© 2012 AOAC INTERNATIONAL
12
12
w
w
12
12
12
12
12
12
12
12
AU
AU
23.613
vitamin B12 - 23.655
170.00
165.00
160.00
155.00
150.00
145.00
140.00
135.00
130.00
125.00
120.00
115.00
110.00
105.00
100.00
0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00 16.00 17.00 18.00 19.00 20.00 21.00 22.00 23.00 24.00 25.00 26.00 27.00 28.00 29.00
Minutes
Figure 2011.10C. Typical standard chromatogram.
elute vitamin B
in 2
04
–2
57
min:
see
Table
2011.10D
. Flow
rate:
with the vitamin B
peak areas of the samples and verify that the
1.0 mL/min.
(
f
)
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
vitamin B peak areas of the samples are within the range of the
vitamin B peak areas of the standards.
(
c
)
Calculation of standard concentrations
.
WS = S × P × A/200
where WS = working standard concentration in
g/L; S = amount
of vitamin B standard weighed in mg; P = purity of USP reference
standard in
g cyanocobalamin (vitamin B )/mg of the standard; A
= aliquot of vitamin B internal standard used (0.5, 1, 2, 3, 4, 5, or
10) in mL; and 200 = dilution volume in mL.
(
d
)
Preparation of standard curves
.—(
1
) At each standard
concentration, average the peak area of the standard injected at the
beginning of a set of samples with the peak area of the standard
sample chromatogram and verify that vitamin B
is resolved from
injected at the end of the set of samples. Prepare a standard curve
all other peaks in the chromatograms (Figures
2011.10C
and
D
).
(
b
)
Measurement of peak area
.—Peak areas are measured with
by performing linear least squares (regression) on concentration
versus the average peak areas of the working standards. A standard
a data system. Before calculating the vitamin B
concentrations
curve must have a correlation of at least 0.999 to be considered
of samples, compare the vitamin B
peak areas of the standards
acceptable for sample calculations.
132.00
130.00
128.00
126.00
124.00
122.00
120.00
118.00
116.00
114.00
112.00
110.00
108.00
106.00
104.00
102.00
100.00
0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00 16.00 17.00 18.00 19.00 20.00 21.00 22.00 23.00 24.00 25.00 26.00 27.00 28.00 29.00
Minutes
Figure 2011.10D. Typical standard chromatogram.
