AOAC SPIFAN Nutrients ERP-Final Review (Oct. 2018)

2  B handari et al . : J ournal of AOAC I nternational V ol . 101, N o . x , 2018

folic acid and 1 μg natural food folate (5). DFE is a unit of measurement of folates that reflects the greater bioavailability of folic acid compared with natural folate (5). The U.S. Food and Drug Administration’s new nutrition labeling regulation of May 2016 requires the use of microgram DFEs to declare the amount of total folate (natural folate + synthetic folic acid) in food Nutrition Facts labels (16). Therefore, methods capable of estimating contents of folic acid and natural food folate separately are helpful in correctly labeling of folate on Nutrition Facts labels. The need for an updated reference method for folate was identified by AOAC’s Stakeholder Panel on Infant Formula and Adult Nutritionals (SPIFAN) as a priority in 2011. An LC‒MS/MS method for total folate analysis that incorporates the trienzyme extraction and extract purification by a weak anion exchange (WAX) solid-phase extraction (SPE) was found suitable for the purpose and was approved by SPIFAN as a First Action Official Method 2011.06 (17) in 2011. The method incorporated isotopically labeled folic acid ( 13 C-folic acid) as an internal standard. The method was subsequently updated in selected aspects, including the use of two additional internal standards, labeled 5-methyl-tetrahydrofolic acid ( 13 C-methyl- THF) and 5-formyl-tetrahydrofolic acid ( 13 C-formyl-THF). The method underwent comprehensive single-laboratory validation (SLV) studies twice using the SPIFAN kits, containing a set of infant formulas and adult nutritional products selected as representative subsamples of a wide range of commercially available products. The SLV results were reviewed by SPIFAN in March 2016. The method was recommended to advance to a multilaboratory testing (MLT) study for further evaluation of the method’s precision performance, particularly repeatability and reproducibility of the analytical results. A collaborative study of the method was organized; this paper presents the results of the study and compares the performance to the SMPR 2011.006 (18). MLT Study An MLT study of the updated Official Method 2011.06 for analysis of total folate in infant formula and adult nutritionals using the trienzyme extraction and LC-MS/MS analysis was planned. An extensive campaign was carried out in a variety of ways, personal as well as through AOAC, International Organization for Standardization, International Dairy Fede­ ration, and American Association of Cereal Chemists contacts and meetings, to recruit potential laboratories for the MLT. Formal invitation was sent to more than 40 laboratories around the world by the study director. Initially, 18 laboratories indicated their interest to participate in the study. A detailed protocol of the study including the updated analytical method was sent to potential participants to find out whether they met the requirements of the study. Attempts were made to have the potential participants ask any questions related to the study and the method in the follow-up communications. This effort helped to make sure that the participants had a full understanding of the method and its requirements. METHOD There were some updates to the method as described in First Action Official Method 2011.06 . The updated method used for the MLT study is described in the following section.

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

First Action 2011 Final Action 2017

[Applicable for determination of total folate contents in infant formula and adult nutritionals.] Caution: The 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 treatments with protease, amylase, and rat plasma conjugase (the trienzyme extraction). The extract is purified and concentrated using aWAX SPE. Polyglutamate forms of folates in the sample are deconjugated to monoglutamates during the extraction and are analyzed by LC-MS/MS. Folic acid, 5-CH3-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-CH3-THF ( 13 C-5-CH3-THF) and 5-CHO- THF ( 13 C-5-CHO-THF)] are used as the internal standards. 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 pipettes .—Adjustable (100–1000 μL). ( l )  Vortex mixer. ( m )  24-well SPE vacuum manifold. ( n )  SPE columns. —MixedmodeWAXSPE, 33 μmpolymeric, 60 mg/3 mL; Phenomenex, 8B-S038-UBJ. ( o )  LC column .—Waters ACQUITYUPLC HSS T3 Column (100Å, 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 )  Micro centrifuge tubes .—Polypropylene, 1.5–2.0 mL. ( t )  Low-actinic volumetric flasks. —Assorted sizes. ( u )  Glass pasteur pipettes. ( v )  Volumetric pipettes. —Class A, assorted sizes. ( w )  Repeating pipettes .—5 and 25 mL. ( x )  Glass beakers .—Assorted sizes. ( y )  Amber glass HPLC vials .—With septa and caps. ( z )  Low-actinic glass bottles .—250–500 mL. B. Apparatus and Materials