223 / 258
140
C
ampos
G
iménez
&
M
artin
:
J
ournal of
AOAC I
nternational
V
ol
.
100, N
o
.
1, 2017
using decylamine as an ion-pairing agent in a sodium acetate
buffer solution (pH 5.4) containing TCEP.
B. Apparatus
(a)
Balances.
—With readability of 0.1 mg and 0.01 g.
(b)
pH meter.
—Metrohm 691 (Herisau, Switzerland), or
equivalent.
(c)
Folded paper filters
.—Grade 597
½
.
(d)
Syringe filters
.—0.22 or 0.45 μm pore size.
(e)
Chromatographic system.
—HPLC or UHPLC system
equipped with a quaternary or binary pump, a sample injector, a
UV-Vis detector (or optionally, a photodiode array detector), a
degassing system, and data software.
(f)
UHPLC column.
—Waters Acquity UPLC ethylene
bridged hybrid C
18
column (1.75 μm, 2.1×100 mm; or
equivalent).
(g)
HPLC column.
—LiChrospher RP-18 column (5 μm,
250×4.6 mm; or equivalent).
C. Reagents and Standards
(a)
Acetonitrile.
—HPLC
grade,
Merck
(Geneva,
Switzerland); or equivalent.
(b)
Ascorbic acid.
—>99%, Fluka (Buchs, Switzerland), or
equivalent.
(c)
Decylamine.
—Fluka, or equivalent.
(d)
Phosphoric acid.
—85%, Merck; or equivalent.
(e)
Ultrapure water.
—Resistivity >18 MΩ/cm.
(f)
Sodium acetate trihydrate
.— Merck, or equivalent.
(g)
TCA.
—Merck, or equivalent.
(h)
TCEP.
—Fluka, or equivalent.
(i)
Isoascorbic acid.
—Fluka, or equivalent.
(j)
Orotic acid.
—Sigma, or equivalent.
D. Preparation of Solutions
(a)
Sodium acetate solution (500 mmol/L, pH 5.4).
—In a
500 mL volumetric flask, weigh 34.0 g sodium acetate trihydrate
and add ~400 mL water for dissolution. Adjust the pH to 5.4
with phosphoric acid (85%) and dilute to volume with water.
(b)
TCA (15%).
—In a 500 mL volumetric flask, weigh 75.0 g
TCA, dissolve the compound, and dilute to volume with water.
(c)
TCEP (250 μg/mL).
—In a 500 mL volumetric flask,
weigh 125 mg TCEP, dissolve the compound, and dilute to
volume with water.
(d)
Mobile phase for UHPLC.
—In a 250 mL flask, mix
0.4 g decylamine, 2.5 mL acetonitrile, 25 mL sodium acetate
solution 500 mmol/L (pH 5.4), and 205 mL water (do not dilute
to volume). Adjust the pH to 5.4 with phosphoric acid (85%).
Add 10 mg TCEP.
(e)
Mobile phase for HPLC.
—In a 1000 mL flask, mix
1.6 g decylamine, 80 mL acetonitrile, 100 mL sodium acetate
solution 500 mmol/L (pH 5.4), and 820 mL water (do not dilute
to volume). Adjust the pH to 5.4 with phosphoric acid (85%).
Add 50 mg TCEP.
E. Preparation of Standards
Note
: Vitamin C is sensitive to light and oxygen. Conduct
operations under subdued light conditions, or use amber
glassware. Keep all solutions away from direct light.
(a)
Ascorbic acid stock solution (500 μg/mL)
.—In a 25 mL
amber glass volumetric flask, weigh 12.5 mg ascorbic acid.
Dissolve and dilute 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)
.— In a 10 mL amber glass volumetric flask, pipet
1 mL stock solution. Dilute 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)
.—In 10 mL amber glass
volumetric flasks, pipet 0.1, 0.2, 0.4, 0.6, 1.0, 1.5, and 2.0 mL
intermediate standard solution. Dilute to volume with mobile
phase to prepare the respective concentrations given above.
F. Sample Preparation
(a)
Reconstitution of powder samples
.—(
1
) Weigh 25 g
powder in a 250 mL brown glass beaker and add 10 mg TCEP.
(
2
) Add 200 g warm water (40°C). Mix well until dissolution
is complete.
Proceed to
(b)
as soon as possible as vitamin C can
degrade rapidly. Do not let the reconstituted samples stand
for >30 min.
(b)
Extraction.
—(
1
) Weigh 2 g liquid or reconstituted
sample in a 10 mL amber glass volumetric flask.
(
2
) Add 4 mL TCEP solution and 2 mL TCA (15%).
(
3
) Dilute to volume with water.
(
4
) Filter the solution through a folded paper filter.
(
5
) Transfer 1 mL filtrate to a 10 mL amber glass volumetric
flask containing 1 mL acetate solution (pH 5.4) and dilute to
volume with mobile phase.
(
6
) Filter ~2 mL through a 0.22 or 0.45 μm membrane into
an HPLC vial.
(
7
) Proceed to chromatographic analysis using either
UHPLC conditions in
G(a)
or HPLC conditions in
G(b)
.
G. Analysis
(a)
UHPLC 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 for UHPLC.
—
See
D(d
): 0.4 decylamin,
2.5 mL acetonitrile, 25 mL sodium acetate 500 mmol/L (pH 5.4),
205 mL water, and 10 mg TCEP (pH 5.4).
(
7
)
Detection wavelength.
—265 nm.
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)
HPLC conditions.
—(
1
) Injection volume.—25 μL.
(
2
)
Autosampler temperature.
—10°C.
(
3
)
Column temperature.
—25°C.
(
4
)
Flow rate.
—1.0 mL/min.
(
5
)
Run time.
—20 min.
(
6
)
Mobile phase for HPLC.
—
See
D(e
): 1.6 g decylamine,
80 mL acetonitrile, 100 mL sodium acetate 500 mmol/L
(pH 5.4), 820 mL water, and 50 mg TCEP (pH 5.4).
(
7
)
Detection wavelength.
—265 nm.
Note
: At the end of each analytical series, rinse the column
with acetonitrile–water (1+1, v/v) for 60 min at 1.0 mL/min.
223