Review Team (February 2016)

Bio-02 (February 2016) FOR ERP USE ONLY DO NOT DISTRIBUTE

5. SINGLE LABORATORY VALIDATION 5.1 Methodology

The sample is dispersed in phosphate buffered saline and autoclaved at 121±2 ⁰ C for 25 minutes. The sample is cooled to room temperature and then diluted to 100mL in a volumetric flask. The extract is centrifuged and filtered using a Whatman glass microfiber filter paper. Clear filtrate is collected for clean-up and extraction. Biotin immunoaffinity column is mounted onto a SPE manifold. A disposable syringe barrel is connected to the affinity column 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 phosphate buffered saline followed by water. Air is passed through the column to remove residual liquid. Biotin from the column is eluted with methanol and collected in a reacti-vial. The eluent is evaporated to dryness using a heating block set at 85±5°C and the sample is re-constituted in 1mL of water . The biotin in the reconstituted sample is quantified by HPLC using a UV detector set at 200nm. Refer to Appendix 1 for detailed methodology 5.2 Specificity / Blanks Reagent blanks and placebos were taken through the sample preparation procedure and analysed. There was no response (interference) observed in the reagent blanks at the retention time of biotin (Appendix 2). Infant elemental powder showed no response around the retention time of biotin (Appendix 3). However for placebos; child formula powder, adult nutritional RTF high fat and infant formula RTF milk based, a response around the retention The response (peak) of biotin in the samples was identified by absolute retention time with reference to external standards. Refer to Appendix 5 for typical standard chromatogram and Appendix 6 for a typical sample chromatogram. 5.4 Linearity and range A linearity check with six levels of working standards (1.0μg/100mL, 2.5μg/100mL, 5.0μg/100mL, 7.5μg/100mL, 10.0μg/100mL and 20.0μg/100mL) was carried out to establish linearity of the method. The range of the working standards covers approximately 0.4% to 400% of the biotin concentration of the infant formula and adult nutritional products in the SLV test materials kit. The correlation coefficients (r 2 ) of all the calibration generated during the validation were not less than 0.998 (Appendix 4). 5.5 Accuracy / Recovery Accuracy of the method was validated using standard reference material (SRM 1849a), in- house reference material (IRM) and spiked recovery studies. Analysing the samples and comparing the analytical result to the known or added value shows the accuracy of the method. time of biotin was detected. 5.3 Identification of biotin

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