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Method development and analysis of retail foods

for annatto food colouring material
a b b b
M. J. Scotter , L. Castle , C. A. Honeybone & C. Nelson
a
Ministry of Agriculture, Fisheries and Food, Central Science Laboratory, Sand
Hutton, York YO41 1LZ, UK; m.scotter@csl.gov.uk
b
Ministry of Agriculture, Fisheries and Food, Central Science Laboratory, Sand
Hutton, York YO41 1LZ, UK
Published online: 10 Nov 2010.

To cite this article: M. J. Scotter , L. Castle , C. A. Honeybone & C. Nelson (2002) Method development and
analysis of retail foods for annatto food colouring material, Food Additives & Contaminants, 19:3, 205-222, DOI:
10.1080/02652030110085386

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 Food Additives and Contaminants , 2002, Vol. 19, No. 3, 205 ± 222
Method development and analysis of retail foods for
annatto food colouring material
M. J. Scotter*, L. Castle, C. A. Honeybone and
C. Nelson
Ministry of Agriculture, Fisheries and Food, Central Science
Laboratory, Sand Hutton, York YO41 1LZ, UK
(Received 9 April 2001; accepted 25 July 2001)
Downloaded by [New York University] at 05:32 23 June 2015
Analytical methods for the determination of the
permitted food colouring annatto (E160b) have
been developed or re®ned to encompass the wide
range of food commodity types permitted to contain
it. Speci®c solvent extraction regimens have been
used depending upon the food commodity analysed
and HPLC analysis techniques coupled with spectral
con®rmation have been used for the determination
of the major colouring components. Qualitative and
quantitative data on the annatto content of 165
composite and two single retail food samples covering
a wide range of foods at levels above the limit of
quanti®cation (0.1 mg kg 1 ) is reported. Quantitative
results are given for the major colour principals 9 0 -cis-
bixin, 9 0 -cis-norbixin and trans-bixin. Semi-quantita-
tive results are given for the minor bixin and norbixin
isomers monocis- (not 9 0 -), di-cis- and trans-norbixin,
for which authentic reference standards were not avail-
able. Repeat analyses (n ˆ 4 9) of 12 diŒerent types
of food commodity (covering the permitted range)
spiked with annatto at levels between 1.7 and
27.7 mg kg 1 gave mean recoveries between 61 and
96%. The correspondin g relative SDs (RSD) were
between 2.1 and 7.9%.
Keywords : food, additives, research,
chromatography, analysis, bixin, norbixin
* To whom correspondence should be addressed.
m.scotter@csl.gov.uk
Food Additives and Contaminant s ISSN 0265±203X print/ISSN 1464±5122 online # 2002 Taylor & Francis Ltd
http://www.tandf.co.uk/journals
DOI: 10.1080/0265203011008538 6
Introduction
Annatto extracts (E160b) are natural carotenoid col-
ouring agents obtained from the outer coats of the
seeds of the tropical shrub Bixa orellana. The major
colouring component of annatto is the diapo-carote-
noid 9 0 -cis-bixin (methyl hydrogen 9 0 -cis-6,6 0 -diapo-
carotene-6,6 0 -dioate, C25 H30 O4 ), the monomethyl
ester of the dicarboxylic acid cis-norbixin (Preston
and Rickard 1980). Cis-bixin is soluble in most polar
organic solvents to which it imparts an orange colour
but is largely insoluble in vegetable oil. It may be
readily converted to the all-trans isomer due to its
instability in the isolated form in solution. Trans-
bixin is the more stable isomer and has similar
properties to the cis-isomer but is red in solution
and soluble in vegetable oil. Trans-bixin may form
only some 17% of the mixture. Water-soluble 9 0 -cis-
norbixin (C24 H28 O4 ) can be isolated from annatto
seeds by agitation in aqueous alkali at <70°C or
formed by alkaline hydrolysis of cis-bixin to give
either the sodium or potassium salt. The dicarboxyli c
acid is soluble in polar solvents to which it imparts an
orange colour. Cis-norbixin is readily converted to
the trans-isomer, which is red in colour but only
sparingly soluble in chloroform and 0.1 m sodium
hydroxide (Preston and Rickard 1980). Crystalline
bixin products of 80±97% purity may be obtained
by extraction of annatto seed with certain permitted
organic solvents and subsequent production of a
solvent-free product, which is then processed to give
a range of high purity oil- and water-soluble annatto
HPLC,
formulations (Levy and Rivadeneira 2000).
Collins (1992) has reviewed the chemistry and appli-
cations of oil- and water-soluble annatto colours.
Because of its ability to bind strongly with protein,
annatto is especially suited for the colouring of meat,
®sh and cheese. However, annatto has widespread use
in the colouring of commodities including ¯our and
e-mail: sugar confectionery, extruded or puŒed breakfast
cereals, snacks including extruded snacks, sauces,
 206 M. J. Scotter et al.

