CHAPTER 24

Biotin Analysis in Dairy

Products
DAVID C. WOOLLARD*a AND HARVEY E. INDYKb
a
New Zealand Laboratory Services, 35 ORorke Rd, Penrose,
Auckland 1642, New Zealand; b Fonterra Co-operative Group Ltd,
PO Box 7, Waitoa 3341, New Zealand
*Email: david.woollard@nz.bureauveritas.com

24.1 Introduction
Biotin is an essential micronutrient for all living mammals because of its role as
a cofactor of carboxylation and decarboxylation enzymes. Human cells have
four biotin-dependent carboxylases that catalyse key reactions in gluconeo-
genesis, amino acid catabolism and fatty acid synthesis. Mammalian cells
cannot biosynthesize biotin, and thus it becomes an essential component of the
diet and is therefore classied as a vitamin. Biotin is widely distributed in the
human diet, either mostly free in the case of milk, vegetables and fruit, or partly
bound in animal tissues and plant seeds. Protein-bound biotin is generally
formed via covalent linkage to lysine residues, and gastrointestinal (GI) tract
proteases can release biotin in the form of biocytin (e-N-biotinyl-L-lysine).
The biotin content of cows milk is considered to be modest at approximately
2 mg/100 g. In the context of this discussion it is also relevant to note that biotin
is central to several metabolic pathways involved with mammalian milk
biosynthesis.
The structure of biotin has three asymmetric carbons and can therefore exist
as eight potential stereoisomers, but only the d-biotin form is both biologically

Food and Nutritional Components in Focus No. 4

B Vitamins and Folate: Chemistry, Analysis, Function and Eects
Edited by Victor R. Preedy
r The Royal Society of Chemistry 2013
Published by the Royal Society of Chemistry, www.rsc.org

377
378 Chapter 24
Table 24.1 Daily intake of biotin to meet nutritional needs.a
Life stage and gender Amount (mg)
Babies: birth to 6 months 5
Infants: 712 months 6
Children: 13 years 8
Children: 48 years 12
Children: 913 years 20
Teens: 1418 years 25
Adults 30
Pregnant women 30
Breast-feeding women 35
a
Estimated recommended human daily intake required as a function of age
(source: Institute of Medicine).

active and found in nature, and is the only form of the parent vitamin that
requires consideration. Biotin rarely exists in the free form in animal tissues, the
majority being bound as biocytin within apoenzyme protein structures via an
amide link between its terminal carboxyl group and protein lysine residues.
Clinical deciencies of biotin in humans are not common in view of the low
dietary intake levels required (see Table 24.1), but have been reported when
prolonged and excessive amounts of raw egg white are consumed. However, in
infants, biotin deciency can occur as a consequence of insucient production of
biotinidase, the enzyme required to release bioavailable biotin from biocytin.
Rare genetic deciencies of biotinidase are also known. Obvious cutaneous
symptoms include hair loss and skin rashes, but many neurological problems can
develop. Deciency during pregnancy is considered to be tetragenic in the new-
born. Biotin-producing microorganisms within the intestine can contribute to the
human biotin pool and complicate the evaluation of nutritional need.
As with all nutritional studies, reliable analytical methods are a pre-requisite
for population-based policies related to Recommended Daily Allowances
(RDA), total diet surveys and label claims, as well as supporting clinical and
food compliance testing of biotin and its biomarkers. In general, for an esti-
mation of total biotin in food, a proteolytic step is required to release the
bioactive biocytin, which may then be included with free biotin in the analysis.
The specic extraction procedures employed for the release of matrix-bound
biotin remain a major analytical challenge and in fact may inuence results
even more than the end-point analytical measurement technique. Several
extraction strategies developed for the analysis of bound biotin have, depending
on sample type, most commonly employed high temperature mineral acid
digestion and/or ambient temperature enzymatic hydrolysis techniques.
The rst biotin tests for foods relied on microbiological principles, although
some non-specic colorimetric tests were developed for biotin in pharma-
ceutical preparations. This chapter summarizes the important analytical
techniques that have been employed, or are in use today, with emphasis
on dairy products. The analytical techniques reported for the determination of
biotin content can be categorized under (i) microbiological, (ii) biological,
Biotin Analysis in Dairy Products 379
Table 24.2 Overview of the attributes and limitations of the principal analy-
tical strategies available for the analysis of biotin in foods.
Principle Key steps Advantages Limitations
Microbiological Extraction of biotin Low equipment Test microorganism
assay (MBA). and biocytin from costs. Versatile, vulnerable to
Growth of sample. Incubate sensitive, reference growth inhibition
biotin-dependent with microorgan- Infant Formula or stimulation
microorganism ism and measure Council method. from matrix com-
proportional to turbidity. ponents. Highly
biotin in sample manipulative,
extract. time-consuming.
High performance Extraction of High specicity for High equipment
liquid chromato- biotin forms, biotin. Proven costs
graphy (HPLC). purication, and commonly
Chromatographic chromatographic available
separation of bio- separation, technology.
tin species with post-column
quantitation based derivatisation -
on detector uorescence, or
response. MS detection.
Ligand-binding Extraction of biotin Rapid, specic High equipment
assays, relying on species. Direct or inexpensive equip- costs if non-
biospecic mole- indirect detection ment costs if labelled platform
cular recognition by biotin-specic labelled technique. utilised.
of biotin. Labelled antibody or bind- Proprietary
or non-labelled ing protein in kit-based assays
platforms either a labelled or available.
available. non- labelled het-
erogenous format.

