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AOAC Final Action Method 2016.02 (Biotin) Page 1 of 16
Determination of total biotin by liquid chromatography coupled with immunoaffinity column
clean-up extraction: Multi Laboratory Testing, Final Action 2016.02
First Action 2016
Final Action 2017
George Joseph and Ranjani Devi
AsureQuality Ltd, PO Box 41, Shortland Street, Auckland 1140, New Zealand
Elaine C. Marley and David Leeman
R-Biopharm Rhône Ltd, West of Scotland Science Park, Glasgow, Scotland G20 0XA
Corresponding author’s email:
A collaborative study was carried out on AOAC First Action Official Method 2016.02: Determination of total
biotin by liquid chromatography coupled with immunoaffinity column clean-up extraction. Subsequent to the
successful method optimisation and analysis practice samples by 12 laboratories covering 10 different
countries, 9 laboratories completed the analysis 12 pairs of blind duplicates before the due date of the
study. Carefully selected SPIFAN matrices were used for the multi laboratory testing.
The sample is dispersed in phosphate buffered saline (PBS) and autoclaved at 121±2⁰C 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 and 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.
Biotin / Vitamin H / Vitamin B7 is a B group vitamin involved in the production of energy and functions as a
coenzyme in bicarbonate-dependent carboxylation reactions. Biotin exists as free biotin and in protein-
bound forms in foods. Signs of biotin deficiency are quite evident in humans who consume raw egg white
over long periods. Raw egg white contains avidin, a protein that has shown to bind biotin in the small
intestine and prevent its absorption. Clinical findings of biotin deficiency include dermatitis, conjunctivitis,
alopecia and central nervous system abnormalities.
Biotin (Hexahydro-2-oxo-1H-thieno[3,4-d]imidazol-4-pentanoic acid; CAS 58-85-5) is the fusion of an
imidazolidone ring with a tetrahydrothiophen group linked to a valeric acid side chain. The chemical formula
of biotin is C 10H16N2O3S with a molecular weight of 244.31.
Biocytin (N6-[5-[(3aS,4S,6aR)-Hexahydro-2-oxo¬-1H-thieno[3,4-d]imidazol-4-yl]-1-oxopentyl]-L-lysine; CAS
576-19-2) is a bound form of biotin (the linked molecule is lysine) is also biologically active in human
system. The chemical formula of biocytin is C16H28N4O4S with a molecular weight of 372.48.
Naturally active forms of biotin in foodstuffs are d-biotin and d-biocytin though the fortified form is
exclusively d-biotin. These two forms should therefore be determined in order to estimate the total biotin
nutritional value.
2016.02 (BIOTIN) FINAL ACTION REVIEW
FOR ERP USE ONLY
DO NOT DISTRIBUTE
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