Table 1. Permitted uses of annatto and maximum levels
of addition (EC 1994).
Food commodity type
Margarine, minarine, other fat emulsions
and fats essentially free from water
Decorations and coatings
Fine bakery wares
Edible ices
Liqueurs, including forti®ed beverages with
<15% alcohol by volume
Flavoured processed cheese
Ripened orange, yellow and broken white
cheese; un¯avoured processed cheese
Desserts
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rather, on the total pigment content, usually
expressed as the major component,, i.e. bixin or
norbixin. This is due largely to the shortcomings of
Maximum
permitted available analytical methods, which have generally
level (mg kg 1 ) relied on essentially qualitative techniques and/or
spectrophotometric methods that may be prone to
interference (Scotter et al. 1994). Current research on
10 methods of analysis for annatto have focused largely
20
10 on colour formulations and their thermal stability,
20 where developments in HPLC techniques have en-
abled the separation and determination of the major
10 and minor geometric isomers of both bixin and
15 norbixin (Scotter et al. 1994, 1998, Scotter 1995).
The analysis of food commodities for annatto has
15
10 been concerned mainly with dairy produce and high-
fat foods where annatto has well-established usage,

`Snacks’: dry, savoury potato, cereal or

starch-based products: extruded or expanded
savoury snack products 20
Other savoury snack products and savoury
coated nuts 10
Smoked ®sh 10
Edible cheese rinds and edible casings 20
Red Leicester cheese 50
Mimolette cheese 35
Extruded, puŒed and/or fruit-¯avoured breakfast
cereals 25
seasonings and pickles, dairy and savoury products,
desserts and soft drinks.
The use of food colours in the UK is controlled by a
European Community Directive (EC 1994), which
contains a list of permitted colours, a list of foodstuŒs
to which these colours may be added, and, where
appropriate, maximum limits on the level of addition.
The permitted uses of annatto and the maximum
levels of addition are given in table 1. Annatto
extracts are listed amongst those colours that may
be used singly or in combination in certain foods up
to the maximum levels speci®ed (on a ready-for-
consumption basis). Annatto was reported to be the
most commonly consumed natural colour additive in
the UK (MAFF 1987, 1993) where the per capita
consumption was estimated to be 0.065 mg kg 1
b.w. day 1 based on pure colouring component, re-
presenting some 12.5% of the acceptable daily intake
(ADI).
There is very little analytical data available on the
annatto content of foods. Much of the available data
are non-speci®c in that they do not provide details on
the individual colouring components of annatto but,
such as cheese and dairy spreads, and in some ®ne
bakery wares (Corradi and Micheli 1981, Smith et al.
1983, Lancaster and Lawrence 1995). In contrast,
minimal information is available on methods of ex-
traction for the many other types of food commodity
in which annatto is permitted, such as smoked ®sh,
extruded snack foods, decorations and coatings,
desserts and liqueurs. Validated methods of analysis
are therefore required which encompass all food types
to which annatto may be added, with due regard to
the wide range in composition and formulation of
annatto additives used, i.e. both oil-soluble (bixin)
and water-soluble (norbixin) types and stabilized
forms.
Corradi and Micheli (1981) analysed drinks and
syrups by mixing with water acidi®ed with acetic
acid and extracting annatto into diethyl ether. High
fat products, e.g. butter and margarine were
dissolved in petroleum spirit, annatto was partitioned
into aqueous ammonaical ethanol and then acidi®ed
with acetic acid and back-extracted with diethyl
ether. For foods containing fat and protein, e.g.
yoghurt, cheese and pastries, samples were ground
with sand before extraction. Annatto has been
extracted from meats (McNeal 1976) and from
whey solids with dilute ammonium hydroxide
(Hammond et al. 1975), where proteins were pre-
cipitated by the addition of ethanol and
phosphate buŒer. Annatto was analysed in milk
and ice cream by precipitation with boiling acetic
acid and extraction of the whey with diethyl ether,
and the colour has been extracted from macaroni
and noodles using 80% ethanol followed by back-
extraction into diethyl ether under alkaline conditions
(AOAC 1980).
 Method development and analysis of retail foods for annatto food colouring material
Smith et al. (1983) determined annatto in margarine,
cheese and boiled sweets using techniques similar to
those reported earlier (Corradi and Micheli 1981)
but with modi®cations for measurement by
spectrophotometry and HPLC. Margarine samples
were saponi®ed to enable separation of fat and
to convert any bixin to norbixin, thereby facilitating
its extraction into and puri®cation in aqueous
media. However, HPLC peak shapes and resolution
were poor. A method for the determination of
annatto in cheese has been reported in which a
simple acetone extraction is used followed by
derivative spectrophotometry and HPLC (Luf and
Brandl 1988).
More recently, other workers have developed
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improved methods for the extraction of annatto
from high-fat foods, dairy products and candy
by pre-extraction of fat and extraction of annatto
into ethanolic aqueous ammonia (Lancaster and
Lawrence 1995). HPLC was used to measure
both the cis- and trans-isomers of bixin and
norbixin but peak resolution was poor and no
estimates of absolute or isomeric purity were given
for the reference materials that were used for cali-
bration. Recovery of norbixin from spiked cheese
samples was 93% over the range 1±110 mg kg 1 ,
and the recovery of bixin from spiked cereal
wafer averaged 93% (0.1±445 mg kg 1 ). The recovery
of norbixin from hard candies was 88%. Spectro-
photometric (derivative) and HPLC techniques have
been used to quantify annatto in the presence of other
carotenoids, based on the procedure described by
Brockmann (1984) for the analysis of certain baked
goods. The cis- and trans-isomers of bixin and nor-
bixin were not identi®ed separately under the stated
conditions.
It is clear that available methods are either
purely qualitative or not su ciently broad enough
in scope to cover all of the food commodities
permitted to contain annatto. The method described
by Lancaster and Lawrence (1995) goes some
way towards addressing these problems. Whilst they
reported good extraction e cacy and repeatability
for certain high-fat dairy products, margarine
and hard candy, the methodology was somewhat
labour intensive, and the separation of annatto
colouring components by HPLC was minimal.
207
Materials and methods
Reagents
All reagents were of recognized analytical grade un-
less speci®ed otherwise. Acetic acid, acetonitrile,
methanol and water were HPLC grade.
Annatto standard materials
Standard materials of the 9 0 -cis-isomers of bixin and
norbixin, and the trans-isomer of bixin, were prepared
according to a published procedure (Scotter et al.
1994). The colour content and isomeric purity of the
standards was determined using spectrophotometry
and HPLC respectively. Stock and series dilution