(iii) chromatographic and physicochemical or (iv) biospecic ligand-binding

principles. The three principal analytical techniques are briey compared in
Table 24.2. Recommended comprehensive literature reviews are provided in the
reference list (Eitenmiller et al. 2007; Frappier 1993; Livaniou et al. 2000; Ploux
2000).

24.2 Microbiological Methods

These highly sensitive methods for the determination of biotin in complex
matrices were among the rst to be developed and are based on the principle
that growth of specic microorganisms is dependent on the presence of limiting
amounts of biotin in the culture media (Livaniou et al. 2000). Although these
methods have the advantage of low cost, they are sensitive to many factors that
can compromise results and require two days for completion.
Since the microorganisms respond only to free biotin, bound forms must be
treated prior to analysis to release biotin in its free form. Following extraction
of biotin from the sample by high-temperature acidic and/or ambient enzy-
matic strategies, the methodology relies on the incubation of the food extract at
multiple dilution levels with the microbiological culture in a biotin-decient
380 Chapter 24
growth medium. Quantication of culture growth, typically after 1824 h, is
generally achieved by measurement of turbidity at 540660 nm, with response
calibrated against added biotin solutions of known concentration and inter-
polation of the growth responses from unknown sample extracts.
Several microorganisms have been applied to the development of micro-
biological assays for biotin, with Lactobacillus plantarum employed pre-
dominantly, and less commonly, Saccharomyces cerevisiae. L. plantarum has
been mainly used for the analysis of dairy-based food products (Angyal 1996;
Bell 1974; Blake 2007; Eitenmiller et al. 2007; Hoppner and Lampi 1992).
However, fatty acids are growth stimulatory and lipids therefore need to be
solvent extracted prior to acid hydrolysis. Although AOAC International does
not provide a collaboratively validated method for biotin, the International
Formula Council does recommend a method based on L. plantarum. Inter-
nationally accepted compendia methods generally describe tube-based assays
which can be automated using a programmable dispenser. However, these
methods have more recently been successfully deployed in a 96-well microtitre
plate format.
In general, microbiological assay (MBA) is acknowledged to be limited by
poor precision, with measurement uncertainties of up to 20% reported. Ana-
lytical failures can originate from either poor growth or contamination issues.
The questionable accuracy of microbiological methods and the view that the
method tends to overestimate biotin content are signicant concerns given that
existing biotin data in food composition tables are predominantly based on
these methods. Despite poorly dened standardization of methods between
laboratories and issues related to dierent growth responses, the micro-
biological assay for biotin nonetheless remains the gold standard reference
method, since the method yields a single biological response to biotin activity.
A recent development is the commercial availability of proprietary micro-
biological assay kits that incorporate microtitration plates pre-coated with lyo-
philized microorganism, thereby avoiding the problems of maintaining stock
cultures (VitaFasts, R-Biopharm). Incubation at 37 1C for 4448 h is followed
by measurement of growth using a microplate reader and construction of a dose
response calibration curve. Dairy products must rst be treated to remove fat
and protein that would otherwise interfere with the turbidimetric measurements.
This is typically achieved by the addition of Carrez I (150 g/L potassium hexa-
cyanoferrate(II)-3-hydrate) and Carrez II (300 g/L zinc sulfate-7-hydrate)
reagents, followed by centrifugation. This method has recently received ocial
AOAC Research Institute status based on a comprehensive single-laboratory
validation report. It has the potential to oer signicant advantages for routine
testing laboratories, despite the costs associated with the kits.

