AOAC Final Action Methods in 2018

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AOAC INTERNATIONAL Official Methods Board (OMB)

Methods Book (Awards) Final Action in 2018 April 30, 2020

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

dboyd@aoac.org 301.924.7077 x126

( x ) Glass beakers .—Assorted sizes. ( y ) Amber glass HPLC vials .—With septa and caps. ( z ) Low-actinic glass bottles .—250–500 mL. C. Reagents and Standards

50.1.29

AOAC Official Method 2011.06 Total Folate in Infant Formula and Adult Nutritionals Trienzyme Extraction and LC-MS/MS Quantitation

Chemicals used are LC-MS grade unless specified. Product numbers and suppliers, when listed, reflect those used in validation. Equivalent chemicals can be used. Chemicals used as standards and internal standards: All chemicals used in preparation of standards are stored at a minimum of –20°C or as directed by the manufacturer. Because folate compounds are light sensitive, all samples and standards must be prepared, handled, and stored in the dark or under yellow- shielded lighting or UV-filtered lighting. If standards and samples must be transported through or into an area without UV-filtered lighting, they must be tightly wrapped in foil. Chemicals and/or reagents equivalent to those listed can be used. ( a ) Folic acid (FA). —Schircks Laboratories, Cat. No. 16.203. ( b ) (6R,S)-5-methyl-5,6,7,8-tetrahydrofolic acid, calcium salt (5-CH 3 -THF). —Schircks Laboratories, Cat. No. 16.235. ( c ) (6S)-5-formyl-5,6,7,8-tetrahydrofolic acid, calcium salt (5-CHO-THF). —Schircks Laboratories, Cat. No. 16.221. ( d ) Pteroyltri-gamma-L-glutamic acid (folic acid triglutamate) (Pte- Glu3). —Schircks Laboratories, Cat. No. 16.253. ( e ) 13 C 5 -folic acid (internal standard) ( 13 C 5 -FA) .—IsoSciences (Ambler, PA, USA) Cat. No. 14139. ( f ) 13 C 5 -(6S)-5-methyltetrahydrofolic acid, calcium salt (internal standard) ( 13 C 5 -CH 3 -THF). —IsoSciences, Cat. No. 14168CA. ( g ) 13 C 5 -(6S)-5-formyltetrahydrofolic acid, calcium salt (internal standard) ( 13 C 5 -CHO-THF) .—Merck & Cie. Other chemicals: Store chemicals per manufacturer guidelines or set conventions. Chemicals listed were used in the validation study. Equivalent chemicals and enzymes could be used from alternate sources. ( h ) α-Amylase. —From Aspergillus oryzae, powder, ≥150 units/ mg protein; Sigma-Aldrich, Cat. No. A9857. ( i ) Protease. —From Bacillus licheniformis , Subtilisin A, lyophilized powder; Sigma-Aldrich, Cat. No. P3910. ( j ) Conjugase (male Sprague Dawley rat plasma) with lithium and heparin (not filtered). —BioIVT (Westbury, NY, USA), Cat. No. RAT00PLLHMNN. ( k ) Ammonium hydroxide solution. —Certified ACS Plus, 28–30%, w/w, Aqueous Ammonia; Fisher Scientific, Cat. No. A669-500. ( l ) Sodium phosphate, dibasic anhydrous. —Granular or powder/certified ACS, ≥99%; Fisher Scientific, Cat. No. S-374- 500. ( m ) Methanol .—LC-MS grade, 99.9% minimum; Fisher Scientific, Cat. No. A456-4. ( n ) Glacial acetic acid .—Certified ACS; Fisher Scientific, Cat. No. A38S-212. ( o ) Sodium hydroxide .—Pellets/ACS certified, ≥97%; Fisher Scientific, Cat. No. S318-1. ( p ) Ammonium acetate. —Crystalline/certified ACS, ≥97%; Fisher Scientific, Cat. No. A637-500. ( q ) 2-Mercaptoethanol .—Electrophoresis grade, ≥98%; Fisher Scientific, Cat. No. BP176-100; Sigma-Aldrich, M6250. ( r ) Ascorbic acid .—White crystalline powder, ≥99%; Fisher Scientific, Cat. No. BP351-500. ( s ) Tris-(2-carboxyethyl)phosphine hydrochloride (TCEP- HCl) .—≥98%; Fisher Scientific, Cat. No. AC36383.

First Action 2011 Final Action 2018

Codex-Adopted AOAC-ISO Method (Applicable for determination of total folate contents in infant formula and adult nutritionals.) Caution: Method uses commonly used solvents and reagents. Refer to appropriate manuals or safety data sheets to ensure that safety guidelines are applied before using chemicals. Use all appropriate personal protective equipment and follow good laboratory practices. A. Principle Folates in a sample are extracted in a buffer (pH 6.0) containing internal standards by treatmentswith protease, amylase, and rat plasma conjugase (trienzyme extraction). Extract is purified and concentrated using a weak anion exchange (WAX) SPE. Polyglutamate forms of folates in the sample are deconjugated to monoglutamates during extraction and analyzed by LC-MS/MS. Folic acid, 5-CH 3 -THF, and 5-CHO-THF are quantified, and total folate is estimated and expressed as folic acid. Isotopically labeled folic acid [( 13 C-folic acid), 5-CH 3 -THF ( 13 C-5-CH 3 -THF), and 5-CHO-THF ( 13 C-5-CHO- THF)] are used as the internal standards. B. Apparatus and Materials Equipment and supplies equivalent to those listed can be used. ( a ) Precision balance .—Reading 0.1 mg. ( b ) LC system .—Agilent 1290 Infinity Binary or Dionex 3000 Ulimate LC. ( c ) Mass spectrometer .—Sciex 6500 or 5500 Triple Quad. ( d ) pH meter. ( e ) Refrigerator .—Capable of cooling to 4°C; explosion proof. ( f ) Freezer. —Capable of cooling to –20 and –70°C; explosion proof. ( g ) Water bath with shaker. ( h ) Fume hood. ( i ) Centrifuge. —Capable of up to 1400 × g ; Sorvall Legend X1R and rotor F15-8x50c with radius (Rmax 10.4 cm). ( j ) Microcentrifuge .—Capable of up to 9500 × g . ( k ) Mechanical/electronic pipets .—Adjustable (100–1000 μL). ( l ) Vortex mixer. ( m ) 24-well SPE vacuum manifold. ( n ) SPE columns. —Mixed mode WAX SPE, 33 μm polymeric, 60 mg/3 mL; Phenomenex, 8B-S038-UBJ. ( o ) LC column .—Waters ACQUITY UPLC HSS T3 Column (100 Å, 1.8 μm, 2.1 × 50 mm). ( p ) Glass test tubes .—16 × 125 mm. ( q ) Centrifuge tubes. —50 mL polypropylene. ( r ) Syringe filters. —0.45 μm PVDF with a filtration area of about 3 cm 2 . ( s ) Microcentrifuge tubes .—Polypropylene, 1.5–2.0 mL. ( t ) Low-actinic volumetric flasks. —Assorted sizes. ( u ) Glass Pasteur pipets. ( v ) Volumetric pipets. —Class A, assorted sizes. ( w ) Repeating pipets .—5 and 25 mL.