calibration standards were prepared in methanol
and stored in the dark at 5°C before use. Solutions
were purity checked weekly and were discarded after
1 month.
High-performance liquid chromatography (HPLC)
HPLC analysis was carried out using a Hewlett-
Packard 1090M series II DR5 ternary pumping
system with integral variable volume autosampler,
column oven (35°C) and model 1040 series II photo-
diode array detector with HP Pascal workstation
(Hewlett Packard, Bracknell, UK). The column used
was a 250 4.6 mm RPB 5 mm (octyl-/octadecylsilane ,
fully end-capped 14% carbon loading; HiChrom,
Theale UK) and the mobile phase consisted of solvent
A (acetonitrile) and solvent B (0.4% v/v aqueous
acetic acid) delivered at 65%A:35%B at 1 ml min 1 .
UV/VIS detection was carried out at 455 10 nm
bandwidth for annatto with a reference wavelength
of 600 4 nm bandwidth. All samples were ®ltered
through a 0.2 mm membrane ®lter before analysis. The
system was calibrated using mixed reference stan-
dards of all-trans-bixin, 9 0 -cis-bixin and 9 0 -cis-norbix-
in. Calibration standards were prepared in methanol
over the concentration range 0±10 mg l 1 and con-
tained butylatedhydroxytoluen e as antioxidant at
150 mg l 1 . Calibration graphs were constructed
for each analysis batch by plotting concentration of
standard (mg l 1 ) against integrator peak area. Linear
regressio n analysis was carried out on calibration sets
where the R2 obtained were typically 5 0.99. The
 208 M. J. Scotter et al.
concentration of analytes in the sample extracts and
subsequently in the samples was determined from the
calibration line regression equation.
Sampling regimen
A comprehensive list of annatto-containing foodstuŒs
(sorted by product types within a speci®ed food
group) covering the scope of those foods permitted
to contain annatto was obtained from the Food
Additives Information System (FAIS) held by the
UK Food Standards Agency.
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Production of composites
A total of 787 food commodities were obtained from
retail outlets from June to November 1998 and used
to prepare 165 composite and two single samples.
Where possible, each composite contained at least one
product from each of three sampling areas in the UK:
London and the South East, Norwich and York.
Each commodity was assigned to a composite of
similar products. Each composite contained up to
six (except in one case, 12) individual samples, de-
pending on commodity type and product availability.
Similar products were assigned to each composite,
e.g. vanilla ice cream, custard powder or ready meal.
For compound food products such as certain types of
®ne bakery wares and ready meals, composites were
assigned by taking into account the product type and
the ingredient which was speci®ed to contain annatto,
e.g. margarine in a pastry product, cheese in pizza,
although this was not always readily ascertained from
the packaging.
Generally, composites were prepared by taking an
equal mass of each assigned commodity, homogeniz-
ing in a domestic food processor and blending with
the other homogenized samples. Commodities that
were of a highly heterogeneous nature, e.g. ®sh and
compound foods were homogenized whole before
subsampling. About 100 g of each composite was
retained for analysis and stored at ¯ 20°C.
The following regimens were used for speci®c
commodities.
. Cheese: a 50 g subsample of each commodity was
®nely grated, blended with the other grated sub-
samples and stored overnight at ¯ 20°C. Whilst
still frozen, the composite was grated again to
produce a ®ne crumb.
. Margarine: commodities were allowed to reach
room temperature before a 50 g subsample of
each was blended with the other subsamples.
Hand mixing was used to avoid phase separation
of reduced fat spreads.
. Ice cream and edible ices: a 50 g subsample of each
commodity was homogenized before compositing,
taking necessary steps to avoid complete thawing.
. Yoghurt, dried dessert mixes, custard powders,
extruded snacks, biscuits, cake decorations, and
breakfast cereals: a 50 g subsample of each com-
modity was homogenized before compositing.
Dessert mix concentrates and custard powders
were analysed as purchased and not prepared
according to the manufacturer’s instructions. This
was because the extraction regimens that were
speci®cally developed for such dry commodities
allowed for a more rapid throughput of samples
and improved sensitivity. As-consumed dilution
factors were incorporated into the ®nal results.
. Cakes and composite foods: a representative
portion or the whole commodity was homogenized.
A 50 g subsample was then taken for compositing.
. Fish: the whole sample along with any exudate was
homogenized and a 50 g subsample taken for com-
positing.
. Jellies: a representative portion of each commodity
was taken and combined before preparation
according to the manufacturer’s recipe.
Crystalline and powdered jelly concentrates were
analysed as purchased and not prepared according
to the manufacturer’s instructions before analysis
and as-consumed dilution factors were incor-
porated into the ®nal results. Speci®c extraction
regimens were developed for these commodities,
which allowed for a more rapid throughput of
samples and improved sensitivity.
Sample preparation and extraction
Brief descriptions of extraction regimens listed by
food commodity type are given below. Extractions
were carried out in a concerted manner with mini-
mum delay between stages. Extracts of norbixin are
signi®cantly unstable and degrade rapidly, especially
in the light. Containers were therefore covered with
aluminium foil to protect from light where necessary.
All samples were analysed as ready for consumption
 Method development and analysis of retail foods for annatto food colouring material
except for (1) samples that required cooking were not
cooked before analysis and (2) samples such as desert
mix concentrates and jellies which required further
preparation (according to manufacturers instruc-
tions) before consumption were analysed as concen-
trates. In all cases, the results are expressed on an `as
consumed’ basis by applying appropriate dilution
factors were necessary.
Regime 1: cheese, cheese products and cheese-based
compound foods
The sample (3 0.01 g) was accurately weighed into
a 250 ml centrifuge bottle to which was added
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50 ml hexane, 20 ml ethanol:water:ammonia (100:35:
15 v/v/v) solution and 1 ml 0.1% BHT solution in
methanol. The mixture was blended for 10±15 s using
an Ultra-Turrax homogenizer, the bottle stoppered
and shaken vigorously for 1 min. The stopper was
removed occasionally to prevent pressure build-up.
The mixture was then centrifuged at 2500 rpm for
10 min and the upper hexane layer removed by
vacuum aspiration. About 3 g Celite 545 ®lter-aid
and 50 ml hexane were added, the bottle stoppered
and shaken vigorously for 1 min, centrifuged as
before and hexane layer removed.
The ethanolic extract was decanted into a second
centrifuge bottle containing 1 ml 0.1% BHT in meth-
anol. A further 20-ml portion of aqueous ammonaical
ethanol solution was added to the residue in the ®rst
