AOAC Final Action Methods in 2018

( 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.

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