AOAC SPIFAN ERP & Working Group Method Reviews (July 10, 2019)

Figure 2011.10C. Typical standard chromatogram.

oven for at least 1.5 h, but for no more than 4 h. After at least 1.5 h, remove samples from the oven and cool to room temperature. 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

in 23–30 min and

an appropriate gradient to elute vitamin B 12

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

Figure 2011.10D. Typical standard chromatogram.

© 2016 AOAC INTERNATIONAL