centrifuge bottle and the mixture was homogenized
and centrifuged as before. The ethanolic extracts were
pooled together in the second bottle. This stage was
repeated until no further colour could be extracted.
Acetic acid solution (11%, 50 ml) was added to the
combined ethanolic extracts, the bottle stoppered and
the solution mixed. Chloroform:acetic acid solution
(98.5:1.5 v/v, 45 ml) was added, the bottle stoppered
and shaken with venting. The mixture was centrifuged
at 2500 rpm for 10 min and the upper aqueous layer
removed by vacuum aspiration. The chloroform was
decanted through a glass wool pledget contained in
the neck of a glass ®lter funnel, into a 250 ml rotary
evaporating ¯ask containing 1 ml 0.1% BHT in
methanol solution.
The residue in the centrifuge bottle was rinsed with
20 ml chloroform:acetic acid solution, which was sub-
sequently decanted into the rotary evaporating ¯ask.
The glass wool was rinsed with a few millilitres of
209
chloroform:acetic acid if necessary to remove any
colour. The solvent was removed to near dryness
using vacuum rotary evaporation (440°C) taking
care not to allow the extract to dry.
The concentrated extract was quantitatively trans-
ferred to a 25 ml volumetric ¯ask with methanol
and diluted to the mark. After mixing, a 2-ml aliquot
was transferred to a glass syringe ®tted with a micro-
®lter (0.2 mm) and subsequently ®ltered into a vial for
analysis by HPLC.
Regime 2: custard powder and low-fat dessert dry
mixes
The sample (3 0.01 g) was accurately weighed into a
250 ml centrifuge bottle and 3 g Celite 545 ®lter-aid
added along with 1 ml BHT in methanol solution and
20 ml aqueous ammonaical ethanol solution. The
bottle was stoppered and shaken vigorously for
1 min, removing the stopper occasionally to prevent
pressure build-up. The mixture was then centrifuged
at 2500 rpm for 10 min.
The ethanolic extract was decanted through a glass
wool pledget contained in the neck of a glass ®lter
funnel, into a 250 ml separating funnel and the neck
of the bottle rinsed with a few drops of ethanol
solution. Aqueous ammonaical ethanol solution
(20 ml) was added to the centrifuge bottle and the
extraction repeated until no further colour could be
extracted.
To the combined ethanolic extracts in the separating
funnel was added 20 ml 50% acetic acid solution,
after which the funnel was stoppered and the contents
mixed. Chloroform:acetic acid solution (40 ml) was
added and the funnel again stoppered and shaken,
with venting. The chloroform layer was transferred to
a 250 ml rotary evaporation ¯ask containing 1 ml
BHT in methanol solution.
Any colour remaining in the aqueous layer was
treated with further portions of 10 ml 50% acetic acid
and 20 ml chloroform:acetic acid solution as above.
The chloroform extracts were combined in the rotary
evaporation ¯ask and the solvent removed to near
dryness as above, taking care not to allow the extract
to dry. The extract was then treated as in Regime 1
for analysis by HPLC.
 210 M. J. Scotter et al.
Regime 3: desserts, cake decorations, ®ne bakery
wares (including cakes and biscuits), extruded
snacks and breakfast cereals
The ®nely homogenized sample was accurately
weighed (10 g 0.01 g) into a 250 ml centrifuge bottle,
except for custard powder where 3 0.01 g sample
was taken. The samples were then extracted as
follows.
About 3 g Celite 545 ®lter-aid, 1 ml BHT in methanol
solution and 20 ml aqueous ammonaical ethanol
solution were added to the sample. The centrifuge
bottle was stoppered and shaken vigorously for 1 min,
removing the stopper occasionally to prevent pressure
build-up. The mixture was then centrifuged at
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2500 rpm for 10 min.
For fat-containing commodities such as cake, biscuit,
extruded snack and breakfast cereals, a preliminary
fat wash was required. To the other reagents in the
centrifuge bottle was added 50 ml hexane. The bottle
was then stoppered and shaken vigorously for 1 min,
removing the stopper occasionally to prevent pressure
build-up. The mixture was centrifuged at 2500 rpm
for 10 min and the upper hexane layer removed by
vacuum aspiration . A further 50 ml hexane was added
to the centrifuge bottle and the fat extraction re-
peated. The extracts were then treated as in Regime
1 following removal of the hexane layer.
Regime 4: margarine, fat-based emulsions and
spreads, butter and fat-based compound foods
The sample (8 0.01 g) was accurately weighed into a
50 ml centrifuge tube and 2 g Celite 545 ®lter-aid,
1 ml BHT in methanol solution and 15 ml ethanol
solution added. After mixing with a glass rod, 10 ml
hexane was added, the tube stoppered and shaken
vigorously for 1 min, removing the stopper occasion-
ally to prevent pressure build-up. The tube was then
centrifuge at 2500 rpm for 10 min and the upper
hexane layer removed by vacuum aspiration. A fresh
10 ml portion of hexane was added and the fat
extraction repeated.
The ethanolic extract was transferred with a Pasteur
pipette through a glass wool pledget contained in
the neck of a glass ®lter funnel, into a 100 ml separat-
ing funnel. Aqueous ammonaical ethanol solution
(15 ml) was added to the centrifuge tube and the
extraction repeated until no further colour could be
extracted.
To the combined extracts in the separating funnel was
added 10 ml 50% acetic acid solution whereupon
the funnel was stoppered and the contents mixed.
Chloroform:acetic acid solution (20 ml) was added
and the funnel once more stoppered and shaken with
venting. The chloroform layer was transferred to a
100 ml rotary evaporation ¯ask containing 1 ml BHT
in methanol solution.
Any colour remaining in the aqueous layer was
treated with further addition of 10 ml 50% acetic acid
and 20 ml chloroform:acetic acid solution and the
combined chloroform extracts transferred to a rotary
evaporation ¯ask. The solvent was removed and the
remaining extract treated as in Regime 1.
Regime 5: ®sh, including brine-cured or smoked
`white’ ®sh (e.g. cod and haddock) and `oily’ ®sh
(e.g. herring and mackerel); ice cream, ice cream-
based confectionery, yoghurt and other dairy
desserts
Fish. About 5 g of sample homogenate was
accurately weighed into a beaker along with 5 g
Celite 545 ®lter-aid. The sample was then mixed to
absorb any liquid and carefully transferred to a
mortar. The mass was intimately ground to a friable
consistency, whereupon 0.2 ml 2 m hydrochloric
acid was added and the grinding continued until
the mixture was homogeneous. The mixture was
transferred to a 250 ml centrifuge bottle and
200 mg ascorbyl palmitate, 50 ml hexane and
25 ml acetonitrile added.
Ice cream, ice cream-based confectionery, yoghurt and
other dairy desserts. The sample (5 0.01 g) was
accurately weighed into a 250 ml centrifuge bottle
whereupon 3 g Celite 545 ®lter-aid, 1 ml BHT
solution in methanol and 0.2 ml 2 m hydrochloric
acid were added. The contents were mixed well with
a glass rod and 25 ml acetonitrile and 50 ml hexane
added.
For both regimens:
. the bottle was then stoppered and shaken vigor-
ously for 1 min. The mixture was centrifuged at
2500 rpm for 10 min and the upper hexane layer
 Method development and analysis of retail foods for annatto food colouring material 211