24.3 Biological Methods

These earliest developed methods were mainly applied to the determination of
biotin in foods and are generally considered to be less sensitive and far less
Biotin Analysis in Dairy Products 381
technically practical than microbiological assays (Frappier 1993). Biotin
bioassays were based on using rats or chicks as test subjects. The rat method
exploited the inclusion of raw egg white (or avidin) in the diet to induce biotin
deciency over several weeks. The gain in animal weight was then followed
after feeding of the diet with calibration against pure biotin, with weight gain
related to the logarithm of the biotin dosage.
Bioassays also include methods that determine biotin indirectly through its
biological function in the test animal. Thus, measurement of the activity of
biotin-dependent pyruvate carboxylase in the blood has been utilized as an
indicator of biotin content.

24.4 Chromatographic and Physicochemical Methods

Simple chemical laboratory procedures for biotin are restricted to pure mate-
rials as described in pharmacopeia monographs using alkaline titration. Simi-
larly, spectral methods for determining native biotin are unknown in most
samples except for highly puried materials as it has no useful absorption
above 210 nm. To obtain suitable sensitivity and selectivity, biotin must rst be
derivatized. For example, the disruption of a p-hydroxy-azobenzene-2 0 -
carboxylic acid (HABA) avidin complex by biotin, which binds to avidin with
higher anity, can be followed titrimetrically or spectroscopically to give useful
determinations of biotin.
As recently as 2008, a reliable colorimetric method has been reported for
biotin in pharmaceuticals based on its catalytic impact on the azidetriiodide
reaction (Walash et al. 2008). However, biotin is best chromatographically
separated from other compounds to allow practical determinations in all but
the simplest matrices. This is particularly true for biological and food materials.
Reviews such as Livaniou et al. (2000) summarize many of the historical
physico-chemical techniques.
Following chromatographic separation, spectrometry has been used because
the vitamin can be visualized in isolation from potential interferences. For
example, planar chromatographic techniques, followed by spraying with an
acidic solution of p-dimethylaminocinnamaldehyde has found signicant
practical use, since the intense red colour (533 nm) is specic to the ureido
ring of biotin. Thin-layer chromatography (TLC) on silica-gel plates has
been applied to biotin detection in multivitamin premixes and capsules,
although this technique is not used greatly in the modern era. Ploux (2000) has
reviewed some of the past applications with respect to TLC developing solu-
tions and other visualization sprays such as o-toluidine-potassium iodide. TLC
still has a place in some investigational laboratories as a tool for biochemical
studies.
Ion chromatography (IC) using Dowex 1X2 has played an important role in
biotin determinations of the past, being an essential component of biotin dis-
covery and purication. Nowadays, high-performance IC platforms with pre-
parative columns have replaced earlier techniques and remain an important
382 Chapter 24
tool for investigators of biotin biochemistry. The various fractions can be
collected into tubes or wells, and subsequently studied for composition.
Gas chromatographic platforms have had a minor role in detection of the non-
volatile and labile biotin vitamin. However, a biotin-silyl ester has been reported
though it has not been used widely outside the pharmaceutical industries. The
carboxylic acid and amido groups of biotin can be exploited to produce volatile
silyl esters or N-silanization compounds, with bis-trimethylsilyl acetamide the
preferred reagent. Such derivatives are readily separated from inferences on OV-
17 capillary columns, although reports state that oxidation products of biotin
can co-elute. The silyl-ester is sensitive enough for ame ionization detection
(FID) in simple applications, but is challenged for sensitivity in biological studies.
Some researchers question the repeatability of this reaction and have proposed
other options. Nevertheless, gas chromatography (GC) and gas chromato-
graphymass spectrometry (GC-MS) applications have not become a com-
monplace analytical technique for biotin and certainly not within food industries.
Similarly, capillary electrophoresis has been used for high concentration samples,
but has not been applied to foods.
The rst step for the chromatographic determination of biotin in foods is to
release the analyte from its protein-bound environment where it may be
covalently linked to lysine residues (biocytin, e-N-biotinyl-L-lysine). The vita-
min is stable enough to survive, at least in part, the elevated temperatures
associated with autoclaving required for protein cleavage, in order to release
biocytin. The degree of acid hydrolysis varies with the food matrix, with meat
