AOAC Final Action Methods in 2017

C ampos G iménez & M artin : J ournal of AOAC I nternational V ol . 101, N o . 4, 2018  1115

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Figure 2014.02. Example chromatograms on selected products and standard solutions. Color figures are available online as supplemental information at: http://aoac.publisher.ingentaconnect.com/content/aoac/jaoac

( f )  Calibration .—Plot peak responses against concentrations (in nanograms per milliliters). Perform regression analysis. Calculate slope and intercept. ( g )  Quantitation (liquid and powder samples) .—Calculate the concentration of vitamin B 12 in μg/100 g of product as follows: A I W W V V S W m V − ( ) × + ( ) × × × × × × × 1 2 1 3 1 2 100 100 where A = response (height or area) of the peak obtained for the sample solution, I = intercept of the calibration curve, S = slope of the calibration curve, W 1 = weight of powder sample used for reconstitution (25 g), W 2 = weight of water used for reconstitution (200 g), m = weight of sample suspension (60 g), V 1 = volume of the test solution (volume used to dissolve the test portion) in mL (100 mL), V 2 = volume of the aliquot of the sample solution loaded onto the affinity column (9 mL), and V 3 = volume in which the aliquot of the sample solution is reconstituted after immunoaffinity cleanup (0.3 mL). ( h ) Quantitation (amino-acid-based products) .—Calculate the concentration of vitamin B 12 in the sample in μg/100 g of product as follows:

portion) in milliliters (100 mL), V 2 = the volume of the aliquot of sample solution loaded onto the affinity column (9 mL), V 3 = the volume in which the aliquot of sample solution is reconstituted after immunoaffinity cleanup (0.3mL), and D = the dilution factor (e.g., 2). ( i )  Reporting .—Report results with two decimal points as cyanocobalamin in μg/100 g of product.

G. Example Chromatograms See Figure 2014.02 .

Collaborative Study Protocol

Part 1 All participant laboratories received two practice samples and were asked to analyze each of them in duplicate (two extractions from each reconstituted sample). Any deviation from the written method was to be recorded and reported. Results were communicated to the study director by using the electronic template provided with the protocol. The participants were asked to report the final cyanocobalamin results, the peak responses for standard curves and samples, and the different masses used during sample preparation. After review by the study director, results within a range of expected levels (average ± 2 × standard deviation of reproducibility) identified the laboratories qualified for the second part of the study.

W W W V V D S W × × × × × × + + ( ) 1 2 3 1 3 m V × × 2 1000 1

100

( 

) −



C

A A I blank sample −

×

=

sample

where A blank = the response (height or area) of the peak in the blank, A sample = the response (height or area) of the peak in the sample, I = the intercept of the calibration curve, S = the slope of the calibration curve, W 1 = theweight of sample used for reconstitution (25 g), W 2 = the weight of water used for reconstitution (190 g), W 3 = the weight of skimmed milk powder used for reconstitution (10 g), m = the weight of sample suspension (60 g), V 1 = the volume of the test solution (volume used to dissolve the test

Part 2

All qualified laboratories received a second shipment containing 20 coded products, corresponding to 10 products in blind duplicates. The samples were a set of infant formula and adult nutritional products, representing a wide range of commercially available products. The laboratories were asked