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Dilute to volume with laboratory water. Filter an aliquot of each
standard and prepared sample through a 0.45
m
m syringe filter into
an autosampler vial.
(
b
)
HPLC analysis.—
(
1
)
System setup and configuration.—See
Figures
2011.10A
and
B
for configurations.
(
2
)
Instrument
operation
conditions.—
(
a
)
Run
time
.—30–35 min.
(
b
)
Injection volume
.—900 μL to 2.0 mL.
(
c
)
System configuration.—See
Table
2011.10E
.
(
d
)
Isocratic pump.—
Mobile phase D: 2.5% acetonitrile.
Flow rate: Adjust so that vitamin B
12
elutes from the size-
exclusion column between 10.5 and 14.5 min. Typical flow rates,
1.1–1.2 mL/min.
Note
: To determine an appropriate flow rate,
connect the size-exclusion column directly to the UV-Vis detector
and inject the high standard. Adjust flow rate as necessary so that
vitamin B
12
elutes between 10.5 and 14.5 min.
(
e
)
Gradient pump
.—Mobile phase compositions: mobile
phase A, 0.4% TEA in laboratory water, pH 5–7; mobile phase B,
0.4% TEA and 25% acetonitrile in H
2
O, pH 5–7; mobile phase C,
0.4% TEA and 75% acetonitrile in H
2
O, pH 5–7. Determine
an appropriate gradient to elute vitamin B
12
in 23–30 min and
resolve vitamin B
12
from riboflavin using the information in
Table
2011.10F
.
(
f
)
Gradient pump flow rate
.—1.0 mL/min.
(
g
)
Detector settings
.—Detection wavelengths and bandwidth,
550 and 10 nm, respectively.
(
3
)
HPLC of standards and samples.—
Make 3–4 injections of
a working standard and verify the precision of those injections is
≤3%.
If the system is working properly, inject a set of 3–6 working
standards once, followed by a control sample, a set of 1–14
samples, and another set of 3–6 working standards. Every set of
1–14 samples should be bracketed by standards of appropriate
concentration.
F. Calculations
(
a
)
Chromatography
.—Visually inspect each standard and
sample chromatogram and verify that vitamin B
12
is resolved from
all other peaks in the chromatograms (Figures
2011.10C
and
D
).
(
b
)
Measurement of peak area
.—Peak areas are measured with
a data system. Before calculating the vitamin B
12
concentrations
of samples, compare the vitamin B
12
peak areas of the standards
with the vitamin B
12
peak areas of the samples and verify that the
Figure 2011.10D. Typical standard chromatogram.
Figure 2011.10C. Typical standard chromatogram.
Candidates for 2016 Method of the Year
343