AOAC OMA# 2011.06 Final Action Review-OMB

S zpylka et al .: J ournal of AOAC I nternational V ol . 95, N o . 6, 2012  1547

INFANT FORMULA AND ADULT NUTRITIONALS

Determination of Total Folates in Infant Formula and Adult Nutritionals by Trienzyme Extraction and UPLC-MS/MS

Quantitation: First Action 2011.06 J ohn S zpylka 1 , J on D e V ries , A ndrea C heney , and S teve H ouse General Mills/Medallion Laboratories, 9000 Plymouth Ave North, Minneapolis, MN 55427

method. The method was adopted under the AOAC Board of Directors-approved alternative pathway. This process allows the method to be used by laboratories for approximately 2 years to gather additional information. The information will be reviewed along with any feedback provided on the method in order for the ERP to decide if the method can be recommended to the AOAC Official Methods Board for Final Action status (1). AOAC Official Method 2011.06 Total Folates in Infant Formula and Adult Nutritionals Trienzyme Extraction and UPLC-MS/MS Quantitation First Action 2011 (Applicable to the determination of folate content of infant formula and adult nutritionals.) A. Principle A homogenized sample is accurately weighed into a 50 mL centrifuge tube. An appropriate amount of internal standard (IS) and phosphate buffer, pH 6, are added. The sample is placed in a shaker bath set at 50°C to disperse the matrix. α-Amylase and protease enzymes are added to further break down the matrix for extraction of the folates. Rat plasma conjugase is used to deconjugate the polyglutamate forms of folates to monoglutamates. The sample is centrifuged and the supernatant is filtered through a 0.45 µm filter. The filtrate is cleaned using weak anion exchange (WAX) SPE. The folates are eluted into methanol containing 10% formic acid and 1% ascorbic acid and analyzed by UPLC-MS/MS. Six folate vitamers are quantified and the total folates determined and expressed as folic acid. Isotopically labeled folic acid ( 13 C-folic acid) is used as the IS. Equivalent equipment and vendors can be used. ( a ) pH meter.— Mettler Toledo (Cat. No. E-33253; VWR Scientific Products; www.vwrsp.com). ( b ) UPLC system .—Acquity (Waters Corp.; www.waters. com). ( c ) Mass spectrometer .—Waters Quattro Premier (Waters Corp.). ( d ) UPLC column .—HSS T3, 2.1 × 150 mm, 1.8 µ m particle size (Part. No. 186003540; Waters Corp.). ( e ) 96-Well SPE vacuum manifold .—Part No. 228008 (Porvair Sciences; www.porvair-sciences.com). ( f ) 96-Well SPE cartridges .—60 mg 96-well plate, Strata B. Apparatus and Materials

Submitted for publication June 7, 2012. The method was approved by the Expert Review Panel on Infant Formula and Adult Nutritionals as First Action. See “Standards News,” (2011) Inside Laboratory Management, July/August issue. The AOAC Stakeholder Panel on Infant Formula and Adult Nutritionals (SPIFAN) invites method users to provide feedback on the First Action methods. Feedback from method users will help verify that the methods are fit for purpose and are critical to gaining global recognition and acceptance of the methods. Comments can be sent directly to the corresponding author. Corresponding author’s e-mail: John.Szpylka@silliker.com 1  Current address: Silliker Laboratories, 111 E. Wacker Dr, Suite 2300, Chicago, IL 60601 DOI: 10.5740/jaoacint.CS2011_06 After supporting data on this method were collected, the information was reviewed by an Expert Review Panel (ERP). After the review was completed, the ERP, based upon the currently available validation data, decided that this method should be adopted as an AOAC Official First Action The method for “Determination of Total Folates in Infant Formula and Adult Nutritionals by Trienzyme Extraction and UPLC-MS/MS Quantitation” was submitted to the Folate Working Group for consideration for adoption as Official First Action by AOAC INTERNATIONAL. This method uses ultra performance liquid chromatography/tandem mass spectrometry (UPLC-MS/MS) to determine the total folates in infant formulas and adult nutritionals after trienzyme digestion. Deconjugation of the various folate polyglutamates to folate monoglutamates is achieved by using rat plasma conjugase after the sample digestion with protease and α-amylase during the trienzyme digestion process. This method shows linearity of folate concentrations in the range of 10–19 100 μg/100 g. Extension of the range to cover folate concentrations of 5–2 000 000 μg/100 g can be achieved with appropriate adjustment of the sample weight and SPE cleanup loading volume. The recoveries ranged from 94.10 to 101.34%. F olates play an important role in good health. Because of this, it has become increasingly necessary to have reliable and accurate methods for determining the levels of total folates in infant formula and adult nutritionals. The method for the “Determination of Total Folates in Infant Formula and Adult Nutritionals by Trienzyme Extraction and UPLC-MS/MS Quantitation” has been submitted as a suitable alternative to microbiological assay methods, which are time-consuming and relatively complex.