6. AOACSPIFANMethods-2018Awards

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230 M c M ahon : J ournal of AOAC I nternational V ol . 99, N o . 1, 2016

Acetate and both fortified with α-tocopherol acetate) were used by the participating laboratories so that the laboratories could become familiar with the analysis procedure. The results were submitted to the Study Director for approval prior to commencing the collaborative study. Results within a range of expected levels indicated that the laboratory was capable of successfully running the analysis. The same practice samples were used as QC samples during the analysis of the study samples. Most of the laboratories submitted practice sample results that met the acceptance criteria. A couple of laboratories submitted results just outside the acceptance limits but were accepted to participate in the study as they met the acceptance limits for at least three of the four analytes.

was calculated as 4.90%

For total vitamin E as TEs, RSD r

was 5.68%.

and RSD R

Soy Product

For retinyl palmitate, RSD r For α-tocopherol acetate, RSD r

was 6.84% and RSD R

was 9.66%.

was calculated as 1.67% and

was 6.47%. For α-tocopherol, RSD r

RSD R

was calculated as 7.89% and RSD R

was 8.74%. For total vitamin E as TEs, RSD r

was calculated as 2.07%

and RSD R was 4.22%. For two blind duplicate samples of the milk-based hydrolyzed protein formula, where the repeatability was observed as being very high (up to 50% RSDs), repeat analysis was performed by a selection of laboratories. All initial results were confirmed. Repeatability obtained at the SLV stage (duplicates over 6 separate days) was very good for this matrix, 5.4 and 5.78% for cis and trans retinyl palmitate and 5.5 and 10.2% for α-tocopherol acetate and α-tocopherol. Results obtained for this sample were deemed invalid due to the material being expired at time of testing following an investigation and confirmation that other study directors encountered similar difficulties and following a discussion at the September 2014 ERP meeting. The results for this sample have been removed from the AOAC Final Action Method document. AOAC INTERNATIONAL Interlaboratory Study Workbook Revision 2.1 (6) was used to perform the statistical evaluation of the data. Outliers were detected and reviewed prior to exclusion from the data set. Where possible, detected outliers were reviewed by the participating laboratories for possible transcription or calculation errors. Across the four anaytes, 13-cis and all-trans Vitamin A, and α-tocopherol acetate and α-tocopherol, laboratories 4–8 and 10–12 had no statistical outliers. Laboratory 13 had one outlier, laboratories 1 and 15 had 2 outliers, laboratory 2 had 3 outliers, laboratory 3 had 4 outliers, laboratories 9 and 14 had 14 outliers. The statistical summary (Tables 1-20) lists the outliers and the reasons for removal (Cochran test outlier or Single Grubbs test outlier), as well as Cochran test or single Grubbs test outliers. An invitation was sent to all participating laboratories to comment on the performance of the method in their laboratories. In general, the comments were very positive. Laboratory 3 indicated that sample preparation was easy. According to the laboratory, it is not necessary to prepare the working standard (6 point curve) fresh daily, as the reagent used to prepare the stock and the working standards is the same. Laboratory 3 also indicated that the papain solution should be sonicated to ensure it is fully dissolved. Laboratory 4 changed the dilution of the standards to be within the working range of the spectrophotometric measurements and HPLC calibration curves. It used an injection volume of 35 µL instead of 50. An injection volume between 20 and 100 μL can be used with the method, depending on sensitivity. The laboratory indicated that 35 µL presented areas (or heights) to give optimum quantification and optimization. Laboratory 5 commented that the sample preparation procedure is

Milk-Based Products

For retinyl palmitate, RSD r

was calculated in a range of

in a range of 6.51–10.52%.

1.06–4.72% and RSD R

was 16.60% and RSD R

was

For retinyl acetate, RSD r 22.61%. For α-tocopherol acetate, RSD r 0.60–3.84% and RSD R For α-tocopherol, RSD r 1.57–5.78% and RSD R For total vitamin E, RSD r

was calculated in a range of

in a range of 4.15–8.67%.

was calculated in a range of

in a range of 5.68–12.47%.

as tocopherol equivalents (TEs)

was calculated in a range of 0.81–3.74% and RSD R of 3.84–7.17%. Note : One milligram of α-tocopherol acetate is equal to 0.671 mg TEs. One milligram of α-tocopherol is equal to 1 mg TEs. in a range

Low Fat Product

For total vitamin A palmitate, RSD r was 21.73%. For α-tocopherol acetate, RSD r

was 15.78% and RSD R

was 2.11% and RSD R

was

8.50%.

was 8.90% and RSD R

was 43.56%.

For α-tocopherol, RSD r For total vitamin E, RSD r

as TEs was 2.71% and RSD R

was

10.78%. The concentration of α-tocopherol was relatively low in the product: 0.14 mg/100 g ready to feed (RTF).

Hydrolyzed Protein and Elemental Products

For retinyl acetate, the partially hydrolyzed soy-based product gave an RSD r of 2.30% and an RSD R of 11.93%. For α-tocopherol acetate, RSD r was calculated as 3.65% and RSD R was 11.25%. For α-tocopherol, RSD r was calculated as 1.67% and RSD R was 11.94%. For total vitamin E as TEs, RSD r was calculated as 5.46% and RSD R was 10.15%. For retinyl palmitate, the elemental product gave an RSD r of 15.13% and an RSD R of 16.25%. For α-tocopherol acetate, RSD r was calculated as 3.38% and RSD R was 6.66%. For α-tocopherol, RSD r was calculated as 15.48% and RSD R was 17.44%.