requiring 2 h at 121 1C in 6 M sulphuric acid. Such harsh conditions will pro-
mote some oxidation of biotin to its sulfone and sulfoxide, so compromises are
often made in acid strength and heating regimen to achieve optimum recov-
eries. Hot hydrochloric acid is very damaging to biotin loss and so is rarely
used. Plant materials are more easily hydrolysed and therefore are typically
subjected to milder conditions (e.g. 2 M sulphuric acid), while some dairy
products require minimal extraction other than mild heating to aect dis-
solution. Papain or trypsin enzyme digestion will break peptide bonds without
risking damage to biotin and has become a preferred method for food product
digestion. Coupled with takadiastase, the food matrix can be dispersed and
solubilized, thereby releasing biotin. Although biocytin itself is not hydrolysed,
this is unimportant provided that the method can uniquely separate biocytin
from biotin (Gimenez et al. 2010; Lahely et al. 1999).
Although high-performance liquid chromatography (HPLC) separations of
biotin and its analogues have been reported using many chromatographic
modes, reversed-phase chromatography remains at the forefront, irrespective
of which detection method is employed. Anion-exchange chromatography with
electrochemical detection has not gained the same prominence, despite its
apparent usefulness. Biotin, its metabolites and oxidation products are readily
separated using aqueousorganic gradients.
Although a water-soluble vitamin, biotin is suciently non-polar to exhibit
good retention on reversed-phase columns. Depending on the pH of the mobile
phase, biotin elutes from C8 and C18 columns well separated from other
Biotin Analysis in Dairy Products 383
B vitamins, but typically between folic acid and riboavin. For direct low-
wavelength ultraviolet (UV) detection, the organic solvent must be acetonitrile
and acidication should be made with triuoroacetic acid to avoid high
background absorption by the mobile phase. In recent times, ultra-HPLC
equipment has been available, thereby improving the sensitivity and speed of
analysis achievable with sub-3 mm particles. This important advance provides
an improved strategy towards overcoming the risks of chromatographic
interferences. For conventional HPLC platforms, chromatographic columns
are recently available with solid particle cores that mimic ultra-HPLC, without
the associated high system back-pressures.
Since it is unpractical to use direct UV detection, biotin is best quantitated
following derivatization to a suitable chromophore or uorophore. One of the
early advances was an o-line coupling with the horseradish peroxidaseavidin
binding assay, whereby fractions were collected and subjected to uorimetic
determination. This method was used to survey a wide range of foods and
subsequently reveal that original MBA-derived data for the biotin content of
many foods was questionable (Staggs et al. 2004). Pre-column derivatization
has been used with equal success, yielding the nanogram sensitivities to analyse
this vitamin. 9-Anthryldiazomethane (ADAM) forms a uorescent ester that is
subsequently separated by reversed-phase HPLC (lex 365 nm, lem 425 nm).
Similarly, biotin esters with panacyl bromide in the presence of crown ether
are uorescent (lex 380 nm, lem 470 nm). 4-Bromomethylmethoxycoumarin
derivatives of biotin are UV absorbing and provide another useful analytical
method for biotin. One of the complications of pre-column chemistries can be
the need for signicant clean-up of the extract prior to injection.
From an operational viewpoint, the post-column reaction of biotin with
avidin labelled with uorescein isothiocyanate (avidinFITC) has become the
simplest HPLC method for the quantitation of biotin and biocytin, and has
allowed routine testing laboratories to generate quality control (QC) data
(Lahely et al. 1999). Typical chromatograms are shown in Figure 24.1 of a
standard and a milk powder extract. The analytical time is only a few minutes
because of the absence of interfering uorescent substances. Biocytin, if pre-
sent, elutes in front of biotin and can be concurrently detected.
An interesting feature of the reaction is its non-linearity which can confuse
investigators, but is easily overcome using a quadratic t to multiple (ve or
more) calibration points as shown in Figure 24.2 This method has been
validated among ten laboratories to become a European Norm (EN15607;
European Committee for Standardization 2009) and has also been further
developed by others with modied sample preparation and HPLC congura-
tions to gain greater robustness (Campos-Gimenez et al. 2010; Thompson et al.
2006). Such facile methodology should catalyse accumulation of reliable biotin
data and promote enhanced nutritional and food compositional studies.
Streptavidin-FTIC can replace avidin-FTIC and exhibits enhanced reagent
stability, although at greater cost. Nonetheless, this HPLC technique is no
more expensive in time and consumables compared with alternative testing
strategies.
384 Chapter 24

