AOAC OMA# 2011.06 Final Action Review-OMB

1548  S zpylka et al . : J ournal of AOAC I nternational V ol . 95, N o . 6, 2012

Table 2011.06A. Sample chart

Sample weight to use, g (±10%)

Amount IS to add, μg

Amount buffer to add, mL

Amount to be loaded on SPE plate, mL

Est. total folates, μg/100 g

≥5 to <10

2.0 1.0 0.1 0.1 0.1

5 5 5

10 10 10 10 10

10

10 to about 20000 >20000 to <200000 >200000 to <2000000

5–10

5

10

1–2

Matrix that suppresses internal standard (grains, flours, brans, meals)

5

5–10

( o )  Acetic acid, glacial.— Cat. No. MK314146 (VWR Scientific Products). ( p )  Sodium hydroxide.— Cat. No. MK770810 (VWR Scientific Products). ( q )  Ammonium formate.— Cat. No. 17843-250G (Sigma- Aldrich). ( r )  Formic acid, 98%.— Cat. No. EM-FX0440-7 (VWR Scientific Products). ( s )  Ammonium acetate.— Cat. No. JT0599-8 (VWR Scientific Products). ( t )  2-Mercaptoethanol.— Cat. No. EM-6010 (VWR Scientific Products). ( u )  Ascorbic acid.— Cat. No. MK185210 (VWR Scientific Products). ( v )  Phosphoric acid.— Cat. No. EM-PX1000-6 (VWR Scientific Products). ( w )  Water .—Optima (Cat. No. W7-4; Fisher Scientific). D. Reagent Preparation ( a )  Folates stock solution solvent .—100 mM ammonium acetate buffer with 25% ascorbic acid and 1% mercaptoethanol, pH 5.5. ( 1 ) Weigh approximately 1.2 g ammonium acetate and transfer into a 1000 mL beaker [use deionized (DI) water to completely transfer if necessary]. ( 2 ) In a fume hood, slowly add approximately 700 mL DI water and approximately 4.8 mL glacial acetic acid. ( 3 ) Add 250 g ascorbic acid and 10 mL 2-mercaptoethanol. Stir to completely dissolve using a stir bar. Add concentrated ammonium hydroxide dropwise as necessary to dissolve completely. ( 4 ) Check the pH of the solution and adjust to pH 5.5, if necessary, using concentrated ammonium hydroxide or glacial acetic acid. (The ratio of ammonium acetate:glacial acetic acid will give a pH close to 5.5 but the ascorbic acid addition will lower the pH.) ( 5 ) Transfer solution into a 1 L volumetric flask and bring to volume with DI water. ( b )  Folate intermediate standard solution solvent. —10 mM ammonium acetate buffer with 1% ascorbic acid, pH 5.5. ( 1 ) Weigh approximately 1.2 g ammonium acetate and transfer into a 1000 mL beaker (use DI water to completely transfer if necessary). ( 2 ) In a fume hood, add approximately 700 mL DI water and approximately 4.8 mL glacial acetic acid. Add 10 g ascorbic acid and stir until completely dissolved. ( 3 ) Adjust the pH to 5.5 if necessary with concentrated ammonium hydroxide or glacial acetic acid.

X-AW, 33 µm polymeric WAX (Part No. 8E-S038-UGB; Phenomenex Corp.; www.phenomenex.com). ( g ) 96-Well SPE collection vessel .—Part No. AHO-7194-S (Phenomenex Corp.). ( h ) Scintillation vials with perforated lids .—Part No. 600000671CV (Waters Corp.). ( i ) Reservoir .—12-Well (Cat. No. RRI-3019; Phenix Research Products; www.phenix1.com). ( j ) Refrigerator .—Capable of cooling to 4°C, explosion- proof (RCA, or equivalent). ( k ) Freezer .—Capable of freezing to –70°C, explosion- proof (RCA, or equivalent). ( l ) Water bath with shaker .—Model MW1130 A (Blue M Electric Co.; www.Blue-M.com). ( m ) Centrifuge .—Clinical, capable of up to 2000 rpm (International Equipment Co., Needham Heights, MA, or equivalent). Equivalent reagents and vendors can be used. ( a ) Folic acid (standard) .—Cat. No. 16.203 (Schircks Laboratories; www.Schircks.com). ( b ) 13 C-folic acid (IS) .—Cat. No. CLM-7861-0 (Cambridge Isotope Laboratories; www.isotope.com/cil) . ( c ) (6R,S)-5-methyl-5,6,7,8-tetrahydrofolic acid, calcium salt.— Cat. No. 16.235 (Schircks Laboratories). ( d ) (6S)-5,6,7,8-tetrahydrofolic acid.— Cat. No. 16.207 (Schircks Laboratories). ( e ) (6S)-5-formyl-5,6,7,8-tetrahydrofolic acid, calcium salt.— Cat. No. 16.221 (Schircks Laboratories). ( f ) 10-Methylfolic acid.— Cat. No. 16.211 (Schircks Laboratories). ( g ) 10-Formylfolic acid.— Cat. No. 16.215 (Schircks Laboratories). ( h ) Pteroyltri-γ-L-glutamic acid (folic acid triglutamate).— Cat. No. 16.253 (Schircks Laboratories). ( i ) α-Amylase.— 5 000000 Units (Cat. No: A6211; Sigma- Aldrich; www.sigma-aldrich.com/order). ( j )  Protease from Bacillus sp. (or Pronase E).— Cat. No. P3910 (Sigma-Aldrich). ( k )  Conjugase (male rat plasma with lithium heparin).— Lampire, or equivalent. ( l )  Ammonium hydroxide.— Cat. No. MK325614 (VWR Scientific Products). ( m )  Sodium phosphate, dibasic salt.— Cat. No. JT3827-01 9 (VWR Scientific Products). ( n )  Methanol.— Optima LC/MS, 99.9% (Cat. No. A456-4; Fisher Scientific; www.fishersci.com). C. Reagents