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

Table 2011.18B. Single-laboratory validation: Accuracy (spike recovery) data for myo-inositol

No. of replicates (duplicates on multiple days)

Native level (mg/100 g RTF)

Spike level (mg/100 g RTF) Recovery, % RSD

Sample type

Unbound (free) myo-inositol

Child formula powder, unfortified Infant formula powder soy-based

12 12 12 12

0.0426

1.89 14.9 15.2 1.74 1.09

101 98.2 102 93.9 93.2 90.2 101 96.3 77.1 87.3 90.0 81.9 79.9 72.8 75.7

2.78 2.47 3.00 3.00 1.54 3.14 2.64 3.27 13.2 6.88 15.7 0.21 7.51 5.18 3.23

4.18 3.35 0.00 0.00 0.00

Infant formula powder partial hydrolyzed milk-based

Infant elemental powder, unfortified Infant elemental powder, unfortified Infant elemental powder, unfortified Adult nutritional powder milk-based Adult nutritional RTF high protein

6 6

0.390

12 12

0.409 0.042

65.0 61.4

Myo-inositol bound as phosphatidylinositol

Infant formula (NIST SRM 1849a) Infant elemental powder, unfortified

6 6 6 6 6 6 6

11.1 a 0.00 0.443

7.3

0.294 0.340

Child formula powder

Infant formula powder soy-based

2.48

3.22

Infant formula powder partial hydrolyzed milk-based Infant formula powder partial hydrolyzed soy-based

0.247

0.152

1.94 1.43

2.47 1.54

Adult nutritional powder milk-based

a  Results in mg/kg powder.

( b ) Centrifuge . ( c ) Desiccator .

C. Chemicals and Solvents ( a ) Acetic acid .—Glacial, ACS.

( d ) N-evap.— With water bath or equivalent . ( e ) Oven .—Capable of maintaining 120°C. ( f ) pH meter .—With pH 4 and 7 buffers. ( g ) Stir plate .—Multiposition with stir bars. ( h ) Vacuum manifold. ( i ) Vortex mixer.

( b ) Chloroform .—High-purity, HPLC grade. ( c ) Diethyl ether .—Anhydrous, HPLC grade. ( d ) Drierite (desiccant) .—Anhydrous calcium sulfate, 8 mesh. ( e ) Helium .—Zero grade or equivalent. ( f ) Hexane .—HPLC grade. ( g ) Hydrochloric acid .—Concentrated (36–38%), ACS. ( h ) Laboratory water.— ≥15 MΩ∙cm. ( i )  Metaphosphoric acid .—ACS. ( j ) Methanol .—HPLC grade. ( k ) Myo-inositol .—USP reference standard, official lot; store desiccated. See standard label for purity. ( l ) Sodium chloride.— ACS. ( m ) Sodium hydroxide .—50% (w/w), low carbonate form. D. Preparation of Reagents and Standard Solutions All solutions can be scaled up or down for convenience provided good laboratory practices are observed. Solutions can be stored at 2–30°C in tight, inert containers unless otherwise noted. ( a ) Myo-inositol stock standard solution (approximately 2000 mg/L).— Accurately weigh approximately 0.100 g myo- inositol and quantitatively transfer to a 50 mL volumetric flask. Dilute to volume with water. Mix well. Store refrigerated. Expiration: 3 months. ( b ) Myo-inositol intermediate standard solution (approximately 200 mg/L).— Dilute 10.0 mL stock standard to 100 mL with laboratory water and mix well. Discard after use. ( c ) Myo-inositol working standard solutions (approximately 4, 2, 1, 0.5, 0.2, and 0.05 mg/L).— Dilute 2.0, 1.0, and 0.5 mL myo- inositol intermediate standard to 100 mL with laboratory water (4, 2, and 1 mg/L). Dilute 0.5 mL myo-inositol intermediate standard

( j ) System HPLC .—Corrosion-resistant components including an autosampler, isocratic pumps (2), 6-port switching valve, pulsed amperometry detector with a gold electrode and PEEK or Teflon 0.007–0.01 in. id tubing. Autosampler capable of injecting 20 µ L. ( k ) Columns .—Dionex CarboPac MA1 (4 × 250 mm) P/N 44066, MA1 (4 × 50 mm) P/N 44067, and PA1 (4 × 50 mm) P/N 43096, or equivalent (www.thermofisher.com/dionex/). B. Materials ( a ) Beakers .—Assorted sizes. ( b ) Centrifuge tubes .—50 mL with Teflon-coated caps. ( c ) Syringe filters .—Nylon, 0.45 and 0.2 µm. ( d ) Filter paper .—Whatman 2V or equivalent (www.whatman. com). ( e ) Erlenmeyer flasks .—50 or 125 mL or equivalent. ( f ) Volumetric flasks .—Assorted sizes. ( g ) Funnels .—Suitable for use with filter paper. ( h ) Pipets .—Volumetric (Class A) and mechanical; assorted sizes. ( i ) Solid-phase extraction (SPE) cartridges.— Silica, 1 g (J.T. Baker P/N 7086-07; www.avantormaterials.com) or equivalent. ( j ) Syringes .—1 mL disposable and 25 mL gas-tight glass with 4 in. stainless steel needles.

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