800

3.181
600
mAU

400

200

0
0 1 2 3 4 5
min
B

800
3.241
600
mAU

400

200

0
0 1 2 3 4 5
min

Figure 24.1 HPLC chromatogram of biotin standard with post-column avidin reac-
tion. Note the fast chromatography permitted with this sensitive and
specic reaction. Detection is using uorescence. A: biotin standard,
0.04 ug/mL. B: supplemented milk powder extract with 12 ug/100 g.

High-performance anity chromatography has recently been reported with

trypsin-modied avidin supported on 5 mm silica. While the separations were
successful and a wide range of foods were studied, elution times were 80
minutes and ADAM post-column reactions were still required (Hayakawa et al.
2009). However, such anity columns within a solid-phase extraction (SPE)
platform make realistic choices for sample preparation, whereby the biotin can
be puried and concentrated prior to reversed-phase HPLC. R-Biopharm has
recently developed a commercially available antibody-based immunoanity
column to bind biotin from aqueous extracts, providing an excellent technique
to clean up complex samples.
The next generation of HPLC detectors, namely mass spectrometers, are now
available and being deployed for water-soluble vitamin determinations in
dietary supplements with electrospray ionization interface (ESI) (Holler et al.
2006). There remain obstacles to overcome for multivitamin analyses in com-
plex food matrices, as co-elution of vitamins or excipients can compromise
Biotin Analysis in Dairy Products 385

12000

y = 437277x2 + 70206x 37.9

10000
Fluorescent Detector Response

R2 = 0.9999

8000

6000

4000

2000

0
0.00 0.02 0.04 0.06 0.08 0.10
mg/L Biotin

Figure 24.2 Non-linear calibration curve for biotin by post-column avidin reaction.
The curve ts very well to a quadratic equation. It is important to have
many data points to describe the curvature unless a narrow concentra-
tion range is selected and sample extracts diluted into this range.

ionization eciency. These issues can be resolved using isotope dilution tech-
niques when deuterated internal standards become available. Nevertheless, the
use of positive ESI and [2H2]-biotin has successfully allowed biotin levels to be
characterized in NIST 3280 multivitamin tablets (Nelson et al. 2006). Although
for food samples, sample preparation restrictions will limit LC-MS ability to
quantitatively analyse all total B vitamins in a single run, gains are currently
being made to detect free (non-bound) vitamins in supplemented infant formula.

24.5 Ligand-binding Methods

24.5.1 Labelled Techniques
During recent decades, many biospecic ligand-binding assay variants have
been applied to the determination of biotin in biological matrices, including
foods (Bitsch et al. 1989; Eitenmiller and Landon, 2007; Finglas et al. 1986;
Finglass and Morgan 1994; Livaniou et al. 2000; Mock et al. 1992; Ploux 2000).
Such methods include protein-binding assays and immunoassays, which both
share similar procedural principles, but dier in that they utilize either a biotin-
specic protein or antibody. In the case of antibody-based immunoassays, the
small molecular mass of biotin requires its coupling as a hapten in order to
stimulate the production of antibodies in the host animal that can then be
utilized in a concentration assay. Despite being more prevalent in the clinical
diagnostic eld, labelled biospecic protein-binding and immunoassay methods
386 Chapter 24
have also provided an alternative analytical strategy for food analysis and oer
unique advantages. In general, these methods have the attributes of high sen-
sitivity, specicity and speed, and are relatively simple to implement. They can
therefore be easily adopted by the food industry for routine compliance testing
of product and represent potentially useful alternatives to traditional micro-
biological assays and contemporary HPLC strategies.
To-date, all reported protein binding assays utilize the glycoprotein avidin,
or its structurally related streptavidin, which both exhibit an extremely high
anity and specicity for biotin. The unique features of the avidin/streptavidin
biotin interaction system have been widely exploited, including application to
the quantitation of biotin in foods. Both antibody-based immunoassays and
avidin-based protein binding assays for biotin have been reported utilizing the
96-well microtitration plate format and applied to foods, including milk. Each
oers dierent analytical attributes as a consequence of the dierent anities
and specicities for biotin and related structures. The majority of such assays
are formatted utilizing a heterogeneous solid-phase platform and require
radioisotopes, enzymes, and chemiluminescent and uorescent substances as
labelling systems to signal binding levels. Although radioisotope-based assays
are commonly applied for clinical diagnostics, they are less desirable in the food
analysis environment, where enzyme-linked formats are generally preferred.
Although avidin-based protein binding assays are highly specic for biotin,
they cannot discriminate between biotin and biotin metabolites that bind with
typically lower anity. In such circumstances, a chromatographic separation
prior to a binding assay end-point has overcome this limitation and has been
applied to the analysis of human milk.
There are a number of commercially available and proprietary protein-
binding kits utilizing the enzyme protein binding assay (EPBA) format for
measurement of biotin in biological samples and foods (e.g. Ridascreens,
R-Biopharm). As with microbiological assays, dairy samples must be depleted of
fat and protein interferences prior to analysis. While kit assays are most com-
monly applied to the measurement of free, rather than total biotin, information
on cross-reactivity towards biocytin and biotin-4-amidobenzoate is frequently
provided. In one kit, avidin is bound to the surface of microtitre wells into which
sample extracts or standards and a biotin-alkaline phosphatase conjugate are
dispensed. The conjugate competes with endogenous free biotin for avidin
binding sites. Incubation is allowed for one hour and the wells washed with Tris
buer to remove unbound material. A self-indicating phosphatase substrate is
added and incubated at 37 1C, resulting in the development of a yellow colour.
The reaction is stopped after 30 minutes and the spectral absorbance of the wells
read at 405 nm. The concentration of biotin is indirectly related to the colour
intensity and a non-linear calibration determines the unknown concentrations.
The samples, typically 510 g are initially warmed in water (40 1C) to solubilize
biotin and adjusted to pH 67 if necessary, followed by equal volumes of the two
Carrez solutions. The extract is diluted to a dened volume and centrifuged to
produce a clear supernatant. 100 mL is required for most EPBA tests. Further
clarication with a 0.45 mm lter is sometimes necessary.
Biotin Analysis in Dairy Products 387
Several alternative assay format variants have been reported, including an
EPBA whereby sample extracts and standards are incubated with a streptavi-
dinperoxidase conjugate, and dispensed in microtitre wells coated with
biotinalbumin conjugate. After 30 minutes incubation, 3,3 0 ,5,5 0 -tetramethyl
benzidine (TMB) in acidic solution both stop the reaction and create a yellow
colour that is measureable at 450 nm.

