Biacore Journal Number 2 2001

SCIENTIFIC REPORT
PAGE 4
n 1996, the Food and Drug Administration
ruled that cereal and grain products must be
fortified with a synthetic form of folic acid.
The directive followed a recommendation by the
US Public Health Service that all women of child-
bearing potential should consume 0.4 mg folic
acid per day in addition to dietary folates, in
order to reduce the risk of neural tube defects in
the unborn child. It is known that folic acid may
also protect against other diseases including seve-
ral forms of cancer, stroke and vascular disorders.
Food manufacturers and regulatory authoriti-
es require that low concentrations of folic acid
can be measured rapidly and simply. The most
common way to determine the concentration of
folic acid in a food sample is to measure its effect
on bacterial growth, a technique limited by its
inherent lack of speed and low precision. The
technique demands extensive extraction and sam-
ple purification to remove components other than
folic acid that may alter bacterial growth rates.
Although alternative assays, based on liquid
chromatography or the use of vitamin-specific bin-
ding proteins exist, they were developed for the
analysis of clinical samples. They are unsuitable for
food analysis due to differences in sample compo-
sition and a specificity for vitamin forms that exist
exclusively under physiological conditions.
A competition assay for folic acid has been
developed for use with Biacores SPR technology
(Figure 1) and has been validated and compared
with established microbiological assays (1).
Briefly, an excess of specific antibodies is mixed
by an automated procedure with sample or cali-
bration solution containing the free ligand and
the antibody and ligand form a complex. When
injected into the detection unit, non-complexed
antibodies are measured by the biosensor system
when they bind to folic acid immobilized on the
sensor chip. The biosensor response is inversely
proportional to the ligand concentration. After
each analytical cycle, the chip is prepared for fur-
ther use by using regeneration solution that dis-
sociates the antibody:folic acid complex. The
standard extraction procedures described here
are adequate for vitamin analysis of the majority
of fortified foods. However, samples in which cer-
tain specified components are known to be pre-
sent, require modified extraction procedures to
accurately determine vitamin concentrations. The
key features of the assay are stated in Table 1.
The following data is taken from a multi-cen-
ter study by Indyk et al and reported in full in J
AOAC International 83: 1141-1148 (2000) (1).
Determination of vitamin concentrations in food
samples by Biacores SPR technology
Growth response of vitamin-dependent bacteria is the most widely used
method for measuring vitamin content in food products. Although sensitive
and specific, microbiological assays are slow. Biacores SPR technology is an
automated, real-time alternative for the quantification of folic acid, biotin
and vitamin B
12
in routine quality control and nutritional labeling require-
ments.
Harvey Indyk and Alan McWhirter*
Anchor Products, Waitoa, New Zealand and *Biacore AB, Uppsala, Sweden
I
Low non-specific binding
Handles colored and turbid
solutions
No colorometric detection
reagents
Minimal sample preparation
No requirement for highly
skilled personnel
Handles up to 40 samples in a
working day
Table 1
Vitamin analysis by Biacores SPR techno-
logy.
Figure 1
Principle of Biacores competition assay.
Measurment of unbound
vitamin-specific molecules
R
e
s
p
o
n
s
e
(R
U
)
Vitamin concentration
Vitamin Vitamin-specific
molecule
Vitamin Sensor Chip
Vitamin-specific molecules are added to sample extract
Biacore Journal Number 2 2001 PAGE 5
Study set-up
Number of participating individuals 10
Number of laboratories 4
Number of Biacore Q instruments 6
Statistical parameters

Cochrane outliers 1 participant

Repeatability (relative standard deviation) <8 %
Reproducibility (relative standard deviation) <10 %
HORRAT values# 0.26 1.09 for truly homogenous samples
Sample Biacore assay extraction
a
Microbiological assay extraction
b
Infant formula 4,0 3,8
Infant formula 3,8 3,7
Infant formula 6,1 6,2
Infant formula 3,0 3,2
Skimmed milk 4,3 4,2
Skimmed milk 3,0 3,2
Biacore

