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AsureQuality Laboratory Services
APPENDIX 1
DETERMINATION OF BIOTIN BY HPLC-UV COUPLED WITH IMMUNOAFFINITY
COLUMN CLEAN-UP EXTRACTION
1. INTRODUCTION
AsureQuality Auckland Laboratory has initiated a method to facilitate a specific, precise,
accurate and robust procedure for the analysis of biotin from Infant Formula and Adult /
Pediatric Nutritional Formulas. The method also has an assured limit of quantification of
0.1µg/100g (1ppb) based on a simple mathematical relationship between lowest standard
and dilution. The method involves immunoaffinity column (R-Biopharm Rhone, EASI-
EXTRACT biotin column) clean-up and extraction followed by liquid chromatography with UV
detection.
2. PRINCIPLE / 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.
3.
CHEMICALS
1. Laboratory Reagent Grade Water
2. Sodium Dihydrogen Phosphate Dihydrate
3. Disodium Hydrogen Phosphate Dihydrate
4. Sodium Hydroxide
5. Methanol, HPLC grade
6. Acetonitrile, HPLC grade
7. Ortho-phosphoric acid, 85%
8. Phosphate buffered saline (PBS) pH 7.4 (Life Technologies 100100.31 or equivalent)
9. Biotin, purity
≥
99% (Sigma B4501 or equivalent)
4. REAGENTS
1.
2 M Sodium Hydroxide
: Weigh 80g of sodium hydroxide into a 1L volumetric flask, dissolve
in water and make up to mark.
2.
0.15 M Sodium Phosphate Buffer
: Weigh 9.15g of
sodium dihydrogen phosphate
dihydrate and 16.31g of disodium hydrogen phosphate dihydrate into a 1L volumetric flask.
Dissolve in water and make up to mark. Adjust pH to 7 with 2M sodium hydroxide.
Bio-02 w/SLV
FOR ERP USE ONLY
DO NOT DISTRIBUTE