AOAC Final Action Methods in 2017

J OSEPH ET AL . : J OURNAL OF AOAC I NTERNATIONAL V OL . 101, N O . 3, 2018 833

Table 2. SPIFAN matrixes

Sample

Product description

Batch/lot No.

Blind duplicate codes

Practice 1 Practice 2

Infant formula powder, partially hydrolyzed soy-based

410457651Z

SWUO667 URTF231 KDOX966 ECHL425 DYLB360 NSRB999 TJHR217 KGSZ273 LYNY751 RQXQ518 EFXN778 CULF358 FPTE312 XKIP216

SWUO667 URTF231 ATAN351 UOPM297

Infant formula powder, FOS/GOS-based a

50350017W1

MLT-1 MLT-2 MLT-3 MLT-4 MLT-5 MLT-6 MLT-7 MLT-8 MLT-9 MLT-10 MLT-11 MLT-12

Infant formula powder, partially hydrolyzed milk-based

410057652Z

Infant elemental powder

00795RF EV4H2R

Infant formula RTF, milk-based b

HYJU890

Adult nutritional RTF, high-fat

00729RF00

ZMQM883

Infant formula powder, milk-based Infant formula powder, soy-based

4044755861

JSDT587

E10NWZC

OACN211 LTCT316 PZGP859 GVPE615

NIST SRM 1849a

CLC10-b

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Adult nutritional powder, low-fat

00859RF00 00866RF00 4052755861

Child formula powder

Toddler formula powder, milk-based Infant formula powder, milk-based Adult nutritional RTF, high-protein

BFA0941

K16NTAV

GBZC169 DOMY545

00730RF00

a FOS/GOS = Fructooligosaccharide/galactooligosaccharide. b RTF = Ready-to-feed.

AOAC Official Method 2016.02 Determination of Total Biotin Liquid Chromatography Coupled with Immunoaffinity Column Cleanup Extraction

The qualified laboratories were then asked to analyze the MLT samples on two different days following a carefully designed protocol. The results were submitted to the study director for evaluation. Unless otherwise specified in the protocol, all powdered samples were analyzed on a reconstituted basis, using 25 g sample in 225 g water, as stated in the method. An electronic template was provided for data reporting, including system suitability, linearity, peak areas of the standard curve, as well as results of the sample extracts. Furthermore, detailed information on the different weights and volumes used during sample preparation as indicated in the method, as well as raw data [chromatograms of standards and samples; Figures 1 and 2] were requested. Laboratories were asked to report final biotin and biocytin results (µg/100 g) to two decimal places. After data collection, outliers were detected using the Cochran and Grubbs tests. The number and coded identity of statistical outlier laboratories is included in the final report. Average biotin concentration, SD of repeatability (s r ), and RSD of repeatability (RSD r ) values were estimated from blind duplicates of the MLT samples. The blind coded duplicates were analyzed on the same day. SD of reproducibility (s R ), RSD of reproducibility (RSD R ), and Horwitz ratio [HorRat; (RSD R /predicted RSD R ] values were also determined. The analytical method provided to the laboratories for the MLT is the same as the version published in the Journal of AOAC INTERNATIONAL (11), which is codified as AOAC First Action 2016.02 . Extensive details of the sample preparation, chromatography, calculation, and reporting criteria were specified in the method and in the protocol provided to the laboratories. None of the laboratories that participated in the MLT recorded any modifications or deviations from the documented procedure. This information was requested to assess the suitability of the method for further approval as a Final Action method.

First Action 2016 Final Action 2017

Applicable to the determination of total biotin in all forms of infant, adult, and/or pediatric formula (powders, ready-to-feed liquids, and liquid concentrates). Caution : Refer to safety data sheets for all chemicals prior to use. Ensure that all appropriate personal protective equipment is used and follow good laboratory practices .

A. Principle/Methodology

The sample is dispersed in PBS and autoclaved at 121 ± 2°C for 25 min. The sample is cooled to room temperature and then diluted to 100 mL in a volumetric flask. The extract is centrifuged and filtered using a Whatman glass microfiber filter paper (GE Healthcare Life Sciences). Clear filtrate is collected for cleanup and extraction. A biotin IAC is mounted onto an SPE manifold. A disposable syringe barrel is connected to the IAC as a reservoir. The buffer in the affinity column is drained, and the sample filtrate is loaded through the reservoir and allowed to flow through by gravity. The column is washed with PBS followed by water. Air is passed through the column to remove residual liquid. Biotin/biocytin from the column is eluted with methanol and collected in a Reacti-Vial (Cat. No. 13223; Thermo Scientific). The eluent is evaporated to dryness using a heating block set at 85 ± 5°C under a gentle stream of nitrogen, and the sample is reconstituted in 1 mL water. The biotin and biocytin in the reconstituted sample are analyzed simultaneously by HPLC using a PDA detector set at 200 nm. Identification of peaks is based on absolute retention time. Quantification is by multipoint external calibration using peak area responses of the analytes.