AOAC OMA 2021.07 Reviewer Forms (July 25, 2023)

AOAC Stakeholder Program on Infant Formula and Adult Nutritionals (SPIFAN)

AOAC OMA# 2021.07 (Lacto-01) REVIEWER FORMS

July 25, 2023

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 Nutri�on “FINAL ACTION REVIEW” Evalua�on of Method 2021.07 (Lacto-01)

Title: AOAC Official Method 2021.07

Analysis of Bovine Lactoferrin in Infant Formula and Adult Nutri�onal Products Op�cal Biosensor Immunoassay

Author s: Harvey Indyk, Brendon Gill

Reviewer Name: Reviewer 1

Did Method Author Consider ERP’s General Recommenda�ons: Yes • Was the method tested using all the SPIFAN samples for precision and accuracy? No, no SPIFAN matrices containing Lactoferrin were available • Was data provided from a standard reference material? No, no reference material containing Lactoferrin was available • Was the analysis conducted using ready -to- feed (recons�tuted) infant formula?

No, the metho d is only applicable to powdered formula�ons • Was rice- based formula available and was it used? No • Was the method tested using properly characterized standards? Yes • Is there proof of performance through system suitability? Yes

• Is there clarity of calibra�on procedure? Yes

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• Does the method demonstrate reproducibility? To what extent? Eight laboratories par�cipated in MLT but one laboratory was excluded from the final sta�s�cal analysis due to having consistently lower results and user concerns over stability of calibra�on standard between the prac�ce samples and test samples. Seven samples were analyzed as blind-coded duplicates pairs. Each laboratory also did 1 prac�ce sample in duplicates. The submited data for test samples was sta�s�cally analyzed for overall mean, intra -laboratory repeatability (Sr), repeatability rela�ve standard devia�on (RSDr), inter - laboratory reproducibility (SR), reproducibility rela�ve standard devia�on (RSD R ). Both repeatability and reproduc ibility data met SMPR

Did Method Author Consider ERP’s Method Specific Recommenda�ons: Yes, ERP offered several recommenda�ons and comments when evalua�ng the single laboratory valida�on data. They were all addressed at the �me of the method approval as a First Ac�on Official method

Any Revision to Method Scope/Applicability of Method: No The applicability is currently stated as following: Applica ble to the determination of bovine lactoferrin in bovine -milk protein-based, soy protein-based, and amino acid- based, infant formula and adult nutritional powders . Unfortunately, no soy protein or amino acid- based formulas were tested as part of the MLT. They were tested as part of single laboratory valida�on, though. General comments (i.e. safety) about the method: No specific safety concerns are noted • Feedback from Users of the Methods since being awarded First Ac�on Official Methods Status None noted

Addi�onal Pros/Strengths: Easy sample prepara�on The method showed good correla�on with Lactoferrin AOAC method based on LC -UV analysis.

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Addi�onal Cons/Weaknesses Instrumenta�on may not be available in most laboratories.

Suppor�ng Informa�on Raw data for all the samples, sta�s�cal analysis res ults, results of the two methods comparison are all presented in MLT study report • General Comments According to samples descrip�on provided there were no soy-based products or elemental formulas included in MLT study. These products are explicitly listed in the scope of the method and were included in a single laboratory valida�on study of this method. Is there a reason these matrices were not part of the MLT sampl e set?

• Performance Characteris�cs and compared to approved SMPR: Analy�cal Range: 2.6- 200 mg/100 g (meets SMPR)

LOQ: 2.6 mg/100 g (meets SMPR)

Accuracy/Recovery: 96.1% – 109.2 % (determined by spiking 50% and 100 % of typical for�fica�on level, meets SMPR)

Precision (RSD r ): 3.8– 6.8% RSDr (meets SMPR of 6% for all but one sample). Repeatability met SMPR for all the sample during single laboratory valida�on

Reproducibility (RSD R ): 5.7– 8.9% RSD R ( meets SMPR)

ERP Summary/Recommenda�on(s): NA

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• Does the method meet the approved SMPR? Yes

• Has the method been performing well in the laboratory? Yes

• Are there any addi�onal feedback on method performance? Comments from study par�cipants included some notes on sample prepara�on but no significant problems with the method were noted

• Would you recommend designa�ng this method as a Final Ac�on method to the ERP? Yes, I will recommend this method as a Final Ac�on Official Method

Final Comments? Would considera�on of this method for Codex Type II be affected by the fact that MLT only included results from 7 laboratories, which is less than recommended number of MLT par�cipants?

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Expert Review Panel for Infant Formula and Adult Nutrition “FINAL ACTION REVIEW” Evaluation of Method Lacto-01 (2021.07)

Title: Analysis of Bovine Lactoferrin in Infant Formula and Adult Nutritional Products Optical Biosensor Immunoassay Author: Harvey E. Indyk and Brendon D. Gill Reviewer Name: Reviewer 2

Did Method Author Consider ERP’s General Recommendations:

• Was the method tested using all the SPIFAN samples for precision and accuracy?