24.5.2 Non-Labelled Techniques

More recent developments in the biosensor eld have provided novel platforms
which have been increasingly applied to both protein-binding and immu-
noassays for the quantitation of vitamins, including biotin, in a range of foods.
They represent an alternative to conventional ligand-binding assays and other
analytical techniques, providing high-throughput, rapid and cost-eective
strategies that can meet the increasing demands within the food industry with
respect to compliance testing. As with traditional binding assays, the specic
biorecognition of analyte can dramatically reduce the need for extensive sample
preparation. Biosensors are based on the intimate contact of the biorecognition
element with a physicochemical transducer, which functions to convert the
specic interaction with analyte into a measurable signal, thereby facilitating
real-time measurement. Several biorecognition molecular species have been
utilized, including enzymes, antibodies and binding proteins, while electro-
chemical, optical and thermal devices have been used as the transducer.
The most commonly applied biosensor system applied for the analysis of a
wide range of target analytes, including vitamins, is based on the optical
transducer phenomenon of surface plasmon resonance (SPR), although other
detection principles have been exploited (Blake, 2007; Kalman et al. 2006;
Reyes et al. 2001). Such SPR-based optical biosensors are providing an
innovative platform for the application of non-labelled biospecic techniques
to aspects of food composition, safety and compliance, and specically for the
analysis of water-soluble vitamins in foods including folic acid, vitamin B12,
pantothenic acid and biotin. SPR has been successfully integrated into a
commercially available instrument platform and provides for the real-time and
label-free detection of ligand-binding events from which target analyte con-
centration can be derived. In simple terms, detection is based on real-time
binding events between two biospecically interacting molecular species at the
surface of a sensor that moderate an evanescent eld established within the
SPR optical detector under conditions of total internal reection. The change in
SPR angle caused by association or dissociation at the sensor surface is pro-
portional to the mass of bound material and is continuously recorded in the
form of a sensorgram. The advantages of this platform compared with con-
ventional ligand-binding methods include real-time measurement, freedom
from enzyme or radioisotope labelling requirements, rapid analysis times,
minimal sample preparation requirements, sensitivity, specicity and enhanced
precision. The sensor chips used provide low non-specic binding, stability over
many analysis cycles and excellent precision.
388 Chapter 24

4000

4
3000
3
2000
5
Response (RU)

a
b
1000 c
d
e
1 7
0
2

1000

6
2000

3000
100 0 100 200 300
Time (s)

Figure 24.3 Sensorgrams derived from an SPR-biosensor analysis of biotin. Super-

imposed sensorgrams of biotin calibration standards: (a) 1.23 ng/mL; (b)
3.7 ng/mL; (c) 11.1 ng/mL; (d) 33.3 ng/mL; (e) 100 ng/mL. SPR-based
biosensor assay utilizing a biotin specic antibody under inhibition
conditions: (1) baseline with buer ow; (2) sample injection; (3) binding
association phase; (4) end of injection; (5) stable binding response
acquired; (6) surface regeneration; (7) baseline for subsequent cycle.

