AOAC SPIFAN Working Group Revised SMPRs (4th Qtr. 2017)

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

Standard Method Performance Requirements (SMPRs®) Revised by Amino Acids/Fluoride/GOS Working Groups (4th Qtr. 2017)

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

dboyd@aoac.org 301.924.7077 x126

AOAC SMPR 2014.003

Table 1. Method performance requirements a Analytical range 0.2–3.0 b Limit of quantitation (LOQ) ≤0.2 b Repeatability (RSD r ) ≤6% Recovery

Standard Method Performance Requirements for GOS in Infant Formula and Adult/Pediatric Nutritional Formula

90 to 110% of mean spiked recovery over the range of the assay

Reproducibility (RSD R ) ≤12% a  Concentrations apply to (a) “ready-to-feed” liquids “as is”; (b) reconstituted powders (25 g into 200 g of water); and (c) liquid

Intended Use: Reference Method for Dispute Resolution 1 Applicability Determination of galactooligosaccharides (GOS) in all forms of infant, adult, and/or pediatric formula (powders, ready-to-feed liquids, and liquid concentrates). 2 Analytical Technique Any analytical technique that meets the following method performance requirements is acceptable. 3 Definitions Adult/pediatric formula .—Nutritionally complete, specially formulated food, consumed in liquid form, which may constitute the sole source of nourishment [AOAC Stakeholder Panel on Infant Formula and Adult Nutritionals (SPIFAN); 2010], made from any combination of milk, soy, rice, whey, hydrolyzed protein, starch, and amino acids, with and without intact protein. Galactooligosaccharides (GOS) .—Also known as oligogalactosyllactose, oligogalactose, or transgalactooligosaccharides (TOS) produced by transgalactosylation of lactose. General formulae are shown in Figure 1. Infant formula .—Breast-milk substitute specially manufactured to satisfy, by itself, the nutritional requirements of infants during the first months of life up to the introduction of appropriate complementary feeding (Codex Standard 72-1981) made from any combination of milk, soy, rice, whey, hydrolyzed protein, starch, and amino acids, with and without intact protein.

concentrates diluted 1:1 by weight. b  g/100 g reconstituted final product.

Limit of detection (LOD) .—The minimum concentration or mass of analyte that can be detected in a given matrix with no greater than 5% false-positive risk and 5% false-negative risk. Limit of quantitation (LOQ) .—The minimum concentration or mass of analyte in a given matrix that can be reported as a quantitative result. Repeatability .—Variation arising when all efforts are made to keep conditions constant by using the same instrument and operator, and repeating during a short time period. Expressed as the repeatability standard deviation (SD r ); or % repeatability relative standard deviation (%RSD r ). Reproducibility.— The standard deviation or relative standard deviation calculated from among-laboratory data. Expressed as the reproducibility relative standard deviation (SD R ); or % reproducibility relative standard deviation (%RSD R ). Recovery .—The fraction or percentage of spiked analyte that is recovered when the test sample is analyzed using the entire method. 4 Method Performance Requirements See Table 1. 5 System Suitability Tests and/or Analytical Quality Control Suitable methods will include blank check samples, and check standards at the lowest point and midrange point of the analytical range. 6 Reference Material(s) No National Institute of Standards and Technology (NIST) Standard Reference Material® (SRM) 1849a Infant/Adult Nutritional Formula or equivalent is available. 7 Validation Guidance Recommended level of validation: Official Methods of Analysis SM . 8 Maximum Time-to-Result No maximum time. Approved by AOAC Stakeholder Panel on Infant Formula and Adult Nutritionals (SPIFAN). Final Version Date: March 18, 2014.

(ß-D-Gal p -(1→B)) n

-ß-D-Gal p -(1→A)-D-Glc p

(ß-D-Gal p -(1→B)) m

-D-Gal p

where n ≥ 0; m ≥ 1 B = 2,3,4 or 6 If n>0, A=1,2,3,4 or 6 If n= 0, A=1,2,3 or 6

Figure 1. General formulae for galactooligosaccharides which may or may not contain a terminal glucose. Although not obvious from this generalized scheme, branched structures may also exist. Gal p = galactopyranose; Glc p = glucopyranose.

© 2014 AOAC INTERNATIONAL

AOAC SMPR 2014.013

2 Analytical Technique Any analytical technique that meets the method performance requirements is acceptable. It is expected that multiple methods will be required to completely fulfill the requirements. 3 Definitions Accuracy (corresponds to the VIM definition for “trueness”). — The closeness of agreement between the average of an infinite number of replicate measured quantity values and a reference quantity value. Adult/pediatric formula .—Nutritionally complete, specially formulated food, consumed in liquid form, which may constitute the sole source of nourishment [AOAC Stakeholder Panel on Infant Formula and Adult Nutritionals (SPIFAN); 2010], made from any combination of milk, soy, rice, whey, hydrolyzed protein, starch, and amino acids, with and without intact protein. Infant formula .—Breast-milk substitute specially manufactured to satisfy, by itself, the nutritional requirements of infants during the first months of life up to the introduction of appropriate complementary feeding (Codex Standard 72–1981), made from any combination of milk, soy, rice, whey, hydrolyzed protein, starch, and amino acids, with and without intact protein.

