AOAC SN2-02 Reviewer Forms (July 25, 2023)

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

SN2-02 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 Nutrition Evaluation of Method Sn2-02

Title: Animal and vegetable fats and oils and milk and milk products—Fatty acids at the sn-2 position of triacylglycerol molecules- Enzymatic transesterification method Author: Daniëlla Holt, Yomi Watanbe, Hans Cruijsen, Martine van Gool, Pierre-Alain Golay, Francesca Giuffrida, Florence Lacoste, Scott Bloomer, Reviewer Name: Reviewer 1 Summary of Method: Fat is extracted three times from infant formula using a heptane/isopropanol mixture. Triacylglycerols in the test portion are purified by column chromatography using either silica packed glass columns or silica gel-SPE cartridges. The Sn-1(3) positions of the triacylglycerols are selectively hydrolyzed with ethanol using enzymes to yield 2-monoacylglycerols (2-MAG). The 2-monoacylglycerols are subsequently separated by normal phase SPE, transesterified into fatty acid methyl esters and their composition is determined by gas chromatography coupled with flame ionization detection. Method Scope/Applicability: Applicable to the determination of fatty acid content at Sn-2 position of the triglycerides in infant formula and adult nutritionals. General comments about the method: • Method based on AOCS method Ch 3a-19 with an initial fat extraction added. • Method title says milk and milk products, no milk was studied. There is disconnect between title of method and title of validation study. • Section 9.5 doesn’t directly describe the procedure for preparing fatty acid methyl esters but refers user to several standards elsewhere. • SMPR applicability statement defines that method must quantitate fatty acids esterified and Sn2 position. Table 1 defines the scope of these results as g rams of fatty acid per 100 g of reconstituted product. Nowhere in the equations given in the method in section 10, nor in the validation document can I see the results expressed in term of infant formula powder. The tables in SLV seem to express results in terms of the extracted fat. Method Clarity: • Method is clearly written such that users can follow instructions without ambiguity. • Method format not given in AOAC style. • Equations missing to covert fat results to reconstituted powder or even dry powder results. Pros/Strengths: • Determination Sn2 fatty acids based on established technique on standard from AOCS.

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• Monoacylglycerols, diacylglycerols, phospholipids, polar lipids, and free fatty acids prior to enzymatic digestion of triacylglycerols. Cons/Weaknesses Supporting Data General Comment: Precision determined using two different fat extractions using either Heptane-Isopropanol extraction in method, and also the Rose Gottlieb extraction. Precision tables report results as fatty acid in Sn2 position as weight percentage and mole percentage of the fat not in the units of the actual infant formula products which is grams fatty acid in Sn2 position per 100g of reconstituted product. Method Optimization: No optimization given. No data demonstrating the specificity and efficacy of the CALB enzyme which is critical component to this analysis. Should this data be included as part of SPIFAN review? This is based on AOCS standard, do we presume that this was part of their standard development? If this was a new method we would need to see this data. Performance Characteristics: Analytical Range: Annex D contains information about assessment of range based on grams fat at sn2 position per 100g of fat, but these are not converted to limits based on either powder or as SMPR requires, reconstituted powder. LOQ: 0.003g/100g meets the requirements in given in SMPR 2022.004 of less than or equal to 0.003g/100g Accuracy/Recovery: Accuracy was assessed in two ways. The recovery of model triacylglycerides with known fatty acid composition in each of the Sn1, Sn2, and Sn3 positions. This recovery demonstrated accuracy of the enzymatic transesterification technique. Recovery ranged from 90–98%. Recovery in an adult nutritional product was also assessed by comparison with an anhydrous fat milk sample demonstrated the recovery through the fat extraction. The recovery measured for palmitate, stearate and oleate was 98%, 97%, and 98% respectively. Precision (RSD r ): Repeatability values appear to be within SMPR limits, however as the concentration ranges aren’t specified in the same units as the SMPR it is difficult to be sure. Reproducibility (RSD R ): Not required for SLV. System suitability: No system suitability criteria are given in SLV report.

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1. Is the Validation Study Report in a format acceptable to AOAC? No. 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)? No. Equations for calculating final results for infant formula products are missing. If this to become AOAC Official Method, what happens to missing transesterification details recorded in other ISO standards? 3. Are the figures and tables sufficiently explanatory without the need to refer to the text? No. Units need to be included in the tables of the SLV. 4. Are all the figures and tables pertinent? Yes 5. Could some be omitted and covered by a simple statement? n/a 6. Are the references complete and correctly annotated? Yes 7. Does the method contain adequate safety precaution reference and/or statements? No specific safety precautions are given, only generic safety precaution to using hazardous substances

Recommendation:

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Expert Review Panel for Infant Formula and Adult Nutrition Evaluation of Method SN2-02

Title : DETERMINATION OF FATTY ACIDS AT THE SN-2 POSITION OF TRIACYLGLYCEROL MOLECULES IN INFANT FORMULA AND ADULT/PEDIATRIC NUTRITIONALS BY ENZYMATIC TRANSESTERIFICATION METHOD Author: ISO/Friesland Campina Reviewer Name: Reviewer 2 Summary of Method: Fat is extracted from liquid or powdered infant formula using solvents, where necessary. Triacylglycerols in the test portion are purified by column chromatography. The 1(3) positions of the triacylglycerols are transesterified selectively with ethanol using enzymes to yield 2-monoacylglycerols. The 2- monoacylglycerols are subsequently separated by silica-gel chromatography and their fatty acid composition is determined by gas chromatography coupled with Flame Ionisation Detection (FID).

Method Scope/Applicability: Fats and Oils. Infant and Adult/Pediatric Nutritional Formulas. Slight deviation from the SMPR in applicability. SMPR states C4-C24 saturated and unsaturated fatty acids. Method is applicable to C4-C22 saturated and unsaturated fatty acids with melting points below 50C. Not sure that is a deal breaker just wanted to mention it.

General comments about the method: Method is well written and thoroughly validated.

Method Clarity: Method is clearly written with all details covered. The procedure is complex, and each step is clearly explained.

Pros/Strengths: • Applicable to a variety of matrices. • SLV data was collected on numerous matrices and results met SMPR requirements.

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Cons/Weaknesses • Complex sample preparation with numerous steps. Possibly a multi-day extraction? • How much enzyme is needed per sample roughly? Enzyme is fairly expensive from what I gathered.

Supporting Data • General Comment: Method is thoroughly validated with all necessary supporting data provided. Could a method author explain the Model Tag with 90% recovery. Was this expected?

Method Optimization:

• Performance Characteristics: All SMPR requirements were met.

Analytical Range:

LOQ:

Accuracy/Recovery:

Precision (RSD r ):

Reproducibility (RSD R ):

• System suitability: No System Suitability Parameters Listed in Method?

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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: The method meets all the performance requirements established in the SMPR. I recommend accepting this method as first action after the addition of system suitability parameters in the method document.

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