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AOAC Final Action Method 2016.02 (Biotin)
Page 4 of 16
AOAC Official Method 2016.02
Determination of total biotin by liquid chromatography coupled
with immunoaffinity column clean-up extraction
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 safety data sheets for all chemicals prior to use. Ensure all appropriate personal protective
equipment and follow good laboratory practices.
A. Principle / Methodology
The sample is dispersed in phosphate buffered saline (PBS) 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 (GE Healthcare Life Sciences,
Buckinghamshire, UK). 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 immunoaffinity 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 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 re-constituted in 1mL of water. The biotin / biocytin in the reconstituted
sample are analysed simultaneously by HPLC using a PDA set at 200nm. Identification of peaks is based
on absolute retention time. Quantification was by multipoint external calibration using peak area responses
of the analytes. Spectrum scan (200 nm to 350 nm) can be used for the purity and identity confirmation as
required.
B. 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. PBS - pH 7.4 (Cat. No. 10010031 Life Technologies / Thermo Scientific or equivalent)
9. Biotin - Purity
≥
99% (Cat. No. B4501 Sigma Chemical Co., St. Louis, MO, USA, or equivalent)
10. Biocytin - Purity
≥
98% (Cat. No. B4261 Sigma Chemical Co., St. Louis, MO, USA,or equivalent)
C. Reagents
(a)
Sodium Hydroxide
, 2M. -
Weigh 80g of sodium hydroxide in a 1L volumetric flask, then dissolve in
water and make up to the mark.
(b)
Sodium Phosphate Buffer
, 0.15 M. -
Weigh 9.15g of
sodium dihydrogen phosphate dihydrate and
16.31g of disodium hydrogen phosphate dihydrate in a 1L volumetric flask, then dissolve in water
and make up to the mark. Adjust the pH to 7 with 2M sodium hydroxide.
2016.02 (BIOTIN) FINAL ACTION REVIEW
FOR ERP USE ONLY
DO NOT DISTRIBUTE