Q and the Biacore folic acid kit were

used to measure folic acid in cereal samples,
milk-based and soy-based infant formulae, milk
powder and vitamin premix.
STANDARD EXTRACTION PROCEDURE
All reagents in the assay kit are firstly equilibra-
ted to room temperature. Samples are then dis-
persed thoroughly in deionized water and prepa-
red according to the following simple procedure:
Dissolve the sample in water
Sonicate
Heat sample to 100C for 15 minutes
Centrifuge
Filter
High fat content, found in some milk-based
infant formulae, does not interfere with the
extraction.
Accuracy Four preparations of cereals and
one of milk-based infant fomula were analyzed
five times. The measurements were compared to
those obtained using alternative methods in dif-
ferent laboratories. Accuracy, defined as the
agreement between analysis methods, was bet-
ween 88% and 101% for the cereals and 96%
for milk-based infant fomula.
Precision Statistical evaluation was performed
according to the IUPAC/ISO/AOAC International
Harmonization Protocol on Collaborative
Studies. The results are summarized in Table 2.
In addition to folic acid, infant formulae are
also supplemented with biotin and a similar
inhibition assay has also been developed for
this vitamin. Biacore vitamin assays are shown
to correlate well with microbiological assays
and are shown to be faster and more sensitive.
VITAMIN B
12
Vitamin B
12
is a water-soluble vitamin involved in
erythrocyte synthesis and fatty acid metabolism,
lack of which causes severe hematological and
neurological disease. It is found only in foods of
animal origin and may exist in different forms of
which the most stable is cyanocobalamin. Many
food products like cereals and infant formula are
fortified with cyanocobalamin. Vitamin B
12
, like
folic acid and biotin, is usually measured by
microbiological assays despite relatively poor pre-
cision and the fact that certain bacteria can meta-
bolize inactive B
12
as well as unrelated nutrients.
A competition assay adapted to Biacore has been
validated and compared to MBAs (2).
VITAMIN B
12
CONCENTRATION MEASUREMENTS USING
BIACORES SPR TECHNOLOGY
Although the SPR-based vitamin B
12
assay is
a competition assay, the competing protein in
this case is not an antibody, but a modified form
of its intrinsic high affinity partner, vitamin B
12
-
binding protein, R-protein. The Qflex Kit for
vitamin B
12
contains vitamin B
12
-binding pro-
tein, vitamin B
12
derivative, buffers, chips and
disposables. Samples in this evaluation (2) inclu-
ded a range of infant formula powders, milk
powders, fluid milks from various species, beef
and liver. The simple extraction procedure, per-
formed under low level, yellow incandescent
light, is outlined below:
Vortex sample in extraction buffer
Autoclave
Clarify
The microfluidics system is firstly equilibrated
with buffer. A fixed concentration of R-protein is
then equilibrated with the sample and injected
over a sensor surface supporting immobilized
vitamin. A typical sensorgram is represented in
Figure 2, illustrating the significant events of the
experiment; baseline equilibration, association
Table 2
Statistical summary of multi-center study
on the precision of folic acid analysis
using Biacore Q.
When statistical values were calculated
taking into account laboratory and instru-
ment identity, the results of the precision
study were not significantly altered.
# HORRAT value is the reproducibility rela-
tive standard deviation divided by the pre-
dicted value, based on the relative analyte
concentration in mass/mass units.
Biacore Journal Number 2 2001
SCIENTIFIC REPORT
Table 3
Comparison of extraction conditions on
vitamin B
12
(mg/100g). a: 1 g sample in
20ml 0.1 M phosphate buffer (0.05 M
citrate, 0.001g NaCN, pH 4.5) heated to
121C for 25 minutes. b: 1 g sample in
