© 2012 AOAC INTERNATIONAL
AOAC Official Method 2011.18
Myo-Inositol (Free and Bound as
Phosphatidylinositol) in Infant Formula
and Adult Nutritionals
Liquid Chromatography/Pulsed Amperometry
with Column Switching
First Action 2011
The liquid chromatography method with electrochemical (pulsed
amperometry) detection (PAD) allows for the quantitation of myo-
inositol in infant, pediatric, and adult nutritional formulas. The
concentration of myo-inositol is calculated by comparison with
standards of known concentration.
Total mM
yo-inositol, as
defined by AOAC SMPR 2011.007, can be calculated by adding
the free myo-inositol and myo-inositol bound as
phosphatidylinositol data. The method was validated for the
quantitation of free myo- inositol
and myo-inositol from
phosphatidylinositol only
. Repeatability was determined from
duplicate
triplicate
analyses performed on the same day.
Intermediate precision was determined from replicate
determinations performed over multiple days. Accuracy was
determined by
spiked recovery experiments (free myo-inositol
and myo-inositol from phosphatidylinositol) and by
comparison
to a reference microbiological assay
wi t h and expressed as the
percentage of
HPLC results relative to microbiological results
(free myo-inositol)
. Instrument limits of detection and
quantitation were determined statistically from
injections of low-level standards
.and by spiking samples with low
levels of free myo-inositol.
Caution:
Refer to Material Safety Data Sheets (MSDS) of chemicals
prior to use and follow safe handling procedures and the
suggested personal protective equipment.
A. Apparatus
(
a
)
Analytical balance
.—Minimum weighing capacity of at
least 0.0001 g.
(
b
)
Centrifuge
.
(
c
)
Desiccator
.
(
d
)
N-evap.—
With water bath or equivalent
.
(
e
)
Oven
.—Capable of maintaining 1
21
0°C.
(
f
)
pH meter
.—With pH 4 and 7 buffers.
(
g
)
Stir plate
.—Multiposition with stir bars.
(
h
)
Tubing
.—PEEK or Teflon 0.007–.01 in. id.
(
i
)
Vacuum manifold.
(
j
)
Vortex mixer.
(
k
)
System HPLC
.—Corrosion-resistant autosampler, isocratic
pump (2), 6-port switching valve, and pulsed amperometry detector
with a gold electrode. Autosampler capable of injecting 20
L.
(
l
)
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 equivale
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 (
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); assorted sizes.
(
i
)
Solid-phase extraction (SPE) cartridges.—
Silica, 1 g (J.T.
Baker P/N 7086-07,
or equivalent.
(
j
)
Syringes
.—1 mL disposable and 25 mL gas-tight glass with
4 in. stainless steel needles.
