AOAC SPIFAN Nutrients ERP Report (December 7, 2022)

AOAC Stakeholder Program on Infant Formula and Adult Nutritionals

Washington. DC

EXPERT REVIEW PANEL (ERP) (NUTRIENTS) REPORT Wednesday, December 7, 2022

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

dboyd@aoac.org 301.924.7077 x126

AOAC Stakeholder Program on Infant Formula & Adult Nutritionals (SPIFAN) Nutrients Expert Review Panel (ERP)

The AOAC SPIFAN Nutrients Expert Review Panel (ERP) Chair confirms the following ERP Report has been reviewed and

approves the final version of the Official Chair’s Report for Nutrients December 7, 2022

______________________ Signature ______________________ Date

AOAC INTERNATIONAL Stakeholder Program on Infant Formula & Adult Nutritionals (SPIFAN)

“Virtually” Meeting at AOAC INTERNATIONAL 2275 Research Blvd. Suite 300, Rockville, MD, USA Wednesday, December 7, 2022

REPORT OF THE NUTRIENTS EXPERT REVIEW PANEL (ERP) PROCEEDINGS

Expert Review Panel Members (in attendance):

Darryl Sullivan Sean Austin

Eurofins Food Integrity & Innovation (Chair)

Société des Produits Nestlé S.A.

Esther Campos-Giménez Christophe Fuerer Hans Cruijsen/Martine van Gool Philip Haselberger (HMO ONLY)

FrieslandCampina Abbott Nutrition

Karen Schimpf Brendon Gill Estela Kneeteman

Fonterra Cooperative

INTI NIST

Melissa Phillips/Kate Rimmer

Thomas Vennard

Reckitt

Yannis Vrasidas (HMO ONLY)

Independent Consultant

Jinchuan Yang

Waters Corp.

Expert Review Panel Members (not in attendance): Sneh Bhandari

Independent Consultant

Shay Phillips David Woollard

Reckitt

Independent Consultant

AOAC Staff Includes: Delia Boyd Deborah McKenzie Alicia Meiklejohn Tien Milor

Observers: Simone Carron, MXNS Moheb Elwakiel, Triskelion Renee Erney, Abbott Nutrition Jeremy Gauthier, University of Toronto Steve Holroyd, Fonterra Greg Jaudzems, Nestle - NQAC George Joseph, AsureQuality New Zealand

Markus Lacorn, R-Biopharm AG Giampaolo Perinello, MxNS Italy Frank Pruiksma, FrieslandCampina Dustin Starkey, Abbott Nutrition Leo van Stee, Triskelion

I.

WELCOME AND INTRODUCTIONS Darryl Sullivan welcomed all participants to the ERP meeting and the ERP members were introduced along with roll call.

II. REVIEW OF METHODS BY EXPERT REVIEW PANEL (ERP) FOR FIRST ACTION OFFICIAL METHOD SM STATUS For each method, the ERP reviewed and discussed method(s) submitted for First Action Official Method SM status.

III.

NEXT STEPS/FEEDBACK FROM EXPERT REVIEW PANEL Darryl Sullivan provided next steps including recommendations from the Expert Review Panel to method authors.

