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Butler-Thompson et al.: J ournal of AOAC I nternational V ol. 98, N o. 6, 2015  1661

Table 2011.10F. RP column elution gradient

Mobile phase, %

Time, min

A

B

C

0.00 14.5 14.6

90 90

10 10

0 0 0 0

40–60 a 40–60 a

60–40 a 60–40 a

27.0–30

27.1–30.1

0 0

10 10

90 90

29.90–33.00

a  Appropriate gradient conditions must be established with each column to adequately resolve vitamin B 12 and riboflavin and to elute vitamin B 12 between approximately 24 and 30 min. To establish appropriate gradient conditions with a new column, set the gradient composition at 14.6 and 27.0–30.0 min to the midpoint of the allowable range from the table above. Inject the resolution test solution and calculate the resolution (Rs) between vitamin B 12 and riboflavin. Adjust the mobile phase composition between 14.6 and 27.0–30.0 min until Rs is >1.5. After vitamin B 12 elutes from the C 18 or phenyl column rinse the column with 90% mobile phase C for at least 2.8 min.

on concentration versus the average peak area of the working standards. A standard curve must have a correlation coefficient (r) of at least 0.999 to be considered acceptable for sample calculations. ( 2 ) At each working standard concentration, the peak areas of standards injected at the beginning and end of a set of samples should not increase or decrease by more than 10%. (e) Calculation of vitamin B 12 concentrations in samples .— The vitamin B 12 concentration in each injected sample preparation is extrapolated from the vitamin B 12 standard curve prepared as described above. The concentration of vitamin B 12 in each product can then be calculated: C p = C i × D 1 ÷ ss × D 2 ÷ V where C p = product concentration in μg/kg; C i = vitamin B 12 concentration of the injected sample preparation extrapolated from standard curve in μg/L; D 1 = volume of the first dilution in mL (100 mL); ss = sample size in g; D 2 = volume of the second (final) dilution in mL; and V = volume of filtrate loaded onto the cartridge in mL (2, 6).

(Laboratories 1, 7, 9, and 11) had to be excluded because of sample mislabeling. Based on chromatographic profiles and physical appearance of the products, it was determined that some of the adult high-protein RTF samples were mislabeled as adult high-fat RTF samples. Chromatograms of the adult high-protein RTF samples, the adult high-fat RTF sample, and the mislabeled adult high-fat RTF sample are shown in Figures 1–4, illustrating the chromatographic differences between the two products. Although only five of the nine participating laboratories had duplicate results for the adult high-fat RTF because of mislabeling, AOAC recommended keeping the adult high-fat product in the study and calculating repeatability and reproducibility from the data that were available. As a result, repeatability was calculated from duplicate results generated at five laboratories, and reproducibility was calculated from the single and duplicate results from all nine laboratories. Using the AOAC INTERNATIONAL Interlaboratory Study Blind (Unpaired) Replicates workbook, statistical outliers from one laboratory were identified for six of the 12 products analyzed. After removal of outliers, repeatability RSD r ranged from 2.98 to 9.77% and met the SMPR ≤7% for eight of the 12 products analyzed. The RSD R ranged from 3.54 to 19.5% and met the SMPR of ≤11% for seven of the 12 products analyzed. Although repeatability and reproducibility for several products exceeded the requirements in SMPR 2011.005, it was the majority opinion of the ERP that these results were

Results and Discussion

Data from all participating laboratories are summarized in Tables 1 and 2. It should be noted that two of the participating laboratories (Laboratories 6 and 11) only received half of the study samples. It should also be noted that data for the second adult high-fat RTF replicate from four laboratories

Figure 2011.10C. Example chromatogram showing riboflavin and vitamin B 12

resolution.