( t ) Charcoal .—Darco G-60 activated carbon; Fisher Scientific, Cat. No. D127-500. ( u ) Formic acid .—Reagent grade, ≥95%, water ≤2.5%, acetic acid <1%; Sigma-Aldrich F0507. ( v ) Water. —High-purity, suitable for HPLC mobile phase, and resistivity up to 18 megohms. D. Preparation of Reagents and Standard Solutions ( a ) Stock solvent. —15.6 mM ammonium acetate buffer with 25% ascorbic acid and 1% mercaptoethanol, pH 5.5. Weigh 0.6 ± 0.01 g ammonium acetate and transfer into a 500 mL beaker. In a fume hood, slowly add approximately 300 mL HPLC water and 2.4 mL glacial acetic acid. Add 125 g ascorbic acid and 5 mL 2-mercaptoethanol. Stir to dissolve completely. Adjust pH to 5.5 using concentrated ammonium hydroxide (28–30%). Transfer solution into a 500 mL volumetric flask and bring to volume (500 mL) with HPLC water. Mix thoroughly. ( b ) Intermediate solvent .—1.6 mM ammonium acetate buffer with 1% ascorbic acid, pH 5.5. Weigh 0.12 ± 0.001 g ammonium acetate and transfer into a 1 L beaker. In a fume hood, slowly add approximately 700 mL HPLC water and 480 μL glacial acetic acid. Add 10 g ascorbic acid and stir until completely dissolved. Adjust pH to 5.5, if necessary, with concentrated ammonium hydroxide. Transfer solution to a 1 L volumetric flask and bring to volume with HPLC water. Mix thoroughly. ( c ) Solvent for SPE elution and folate standards. —Methanol with 10% formic acid and 1% ascorbic acid. Take 1 g ascorbic acid and 10 mL concentrated formic acid into a 100 mL volumetric flask. Add approximately 50 mL methanol. Sonicate in a water bath (at room temperature) for complete dissolution (about 2 min). Bring to 100 mL volume with methanol. Mix thoroughly. Use this solution in elute folates from SPE sorbent after cleanup to make calibration standards and as a blank in LC-MS/MS analysis. Prepare fresh on day of use. ( d ) Preparation of folate stock standards (500 μg/mL). — Accurately weigh approximately 25 mg of each of folic acid, 5-CH 3 -THF, 5-CHO-THF, and Pte-Glu3 into separate 50 mL low- actinic volumetric flasks. Add approximately 35 mL of the stock solvent to each flask and sonicate in a water bath for about 1 min for complete dissolution of the folate chemical. Add the least amount of ammonium hydroxide solution (28–30%) to aid in the dissolution of folic acid; may take approximately 30–48 drops (1.5–2.4 mL). Make up to 50 mL with folate stock solvent in each of the flasks and mix the contents. Transfer solutions to suitable clean containers separately and store at –20°C or lower (i.e., –70°C for a better stability). Calculate the correct concentration of each of the folate compound’s respective stock solution after adjusting for moisture content in the folate chemical and their respective purity (HPLC) based on the certificate provided by the supplier. Folic acid may have moisture as high as 7.9% and 5-formyl-THF as high as 14.9%. Typical purity (HPLC-based) of the folate compounds is usually 98–99%. The 5-CH 3 -THF and 5-CHO-THF chemicals used are often calcium salts. Calculate the concentration of both stock standard solutions as salt-free form based on the differences in their respective molecular weights. Stock standard concn, μg/mL = [(standard, grams salt-free adjusted for purity and moisture contents) × 10 6 ]/(final volume, mL)

Folate stock standard solutions if stored at –70°C, folic acid, 5-CHO-THF, and Pte-Glu3 are usually stable for 6 months, and 5-CH 3 -THF is stable for 3 months. Solutions may be stable for less time period if stored at –20°C. Purity of the folic acid standard chemical can be confirmed by making its solution in 0.1 M NaOH (pH 13) at a concentration of 10 μg/mL and reading absorbance at 282 nm in spectrophotometer in a cuvette light path length of 1 cm. Pure folic acid E 1% (in 1 cm light path) at 282 nm = 532.2. ( e ) Preparation of folate intermediate standards (20 μg/mL) .— Add about 5 mL of the intermediate solvent to a 10 mL low-actinic volumetric flask. Add 0.4 mL of each of the stock standard solutions of folic acid, 5-CH 3 -THF, and 5-CHO-THF to the same flask. Make up the volume to 10 mL with the intermediate solvent and mix the contents. Concn (μg/mL) of each folate vitamer in intermediate standard solution = (stock standard solution concentration, μg/mL × 0.4 mL)/10 mL Folate intermediate standard solution can be stored at –20°C for 30 days, and can be stable for a longer time at –70°C. ( f ) 13 C 5 -folic acid internal standard stock solution (1 mg/mL) .— The chemical is often supplied in a 1 mg amount. The entire 1 mg amount of labeled folic acid is dissolved in 1 mL of the stock solvent. Folic acid is difficult to dissolve. The addition of 10 μL ammonium hydroxide solution (28–30%) aids in dissolution. Dissolve the chemical in a correct volume of the solvent if its weight is different than 1 mg, so that the final concentration is around 1 mg/mL. Sonication and mixing on a vortex mixer for 1–2 min may help complete dissolution. ( g ) 13 C 5 -(6S)-5-methyl-5,6,7,8-tetrahydrofolate internal standard stock solution (1 mg/mL) .—The labeled methyl-THF may be supplied in a 1 mg amount. The entire 1 mg amount of labeled methyl-THF is dissolved in 1 mL of the stock solvent. Dissolve the chemical in correct volume of the solvent if its weight is different than 1 mg, so that the final concentration is around 1 mg/mL. Dissolve the chemical completely with the aid of a vortex mixer and brief (30 s) sonication. ( h ) 13 C 5 -(6S)-5-formyl-5,6,7,8-tetrahydrofolate internal standard stock solution (1 mg/mL) .—Weigh 10 mg of labeled formyl-THF in a 10 mL low-actinic volumetric flask. Add about 7 mL of the stock solvent. Dissolve the chemical completely with the aid of a vortex mixer and brief (30 s) sonication. Make up the volume to 10 mL with the stock solvent and mix the solution. Folate internal standard stock solutions if stored at –70°C, folic acid, and 5-CHO-THF can be stable for 6 months and 5-CH 3 -THF for 3 months. Solutions may be stable for less time period if stored at –20°C. Concentration of each of the stock solutions should be calculated based on the known purity of the respective folate chemical. ( i ) Internal standard intermediate solution cocktail ( 20 μg/mL of each folate ).—Add about 5 mL of the intermediate solvent into a 10 mL low-actinic volumetric flask. Transfer exactly 0.2 mL of each of the internal standard stock solutions, i.e., 13 C 5 -FA, 13 C 5 -CH 3 - THF, and 13 C 5 -CHO-THF, into the same 10 mL volumetric flask. Make up to 10 mL with the intermediate solvent. Thoroughly mix the solution. Calculate the actual concentrations of each internal standard as the following: Concn (μg/mL) of each folate vitamer in the internal standard intermediate solution = (internal standard stock solution concn in μg/mL × 0.2 mL)/10 mL