15ml 0.3 M acetate buffer, 0.004g NaCN,
pH 4.6) heated to 121C for 15 minutes.
PAGE 6
Sample Biacore Microbiological Radioisotopic Declared vitamin
assay assay B
12
value#
NIST SRM 1846 infant formula 3.98 (7.60, 14) 3.71 (5.75, 15) 3.0 (7.2, 5) 3.9+0.3
NIST SRM 2383 food composite 0.51 (13.7, 8) 0.37 (19.2, 2) 0.35 (14.6, 3) 0.44+0.19
NIST SRM 8435 whole milk 2.38 (9.24, 5) 1.77 (9.98, 2) 1.6 (7.79, 8) 1.7+0.3
Control infant formula 4.82 (4.13, 23) 4.50 (6.39, 77) 3.82 (4.69, 5) 2.0-6.4 (2.0)
Infant formula 7.35 (5.58, 13) 7.2 (3.2, 4) 6.1 (4.2, 5) 1.7-7.0 (2.0)
Infant formula 9.43 (4.28, 12) 9.2 (6.0, 3) 8.9 (8.5, 5) 5.0-13.0 (4.7)
Skimmed milk 3.67 (7.46, 6) 3.0 (1.9, 3) 3.0 (4.37, 5) na
Skimmed milk 5.87 (3.78, 4) 6.1 (2.3, 3) 5.3 (4.30, 6)) na
Goats milk 0.71 (8.80, 5) 0.66 (16.6, 4) 0.46 (14.8, 5) na
Beef (mince) 1.32 (8.77, 4) 1.85 (8.27, 2) 1.47 (3.93, 3) na
Beef (topside) 2.39 (2.40, 4) 2.59 (8.94, 2) 2.73 (4.23, 3) na
Beef (rump) 3.52 (4.73, 4) 3.55 (14.46, 2) 3.86 (5.38, 3) na
Liver (sheep) 102 (7.43, 3) 121 (4.5,2) 91 (6.0,2) na
phase, non steady state response plateau, regene-
ration of sensor surface and stabilization prior to
a subsequent injection cycle. Several food
extracts gave a very low response (ca 15RU) in
the absence of R-protein, compared to a respon-
se under total inhibitory conditions of ca 10RU.
This minimal non-specific binding is largely attri-
butable to the presence of bovine serum albumin
in the buffer. Dose-response calibration curves
established quantitation ranges for cyanocobala-
min of 0.08-2.40 ng/ml. Limits of detection and
quantitation were determined by measuring the
binding response of uninhibited binding protein
and were 0.06 ng/ml and 0.20 ng/ml, respective-
ly. A range of samples was subjected to both
Biacore and microbiological assay extraction
techniques and gave similar vitamin B
12
concen-
tration values (Table 3).
Biacore, microbiological assays and radioiso-
topic assays were compared to test a range of
food products for vitamin B
12
. The data are
summarized in Table 4.
Biacore yielded data statistically equivalent to
the reference microbiological assay, while radio-
isotopic estimations were generally lower.
Estimated values for formulae complied with
expected specification ranges and were typically
higher than declared levels, consistent with for-
mulation overages generally recommended
during infant formula production.
Correlation and absence of significant bias,
compared to alternative methods, has been
established for Biacores SPR technology over a
wide range of food products. Performance,
coupled with other attributes like the lack of
radioactive labelling, rapidity, automation and
cost-efficiency, make Biacore a practical alter-
native to established techniques. The procedure
is robust, and is suitable to be used in food
laboratories for routine surveillance programs,
without the need for a high level of expertise.
References
1. Indyk, H.E., Evans, E.A., Bostrm
Caselunghe, M.C., Persson, B.S., Finglas,
P.M., Woollard, D.C. and Filonzi, E.L.
Determination of biotin and folate in infant
formula and milk by optical biosensor-
based immunoassay
J AOAC International 83: 1141-1148 (2000)
2. Indyk, H.E., Persson, B.S., Bostrm-
Caselunghe, M.C., Filonzi, E.L. and
Woollard, D.C.
The determination of vitamin B
12
in milk,
infant formula and foods by optical biosen-
sor-based protein binding assay
In press (2001)
Biacore Journal Number 2 2001 PAGE 7
SCIENTIFIC REPORT
Table 4.
Comparison of methods for vitamin B
12
content (mg/100 g). Data are expressed
as mean (RSD%, n). #Reference values
reported for NIST SRMs; range (label
claim) reported for formulae.
Figure 2
Sensorgram showing baseline equilibra-
tion, association phase, non steady state
response plateau, regeneration and stabi-
lization.