Standard Method Performance Requirements for Determination of Amino Acids in Infant Formula and Adult/Pediatric Nutritional Formula

Intended Use: Reference Method for Dispute Resolution 1 Applicability Determination of free and/or total proteinogenic L-α-amino acids and taurine (as shown in Table 1) in all forms of infant, adult, and/or pediatric formulas (powders, ready-to-feed liquids, and liquid concentrates). For amino acids sensitive to modification during handling and/or processing (primarily methionine, lysine, and cysteine/cystine), which can result in modified forms, different from the parent amino acids, preference will be given to methods best able to discriminate against these modified forms. Structures to be excluded include, but are not necessarily limited to, methionine sulfone, methionine sulfoxide, cysteic acid, and lysine derived Maillard products. Method authors should specifically discuss, with appropriate supporting data, the ability to determine only parent forms of the target amino acids.

Table 1. List of proteinogenic L-α-amino acids and taurine Amino acid

IUPAC name

CAS Registry No.

L-alanine L-arginine

(2S)-2-aminopropanoic acid

56-41-7 74-79-3 70-47-3 56-84-8 52-90-4 56-89-3 617-65-2 56-85-9 56-40-6 71-00-1 73-32-5 61-90-5 56-87-1 63-68-3 63-91-2 147-85-3 56-45-1 72-19-5 73-22-3 60-18-4 72-18-4 107-35-7

(2S)-2-amino-5-(diaminomethylideneamino)pentanoic acid

L-asparagine a,b L-aspartic acid b

(2S)-2,4-diamino-4-oxobutanoic acid

(2S)-2-aminobutanedioic acid

L-cysteine c L-cysteine c

(2R)-2-amino-3-sulfanylpropanoic acid

(2R)-2-amino-3-[[(2R)-2-amino-2-carboxyethyl]disulfanyl]propanoic acid

L-glutamic acid b L-glutamine a,b

(2S)-2-aminopentanedioic acid

(2S)-2,5-diamino-5-oxopentanoic acid

L-glycine L-histidine L-isoleucine

2-Aminoethanoic acid

(2S)-2-amino-3-(1H-imidazol-5-yl)propanoic acid (2S,3S)-2-amino-3-methylpentanoic acid

L-leucine L-lysine

(2S)-2-amino-4-methylpentanoic acid

(2S)-2,6-diaminohexanoic acid

L-methionine

(2S)-2-amino-4-methylsulfanylbutanoic acid (2S)-2-amino-3-phenylpropanoic acid (2S)-pyrrolidine-2-carboxylic acid (2S)-2-amino-3-hydroxypropanoic acid (2S,3R)-2-amino-3-hydroxybutanoic acid (2S)-2-amino-3-(1H-indol-3-yl)propanoic acid (2S)-2-amino-3-(4-hydroxyphenyl)propanoic acid

L-phenylalanine

L-proline L-serine

L-threonine L-tryptophan

L-tyrosine

L-valine Taurine

(2S)-2-amino-3-methylbutanoic acid

2-Aminoethanesulfonic acid

a Determined only as free amino acids.

b Not reported separately when acid hydrolysis is used.

Generally not reported separately.

c

© 2014 AOAC INTERNATIONAL

Figure 1. Molecular structure of amino acids.

Limit of detection (LOD) .—The minimum concentration or mass of analyte that can be detected in a given matrix with no greater than 5% false-positive risk and 5% false-negative risk. Limit of quantitation (LOQ) .—The minimum concentration or mass of analyte in a given matrix that can be reported as a quantitative result. Proteinogenic L-α-amino acids .—Amino acids that are precursors to proteins. See Figure 1 for molecular structures.

Repeatability .—Variation arising when all efforts are made to keep conditions constant by using the same instrument and operator, and repeating during a short time period. Expressed as the repeatability standard deviation (SD r ); or % repeatability relative standard deviation (%RSD r ). Reproducibility .—The standard deviation or relative standard deviation calculated from among-laboratory data. Expressed as the reproducibility standard deviation (SD R ); or % reproducibility relative standard deviation (%RSD R ). Recovery .—The fraction or percentage of spiked analyte that is recovered when the test sample is analyzed using the entire method. Taurine .—2-Aminoethanesulfonic acid. Taurine is sometimes called an amino acid, but it does not contain a carboxyl group necessary to be classified as an amino acid. It is in fact an acid containing an amino group. See Figure 2 for the molecular structure. 4 Method Performance Requirements See Table 2.

