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6, 2015
Method
A few minor modifications were made to AOAC Official
First Action
2011.18
before it was sent to the multilaboratory
collaborative study participants. These changes included
updating the pulsed amperometric detector (PAD) program
and increasing the sodium hydroxide concentration from
750 mM to 1 M. The PAD program listed in AOAC
2011.18
,
First Action, was the waveform that was used when the method
was originally developed over 15 years ago using equipment
that is now obsolete. After First Action status was granted to
AOAC
2011.18
and before completion of the SPIFAN SLV
and multilaboratory collaborative study, the obsolete PAD
was replaced with a newer model, and the waveform listed in
AOAC
2011.18
was updated based on the recommendations
of the instrument manufacturer. The updated waveform was
added to the multilaboratory collaborative study protocol and to
AOAC
2011.18
, Final Action, and the previous waveform was
removed. It should be noted that whenever changes are made
to the PAD, the accuracy of the detector waveform should be
verified by analyzing an SRM and performing free myo-inositol
spike recovery experiments with an infant or adult formula
containing hydrolyzed protein.
It should also be noted that although the multilaboratory study
protocol specified using a standard (not disposable) gold electrode,
some participating laboratories used disposable gold working
electrodes. The instruction to not use disposable electrodes was
removed from the AOAC
2011.18
Final Action method.
After completion of the multilaboratory collaborative study,
the modifications noted above were incorporated in the Final
Action method along with a few additional modifications based
on study results and feedback from study participants and the
ERP.
For this reason, the method described below is slightly
different than the Official Final Action method currently posted
on the AOAC website.
AOAC Official Method 2011.18
Myo-Inositol (Free and Bound as
Phosphatidylinositol)
in Infant and Pediatric Formula
and Adult Nutritionals
Liquid Chromatography/Pulsed Amperometry
with Column Switching
First Action 2011
Final Action 2014
ISO–AOAC METHOD
The LC 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. Myo-inositol, as
defined by AOAC Standard Method Performance Requirement
(SMPR
®
) 2011.007 (free and bound as phosphatidylinositol), 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 in infant, pediatric, and adult nutritionals.
Repeatability was determined from duplicate analyses
performed on multiple days. Accuracy was determined from
spike recovery experiments (free myo-inositol and myo-inositol
from phosphatidylinositol). Instrument LODs and LOQs were
determined statistically from injections of low-level standards
and by spiking samples with low levels of free myo-inositol.
See
Tables
2011.18 A
–
C
for results of single- and multilaboratory
studies supporting acceptance of the method.
Caution
: Refer to Material Safety Data Sheets (MSDS) of
chemicals prior to use and follow safe handling
procedures and the suggested personal protective
equipment. Chloroform is a hazardous chemical
and should be handled in a fume hood. Perform the
phosphatidylinositol bound myo-inositol extraction
and SPE sample cleanup procedure in a fume hood.
A. Apparatus
(a)
Analytical balance.
—Minimum weighing capacity of at
least 0.0001 g.
(b)
Centrifuge.
(c)
Desiccator.
(d)
N-evap.
—With water bath (Organomation Associates,
Inc., Berlin, MA) 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)
HPLC system.
—Corrosion-resistant components, including
an autosampler, two isocratic pumps, 6-port switching valve,
pulsed amperometry detector with a gold electrode and polyether
ether ketone or Teflon 0.007–.01 in. id tubing.Autosampler capable
of injecting 20 µL
.
(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 equivalen
t (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 2 V 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); assorted sizes.
(i)
SPE cartridges.
—Silica, 1 g (J.T. Baker Inc., Phillipsburg,
NJ; 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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