Folate standard intermediate solution can be stored at –20°C for 30 days, and can be stable for longer at –70°C. ( j ) 2 ppm Internal standard solution for calibration standards (2 μg/mL of each internal standard folate) .—Prepare solution fresh before use. Store at 4°C if necessary but not for more than 6 h. Take 20 mL of the solvent for SPE elution into a 50 mL centrifuge tube. Add 1 mL ammonium hydroxide (28–30%) and mix (neutralized solvent for SPE elution). Prepare fresh before use. Pipet exactly 100 μL of folate internal standard intermediate solution (~20 μg/mL of each folate) into a microcentrifuge tube. Add 900 μL of the freshly prepared neutralized solvent for SPE elution. Mix thoroughly with a vortex mixer for about 30 s. Concn (μg/mL) of each folate vitamer in 2 ppm internal standard solution = internal standard intermediate solution concn in μg/mL × 0.1 mL ( k ) 40 ppb Internal standard solution for calibration standards (40 ng/mL each folate) .—Prepare solution fresh before use. Store at 4°C if necessary but not for more than 6 h. Take 12 mL of the solvent for SPE elution in a scintillation vial or a 50 mL plastic centrifuge tube. Add 600 μL ammonium hydroxide (28–30%; neutralized solvent for SPE elution) and mix. Prepare fresh before use. Pipet about 5 mL of neutralized solvent for SPE elution into a 10 mL low-actinic volumetric flask. Pipet 20 μL of the internal standard intermediate solution (20 μg/mL each folate) into the same 10 mL volumetric flask. Make up the volume to 10 mL with the neutralized solvent for SPE elution and mix thoroughly. Concn (μg/mL) of each folate vitamer in 40 ppb internal standard solution = (concn of folate in internal standard intermediate solution in μg/mL × 0.02 mL)/10 mL ( l ) Preparation of calibration standard solutions .—Prepare solutions fresh before analysis. Store at 4°C if necessary but not for more than 6 h. Take 12 mL of the solvent for SPE elution in a scintillation vial or a 50 mL plastic centrifuge tube. Add 600 μL ammonium hydroxide (neutralized solvent for SPE elution)

and mix. Prepare fresh before use. Solution is also used as an instrument blank in analysis. Calibration standard solutions are prepared as shown in Table 2011.06A . Set up microcentrifuge tubes labeled calibration standard A–G separately. Standards A and B are prepared using folate intermediate standard solution, whereas calibrants C–G are prepared by dilutions of higher concentration calibrants. Add intermediate standard solution as specified in Table 2011.06A to the corresponding calibration tubesAand B.Add 20 μLof the 2 ppm internal standard solution to tubes A and B. Add neutralized elution solvent to tubes A and B as specified in Table 2011.06A . Close the tubes and mix thoroughly by vortex mixer for about 20 s. Calibration standards C–G are prepared by dilutions of the specified calibration standards in Table 2011.06A . Pipet the specified calibration standard in the specified volume. Make up the volume to 1 mL with the 40 ppb internal standard solution as specified in Table 2011.06A . Close the tubes and mix thoroughly by vortex mixer for about 20 s. Calculate the exact concentration of each folate compound, including the internal standard in each calibration standard, by following the dilution of the intermediate standard solution or the respective calibrant in the solution and the used internal standard solution. ( m ) Preparation of substrate solution (Pte-Glu3) to test plasma conjugase activity (20 μg/mL) .—Add about 5mLof the intermediate solvent to a 10 mL low-actinic volumetric flask. Pipet 0.4 mL Pte- Glu3 stock solution (500 mcg/mL). Make up the volume to 10 mL with the intermediate solvent and mix thoroughly. Store at –20°C for 1 month; can be stable at –70°C for longer time periods. Other reagents for folate analysis: ( a ) Protease solution (2 mg/mL) .—Prepare solution fresh before use. Store at 4–8°C if necessary but not for more than 4 h. Dissolve 0.05 g protease in 25 mL water in a 100 mL beaker or Erlenmeyer flask by proper mixing. Each sample requires 1 mL. ( b ) α-Amylase solution (20 mg/mL; 40 units/mL) .—Prepare solution fresh before use. Store at 4–8°C if necessary but not for more than 4 h. Dissolve 0.5 g in about 20 mL water in a 25 mL volumetric flask or a centrifuge tube. Make up the volume to 25 mL. Mix gently for complete mixing. Make sure no foams develop. The amylase solution is transferred to a 50 mL centrifuge tube if not already in

Table 2011.06A. Preparation of folate calibration standard solutions a

Volume of 40 ppb internal standard solution (40 ng/mL), μL

Concn of each folate compound in standard solution, ng/mL

Calibration standard

Volume of intermediate/ calibration standard solution, μL 25 μL intermediate standard solution 10 μL intermediate standard solution 100 μL calibration standard solution A 100 μL calibration standard solution B 100 μL calibration standard solution C 100 μL calibration standard solution D

2 ppm Internal standard solution (2 μg/mL), μL

Volume of neutral elution solvent, μL

Final volume, mL

955

1.0

500

970

1.0

200

900

1.0

900

1.0

900

1.0

900

1.0

50 μL calibration standard solution D

950

1.0

a Internal standard concentration in every calibration standard solution (A–G) = 40 ng/mL of each folate.

