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

Figure 2011.09B. Chromatogram for a sample (NIST 1849 Milk Powder).

( g ) Calculation .—Calculate the concentration of vitamin B 12 , in μ g/100 g, as follows:

residues of methanol are removed by a vacuum. Place the vial in the evaporation dry block. Evaporate the methanolic vitamin B 12 solution under nitrogen at approximately 60°C. Reconstitute the sample in 0.25 mL solution A. Decant the solution into a brown glass crimp-top vial with an integrated glass μ L insert and inject. ( d ) Determination .—Separate the vitamins using the following HPLC conditions: injection volume, 100 μ L; column, ACE 3 AQ (150  3.0 mm); eluent, solution A (0.025% TFA) and solution C (acetonitrile for LC); UV detector, 361 nm; gradient, see Table 2011.09B . ( e ) Identification. —Identify vitamin B 12 peak in the chromatograms of the test solution by comparison with the retention time and UV spectrum of the corresponding peak obtained for the using the external standard method, through comparison of the peak areas of the sample solution with standard solutions of known concentrations. Measure the four working standards before each sample series. Ensure that the sample concentrations are within the linear range of the working standards. standard solution (Figures 2011.09A and B ). ( f ) Quantification .—Quantify vitamin B 12

A c A STD Pr

E x ) . ( ) ( * ) g mL mL mL g mL 100 0 25 9 ( /

STD

Vitamin B 12

, g/100 g =

100

f

100

where c

= concentration of vitamin B 12

in the working standard

STD

solution, μ g/100 mL; A Pr

= peak area for vitamin B 12

in the

sample; A in the working standard solution; E = initial sample weight, g; f = factor (IAC loss); and x = number (generally 1 or 2) of 9 mL, given on the IAC to obtain a concentration of 0.01–0.5 μ g B 12 on the IAC. [ Note : Purification using IAC results in a loss of cyanocobalamin that is specific to each batch of IACs and can be around 20%. These losses are taken into account by the factor f ( see E for determination of f; recovery should be >80%).] References: (1) Application of Immunoaffinity Columns for Analysis of Vitamin B 12 by HPLC by r-Biopharm (2010) Rhône Ltd, Glasgow, UK J. AOAC Int . 95 , 933(2012); AOAC SMPR 2011.005, J. AOAC Int . 95 , 293(2012) STD = peak area for vitamin B 12

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