removed by vacuum aspiration. A fresh 50 ml
portion of hexane was added to the bottle and the
fat extraction repeated;
. the acetonitrile layer was decanted through a ®lter
funnel incorporating a glass wool plug in the stem,
into a 250 ml rotary evaporation ¯ask containing
1 ml BHT in methanol solution. A fresh 25 ml
portion of acetonitrile was added to the centrifuge
bottle, whereupon it was stoppered and shaken
vigorously for 1 min. The mixture was centrifuged
at 2500 rpm for 10 min and the acetonitrile
decanted into a rotary evaporation ¯ask. A fresh
25 ml portion of acetonitrile was added to the
centrifuge bottle and the extraction repeated.
The acetonitrile extraction stage was repeated
until no more colour could be extracted from the
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For validation purposes, a range of food commodities
known not to contain annatto were spiked with either
norbixin or bixin at appropriate levels and analysed
repeatedly to ascertain recovery. Spiking was
achieved by adding the appropriate amount of bixin
or norbixin reference standard to a homogenized
subsample contained in the extraction vessel immedi-
ately before analysis. The annatto levels used for
spiking are appropriate to current usage levels as
determined from known data or from preliminary
analyses. Validation (recovery) data for food matrices
spiked with annatto are given in table 2.
Analysis was carried out on a group basis. A
batch analysis regimen was used in which the
analytical system was calibrated for each batch.