one. Treat amylase solution with charcoal. Add 20 mg charcoal per milliliter of solution (0.5 g in 25 mL). Gently mix on a vortex mixer for about 45 s. Let stand for 5 min at 4–8°C. Filter through a 0.45 μm PVDF syringe filter. Each sample requires 1 mL. ( c ) Male rat plasma conjugase .—The plasma is kept frozen at –20°C or lower until use. Check viability of plasma stored over 3 months. Avoid repeated freezing thawing of plasma to avoid losses in its conjugase activity. Thaw plasma as required. Treat plasma with 20 mg charcoal/mL. Take 5 mL plasma in a tube. Add 100 mg charcoal. Gently mix with vortex mixer for about 30 s. Let stand for 5 min at 4–8°C. Filter through PVDF syringe filter. About 4.2 mL of plasma from 5 mL is usually obtained after charcoal treatment. Prepare fresh. Store at 4–8°C if necessary but not for more than 4 h. Each sample requires 0.3 mL of charcoal-treated plasma. Efficiency of the conjugase must be determined prior to use but only once for every new lot of plasma. ( d ) Checking efficiency of rat plasma conjugase .—Add 30 μL conjugase test solution to a 50 mL centrifuge tube with 10 mL extraction buffer (containing only 13 C 5 -folic acid internal standard, 4 ng/mL) and 50 μL TCEP solution. Mix with a vortex mixer. For the cojugase test blank, add 30 μL intermediate solvent to another 50 mL centrifuge tube with 10 mL extraction buffer and 50 μL TCEP solution. Mix with a vortex mixer. Add 300 μL charcoal-treated conjugase to both tubes, fill headspace with nitrogen, cover, and incubate for 16 h in a shaking water bath at 37°C, with a 60 rpm shaking speed. Follow procedure from step F(a) ( 7 ) for analysis of folates. Using the result obtained and concentration of Pte-Glu3 in the test solution prepared, determine conversion of Pte-Glu3 to folic acid. Conjugase is considered acceptable for use if conversion of Pte-Glu3 to folic acid is ≥90%. The protocol to calculate conversion of Pte-Glu3 to folic acid is located in the Calculations section. ( e ) TCEP solution (1 M) .—Dissolve 1 g TCEP-HCl in 3.5 mL HPLC water in a test tube or a vial. ( f ) Extraction buffer (10 mM phosphate buffer, 1% ascorbic acid, pH 6.0) .—Weigh 0.355 g sodium phosphate dibasic, anhydrous Na 2 HPO 4 , in a 250 mL beaker. Add 2.5 g ascorbic acid and approximately 200 mL HPLC water. Stir u ntil completely dissolved. Adjust pH with phosphoric acid and/or 10 M sodium hydroxide (10 M NaOH) to pH 6.0. Transfer into a 250 mL volumetric flask and dilute to volume with HPLC water. Mix. This can be stored at 4°C for 5 days. Add exactly 0.05 mL of the 20 ppm internal standard intermediate solution to 250 mL buffer. Mix thoro ughly. Internal standard concentration = 4 ng/mL of each folate. Prepare fresh on day of use. Store at 4°C if necessary but not for more than 6 h after addition of internal standards. Each sample requires 10 mL of the extraction buffer. ( g ) HPLC mobile phases (mobile phase A: 1% acetic acid; mobile phase B: methanol) .—For mobile phase A, thoroughly mix 500 mL water (LC-MS) and 5 mL glacial acetic acid. For mobile phase B, use methanol as is (LC-MS). E. Sample Preparation ( a ) Sample processing for homogeneity .—All samples should be as uniform and representative of the product as possible. This should be accomplished by a thorough mixing or stirring of the sample before sampling. Dry samples are reconstituted to liquid samples, and reconstituted liquid samples are used for analysis. ( b ) Reconstitution of powder sample into liquid .—Weigh 25.0 ± 0.1 g of dry sample into a low-actinic glass 250 mL beaker or bottle.

Cover the regular bottle or beaker with aluminum foil if low-actinic bottles or beakers are not available. Note the weights. Add 200.0 ± 0.1 g warm water (40 ± 5°C). Mix well until complete dissolution/ suspension, making sure there are no lumps. Record the final weight for each sample. Calculate weight (grams) of dry sample in the corresponding reconstituted liquid sample in every case. Dry samples are analyzed as reconstituted liquid samples, and ready- to-feed (RTF) samples are analyzed as is. Reconstituted samples, as well as RTF samples, are thoroughly mixed immediately before weighing the aliquot for the analysis. F. Procedure ( a ) Extraction .—( 1 ) Weigh 0.1000–2.000 g of sample, depending on anticipated total folate (in range of 20–500 ng), into a 50 mL centrifuge tube and record to 0.0001 g. ( 2 ) Prepare a method blank containing reagents only. This will be treated like a sample through the entire following method. ( 3 ) Add 10 mL extraction buffer (containing internal standards) and 50 μL TCEP solution to each tube and mix with a vortex mixer. ( 4 ) Add 1 mL protease solution to each tube, fill the headspace with nitrogen, cover, and mix with a vortex mixer. Incubate for 3 h in a shaking water bath at 37°C at 60 rpm shaking speed. ( 5 ) Inactivate the protease by placing the tubes in a boiling water bath (100°C) for 5 min; shake tubes every 1.5 min, and then remove from bath and cool to room temperature. ( 6 ) Add 1 mL α-amylase solution (charcoal treated) and 300 μL rat plasma conjugase (charcoal-treated) to each tube; fill the headspace with nitrogen, cover, and incubate for 16 h in a shaking water bath at 37°C at 60 rpm shaking speed. ( 7 ) Inactivate enzymes after 16 h incubation by placing tubes in a boiling water bath (100°C) for 5 min. Shake tubes every 1.5 min, then remove from bath and cool to room temperature. ( 8 ) Centrifuge tubes at 4–5°C for 10 min at 1400 × g . ( 9 ) Filter supernatant from each tube through a 0.45 μm PVDF syringe filter into separate labeled glass tubes (16 × 125 mm). ( b ) Extract purification .—( 1 ) Set up a 24-well SPE manifold. Set up one SPE tube for every sample and one for the method blank. ( 2 ) Condition each SPE tube as follows, without vacuum: First, condition with 2 mL methanol, and before the SPE sorbent is dry, repeat the first step with 2 mL water. ( 3 ) Load samples into corresponding activated SPE tubes over sorbent, without vacuum. First, load all of the filtered supernatant (about 12 mL) for every sample into the corresponding SPE (no vacuum) tube. Then, wash each SPE tube with 3 mL water with no vacuum. Push out any residual water by gentle push with a pipet bulb. ( 4 ) Elute the analytes from the SPE sorbent. Empty the waste reservoir and place a clean microcentrifuge tube to collect the eluent corresponding to every SPE tube. Add 1 mL of the freshly prepared solvent for SPE elution to each SPE tube. Allow gravity to elute the effluent into the tubes and use a pipet bulb to push out any remaining solvent. Add 50 μL ammonium hydroxide (28–30%) to each tube and immediately mix with a vortex mixer for 20 s. Centrifuge the tubes in a microcentrifuge at 9500 × g for 5 min. Transfer supernatant from each tube into a corresponding HPLC vial for LC-MS/MS analysis. Prepare LC and mass spectrometer for analysis and load vials in autosampler. The sequence of the analysis includes instrument blank, calibration standards (G–A; Table 2011.06A ), instrument blank (neutralized SPE elution solvent), method blank, and all of the samples.

