AOAC SPIFAN ERP & Working Group Method Reviews (July 10, 2019)

( t ) Screw-cap test tubes .—25 × 150 mm and 16 × 100 mm, glass with phenolic cap and PTFE liner (Kimble Chase, Vineland, NJ), or equivalent. ( u ) Scintillation vials .—7 mL (Kimble Chase, or equivalent). ( v ) Glass crystallizing dish .—Kimble Chase, or equivalent. C. Chemicals and Reagents ( a ) Magnesium chloride hexahydrate .—Thermo Fisher Scientific, or equivalent. ( b ) Potassium phosphate monobasic, anhydrous .—ACS grade (Sigma-Aldrich, Milwaukee, WI), or equivalent. ( c ) Potassium phosphate dibasic, anhydrous .—ACS grade (Thermo Fisher Scientific, or equivalent). ( d ) Glycine .—Reagent grade (Sigma-Aldrich, or equivalent). ( e ) Glacial acetic acid .—ACS grade (Thermo Fisher Scientific, or equivalent). ( f ) Potassium hydroxide .—50% (w/w; Thermo Fisher Scientific, or equivalent). ( g ) Sodium hydroxide .—50% (w/w; Thermo Fisher Scientific, or equivalent). ( h ) Purified water .—Siemens (Warrendale, PA), or equivalent. ( i ) Reference standard .—Myo-inositol (Sigma-Aldrich, or equivalent). ( j ) Sodium acetate, trihydrate .—Sigma-Aldrich, or equivalent. ( k ) Hydrochloric acid .—ACS grade (Thermo Fisher Scientific, or equivalent). ( l ) Adenosine-5 ′ -triphosphate (ATP) .—R-ATP (Megazyme, Bray, Co. Wicklow, Ireland), or equivalent. ( m ) Glycerol kinase .—KC-GCROL4, 85 U/mL (Megazyme, or equivalent). ( n ) Phytase .—KC-PHYT2, 12000 U/mL (Megazyme, or equivalent). ( o ) Alkaline phosphatase (bovine intestinal mucosa) .— No. P6774, 43000 U/mL (Sigma-Aldrich, or equivalent). D. Preparation of Reagents ( a ) Glycerol kinase (20 U/mL).— Prepared by diluting enough enzyme solution (85 U/mL) with purified water to make a 20 U/mL solution. The solution was prepared fresh daily just before analysis. ( b ) Phytase (1000 U/mL).— Prepared by diluting enough enzyme solution (12 000 U/mL) with 0.1 M acetate buffer pH 5.0 to make a 1000 U/mL solution. The solution was prepared fresh daily just before analysis. ( c ) Alkaline phosphatase (1000 U/mL).— Prepared by diluting enough enzyme solution (43 000 U/mL) with purified water to make a 1000 U/mL solution. The solution was prepared fresh daily just before analysis. ( d ) Acetate buffer (100 mM, pH 5.0).— Prepared by dissolving 0.6 g sodium acetate trihydrate and adding 0.360 mL glacial acetic acid in a 100 mL volumetric flask. The reagents were dissolved and brought to volume with purified water. The pH was then adjusted to 5.0 with sodium hydroxide. The solution was stored refrigerated for up to 1 week. ( e ) Potassium phosphate monobasic, anhydrous (1 M).— Prepared by dissolving 13.6 g potassium phosphate monobasic anhydrous in a 100 mL volumetric flask and bringing to volume with purified water. The solution was stored at ambient temperature for up to 1 week. ( f ) Potassium phosphate dibasic, anhydrous (1 M).— Prepared by dissolving 34.9 g potassium phosphate dibasic anhydrous in a 200 mL volumetric flask and bringing to volume with purified

AOAC Official Method 2012.12 Free and Total Myo-Inositol in Infant Formula and Adult/Pediatric Nutritional Formula High-Performance Anion Exchange Chromatography with Pulsed Amperometric Detection, Including Total Extraction Using Microwave-Assisted Acid Hydrolysis and Enzymatic Treatment First Action 2012 [Applicable to the determination of free myo-inositol in infant formula and adult/pediatric nutritional formula by HPAEC-PAD. Although not meeting the standard method performance requirement (SMPR) guidelines for bound sources, the method gives two options for a total myo-inositol determination from all bound sources using acid hydrolysis by autoclave or microwave- assisted acid hydrolysis and enzymatic treatment.] Caution: Refer to Material Safety Data Sheets prior to working with any chemical. Use proper personal protective equipment when necessary. A. Principle The method involves using HPAEC-PAD to determine free myo- inositol and total myo-inositol by conventional acid hydrolysis with 6 h incubation in an autoclave or by using a microwave-assisted acid hydrolysis with enzymatic treatment. B. Apparatus ( a ) ICS-3000 system .—Dionex (Sunnyvale, CA), or equivalent. ( b ) Autosampler .—AS (Dionex, or equivalent). ( c ) Electrochemical detector .—ICS-3000 ED (Dionex, or equivalent). ( d ) Working electrode .—ICS-3000 ED Au (Dionex, or equivalent). ( e ) Reference electrode .—ICS-3000 ED Ag/AgCl (Dionex, or equivalent). ( f ) Analytical column .—CarboPac MA1, 250 × 4 mm id (Dionex, or equivalent). ( g ) Guard column .—CarboPac MA1, 50×4 mm id (Dionex, or equivalent). ( h ) Chromatography software .—Empower (Waters, Milford, MA), or equivalent. ( i ) Analytical balance .—Sartorius Model CP225D (Goettingen, Germany), or equivalent. ( j ) Magnetic stir/hot plate .—Corning Model PC-420 (Corning, NY), or equivalent. ( k ) Microwave system .—MARSXpress/Mars5 (CEM, Mathews, NC), or equivalent. ( l ) Microwave liner and cap .—QXP (CEM, or equivalent). ( m ) Microwave vessel .—Omni/XP1500 (CEM, or equivalent). ( n ) Autoclave .—Barnstead/Thermolyne (Thermo Fisher Scientific, Waltham, MA), or equivalent. ( o ) Filters .—Grade A glass fiber (GFA; Whatman/GE, Maidstone, Kent, UK), or equivalent. ( p ) Disposable syringes .—3 mL (BD, Franklin Lakes, NJ), or equivalent. ( q ) Syringe tip filters .—PTFE 0.45 μm (Whatman/GE, or equivalent). ( r ) Magnetic stir bars .—15×1.5 mm (VWR, West Chester, PA), or equivalent. ( s ) pH meter .—Denver Instrument (Bohemia, NY) UB-10, or equivalent.

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