The SLV study incorporated data from intact milk, intact soy, partially hydrolyzed milk, elemental infant formula, toddler milk-based, and low-fat milk based adult nutritional powder matrices. The MLT study included infant formula, follow-on formula, milk-based toddler, and fortified milk powders. It is unclear based on the MLT report the composition of the infant formulas (milk versus soy, intact versus partially hydrolyzed). Were only milk-based formulations included in the MLT?

• Was data provided from a standard reference material?

An SRM for this nutrient is not available.

• Was the analysis conducted using ready-to-feed (reconstituted) infant formula?

The method contains two options for powder matrices – suspension of 0.50 g powder into 10 mL buffer or reconstitution of 25 g powder into 200 g water followed by dilution (4.5 g recon to 10 mL total volume with buffer). It is not clear which option the study participants used. Based on the collaborator comments most chose the first option (0.50 g powder into 10 mL buffer). Did all participants use sample preparation option a?

• Was rice-based formula available and was it used?

The composition of each formula was not fully described in the MLT report. It is assumed that only milk-based formulas were included in the MLT study. Clarification from the study director is requested.

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• Was the method tested using properly characterized standards?

Yes.

• Is there proof of performance through system suitability?

The method contains system suitability that establishes acceptable performance.

• Is there clarity of calibration procedure?

Yes.

• Does the method demonstrate reproducibility? To what extent?

The reported RSD R are within the SMPR. There is a question about the decision to remove the single outlier (Lab 3, sample J-321). This result does not appear to be a statistical outlier and the %RSD R increases significantly when the datapoint is included (omitted is 8.96% RSD, included is 11.34% RSD). What basis was used for the exclusion of the single outlier?

Did Method Author Consider ERP’s Method Specific Recommendations:

Yes.

Any Revision to Method Scope/Applicability of Method: The original method scope included infant, adult, and/or pediatric formulas. The First Action method (2021.07) lists the method applicability as bovine-milk protein, soy protein, and amino acid based infant formula and adult nutritional products. The SLV included milk, soy, and elemental matrices. Is the SLV data sufficient for inclusion of the non-milk categories in the AOAC method scope? Is there a practical need for inclusion of intact lactoferrin in soy and elemental formulas.

General comments (i.e. safety) about the method: • Feedback from Users of the Methods since being awarded First Action Official Methods Status No additional comments.

Additional Pros/Strengths: • No additional comments. Additional Cons/Weaknesses • No additional comments.

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Supporting Information • General Comments

Minor clarification: In the Method Comparison section, Table 4 contains the Degrees of freedom parameter, which is described as n-2, with the value of 13. With 14 samples would the Degrees of freedom be 12? The rest of the statistical values in Table 4 are correct.

Performance Characteristics and compared to approved SMPR:

Analytical Range:

Assuming the reported units are in mg/100 g powder, the MLT study samples constituted a range of 44.2 – 126.0 mg/100 g powder, or 4.9 – 14 mg/100 g recon. The SMPR range is 4-200 mg/100 g recon. The MLT study samples sufficiently describe the lower end of the expected range.

LOQ:

The SMPR LOQ is 4 mg/100 g recon. The MLT study includes samples near this level and demonstrates acceptable method performance at the LOQ.

Accuracy/Recovery:

Accuracy was demonstrated during the SLV and is not a factor assessed during the MLT.

Precision (RSD r ):

All samples, except for infant formula pair S-554/E-252 met SMPR repeatability targets. The RSD r for this samples was 6.8% RSD, while the target is 6% RSD. Viewing the RSD r data in totality, the data indicate acceptable repeatability precision.

Reproducibility (RSD R ):

The RSD R for all samples met the SMPR criteria. Resolution of the question regarding the exclusion of the single outlier (Lab 3, J-321) is recommended before finalizing the Final Action decision.

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ERP Summary/Recommendation(s):

• Does the method meet the approved SMPR?

Most of the data meet the SMPR.

• Has the method been performing well in the laboratory?

Based on the MLT results and apart from Lab 5, the method appears to perform well in the contributor laboratories.

• Are there any additional feedback on method performance?

No

• Would you recommend designating this method as a Final Action method to the ERP?

Prior to Final Action designation the following questions are recommended to be resolved:

• Did all participants use sample preparation option a (0.50 g into 10 mL buffer)? • Can the study director please provide additional information on the matrix categories included in the MLT study, specifically whether the formulation was milk or soy based and if intact or partially hydrolyzed matrices were included?  Were only milk-based formulations included in the MLT? • What criteria were used to exclude the single outlier (Lab 3, sample J-321)? Final Comments? The MLT study results indicate this procedure has acceptable performance to be considered a Final Action method. This study was conducted within a limited geography, with 7 of the 8 participating labs from the same global region. Also, this method utilizes an analytical technique that is less commonly employed in food analysis laboratories. While these factors do not preclude the method from being recommended for Final Action, they should be considered by the ERP.

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