Table 2011.06B. LC gradient Time, min

Table 2011.06D. MRM transitions for different folates and MS parameters for their detection Folate Q1 a , m/z Q3 b , m/z DP c , V EP d , V CXP e , V CE f , V FA 442.2 295.2 75 6 6 20 442.2 176.2 75 6 6 45 5-CH 3 -THF 460.2 313.2 75 6 6 28 460.2 194.2 75 6 6 46 5-CHO-THF 474.2 327.2 75 6 6 25 474.2 299.2 75 6 6 40 13 C 5 -FA 447.2 295.2 75 6 6 20 13 C 5 -5-CH 3 -THF 465.2 313.2 75 6 6 28 13 C 5 -5-CHO-THF 479.2 327.2 75 6 6 25

Mobile phase B a , %

Flow, mL/min

0.00 0.50 5.00 5.05 6.50 6.55 9.00

0 0

0.5 0.5 0.5 0.5 0.5 0.5

100 100

0 0

0.5 a Mobile phase B = methanol. The mobile phase refers to 1% acetic acid in water.

Table 2011.06C. MS source conditions for some of the Sciex Triple Quads

a Q1 = Precursor ion. b Q3 = Product ion. c DP = Declustering potential. d EP = Entrance potential. e CXP = Collision cell exit potential. f CE = Collision cell potential.

Sciex Triple Quad

Source condition parameters

5500

6500 Qtrap 6500 plus

Curtain gas (nitrogen), psi

Collision-activated dissociation Medium Medium

Ion spray voltage, V

5500

calibration curve. This is usually done by the software used for the processing of the MS data. ( d ) Calculation of concentration of folate compounds in sample extracts .—The ratios of peak areas of the detected folate vitamers to the respective internal standard areas for every sample is calculated. The concentration of each of the folate compounds as ng/mL is calculated based on the calibration curve of the respective folate compound. This calculation is usually performed by the software used for processing MS data. The concentration of each of the folate compounds as ng/mL in a sample extract is adjusted by subtracting the amount of respective folate compound detected in the method blank ran in the batch. Corrected folate compound concn in a sample extract, ng/mL = concn of specific folate compound (ng/mL) – concn of respective specific folate compound in the method blank (ng/mL) ( e ) Calculation of folate compounds and total folate in reconstituted or RTF samples (mcg/100 g) .—Estimated folate vitamer concentration (ng/mL) in the extract is multiplied by fold dilutions (if any) and the final volume of the relevant step (volume after SPE elution, 1.05 mL). The total amount of folate vitamer in the sample is adjusted for the aliquot of the sample used for the extraction. The folate vitamer then calculated as μg/100 g of the reconstituted or RTF sample. Table 2011.06E. Conversion of amount of 5-CH 3 -THF and 5-CHO-THF to folic acid equivalent Folate vitamers Formula weight Multiply by to convert to folic acid Example value, mcg/100 g Value as folic acid, mcg/100 g Folic acid 441.4 1 100.0 100.0 5-CH 3 -THF 459.5 0.9606 100.0 96.1 5-CHO-THF 473.4 0.9324 100.0 93.2 Total folate as folic acid Sum as folic acid 289.3

Temperature, °C

700

400

Ion source gas 1 (zero air), psi Ion source gas 2 (zero air), psi

60 50

40 50

( c ) Instrumental analysis .—Instrument blank and calibration standards G–A, as detailed in Table 2011.06A , are analyzed, followed by analysis of an instrument blank, method blank, and sample extracts. The following are LC conditions with Dionex Ultimate 3000 LC/Agilent 1290 LC: LC column, Waters Acquity UPLC HSS T3 (100 Å), 1.8 μm, 2.1 × 50 mm; mobile phases, A = 1% acetic acid and B = 100% methanol; solvent composition, gradient (Table 2011.06B ); column temperature 40°C; autosampler temperature 5 ± 1°C; and injection volume 2 μL. MS source conditions for some of the Sciex Triple Quads are provided in Table 2011.06C . Multiple reaction monitoring transitions for different folates are provided in Table 2011.06D . G. Calculations ( a ) Calculation of ratios of peak areas of the folate compounds to the respective internal standard peak areas .—Calculate the ratios of peak area of each folate vitamer against respective internal standard area for all calibration standards, method blank, and all of the sample extracts. ( b ) Calibration curve .—Acalibration curve is drawn by plotting the concentration of each calibration standard (ng/mL) of a specific folate compound against the corresponding ratio of its peak area to the relevant internal standard peak area. The calibration curves are prepared for all of the folate compounds. The calibration curves are not forced through zero (weighting 1/x). This calculation is usually done by the software used for the processing of MS-acquired data. ( c ) Calculation of concentration of folate compounds in the method blank .—The ratios of peak areas of the detected folate compounds to the respective internal standards are calculated in the method blank. The concentration of each of the detected folate compounds as ng/mL is calculated based on the respective

Figure 2011.06. Chromatogram of folate analysis providing elution pattern of folate vitamers. 5-Methyltetrahydrofolic acid peak is marked as 5-CH 3 - THF, 5-formyltetrahydrofolic acid peak is marked as 5-CHO-THF, and folic acid peak is marked as FA. Color images are available online at http://aoac. publisher.ingentaconnect.com/content/aoac/jaoac.