In view of the low molecular mass of the vitamins, biosensor assays employ
the principle of competitive inhibition. Prior to the analysis for biotin, a biotin
derivative is covalently tethered to the sensor surface. Sample extract or biotin
standard is mixed under equilibrium conditions with an excess of a biotin-
specic antibody, and unbound antibody binds to the surface generating an
SPR binding response in real-time, as illustrated in Figure 24.3.
The relative binding response is interpolated from a calibration curve in
order to compute concentration. As an inhibition assay, the response is
inversely related to biotin concentration and exhibits a sigmoidal dose
response relationship that is typical of most ligand-binding assays. With respect
to specicity, the routine compliance assay is targeted to the quantitation of
free biotin only in nutritional dairy products, and therefore does not include
biocytin (Indyk et al. 2000). However, in milk and supplemented infant for-
mulas, the overwhelming majority of biotin is present in the free form.
It remains speculative as to the potential impact on biotin analysis that may
result from current innovations in the eld of biosensor technologies. New
alternatives to classical biorecognition elements will include articial receptors
such as molecularly imprinted polymers and aptamers, which promise
increased stability. Although they are currently not widely utilized in com-
mercial biosensors, such biosensor innovations are likely to oer future
advantages.
Biotin Analysis in Dairy Products 389
24.6 Biotin Forms and Concentration in Milk
and Dairy Products
A large number of clinical studies, spearheaded by Donald Mock and his
associates (Mock et al. 1992, 1997; Staggs et al. 2004), have been conducted
concerning the distribution of biotin in tissues and biological uids. These have
in turn attempted to assess the extent to which human nutritional requirements
vary with age and gender. Biotin is required in such low quantity that average
diets, whether of animal or vegetable origin, supply sucient vitamin to prevent
widespread deciency states, making it problematical to determine recom-
mended dietary intake (RDI) in humans without deliberate dietary interven-
tion. It is also suggested that microbiological fauna in the intestinal tract
contribute to the host biotin pool. Other measures, such as comparison of
consumed versus excreted biotin, homeostasis of circulatory biotin and use of
radiolabelled (tritiated) biotin have all helped to determine the current RDI for
adults at 2030 mg/day, increasing during pregnancy and lactation to 3035 mg/
day, while other estimates are 1.5 mg/kg body weight across all age groups.
Human milk is the primary agent for infant nutriture and thereby guides the
composition of manufactured infant formula and milk substitutes. The
reported concentration of biotin in human milk is variable with lactation (and
unfortunately between analytical methods), but is more than sucient to
supply the newborn infant with the RDI of 56 mg/day, as evidenced by the
absence of reported deciency syndromes in breast-fed babies. Interestingly,
most biotin in milk is present in a free form and therefore unbound with any
macromolecules. As expected, when milk is separated into its fat and aqueous
fractions, the water-soluble biotin is found predominantly in the skim-milk
phase. Biotin has some lipophilicity and so a small percentage is carried into the
cream as part of the fat-globule membrane. The total concentration of human
milk is not large and somewhat similar to bovine milk. With respect to breast
milk substitutes, it is necessary to ensure the biotin status remains comparable,
thus international guidelines recommend 0.42.4 mg/100 kJ of reconstituted or
ready-to-feed infant formula.
Table 24.3 shows biotin levels in selected consumer dairy products and
compares the milk of the cow with that of the goat and human. When com-
mercial milk is converted by spray-drying into dehydrated powders, the biotin
contents increase proportionately. Skim-milk powder thus becomes a reason-
able source of the vitamin at 1315 mg/100 g. Puried casein contains essentially

Table 24.3 Typical biotin content of species milks and cows milk consumer
products (mg/100 g).a
Cow
Goat Human
Milk Yoghurt Cheese Butter Ice-cream Milk Milk
2.04.3 0.94.0 0.85.9 0.5 1.1 3.23.9 0.40.8
a
Data from cited references and Food Standards Australia New Zealand (2010).
 390

60
20089 Dairy Season 200910 Dairy Season 201011 Dairy Season

50

40

30

Biotin (ug/100 g)
20

10

0
08 08 08 08 08 08 09 09 09 09 09 09 09 09 09 09 09 09 10 10 10 10 10 10 10 10 10 10 10 11 11 11 11 11 11 11 11 11
0 7. 08. 09. 10. 11. 12. 01. 02. 03. 04. 05. 06. 07. 08. 09. 10. 11. 12. 01. 03. 04. 05. 06. 07. 08. 09. 10. 11. 12. 01. 02. 03. 04. 05. 06. 07. 08. 09.
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
21 21 21 22 22 23 23 23 26 26 27 27 28 28 28 29 29 30 30 2 2 3 3 4 4 4 5 5 6 6 6 9 9 10 10 11 11 11

