408 / 471
© 2015 AOAC INTERNATIONAL
(
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.
(
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.
C. Chemicals and Solvents
(
a
)
Acetic acid
.—Glacial, ACS.
(
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
to 200 mL with laboratory water (0.5 mg/L). Dilute 4 and 1 mL
of the 0.5 mg/L myo-inositol working standard to 10 mL with
laboratory water (0.2 and 0.05 mg/L). Expiration: 2 weeks.
Table 2011.18B. Single-laboratory validation: Accuracy (spike recovery) data for myo-inositol
Sample type
No. of replicates
(duplicates on multiple days)
Native level
(mg/100 g RTF)
Spike level
(mg/100 g RTF) Recovery, % RSD
Unbound (free) myo-inositol
Child formula powder, unfortified
12
0.0426
1.89
101
2.78
Infant formula powder soy-based
12
4.18
14.9
98.2
2.47
Infant formula powder partial hydrolyzed milk-based
12
3.35
15.2
102
3.00
Infant elemental powder, unfortified
12
0.00
1.74
93.9
3.00
Infant elemental powder, unfortified
6
0.00
1.09
93.2
1.54
Infant elemental powder, unfortified
6
0.00
0.390
90.2
3.14
Adult nutritional powder milk-based
12
0.409
65.0
101
2.64
Adult nutritional RTF high protein
12
0.042
61.4
96.3
3.27
Myo-inositol bound as phosphatidylinositol
Infant formula (NIST SRM 1849a)
6
11.1
a
7.3
77.1
13.2
Infant elemental powder, unfortified
6
0.00
0.294
87.3
6.88
Child formula powder
6
0.443
0.340
90.0
15.7
Infant formula powder soy-based
6
2.48
3.22
81.9
0.21
Infant formula powder partial hydrolyzed milk-based
6
0.247
0.152
79.9
7.51
Infant formula powder partial hydrolyzed soy-based
6
1.94
2.47
72.8
5.18
Adult nutritional powder milk-based
6
1.43
1.54
75.7
3.23
a
Results in mg/kg powder.
Candidates for 2016 Method of the Year
346