Folate vitamer concn, mcg/100 g, in reconstituted or RTF sample = [vitamer concn, ng/mL, in extract × 1.05 (final volume, mL)]/ [sample aliquot, g × 10] The amount of CH 3 -THF and CH 0 -THF (mcg/100 g) in the samples are calculated as folic acid by multiplying (mcg/100 g) concentration of the folate compound by the corresponding conversion factors provided in Table 2011.06E . Total folate in the reconstituted or RTF sample is calculated by the sum of all folate compound as folic acid (mcg/100 g). Total folate as folic acid mcg/100 g in reconstituted or RTF samples = folic acid (mcg/100 g) + [5-CH 3 – THF (mcg/100 g) × 0.9606] + [5-CHO-THF (mcg/100 g) × 0.9324] ( f ) Calculation of total folate in dry samples (mcg/100 g) as-is basis .—The calculation of total folate (mcg/100 g) in dry samples as-is basis from total folate (mcg/100 g) in reconstituted beverage samples is based on the following relationship:

Folic acid (ng/mL) is also calculated similarly in the conjugase test blank. Blank-corrected folic acid (ng/mL) in the assayed mixture = folic acid (ng/mL) in assayed mixture – folic acid (ng/mL) in conjugase test blank Adjusted folic acid released in whole assayed mixture = blank-corrected folic acid (ng/mL) × final volume of assay mixture after purification (SPE elution volume = 1.05 mL) ( h ) Calculation of percent conversion of Pte-Glu3 to folic acid in the conjugase assay. —Folic acid (ng) released in conjugase assay is converted to folic acid (nanomoles) by using the following equation: Folic acid (nanomoles) in the assayed mixture = folic acid (ng) in assay/441.4 (folic acid molecular weight)

Total folate as-is basis in dry sample analyzed as reconstituted (mcg/100 g) = [ C (mcg/100 g) × V (g)]/ W (g)

Pte-Glu3 (ng) in conjugase assay = 0.03 mL × 20 mcg/mL (in conjugase activity substrate solution) = about 600 ng

where C (mcg/100 g) is total folate in the reconstituted sample, V (g) is the total weight of the reconstituted beverage sample, and W (g) is the weight of aliquot of the powder sample used for reconstitution. ( g ) Calculation of folic acid nanograms released in the conjugase test. —The ratios of peak areas of folic acid and folic acid internal standard are calculated in the assayed solution. Folic acid (ng/mL) in the solution is calculated based on its calibration curve.

Pte-Glu3 (nanomoles) in conjugase assay = Pte-Glu3 (ng) in the assay/699.6 (Pte-Glu3 molecular weight) Each molecule of Pte-Glu3 on hydrolysis yields one molecule of folic acid. % Conversion of Pte-Glu3 to folic acid =

[folic acid (nanomoles) released in the assay/Pte-Glu3 (nanomoles) in the assay] × 100

H. Example Chromatogram See Figure 2011.06 . References: J. AOAC Int . 95 , 1547(2012)

DOI: 10.5740/jaoacint.CS2011_06 (First Action) J. AOAC Int . 101 , 1881(2018) DOI: 10.5740/jaoacint.18-0114 (Final Action) AOAC SMPR 2011.006 J. AOAC Int . 95 , 294(2012) DOI: 10.5740/jaoac.int.11-0442 Posted: December 2018 (Final Action publication); 2019 (Codex- Adopted AOAC-ISO Method)

microwave systems. Never remove hot vessels from microwave; wait until they are near room temperature. Keep microwave door closed while vessels are hot. The door is the primary safety device if a vessel vents. See Tables 2011.14A – I for results of the interlaboratory study supporting acceptance of the method. [ Note : Results shown in Tables 2011.14A – I are slightly different from those shown in J. AOAC Int . 92 , 1484(2009) due to classical statistics applied instead of robust statistics applied in the tables included in J. AOAC Int . 95 , 177(2012).] See Table 2011.14J for results of the interlaboratory study by element for AOAC Stakeholder Panel on Infant Formula and Adult Nutritionals (SPIFAN) fortified products and SPIFAN placebos compared to AOAC Standard Method Performance Requirement (SMPR ® ) 2014.004. A. Principle Test portion is heated at 200°C either with nitric acid in a closed- vessel microwave digestion system (MDC) or with a combination of hydrogen peroxide, nitric acid, and hydrochloric acid in an open- vessel microwave digestion system (MDO). Digested test solution or an appropriate dilution is presented to the inductively coupled plasma-atomic emission spectrometry (ICP-AES) instrument. The instrument is calibrated with acid(s) matched standard calibration solutions. An ionization buffer (cesium) and internal standard(s) (chromium, indium, strontium, and yttrium) are used with or without appropriate in-line addition to minimize easily ionizable element (EIE) effects and to correct for physical and chemical ( Note : ICP-AES equipment must be used by qualified laboratory personnel who are familiar with the operation and maintenance of analytical systems and must follow manufacturer’s directions for safety risk, safety environment, and use of instruments.) ( a ) Microwave . ____ Commercial MDC or MDO designed for laboratory use at 200 ± 20°C, up to 600 psi, with operator selectable output of 0–1800 W microwave power, controlled temperature and pressure ramping capability, and equipped with appropriate vessels with a capacity of minimum 50 mL (e.g., 75 mL). It is recommended that vessel design be selected that will withstand the maximum possible pressure (600 psi) because organic residues of rich-fat or rich-carbohydrate samples, if not given sufficient time to predigest, will generate significant pressure during digestion. ( b ) MDC vessels or MDO flasks or tubes . ( c ) Analytical balance .—Capable of weighing ±0.1 mg. ( d ) Volumetric flasks .—Class A, glass, assorted sizes. ( e ) Volumetric pipets .—Class A, glass, assorted sizes. ( f ) Micropipets .—Adjustable, 0.100–1.000 mL and 1–5 mL with disposable plastic tips. ( g ) Dispenser .—Capable of dispensing 1–10 mL. ( h ) High-density polyethylene bottles .—For storing standard and sample solutions. ( i ) Water baths .—Capable of maintaining 20 ± 2°C and 40 ± 2°C. ( j ) Grinding device . ( k ) ICP-AES spectrometer .—Instrument with axial, radial, or dual view grating configurations and autosampler, capable of determining multiple wavelengths for each element of interest with the required sensitivity. A 3-channel peristaltic pump with interferences. B. Apparatus

