AOAC Final Action Methods in 2017

1116  C ampos G iménez & M artin : J ournal of AOAC I nternational V ol . 101, N o . 4, 2018

could be observed between laboratories using HPLC or UHPLC conditions. Results on practice samples can be found in Table 1, while results of the full statistical evaluation are shown in Table 2. A full set of raw data per laboratory and sample are presented in Tables 3 and 4. Although the main purpose of the study was to collect only precision data, results on the SRM (1849a; 0.45 μg/100 g) were well in agreement with the reference value available (0.482 ± 0.085 μg/100 g), demonstrating the accuracy of the method. Precision results (repeatability and reproducibility) were, in general, well within the limits stated in the SMPR 2011.005 (15). RSD r and RSD R ranged from 1.1 to 6.5% and from 6.0 to 23.8%, respectively. The only matrix showing reproducibility values higher than the requirement (23.4%) was an adult nutritional ready-to-feed high-fat product. This type of matrix already showed a low recovery rate during single laboratory validation (13), and an indication in the method has been added to overdilute this sample prior to extraction in order to improve recovery and precision. Horwitz ratios were all well below 2 (0.17–0.78). These results were submitted to the AOAC expert review panel (ERP) in September 2016. Based on the aforementioned data, the method was recommended to move forward to Final Action status.

to analyze all the samples (single extraction from each liquid or reconstituted powder) on 2 days (10 samples per day). Each sample was assigned to either day 1 or day 2. The blind duplicates were assigned to be analyzed on the same day. Results were communicated to the study director by using an electronic template similar to the one used in Part 1.

Statistical Evaluation

After data collection, outliers were detected by using Cochran andGrubbs tests. The average concentrations, standard deviations, and relative standard deviations of repeatability (RSD r )were estimated from the blind duplicates. Standard deviations of reproducibility, RSD R , and Horwitz ratio values (RSD R /predicted RSD R ) were also estimated. The details on statistical analysis can be found in the “Appendix D: Guidelines for Collaborative Study Procedures To Validate Characteristics of a Method of Analysis” of the AOAC Official Methods of Analysis (14). Twenty-one laboratories located in 13 different countries agreed to participate. Random codes were attributed to the laboratories for identification. Two laboratories did not manage to receive the samples because of restrictions for importation of the samples in their countries. One laboratory did not qualify for the second part of the study, and another one dropped out because of resource availability. One laboratory enrolled later and reported data on powder samples only, which were kindly provided by another participant from the same geographic region. Finally, a total of 18 laboratories managed to provide full results on time for reporting. AOAC Official Method 2014.02 was applied as written; a few laboratories used HPLC instead of UHPLC as described in AOAC Official Method 2011.08 . No significant differences Results and Discussion

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Table 1. Results of practice samples for 17 laboratories a

≤7% ≤11%

Mean μg/100 g

b , % RSD R

c , %

RSD r

Requirements (SMPR 2011.005) Infant formula, powder-milk-based Infant formula, powder-soy-based

0.46 0.43

5.7% 10.1% 3.0% 9.0%

a Results are reported in μg/100 g product as reconstituted (reconstitution rate: 25 g sample + 200 g water). b RSD r = relative standard deviation of repeatability. c RSD R = relative standard deviation of reproducibility.

Table 2. Results of collaborative study samples a

≤7%

≤11%

n b

Requirements (SMPR 2011.005)

Mean, μg/100 g

RSD r , % RSD R , %

Horwitz ratio values

Adult nutritional RTF c , high-fat

17 14 17 17 16 16 17 13 17 14

1.54 0.45 0.41 0.68 0.13 0.21 0.34 0.69 0.26 1.18

6.5 2.7 4.9 1.5 4.4 4.2 3.2 1.1 2.9 2.8

23.4

0.78 0.27 0.27 0.26 0.19 0.21 0.16 0.19 0.17 0.20

SRM 1849a (Infant/Adult nutritional formula)

9.8 9.8 8.8 8.5 8.6 6.0 6.4 6.8 6.1

Soy-based infant formula

Low-fat adult nutritional powder Infant formula powder (FOS d /GOS e )

Infant formula RTF, milk-based

Infant formula powder, part hydrolyzed, milk-based

Infant elemental powder

Infant formula powder, part hydrolyzed, soy-based

Adult nutritional RTF, high-protein

a All results are in μg/100 g of reconstituted product (reconstitution rate: 25 g product + 200 g sample for powders, as is for RTF). b n = Number of la boratories considered in the evaluation (after removal of outliers). c RTF = Ready-to-feed.

d FOS = Fructooligosaccharides. e GOS = Galactooligosaccharides.