REVIEW OF METHODS: AMINO ACIDS

CODIFICATION: Amino-06 METHOD TITLE: Single Laboratory Validation of Determination of Total Taurine in Infant Formula and Adult/Pediatric Nutritionals by Ultra High-Performance Liquid Chromatography with Multiple Reaction Monitoring Mass Spectrometry REVIEWERS: Estela Kneeteman & Tom Vennard VOTE: Motion - To recommended method for First Action Official Methods SM status Estela Kneeteman motioned Tom Vennard second Vote: 8 Yes/0 No/1 Abstain (FrieslandCampina) RECOMMENDATION: Clarifying points and questions: 1. Adding clarification that water is used to bring flasks to volume. 2. For system suitability, additional detail for the system blank (how is “not detected” defined) and criteria for the method blank and initial 6 injections of the CCV. 3. Clarification of number of calibration standards used. The method describes 8 levels, but also states to use 6 levels. 4. Check T7 Taurine Stock Concentration value. Should this value be changed from 5400 to 5000? 5. Specific safety procedures for TCA are recommended, as this reagent is corrosive and a suspected carcinogen. Questions: o What was the suspected cause for the lower Day 2 results for sample SPIFAN DOMY545? o Why was sample SPIFAN DOMY545 eliminated from the inter-lab study? o Have alternative protein precipitation procedures using less hazardous reagents been evaluated? Motion to approve recommendations: Brendon Gill motioned & Tom Vennard second Abstain – FrieslandCampina ADDITIONAL COMMENTS (AMINO-06): Thank you for the thorough validation work. Here are some editorial comments and questions.  Protein precipitation is achieved by TCA precipitation at 2% TCA final, which is much lower than usually done (typically 12%). Did you try other TCA concentrations? Why did you choose 2%?  H(b) and H(e): with what do you use to fill to the mark? Water? This should be described more precisely  Equation 1: the conversion factors are not fully described. There should be: o ug to mg = 10-3 (top) o mL to L = 10-3 (top) o g (sample) to 100g (sample) = 10-2 (bottom) o FINAL is indeed 1 / (1000 x 10) but in fact it is 100 / (1000 x 1000)  Equation 2: I think “sample” is missing after “Vr Total weight of powder or liquid concentrated sample…”  Table 6: footnotes a and b are redundant  Results, D: LOD: is it powder or reconstituted, also for instance Table 12. As some results are given for reconstituted and some other for powders, it would be good to specify it in each table.

CODIFICATION: Amino-07 METHOD TITLE: Analysis of Taurine in Infant Formula and Adult Nutritionals by Hydrophilic Interaction Liquid Chromatography-Tandem Mass Spectrometry (HILIC MS/MS) REVIEWERS: Maria Ofitserova & Estela Kneeteman VOTE: Motion - To recommended method for First Action Official Methods SM status

Maria Ofitserova motioned Estela Kneeteman second Vote: 8 Yes/0 No/1 Abstain (Fonterra Cooperative) RECOMMENDATION: 1. Clarification of point I(g) 2. Clarification of standards E(d)(vi) Motion to approve recommendations: Maria Ofitserova motioned Estela Kneeteman second Abstain – Fonterra Cooperative

REVIEW OF METHODS: FLUORIDE

CODIFICATION: Fluor-04 METHOD TITLE: Fluoride determination in milk, soy, and water-based products using ionselective electrode and direct measurement. REVIEWERS: Melissa Phillips & Jinchuan Yang VOTE: Motion - To recommended method for First Action Official Methods SM status Melissa Phillips motioned Maria Ofitserova second Vote: 8 Yes/0 No/1 Abstain RECOMMENDATION: 1. Address the potential calculation flaw (clarification) o Dilution correction (weight vs volume) o Share data demonstrating equivalence of approaches 2. Requirement to monitor temperature during analysis

Motion to approve recommendations: Jinchuan Yang motioned & Brendon Gill second Abstain – Abbott Nutrition

CODIFICATION: Fluor-06 METHOD TITLE: Quantitative analysis of fluoride in infant formula and adult nutrition using headspace gas chromatography with a flame ionization detector REVIEWERS: Tom Vennard & Brendon Gill

VOTE: Motion – To recommended method for First Action Official Methods SM status Tom Vennard motioned Brendon Gill second Vote: 1 st Vote: 7 Yes/1 No/1 Abstain (Abbott Nutrition) 2 nd Vote: 7 Yes/1 No/1 Abstain (Abbott Nutrition)