AOAC Official Method 2011.14 Minerals and Trace Elements in Milk and Milk Products, Infant Formula,

and Adult Nutritionals Microwave Digestion and Inductively Coupled Plasma-Atomic Emission Spectrometry First Action 2011 Final Action 2013 (Fortified Food Products) Revised First Action 2017 Final Action 2018 (Infant Formula and Adult Nutritionals) [Applicable to determination of calcium (Ca), copper (Cu), iron (Fe), potassium (K), magnesium (Mg), manganese (Mn), phosphorus (P), sodium (Na), and zinc (Zn) in chocolate milk powder, dietetic milk powder 2, infant cereal, peanut butter, wheat gluten, milk, dried milk, butter, cheese, whey, dried whey, infant formula, and adult nutritionals.] Upper limits (mg/kg) for chocolate milk powder, dietetic milk powder 2, infant cereal, peanut butter, and wheat gluten: Ca (24000); Cu (210); Fe (850); K (32000); Mg (7500); Mn (20); Na (16000); P (16000); Zn (320). Limits of quantitation (LOQs; determined as 10*s r of the mean of 10 measurements of the low standard solution, Std1, mg/kg) for chocolate milk powder, dietetic milk powder 2, infant cereal, peanut butter, and wheat gluten: Ca (150); Cu (2); Fe (10); K (200); Mg (50); Mn (0.05); Na (100); P (100); Zn (5). Upper limits (mg/100 g) for ( 1 ) milk and ready-to-feed (RTF) liquids as-is using a typical sample size of 4 g per final analytical solution volume of 25 mL and ( 2 ) reconstituted milk powder, infant formula powders, and adult nutritional powders (25 g into 200 g water), using a typical sample size of mass of the reconstituted slurry per final analytical solution volume of 25 mL: Ca (1280); Cu (1.2); Fe (20); K (2000); Mg (110); Mn (1.0); Na (850); P (800); Zn (18). LOQs (determined as the mean of blanks + 10*s r ; mg/100 g) for ( 1 ) milk and RTF liquids as-is using a typical sample size of 4 g per final analytical solution volume of 25 mL and ( 2 ) reconstituted milk powder, infant formula powders, and adult nutritional powders (25 g into 200 g water), using a typical sample size of mass of the reconstituted slurry per final analytical solution volume of 25 mL: Ca (0.1); Cu (0.003); Fe (0.05); K (1.5); Mg (0.02); Mn (0.001); Na (0.1); P (0.01); Zn (0.003). Limits for nonreconstituted dairy ingredients (butter, cheese, whey powders, whey protein concentrates) shall be adjusted proportionally upward from these values based upon the sample size used for the ingredient. For example, if 0.6 g cheese is digested, the limits will be 4 g/0.6 g = 6.7-fold higher. Caution : Before using chemicals, refer to the supplier guide for chemical safety and/or other adequate manuals or safety data sheets approved by local authorities. Use fume hood and wear full personal laboratory protective clothing, gloves, and appropriate eye protection (safety glasses) when using glassware and preparing standards or test portions with acid solutions. Application of microwave digestion systems involves hot pressurized acid solutions and concentrated acids. Use fume hood and wear full personal laboratory protective clothing, gloves and appropriate eye protection (safety glasses) when using glassware and preparing standards or samples with acid solutions. Follow manufacturer’s directions for safety risk and safety environment of

Table 2011.14A. Interlaboratory study results for calcium (Ca)

Original validation (2011)

Matrix

Mean, mg/kg Rec., % s r a

RSD r

b , % s

R c , mg/kg RSD R

d , % HorRat

No. of labs e Outlier labs f

Chocolate milk powder Dietetic milk powder 2

9732.17 5678.58 5983.54

99 98

152.10

1.56 1.34 3.52 3.26 0.95

264.03 184.11 251.67

2.71 3.24 4.21 6.88 3.51 4.65 5.82 8.19 3.30 5.49 8.20 3.88 7.20 27.8 5.30 10.3

0.68 0.74 0.97 1.06 0.53 1.06 1.05 1.46 0.79 1.14 0.87 1.09 1.82 1.77 1.12 1.69 1.17 1.58 1.35 1.32 1.33 1.13 1.29 1.32 2.05 0.53 1.19 1.3 4.5 0.7

9 9 9 9 7

NS g

75.93

NS NS NS

Infant cereal

100 100 100

210.58

Peanut butter NIST SRM 2387 Wheat gluten NIST RM 8418

409.90 (411) h 367.56 (369) h

13.37

28.20 12.90

3.51

2, 4C

Matrix extension (2017)

Mean, mg/100 g i

SRM 1849a Whole milk

530 (525)

101 ND j

12.7 2.89

2.4

24.7 6.77 8.61 23.7 37.8 1.62 19.2 2.49 15.4

12 11 10

0 0 1 2 0 4 0 0 1 0 0 0 0 0 1 1 0 1 1 0 0 0 0 0 0

116 105 709 689 19.8 494 34.6 55.5 14.5 7.44 34.4 49.5 38.2 68.9 63.2 45.7 69.5 36.7 74.4 48.9 30.3 65.9 99 58

2.48 3.28 1.45 4.86 2.10 1.69 4.29

Whole milk powder

ND 3.45 ND 10.3 ND 33.5 ND 0.42 ND 8.37 ND 1.48

Whey powder

Whey protein concentrate

Butter

Cheese

11 12 11 12 12 12 12 12 11 11 12 10 11 12 12 12 12 12 12

Adult powder, low fat Adult RTF, high fat

ND ND ND

5.1 0.3 7.2

9.3 2.2 7.3

Adult RTF, high fat placebo Adult RTF, high protein Adult RTF, high protein placebo Child milk powder placebo

0.8

10.2

ND 0.167 ND 0.91 ND 2.33 ND 0.98

2.25 2.64

1.102

14.80

2.57 5.27 2.93

7.47

Child milk powder

4.7

10.60

Child elemental powder Infant elemental powder

2.56

7.7

2.7

3.9

6.5

9.50

Infant elemental powder placebo

ND 1.34

2.13

5.18

8.2

Infant PH powder milk Infant PH powder soy Infant powder FOS-GOS

0.4

0.9 3.2

3.8

8.40

ND 2.22 ND 0.774 ND 2.18 ND 2.84 ND 4.14 ND 0.63 ND 1.57

5.52 2.72 4.59 5.81 6.32 1.08 4.73

7.9

2.11 3.76 3.81 8.46 2.08 2.38

7.42 7.92 7.80 12.9 3.56 7.17

Infant powder milk Infant powder soy

Infant RTF milk

Infant RTF placebo

Toddler powder

a s r = Standard deviation for repeatability (within laboratory). b RSD r = Relative standard deviation for repeatability. c s R = Standard deviation for reproducibility (among laboratories). d RSD R = Relative standard deviation for reproducibility. e Reporting laboratories minus outliers. f C = Cochran outlier; SG = Single Grubbs outlier; DG = Double Grubbs outlier. g NS = Not significant. h Value in parentheses = Certified or reference values. i Results expressed as mg/100 g for ready-to-feed liquids as-is or reconstituted powders (25 g in 200 g water) or liquid concentrates (1:1 dilution by weight). j ND = Not determined.

