333 / 435
1066
C
ampos
-G
iménez
et al
.
:
J
ournal of
AOAC I
nternational
V
ol
. 96, N
o
. 5, 2013
(
e
)
Purified water.—
Millipore (Le Mont-sur-Lausanne,
Switzerland) or equivalent.
(
f
)
Sodium acetate trihydrate.—
Merck or equivalent.
(
g
)
TCA.—
Merck or equivalent.
(
h
)
TCEP.
—Fluka or equivalent.
D. Preparation of Solutions
(
a
)
Sodium acetate solution (pH 5.4).
—Into a 500 mL
volumetric flask, weigh 34.0 g sodium acetate trihydrate, add
about 400 mL water, and dissolve completely. Adjust pH to 5.4
with phosphoric acid 85%, and make up to volume with water.
(
b
)
TCA (15%).—
Into a 500 mL volumetric flask, weigh
75.0 g TCA, dissolve, and make up to volume with water.
(
c
)
TCEP (250 µg/mL).—
Into a 500 mL volumetric flask,
weigh 125 mg TCEP-HCl, dissolve, and make up to volume
with water.
(
d
)
Mobile phase for UPLC
®
.—
Into a 250 mL flask, mix
0.4 g decylamine, 2.5 mL acetonitrile, 25 mL sodium acetate
solution (pH 5.4), and 205 mL water. Adjust pH to 5.4 with
phosphoric acid 85%. Add 10 mg TCEP.
E. Preparation of Standards
Note
: Vitamin C is sensitive to light and oxygen; conduct
operations under subdued light or use amber glassware. Keep
all solutions away from direct light.
(
a
)
Ascorbic acid stock solution (500 µg/mL).
—Into a
25 mL amber glass volumetric flask, weigh 12.5 mg ascorbic
acid. Dissolve and make up to volume with TCEP solution. This
solution can be kept for 3 months if stored at 4°C away from
light.
(
b
)
Ascorbic acid intermediate standard solution
(50 µg/mL).
—Into a 10 mL amber glass volumetric flask, pipet
1 mL stock solution. Make up to volume with TCEP solution.
This solution can be used for 1 month if stored at 4°C away
from light.
(
c
)
Ascorbic acid calibration standard solutions (0.5, 1.0,
2.0, 3.0, 5.0, 7.5, and 10.0 µg/mL).
—Into 10 mL amber glass
volumetric flasks, pipet 0.1, 0.2, 0.4, 0.6, 1.0, 1.5, and 2.0 mL of
intermediate standard solution. Make up to volume with mobile
phase to prepare the respective concentrations given above.
F. Preparation of Samples
(
a
)
Reconstitution of powder samples.
—
(
1
) Weigh 25 g powder into a brown glass 250 mL beaker.
Add 10 mg TCEP.
(
2
) Add 200 g warm water (40°C) and mix well until
complete dissolution. Make sure there are no lumps.
(
b
)
Extraction.—
(
1
) Weigh 2 g liquid or reconstituted sample into a 10 mL
amber glass volumetric flask.
(
2
) Add 4 mL TCEP solution and 2 mL TCA 15%.
(
3
) Bring up to volume with water.
(
4
) Filter solution through folded grade 597½ or greater filter
paper (Schleicher & Schuell, Dassel, Germany).
(
5
) Transfer 1 mLfiltrate into a 10 mLamber glass volumetric
flask containing 1 mL acetate solution (pH 5.4). Bring up to
volume with mobile phase.
(
6
) Filter about 2 mL through a 0.22 µm membrane into an
HPLC vial.
G. Analysis
(
a
)
Chromatographic conditions.
—
(
1
)
Injection volume.
—5 µL.
(
2
)
Autosampler temperature.
—10°C.
(
3
)
Column temperature.
—25°C.
(
4
)
Flow rate.
—0.35 mL/min.
(
5
)
Run time
.—4.0 min.
(
6
)
Mobile phase.
—Sodium acetate (25 mmol/L, pH 5.4),
decylamine (1.6 g), acetonitrile (1%), and TCEP (50 mg/L).
Note
: At the end of each analytical series, rinse the column
with acetonitrile
–
water (1 + 1, v/v) for 10 min at 0.4 mL/min.
(
b
)
System suitability test.
—Equilibrate the chromatographic
system for ≥0.5 h. Inject a working standard solution at least
six times and check peak retention times and responses (peak
height or area). Inject working standard solutions on a regular
basis within a series of analyses.
(
c
)
Calibration.
—Make single injections of working
standard solutions at least at the beginning and the end of each
analytical series. Establish the calibration curve (seven points)
by plotting peak response (height or area) versus ascorbic acid
concentration. Perform linear regression. Calculate the slope
(S) and intercept (I) of the calibration curve.
(
d
)
Analysis.
—Make single injections of sample solutions.
(
e
)
Identification.
—Identify the ascorbic acid peak in the
chromatograms of the sample solutions by comparison with the
retention time and UV spectrum of the corresponding peak in
the standard solution.
H. Calculations
Calculate the concentration of vitamin C in mg ascorbic
acid/100 g expressed in product “as is” for RTF (ready-to-feed)
Table 1. Precision results
Level
RSD
r
, % RSR
ir
, %
SRM 1849a infant/adult nutritional formula 8.4
1.7
7.5
Infant elemental powder, mg/100 g
36.8 1.7
1.3
Infant formula powder, milk-based, mg/100 g 11.8 1.4
1.4
Infant formula powder, soy-based, mg/100 g 10.6 1.4
2.0
Adult nutritional RTF, high-fat, mg/100 g
39.3 2.5
5.3
Table 2. Spike recovery results
+50%
+100%
Native level,
mg/100 g
Average,
%
RSD,
%
Average,
%
RSD,
%
Infant elemental
powder
36.8
101 7.2
95 5.8
Infant formula
powder, milk-based
11.8
94
4.7
91 5.0
Infant formula
powder, soy-based
10.6
96 11.6
91 5.6
Adult nutritional
RTF, high-fat
39.3
95
14.5
100
3.9
AOAC OMB Meeting Book
308