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J
oseph
et al
.:
J
ournal of
aoaC I
nternatIonal
V
ol
.
99, n
o
.
4, 2016
OFFICIAL METHODS
Determination of Biotin by Liquid Chromatography Coupled
with Immunoaffinity Column Cleanup Extraction: Single-
Laboratory Validation, First Action 2016.02
G
eorGe
J
oseph
and
r
anJani
D
evi
AsureQuality Ltd, PO Box 41, Shortland St, Auckland 1140, New Zealand
e
laine
C. M
arley
and
D
aviD
l
eeMan
R-Biopharm Rhône Ltd, West of Scotland Science Park, Glasgow, Scotland G20 0XA
Submitted for publication May 9, 2016.
Adopted as a First Action
Official Method
SM
by the Expert Review
Panel on Biotin.
Corresponding author’s e-mail:
george.joseph@asurequality.comApproved March 17, 2016.
DOI: 10.5740/jaoacint.16-0155
S
takeholder
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for
Spifan n
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EthodS
Darryl Sullivan (Chair)
, Covance Laboratories
John Austad
, Covance Laboratories
Sneh Bhandari
, Silliker Laboratories
Esther Campos-Gimenéz
, Nestlé
Adrienne McMahon
, Nestlé
Scott Christiansen
, Perrigo
Hans Cruijsen
, FrieslandCampina
Wil van Loon
, FrieslandCampina
Jon DeVries
, General Mills/Medallion Laboratories
Brendon Gill
, Fonterra
Don Gilliland
, Abbott Nutrition
Karen Schimpf
, Abbott Nutrition
Min Huang
, Frontage Laboratories
Estela Kneeteman
, Instituto Nacional de Tecnología Industrial
Maria Ofitserova
, Pickering Laboratory
Shay Phillips
, Mead Johnson
Guenther Raffler
, Central Laboratories Friedrichsdorf–Eurofins
Kate Rimmer
, National Institute of Standards and Technology
(NIST)
Melissa Phillips
, NIST
David Woollard
, Hill Laboratory
Jinchuan Yang
, Waters Corp.
Introduction
The 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 (1-8). The method also
has an assured limit of quantification of 0.1 μg/100 g (1 part
per billion; ppb) based on a simple mathematical relationship
between lowest standard and dilution. The method involves an
immunoaffinity column (R-Biopharm Rhone, EASI-EXTRACT
biotin column or equivalent) cleanup and extraction followed
by LC–UV set at 200 nm.
A. Principle/Methodology
The sample is dispersed in phosphate-buffered saline
(PBS) and autoclaved at 121 ± 2°C for 25 min. The sample is
cooled to room temperature and then diluted to 100 mL in a
volumetric flask. The extract is centrifuged and filtered using
Whatman glass microfiber filter paper (GE Healthcare Life
Sciences, Buckinghamshire, UK). Clear filtrate is collected
for cleanup and extraction. The biotin immunoaffinity column
is mounted onto an 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 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 reconstituted in 1 mL water. The biotin in the reconstituted
sample is quantified by HPLC–UV set at 200 nm.
B. Chemicals
(a)
Laboratory reagent-grade water.
(b)
Sodium dihydrogen phosphate dihydrate.
(c)
Disodium hydrogen phosphate dihydrate.
(d)
Sodium hydroxide.
(e)
Methanol.
—HPLC grade.
AOAC Official Method 2016.02
Biotin
Liquid Chromatography Coupled with Immunoaffinity
Column Cleanup Extraction
First Action 2016
3