Table 2011.14B. Interlaboratory study results for copper (Cu)

Original validation (2011)

Matrix

Mean, mg/kg Rec., % s r a

RSD r

b , % s

R c , mg/kg RSD R

d , % HorRat

No. of labs e Outlier labs f

Chocolate milk powder Dietetic milk powder 2

5.92 6.19 2.01

NA h 101

0.09 0.19 0.10 0.19 0.13

1.57 3.06 4.98 3.84 2.27

0.29 0.38 0.24 0.35 0.27

4.97 6.12

0.41 0.50 0.81 0.56 0.39

9 9 9 9 9

NS g

NS NS NS NS

Infant cereal

11.72

Peanut butter NIST SRM 2387 Wheat gluten NIST RM 8418

4.98 (4.93) i 5.64 (5.94) i

101

7.08 4.77

Matrix extension (2017)

Mean, mg/100 g i

SRM 1849a Whole milk

1.95 (1.98)

0.053 0.000

2.71

0.099 0.005 0.004 0.024 0.025 0.030 0.007 0.018 0.004 0.016 0.005 0.002 0.005 0.007 0.004 0.005 0.006 0.004 0.008 0.006 0.004 0.003 0.003 ND 0.01

5.09 78.2 71.3 48.5 20.7 56.1 9.97 7.51 ND

0.50 3.27 2.91 2.72 1.33 3.20 0.59 0.54 1.71 2.23 1.13 0.57 0.56 0.49 3.66 0.47 0.53 0.51 0.71 0.48 0.51 2.68 0.59 ND 0.6

0 2 0 0 0 0 1 0 0 0 0 0 1 0 0 1 0 1 0 0 1 0 1 2 0

0.007 0.006 0.049 0.122 0.023 0.054 0.071 0.239 0.009 0.184 0.011 0.006 0.111 0.046 0.082 0.004 0.056 0.063 0.047 0.061 0.068 0.046 0.004 0.029

ND j

4.1

Whole milk powder

ND 0.001 ND 0.004 ND 0.008 ND 0.004 ND 0.002 ND 0.004 ND 0.002 ND 0.006 ND 0.001 ND 0.001 ND 0.005 ND 0.003 ND 0.003 ND 0.001 ND 0.001 ND 0.004 ND 0.002 ND 0.002 ND 0.004 ND 0.001 ND 0.001 ND 0.002 ND ND

20.7

Whey powder

8.4

Whey protein concentrate

6.24

Butter

ND 7.6

6 9

Cheese

Adult powder, low fat

2.57 1.82

12 12 12 12 12 11 12 12 11 10 12 12 11 12 11 10 12 8

Adult RTF, high fat

Adult RTF, high fat placebo

Adult RTF, high protein

3.37 6.39

8.7

Adult RTF, high protein placebo

Child milk powder placebo

10.37

27.4

Child milk powder

4.21 7.52 3.84 25.9 1.73 5.82 4.07 2.71 5.16 2.34 32.9 5.25

9.0

Child elemental powder Infant elemental powder

10.1

8.2

Infant elemental powder placebo

Infant PH powder milk Infant PH powder soy Infant powder FOS-GOS

8.1 9.1

9.18 12.3 8.12

Infant powder milk Infant powder soy

Infant RTF milk

9.1

Infant RTF placebo

Toddler powder

11.3

g NS = Not significant. h NA = Not available. i Value in parentheses = Certified or reference values. i Results expressed as mg/100 g for ready-to-feed liquids as-is or reconstituted powders (25 g in 200 g water) or liquid concentrates (1:1 dilution by weight). j ND = Not determined.

Table 2011.14C. Interlaboratory study results for iron (Fe)

Original validation (2011)

Matrix

Mean, mg/kg Rec., % s r a

RSD r

b , % s

R c , mg/kg RSD R

d , % HorRat

No. of labs e Outlier labs f

Chocolate milk powder Dietetic milk powder 2

164.77

6.72 2.51 1.36 0.65 0.93

4.08 3.02 1.81 4.17 1.89

11.25

6.83 5.56 4.42

0.92 0.68 0.53 1.18 0.35

9 9 9 8 8

NS g

83.11 75.18

102

4.62 3.32 1.95 1.54

NS NS 6C 2C

Infant cereal

94 95 91

Peanut butter NIST SRM 2387 Wheat gluten NIST RM 8418

15.65 (16.4) h 49.29 (54.3) h

12.45

3.12

Matrix extension (2017)

Mean, mg/100 g i

SRM 1849a Whole milk

17.4 (17.6)

0.227 0.000

1.3

0.755

4.34

0.59 1.69 2.58

0 2 0 0 2 1 3 1 0 1 0 2 3 1 1 2 2 1 1 0 1 1 0 3 1

0.016 0.033 0.338 0.662

ND j

16 21 37

0.01 0.02

35 49 59

5 7 9 7 4 5

Whole milk powder

ND 0.01 ND 0.13 ND 0.021 ND 0.02 ND 0.015 ND 0.19 ND 0.074 ND 0.126 ND 0.04 ND 0.001 ND 0.048 ND 0.026 ND 0.053 ND 0.001 ND 0.015 ND 0.035 ND 0.014 ND 0.042 ND 0.048 ND 0.027 ND 0.002 ND 0.019 ND ND

Whey powder

0.2

4.4

Whey protein concentrate

3.13

0.11

16.3

1.35

Butter

0.03

Cheese

0.174 0.615

0.030 0.058 0.312 0.141 0.169 0.075 0.006 0.093 0.104 0.066 0.003 0.101 0.081 0.082 0.084 0.081 0.107 0.003 0.096

1.34 0.78

Adult powder, low fat

2.41 7.81

9.44 12.8

11 12 11 12 10 11 11 10 10 11 12 11 11 12 6 6

Adult RTF, high fat

2.43

1.3

Adult RTF, high fat placebo

0.454

2.44 0.81 1.22 0.81 0.72 1.21 0.52

Adult RTF, high protein

2.05

6.14

8.26 15.4

Adult RTF, high protein placebo

0.489 0.044

8.2 3.3

Child milk powder placebo

Child milk powder

1.17

4.11 3.61

Child elemental powder Infant elemental powder

0.724 1.132 0.028 1.008

14.3

4.7

5.8

Infant elemental powder placebo

2.836

11.791

0.608

Infant PH powder milk Infant PH powder soy Infant powder FOS-GOS

1.45 2.88 2.58 3.42 3.81

9.97

0.88

1.23

6.6

0.6

0.543

1.21 0.62 0.59 0.73 0.82 1.02

Infant powder milk Infant powder soy

0.24 1.26 1.35

6.81 6.48

Infant RTF milk

7.9

Infant RTF placebo

0.018 0.808

Toddler powder

2.38

11.9

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