Figure 2. Molecular structure of taurine.

© 2014 AOAC INTERNATIONAL

Table 3. NIST 1849a reference values for amino acids and taurine Amino acid Value, mg/100 g Expanded uncertainty Coverage factor

Table 2. Method performance requirements a Parameter

Minimum acceptable criteria

Analytical range

0.4–2500 b

Limit of quantitation (LOQ)

≤0.4 b

Alanine Arginine

0.455 0.400 1.070

0.021 0.029 0.057

2.31 2.31 2.31 2.00 2.31 2.31 2.31 2.31 2.31 2.31 2.31 2.31 2.31 2.00 2.31 2.45 2.31 2.31

Recovery

0.4–5.0 b

80 - 110%

Aspartic acid a

90 - 110%

>5.0

Cystine b

0.1286

0.0071

Repeatability (RSD r )

Glutamic acid c

2.59

0.27

≤10%

0.4–5.0 a

Glycine Histidine Isoleucine

0.241 0.315 0.660 1.261 1.010 0.580 1.195 0.720 0.640 0.184 0.510 0.0366 0.7600

0.019 0.036 0.071 0.050 0.071 0.021 0.086 0.030 0.022 0.010 0.043 0.0018

≤7%

> 5.0

Reproducibility (RSD R )

0.4–5.0 b

≤15% ≤10%

Leucine

> 5.0

Lysine

a Concentrations apply to: ( a ) ‘ready-to-feed” liquids “as is”; ( b ) reconstituted powders (25 g into 200 g water); and ( c ) liquid concentrates diluted 1:1 by weight using water.

Phenylalanine

Proline Serine Taurine

mg/100 g reconstituted final product.

b

Threonine Tryptophan

5 System Suitability Tests and/or Analytical Quality Control Suitable methods will include blank check samples, and check standards at the lowest point and midrange point of the analytical range. 6 Reference Material(s) NIST Standard Reference Material® 1849a Infant/Adult Nutritional Formula, or equivalent. SRM 1849a is a milk-based, hybrid infant/adult nutritional powder. One unit of SRM 1849a contains 10 packets each containing approximately 10 g of material. The SRM 1849a certificate values for amino acids and taurine are reference values and are for total content (Table 3). Other reference materials may be specified. 7 Validation Guidance Recommended level of validation: Official Methods of Analysis SM . 8 Maximum Time-to-Result No maximum time. Approved by Stakeholder Panel on Infant Formula and Adult Nutritionals (SPIFAN). Final Version Date: September 6, 2014. Effective Date: November 13, 2014. Revised: March X, 2018.

Tyrosine

Valine

0.11

Sum of Asp+Asn.

a

Sum of CSSC and Cys.

b

Sum of Glu+Gln.

c

© 2014 AOAC INTERNATIONAL

Memorandum Date:

November 16, 2017

To:

Fluoride Working Group

Re: Revisions to Method Performance Table in SMPR 2014.016 The current LOQ requirement of 5 µg / 100g specified in SMPR 2014.016, Determination of Fluoride in Infant and Adult/Pediatric Nutritional Formula (attachment 1) is proving difficult to achieve with current technology. Codex Standard 72 – 1981 specifies a maximum level of fluoride of 100 µg / 100Kcal, which is equivalent to 65 µg / 100g. Methods with LOQs higher than 5 µg / 100g could still be useful for the determination of fluoride. The Fluoride Working Group proposes to: 1) adjust the LOQ from 5 µg / 100g to 30µg / 100g; 2) adjust the lower range band [for recovery, RSDr, and RSDR] from 5 – 25 µg / 100g to 30 - 100 µg / 100g. 3) adjust the upper range band [for recovery, RSDr, and RSDR] from 100 µg / 100g to 200 µg / 100g.

30 – 200*

Analytical Range Limit of Quantitation

30*

Ranges

Parameter

30 - 100* 80 – 120

>100*

Recovery (%)

90 – 110

% RSD r % RSD R

≤ 8

≤ 5

≤ 15

≤ 10

* µg/100g.

Concentrations apply to: a) ‘ready-to-feed” liquids “as is”; b) re- constituted powders (25 g into 200 g of deionized water); and c) liquid concentrates diluted 1:1 by weight with deionized water.

Summary of Next Steps: 1) Review cleaned up table with Fluoride Working Group. Soon. 2) Revise SMPR 2014.016. December 2017.

3) Post on AOAC website for public comment. January 2018. 4) Collect, compile and address public comments. Feb 2018. 5) Present proposed revisions to SMPR 2014.016 to SPIFAN in during the March 2018 mid-year meeting.

ATTACHMENT 1