sample. The glass wool plug in the stem of the ®lter
funnel was rinsed with a few millilitres of aceto-
nitrile; and
. the volume of the pooled acetonitrile extracts
was reduced by rotary evaporation (as above) to
ca 20 ml, initially at a vacuum setting of 200
mbar to avoid foaming. The vacuum was then
reduced further if required until the extract volume
had been reduced to 1±2 ml. The concentrated
extract was then quantitatively transferred to a
25 ml volumetric ¯ask containing 1 ml BHT sol-
ution with methanol and diluted to the mark,
mixed well, micro®ltered and analysed by HPLC
as above.
An appropriate in-house reference material
(IHRM) sample, a reagent blank and one sample
analysed in duplicate were included in each
batch along with a set of calibration standards.
If the results for the analyses of the reagent blank,
the duplicate sample or the IHRM fell outside pre-
determined limits (essentially 2 SD units calculated
from repeat analysis of validation samples), the batch
was repeated.
Validation data for the IHRM’s, i.e. cheese, custard
powder, ice cream and margarine are presented in
table 3. Each IHRM was utilized where appropriate
to the extraction method used in the analysis batches.

Table 2. Recovery data for food matrices spiked with

Quality control annatto.
Spiking Mean
Method validation. It was apparent that the level recoverye RSD
relatively low levels of annatto used in commodities Food matrix n (mg kg 1 ) (%) (%)
such as ice cream and yoghurt are representative of a Breakfast cereal 7 22.2 79 7.7
large proportion of those commodities identi®ed Cheese (1) 4 22.4 75 3.5
in the Food Standards Agency Food Additives Cheese (2) 7 3.0 90 2.6
Database (12 and 60% for ice cream and desserts Custard powdera 9 27.7 82 7.9
respectively) . Whilst very low limits of determination Dessertsb 7 13.9 85.6 5.1
were not a primary consideration of this project, Extruded snacks 8 9.0 61 6.1
methods were developed with su cient scope to Fish 6 8.5 64 4.2
Ice cream 0.8 96 2.1
allow for the determination of annatto down to Jellyc 7 5.7 83 7.1
0.1 mg kg 1 , depending upon the food commodity Margarine 8 1.4 94 6.1
analysed. The limit of detection (LOD) of the Popcornd 6 20.8 91 4.3
method was 0.01 mg kg 1 calculated from an analyte Cake 7 13.9 96 2.4
peak with a signal-to-noise ratio ˆ 5. The limit Yoghurt 6 1.7 92 4.8
of quanti®cation (LOQ) was 0.1 mg kg 1 calculated a
Spiked corn¯our was used to simulate custard powder; b dry mixes;
from an analyte peak with a signal-to-noise c
prepared before use according to manufacturers instructions; d micro-
ratio ˆ 10. waveable; e found/added 100.
 212 M. J. Scotter et al.

Table 3. In-house reference materials analysis data. A spectral bandwidth of 10 nm was applied to
extend the monitoring wavelength band to
Number of Mean annatto
IHRM replicates content (mg kg 1 ) RSD (%)
455 5 nm to ensure sensitive detection of trans-,
monocis- and dicis-isomers without compromising
Cheese 8 34.3 4.8 on selectivity. A reference wavelength of 600 4 nm
Custard powder 11 59.4 1.1 bandwidth was used to correct for background
Ice cream 7 3.2 4.5 absorbances and baseline drift. The HPLC-PDA
Margarine 9 2.6 8.9
separation of a standard mixture is given in ®gure 1
with the corresponding photodiode array spectra in
®gure 2.
A ¯ow cell of 10 mm ¯ow-path length was used in
Where there was no closely matched IHRM available
for a particular batch, a surrogate was used, e.g. ice conjunction with an optical slit width of 4 nm. This
has been shown in previous studies to give the best
cream IHRM was used for ®sh.
compromise between signal-to-noise (sensitivity) and
spectral resolution (Scotter et al. 1994). Con®rmation
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Optimization of HPLC analytical selectivity and
sensitivity. The monitoring wavelength of the
photodiode-array detector was optimized at 455 nm
to detect all bixin and norbixin isomers simul-
taneously at wavelengths close to their correspond-
ing peak maxima, thus providing good sensitivity.
of analyte peaks was achieved through peak retention
time matching and comparison of analyte and stan-
dard spectra, facilitated by the use of a spectral
library. Both the ¶max shift and cis-peak measurement
provided the means by which the diŒerent stereo-
isomers could be identi®ed.

