Ingredients by Liquid Chromatography – Tandem Mass Spectrometry: Single Laboratory Validation Author: ADELLE K. NANCEKIVELL, ELSA M. LOTZ, LISA A. GRAHAM; AsureQuality Ltd Reviewer Name: Reviewer 2 3 ), followed by extraction with acetonitrile and 2% formic acid in water. After centrifugation, the supernatant is subjected to a weak anion exchange solid phase extraction, eluted with 1.75% ammonia in acetonitrile then concentrated. The extract is analysed by LC-MS/MS in multiple reaction monitoring (MRM) by electrospray ionisation (ESI) in negative ion mode. Analytes are reported in terms of the free anion. Quantification is performed by isotopic dilution using 18 O 3 labelled chlorate and 18 O 3 labelled perchlorate as internal standards, against solvent-based calibration standards. Was Method Scope/Applicability Updated? Yes. Applicable to the quantitative determination of Chlorate and Perchlorate in infant/adult formulas and their ingredients, baby foods and their ingredients by liquid chromatography-tandem mass spectrometry (LC- MS/MS). The method analyses chlorate and perchlorate in terms of the free anion. The limit of quantification (LOQ) of all analytes is 0.005 mg/kg or less. Application of this method to matrices not covered by the scope of the application requires additional validation. Please Provide a Brief Summary of the Resubmitted Method: Samples are spiked with isotopically labelled internal standards ( 18 O

Has the resubmitted method met the requirements outlined as per the ERP recommendation/comment(s)? See summary below from the Dec 2021 ERP Meeting. ERP Requirement Author Response

Request to see the chromatograms at the LOQ levels for each individual transition in each representative matrix, corresponding matrix blanks and all calibration solutions

Examples provided of chromatograms for CS1 and 4 samples are shown. I cannot tell which samples these chromatograms correspond to, and I have concerns about the approach to integration in some of the samples where significant coelution is evident. This does not encompass the totality of what was requested ( each transition in each matrix, corresponding matrix blanks, and all calibration solutions ).

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ERP Requirement

Author Response

Clarify the calculation of the results and consider simplified formula based on internal standard ratios (an example provided to the method authors) Update the references based on reviewer comments and also ion ratio requirements Please reference Reviewer 2 comments for coverage of matrices. None of the matrix categories are fully covered. Based on the SMPR, in table 1 (footnote) please check requirements for validation. Specifically, infant/adult formula category is important. Whey protein concentrate is not the same as whey protein hydrolysate. Table 11 – validation results have to be provided for each representative matrix and concentration level individually Validation should be done at the LOQ and at 10x LOQ as the minimum. Follow the SMPR for LOQ determination.

This appears to have been updated as recommended (manuscript page 12).

2 additional references have been added. Other reviewers will need to comment on whether this has been addressed to their satisfaction. Ion ratio requirements have been added (Table 6) No additional sample data has been provided, and thus still no matrix category is fully covered. The table from my Dec 2021 review is included at the end of this form to assist in discussions at Mar 2022 meeting. Table 11 has been updated to include individual results for each matrix and concentration level. No additional validation data is provided to indicate that the validation has been conducted at these levels as requested. Suggest that the authors provide an additional table with the same data as currently given, but organized to highlight the spike level with respect to the LOQ. LOQs have been summarized in Table 9 I found no additional information about the concentration of chlorate and perchlorate in the blanks used for validation.

Provide summary tables for the LOQs

Concentration in blanks used for validation needs to be provided

ERP Recommendations

Author Response

Consider improving chromatography to achieve narrower and symmetrical peak shapes (peak symmetry below 1.5) If isocratic elution is used, consider a gradient elution

No changes to the chromatography are apparent.

This appears to have been clarified as an isocratic elution but no comment about potential use of gradient.

Please provide documentation: Provide General comments: Some of the data and tables have been reorganized for clarity, but much of the critical validation results are still missing. Final Recommendation: This method is highly promising but needs validation using additional matrices to meet one or more of the matrix categories listed in SMPR 2021.001 (Table 1) prior to consideration for First Action.

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Matrix Table from Dec 2021 Review Matrix Category

Matrix Sub-Category

Minimum Representative Matrices Cereal-based Fruit/vegetable-based Meat-based Cereal-based Fruit/vegetable-based Meat-based Whole milk powder Whey protein concentrate Soy protein isolate Bovine One additional species Milk-based Plant-based

SMPR Met?

Additional Validation Data Needed for

Baby food

All parameters All parameters All parameters LOQ -- All parameters LOQ, RSDr All parameters -- All parameters All parameters -- All parameters All parameters LOQ, RSDr All parameters All parameters All parameters

Baby food ingredients Infant/adult formulas

Animal-based milk powdered protein sources

Plant-based protein sources

NO NO NO NO

Liquid milk

Infant/adult formula ingredients

Fat-based

Oil/fat

Carbohydrate-based

Lactose Maltodextrin Oligosaccharides

Mineral- and vitamin-based (premixes)

Any

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Expert Review Panel for Infant Formula and Adult Nutrition Evaluation of Method Chlorate-01 (Resubmission)

Please Provide a Brief Summary of the Resubmitted Method:

Was Method Scope/Applicability Updated? Yes, the applicability statement was updated to include all matrices the method is validated to analyze as well as the LOQ.