RECOMMENDATION: 1. Additional system suitability criteria including: o Calibration (r2 and residual of each calibration level) o Chromatographic (resolution and asymmetry) 2. Augmenting the safety statement to clearly describe the risks of TMCS and consider adding statement to use TMCS in hood (per SDS sections 5.2 and 7.1) 3. Utilize the term trimethylchlorosilane in future publications Additional comments: 1) Section 4.9 System suitability: This CM62 contains Fluoride at concentration of 1.6 mg/kg, about 160 ug/100g. The SMPR requirement for this level is 90-110% recovery and less than 5% in RSDr. In this regard, the 15% level specified in the system suitability for the deviation from the nominal value and for the relative standard seems too wide. 2) In the specificity requirement, 2.5% in RT is used as a limit. When this 2.5% limit is used to assess the extra peaks in Figure 5, it seems that both the adjacent peaks (RT 9.434 min and RT 9.853 min) are within the 2.5% limit of the fluoride peak (RT 9.626 min). I am wondering how this 2.5% limit was established as the specificity limit in RT.

Motion to approve recommendations: Tom Vennard motioned & Brendon Gill second Abstain – FrieslandCampina

REVIEW OF METHODS: HMO

CODIFICATION: HMO-02 METHOD TITLE: Determination of Seven Human Milk Oligosaccharides (HMO) in Infant Formula and Adult Nutritionals REVIEWERS: Yannis Vrasidas & Philip Haselberger VOTE: Motion - To recommended method for First Action Official Methods SM status Yannis Vrasidas motioned Philip Haselberger second Vote: 9 Yes/0 No/1 Abstain RECOMMENDATION/COMMENTS: 1. 6’-SL does not meet LOQ in soy matrices 2. DFL does not meet LOQ in extensively hydrolyzed formula and bovine formula with FOS, but it is still within proposed analytical range. 3. Potential high bias for LNnT and LNT in Elemental formulas at low concentrations (may impact effective LOQ in that matrix) 4. DFL may not meet LOQ in formulas containing polydextrose

Motion to approve recommendations: Yannis Vrasidas motioned Philip Haselberger second Abstain – Société des Produits Nestlé

CODIFICATION: HMO-04 METHOD TITLE: Validation of an LC-MS method for the analysis of milk oligosaccharides

REVIEWERS: Philip Haselberger & Yannis Vrasidas VOTE: Motion - ERP takes no action on this method Yannis Vrasidas motioned Philip Haselberger second Vote: 10 Yes/0 No/0 Abstain RECOMMENDATION/COMMENTS: 1. No further action.

Motion to approve recommendations: No action taken

CODIFICATION: HMO-05 METHOD TITLE: Method for the Determination of 2’-Fucosyllactose (2’-FL) , 3-Fucosyllactose (3-FL) , 6’-Sialyllactose (6’-SL), 3’-Sialyllactose (3’-SL), Lacto-N-Tetraose (LNT), and Lacto-N-neoTetraose (LNnT) by High Performance Anion Exchange Chromatography, with Pulsed Amperometric Detection (HPAEC-PAD)

REVIEWERS: Philip Haselberger & Yannis Vrasidas VOTE: Motion - To recommended method for First Action Official Methods SM status Sean Austin motioned Brendon Gill second Vote: 9 Yes/0 No/1 Abstain RECOMMENDATION/COMMENTS: 1. Do not include matrices fortified with DFL (out of scope) 2. Update on references in the method 3. Mixture of Salt form with free form (for clarification) a. Consider alternative of using nitrogen instead of helium (or both)

Motion to approve recommendations: Sean Austin motioned Brendon Gill second Abstain – Abbott Nutrition

REVIEW OF METHODS: BIOTIN

CODIFICATION: 2016.02 METHOD TITLE: Determination of biotin by UPLC coupled with Immunoaffinity Column clean-up extraction REVIEWERS: Estela Kneeteman & Karen Schimpf VOTE: Motion – No action to be taken by the ERP for this method. The ERP will consider this method in a future submission as a “new” method Estela Kneeteman motioned Karen Schimpf second Vote: 9 Yes/0 No/1 Abstain

RECOMMENDATION/COMMENTS: 1. Additional recommendations 2. See data on the goat matrices

3. Samples prepared with the enzymes 4. Need to see actual spiked recovery 5. Bias against the current method 6. See where it eludes (Biocytin in the UPLC chromatography)

Motion to approve recommendations: Brendon Gill motioned Tom Vennard second Unanimous