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5841 v2

Page 6 of 9

AsureQuality Laboratory Services

5. SINGLE LABORATORY VALIDATION

5.1 Methodology

The sample is dispersed in phosphate buffered saline and autoclaved at 121±2

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

time of biotin was detected.

5.3 Identification of biotin

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.

Bio-02 (February 2016)

FOR ERP USE ONLY

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