Has the resubmitted method met the requirements outlined as per the ERP recommendation/comment(s)? The SLV was updated to include results from more matrices along with updated LOD’s that meet the SMPR. Still have concerns about the chromatography as the peak shape is broader and has more tailing than desired. CHLORATE-01 ERP REQUIREMENTS: Request to see the chromatograms at the LOQ levels for each individual transition in each representative matrix, corresponding matrix blanks and all calibration solutions: It seems like there are interfering peaks in the chlorate transitions for the samples. I’m not sure you can accurately quantitate results with the coelution that is shown in that chromatography. Would like further information on what is shown. Clarify the calculation of the results and consider simplified formula based on internal standard ratios (an example provided to the method authors): Is forcing the calibration curve through zero a common practice in contaminant methods? This question is more for my information than questioning the method. Update the references based on reviewer comments and also ion ratio requirements: Ion Ratio requirements added. Table is confusing though. The MRM ratios are in %. Is that common practice? Please reference Reviewer 2 comments for coverage of matrices None of the matrix categories are fully covered: New matrices added but still missing a couple key matrices specified in the SMPR including Plant-based formula and ingredients.

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Based on the SMPR, in table 1 (footnote) please check requirements for validation Specifically, infant/adult formula category is important: Provided more data on infant/adult formula products and ingredients. Whey protein concentrate is not the same as whey protein hydrolysate: Still no WPC Table 11 – validation results have to be provided for each representative matrix and concentration level individually: Table 11 was updated with more detail and additional results for each matrix. Validation should be done at the LOQ and at 10x LOQ as the minimum: Spiking studies conducted at these 2 levels for many matrices. Provide summary tables for the LOQs: Summary tables were added to manuscript Follow the SMPR for LOQ determination Concentration in blanks used for validation needs to be provided: Inherent levels provided in results tables. CHLORATE-01 ERP RECOMMENDATIONS: Consider improving chromatography to achieve narrower and symmetrical peak shapes (peak symmetry below 1.5) If isocratic elution is used, consider a gradient elution

Please provide documentation:

Provide General comments: I have questions about the chromatograms that were shared with the reviewers. It looks like there are interfering peaks in the samples. There is also a lot of noise in the low standard for chlorate as well. No blank chromatograms were shared as requested.

Final Recommendation: Promising results but would like some clarity on the chromatographic interferences shown in the samples. Would like to see data on Plant-Based infant formula and ingredients as well before making a decision.

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Expert Review Panel for Infant Formula and Adult Nutrition Evaluation of Method Chlorate-01 (Resubmission)

Was Method Scope/Applicability Updated?

It was partially (please see General comments below).

Has the resubmitted method met the requirements outlined as per the ERP recommendation/comment(s)?

It has partially:

- Validation experimental design and results of the study are better described although they do not fully meet the SMPR requirements yet (please, see General comments below). - Calculations have been significantly simplified, although they still need some more clarifications (please, see General comments below).

Please provide documentation:

Provide General comments: 1.- Calculations

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Calculations has been simplified as per ERP recommendations; however, they need further clarifications. For instance, equation (c), please specify how C” is calculated?

Other considerations are that after the equation (c), V1 units are mg/L and no units are specified for C2.

2.- Validation Limit of Quantifications for kiwifruit (used as representative matrix for baby food and baby food ingredients) and lactose (representative matrix for infant/adult formula ingredients) were estimated as 3x SD of the results of seven replicates x 3.14 (99% confidence, one-side t-test). However, in the SMPR it is well stated that “LOQ is determined as the lowest spiking level that meets recovery and repeatability requirements” using either matrix blanks (< 30% contamination, subtracted) or if they are not available, spiking at 2 levels in the range 3-10x the analyte level in the matrix and extrapolating of S/N obtained for a concentration level corresponding to S/N 10. (that late option was chosen for IF, used as representative matrix for infant/adult formula). Furthermore, only 3 matrices (kiwifruit, IF, lactose) have been used as representative for the four matrix categories. In the SMPR the list of “Minimum representative matrices” includes a list of about 15 different matrices. Repeatability of the method was estimated only for the 3 representative matrices already mentioned for estimation of LOQs (kiwifruit, IF, lactose). Intermediate reproducibility has been estimated pooling 129 determinations in different matrix categories and subcategories, which gives a conservative value (as the RSD ip is going to be higher across categories than for a single category). However, it has been done by pooling results at different concentration levels, which may hide a lower performance of the method at low concertation level, and in any case does not give the figure at the LOQ concentration levels as required in the SMPR. Additionally, the number of determinations for each matrix can vary from 1 to 11, which gives more significance to some commodities that to others. Additionally, performance of the method (recovery) for most of the matrices including all the baby food and all the baby food ingredients (except kiwifruit), have been assessed at only at one concentration level. However, the SMPR clearly state that each matrix should be validated at least at the LOQ and 10x LOQ concentration levels. There is a need to clarify what of the ingredient matrices used for validation are minor or major ingredients for infant/adult formula (finished powdered product), as this will imply either 0.01 or 0.1 mg/kg target LOQ according to the SMPR. For instance, whey protein hydrolysate has been validated at 0.05 and if consider as major ingredients (as it is in the case of hypoallergenic IF) it should have been validated at 0.01 mg/kg. Please, indicate why for protein hydrolysate determinations at each level was divided in two (i.e. chlorate 0.05 (x11) and after (x4); 0.2 (x4) and then 0.2 (x4); 0.1 (x4) and then 0.1 (x4)). Same for lactose (0.005 (x7), then 0.005 (x1).

Other comments:

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- In sample preparation, please explain why stones from fruit and cobs from corn are included in the sample weight. They are not edible part of the fruit so it is fine to remove then before homogenization, but then it should not be included for the final results.

- In table 7, please specify which matrix is the procedural calibration corresponding to.

- In the manuscript it is stated that the chromatographic column performance decrease with use. Please specify how many matrix injections (around) could be performed with a new column. Was the chromatogram in figure 3 obtained with a new column? The peak shape improved significantly from the chromatogram showed in previous submission, what is the difference between the old and the new one?

Final Recommendation:

Performance of the method was not proved to meet the requirements defined in the SMPR, therefore the method can not be recommended further for first action.

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