OMB Meeting Book_9-11-14
170.00
165.00
160.00
155.00
150.00
145.00
140.00
135.00
AU AU
130.00
12 23.613 vitamin B12 - 23.655
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.
in 2 04 –2 57 min: see Table 2011.10D . Flow rate:
elute vitamin B
with the vitamin B
peak areas of the samples and verify that the
12
12
12 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 . 12
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.
WS = S × P × A/200
w
w 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 injected at the end of the set of samples. Prepare a standard curve by performing linear least squares (regression) on concentration versus the average peak areas of the working standards. A standard curve must have a correlation of at least 0.999 to be considered 12 12
F. Calculations
( a ) Chromatography .—Visually inspect each standard and
sample chromatogram and verify that vitamin B
is resolved from
12
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
concentrations
12
of samples, compare the vitamin B
peak areas of the standards
acceptable for sample calculations.
12
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
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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.
© 2012 AOAC INTERNATIONAL
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