mAU
90 4

80

3
70

60

50

40

30 6

20

10
12
5 9
7 8
0

Retention time (min)

10 15 20 25 30 35

Figure 1. HPLC-PDA separation of annatt o colour principles. Conditions as given in text. Assignment of peaks: 1Ð
Trans-norbixin; 2ÐDicis-norbixin; 3Ð9 0 cis-norbixin; 4ÐTrans-bixin; 5 and 9ÐDicis-bixin isomers; 6Ð9 0 -cis-
bixin; 7Ð15-cis-bixin*; 8Ð13 0 -cis-bixin* (tentative).
 Method development and analysis of retail foods for annatto food colouring material 213

The steps taken to achieve this involved minimizing

Rnorm
(a) 1 the number of times that extracts were transferred
80
2 between diŒerent sets of glassware, reducing evapora-
3 tion times and temperatures when removing solvents,
60 and by carrying out analyses under subdued light
conditions.
40

20

0
Developments made to the Lancaster and Lawrence
procedure
300 350 400 450 500 nm

Sample sizes were generally reduced (from 20 g) to

Rnorm
( b) 1 minimize protein precipitation in the later stages of
2 extraction. Hexane was preferred to petroleum spirit
80
3 for the removal of fat largely because of availability .
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60 An Ultra-Turrax homogenizer was used to give im-

40
20
0 tated by the use of a vacuum aspiration system, rather
300 350 400 450 500
Figure 2. Photodiode array spectra of annatto colour
principles. (a) Norbixin isomers: 1ÐDicis-; 2Ð9 0 -cis-
and 3ÐTrans-. (b) Bixin isomers: 1Ð13 0 -cis- (tenta-
tive); 2Ð9 0 -cis-; 3ÐTrans-.
Results and discussion
Method development
The main factors associated with development of
methods for annatto analysis in the food commodities
tested have been the stability of the analyte (i.e.
isomeric as well as oxidative stability), e cacy of
extraction (especially from protein-containing foods),
fat content of sample (i.e. requiring a preliminary fat
extraction step) and precipitation of protein during
extraction. In most cases, the best attributes from the
published and in-house methods were combined and
streamlined where necessary without compromising
e cacy. To this end, considering the numbers of
samples to be analysed in this study, methods were
improved in general terms by reducing the analysis
time and minimizing exposure of extracts to heat,
saponi®cation, light and pro-oxidative agents.
proved homogenization of sample (especially cheeses
and compound foodstuŒs) with extraction solvent(s).
Removal of hexane/fat from the sample was facili-
nm than by the use of pipettes. Centrifugation speed was
increased to 2000±2500 rpm to facilitate decantation
and to improve interface integrity during solvent
partition, especially where interfacial precipitation
of protein occurred.
Colour fading was observed in some food extracts so
BHT was added as antioxidant. BHT is reported to
have a protective eŒect on carotenoids in solution by
acting as a free-radical scavenger and is recommended
as an additive in solvents used for their extraction and
isolation from food materials and serum (Scott 1992).
In an eŒort to reduce analysis time and exposure of
extracts to glassware, the third stage of the centrifu-
gation procedure was omitted. This did not appear to
have a signi®cant eŒect on extraction e cacies.
Annatto and curcumin in ®sh
In our laboratory, extensive work has been conducted
on annatto chemistry as well as analytical methods
and purity speci®cations for annatto (Scotter et al.
1994, 1998, 2000, Scotter 1995), from which a method
for the extraction and determination of annatto (and
curcumin) in smoked ®sh has recently been developed
(Scotter, unpublished data). The extraction is based
on the ¯uorimetric determination of curcumin in
yoghurt and mustard as described by Navaz Diaz
and Ramos Peinado (1992). Since mixed annatto and
 214 M. J. Scotter et al.
curcumin colour formulations are often used com-
mercially to colour ®sh and other commodities, a
method was required that could determine them
simultaneously. However, curcumin is reported to
be highly unstable under alkaline conditions
(Tùnessen and Karlsen 1985) hence the Lancaster
and Lawrence (1995) procedure for annatto, which
requires the use of ethanolic aqueous ammonia as the
extracting solvent, was not suitable.
To facilitate homogenization of the ®sh, the sample
was ground in a pestle and mortar in the presence of
Celite ®lter aid. A small amount of hydrochloric acid
solution was added to promote release of the colours
from the food matrix and to neutralize the eŒects of
any bases present. Ascorbyl palmitate was added to
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the sample at the extraction stage since signi®cant
colour fading was observed in ®sh extracts, which
could only be partially ameliorated by the addition of
BHT. According to Najar et al. (1988) light is the
most destructive agent for annatto extracts, followed
by the pro-oxidant benzoyl peroxide. Air was re-
ported to be much less eŒective in promoting colour
loss and ascorbyl palmitate eŒectively retarded the
destructive eŒect of light. Ascorbyl palmitate is an
eŒective antioxidant for fats and oils and was selected
because of the similarity of oxidative mechanisms for
lipids and for carotenoids. Moreover, it is also used as
an antioxidant in oil-soluble vitamin analysis. This is
particularly important during initial extraction of
food matrices where any number of chemical pro-
oxidants may be present. Addition of ascorbyl palmi-
tate to the sample homogenate during the initial
extraction stage improved extraction e ciency and
sample extract integrity to an acceptable level.
Owing to the high oil content of ®sh such as mackerel
and herring, a fat extraction procedure similar to
that described in the Lancaster and Lawrence method
was introduced. In order to facilitate removal of the
hexane/fat mixture, a centrifugation procedure was
also introduced in place of the original ®ltration. A
minimum of three portions of acetonitrile was re-
quired to remove all of the colour from most samples.
These were subsequently pooled before concentration
by rotary evaporation. As in the modi®ed Lancaster
and Lawrence method, qualitative and quantitative
analysis was performed using HPLC with photodiode
array detection instead of spectro¯uorimetry, since
the amount of water required in the mobile HPLC
phase to eŒect a suitable separation of the compon-
ents severely quenched any ¯uorescence of the curcu-
minoids. This method was also developed for the
analysis of ice cream, ice cream-based confectionery,
yoghurt and other dairy products.
The method developed for ®sh analysis, was notice-
ably more rapid than the Lancaster and Lawrence
procedure and it utilized HPLC with photodiode
array detection. It also allowed both the 9 0 -cis-
and trans-isomers of bixin and the 9 0 -cis-isomer
of norbixin to be determined quantitatively, and
other minor cis-isomers of bixin and norbixin semi-
quantitatively.
Conclusions
Extensive development has been undertaken on
methods for annatto extraction from a wide range
of food types for which published methods were not
available for many of them, by adapting two pub-
lished procedures. Reference materials of 9 0 -cis-bixin
and norbixin, and trans-bixin were prepared and
assayed according to published procedures and used
to determine the e cacy of extraction and to calibrate
HPLC systems. The determination of annatto colour
principles and minor isomers was improved in general
terms to meet the requirements of the speci®c extrac-
tion procedures.
The reverse-phase HPLC system used allowed for
qualitative identi®cation and quantitation of colour
principles and minor cis-isomers of bixin and norbix-
in. For the major colour principals 9 0 -cis-bixin, 9 0 -cis-
norbixin and trans-bixin are quantitative but since
authentic reference standards were not available for
the minor bixin and norbixin isomers monocis- (not
9 0 -), di-cis- and for trans-norbixin, appropriately
matched surrogate standards were used, hence the
®gures given for these analytes are to be regarded as
semi-quantitative only. The analyte peaks for these
compounds were identi®ed from their characteristic
UV-VIS spectra and relative retention times (Scotter
et al. 1994).
A total of 165 composite samples and 2 individual
samples have been analysed and the results presented
in table 4. The variability of the results are reported
for each commodity type and have been estimated
from the analytical result the relative SD (RSD%)
calculated from the repeat analyses of spiked com-
modities and IHRMs (tables 2 and 3). The results
have not been corrected for recovery but have been
adjusted for the analysis of concentrates such as
 Method development and analysis of retail foods for annatto food colouring material 215