Figure 24.4 Seasonal trend in biotin levels in skim-milk powder (mg/100 g) over three consecutive production seasons in New Zealand
(20082011) resulting from an extensive pasture grazing dairy practice. Biotin concentration was estimated using a biosensor-
based immunoassay.
Chapter 24
Biotin Analysis in Dairy Products 391
no biotin, as it is removed during the precipitation and washing processing
stages. Although the whey fraction of processed milk contains the majority of
the endogenous milk biotin, puried whey proteins lose most of their associated
biotin during ultraltration, since in milk, biotin is not covalently bound to this
fraction.
Countries with high biotin intake, as shown in total diet surveys, generally
consume more meat, eggs and sh, which represent the predominant sources of
the vitamin (Murakami et al. 2008). However, cheese contains measurable
biotin across a range of concentrations (approximately 16 mg/100 g) and can
contribute signicant dietary biotin in some European societies where cheese
consumption is high. On a solids basis, cheese has less biotin than its raw
material (milk), so speculation that starter microorganisms can contribute
additional biotin during production is probably false and, in fact, may consume
biotin. However, hard cheeses such as Cheddar and Gruyere contain lesser
amounts of biotin than soft cheeses (e.g. Brie). Similarly, yoghurt and other
cultured dairy products have diminished biotin levels as compared to the parent
milk on a solids-non-fat basis.
Recent work in New Zealand has shown a seasonality of biotin levels in
bovine milk, as illustrated in Figure 24.4. Extensive pasture grazing and animal
husbandry practices based on herd calving scheduled to maximize lactating
cows at peak grass growth result in a minor but consistent seasonal trend.

Summary Points
 This chapter focuses on the analysis of biotin in dairy products.
 Biotin is a member of the B group of water-soluble vitamins.
 As a vitamin, biotin is an essential component of the human diet.
 Levels of biotin in dairy products formulated for infant nutrition are
rigorously regulated.
 Reliable analytical methods are essential to support the formulation and
compliance of manufactured dairy products.
 The attributes and limitations of dierent analytical platforms for the
measurement of biotin are reviewed.

Key Facts
 Biotin is a vitamin required in very small (microgram) quantities as a
cofactor to essential enzyme reactions.
 Biotin is hard to detect in dairy and food samples, dierent methods often
giving dierent answers.
 Detection using microbiological organisms has traditionally been used,
but is currently being replaced by modern chromatographic and immu-
nochemical techniques.
 Biotin levels in dairy products are only moderate, and insucient to
supply human daily needs. However, since it is ubiquitous in many foods,
deciency states are uncommon.
 Infants fed on dairy-based formulae need additional biotin added to their food.
392 Chapter 24
Denitions of Words and Terms
Affinity chromatography. A separation technique that exploits the high biological
specicity and anity of specic naturally occurring proteins for the analyte.
Apoenzyme. An enzyme devoid of its cofactor.
Avidin, streptavidin. Avidin is a protein expressed in the white of the reptilian,
amphibian and avian egg with very high binding anity for biotin. Strep-
tavidin is a structurally related protein expressed in certain bacteria that
exhibits comparable anity for biotin.
Biosensor. An analytical device for the detection of an analyte that combines a
biospecic detection element with a physicochemical detector that may be
based on a number of principles.
Chromatography. A group of analytical techniques whereby complex mixtures
of compounds are both separated into individual forms and then sequentially
detected by a range of techniques. The techniques described in this chapter
are predominantly based on a high-pressure liquid chromatographic (HPLC)
platform.
Cofactor. A non-protein compound that is essential for facilitating the biolo-
gical activity of an enzyme.
Enzyme. A protein that catalyses the conversion of one biologically active
compound to another.
Ligand-binding. The biospecic binding interaction between an analyte and a
biological macromolecule, usually a protein. Such an interaction is non-
covalent, usually reversible and either crucial for biological activity, or can be
exploited for analytical purposes.
Mass spectrometry. An analytical detection technique that measures the mass-
to-charge ratio of charged particles, and provides conrmatory identication
of target analytes separated by chromatography.
Stereoisomers. Compounds that have the same molecular formula and
sequence of bonded atoms, but dier in the three-dimensional orientations of
those atoms.
Vitamin. An organic compound that the body cannot biosynthesize yet is
essential for normal growth and development and therefore must be derived
from the diet.

List of Abbreviations
ADAM 9-anthryldiazomethane
EPBA enzyme protein binding assay
ESI electrospray ionisation interface
FID ame ionization detection
FITC uorescein isothiocyanate
GC gas chromatography
GC-MS gas chromatographymass spectrometry
GI gastrointestinal
HABA p-hydroxy- azobenzene-2 0 -carboxylic acid
Biotin Analysis in Dairy Products 393
HPLC high-performance liquid chromatography
IC ion chromatography
MBA microbiological assay
QC quality control
RDA Recommended Daily Allowance
RDI recommended dietary intake
SPE solid-phase extraction
SPR surface plasmon resonance
TLC thin-layer chromatography
TMB 3,3 0 ,5,5 0 -tetramethyl benzidine
UV ultraviolet

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