Table 4. Annatto content of selected foods.a c

Data type
1
Quantitative (mg kg ) Semi-quantitative (mg kg 1 †

Composite 9 0 -cis- trans- 9 0 -cis- Other bixin trans- Other norbixin

number Composite description Bixin Bixin Norbixin isomers Norbixin isomers

Red Leicester cheese (maximum permitted level 50 mg kg 1 ) variability of resultsd ˆ 5%

001 Red Leicester ND ND 14.1 ND 15.6 4.1
002 Red Leicester ND ND 16.4 ND 16.0 3.9
003 Red Leicester ND ND 16.6 ND 17.0 3.9
004 Red Leicester, low fat ND ND 9.0 ND 12.0 2.7
Flavoured processed cheese (maximum permitted level 15 mg kg 1 ) variability of resultsd ˆ 5%
008 Double Gloucester, with herbs ND ND 1.3 ND 2.6 0.2
013 Edam with onion and garlic ND ND 0.4 ND 2.4 0.2
022 Cheese slices ND ND 0.3 ND 0.4 0.1
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023 Processed cheese snack ND ND 1.4 ND 3.2 0.5

024 Cottage cheese, ¯avoured ND ND 0.3 ND 0.4 0.1
030 Cheese spread ND ND 0.1 ND 0.1 ND
033 Coloured Cheddar, ¯avoured ND ND 9.1 ND 10.3 2.0
164 Microwaveable cheese sauce 0.4 0.6 2.8 ND 0.8 ND
Ripened orange, yellow and broken-white cheese; un¯avoured processed cheese (maximum permitted level 15 mg kg 1 ) variability of
resultsd ˆ 5%
005 Double Gloucester ND ND 4.0 ND 4.5 0.7
006 Double Gloucester ND ND 3.0 ND 3.6 0.7
007 Double Gloucester ND ND 2.9 ND 6.1 0.6
009 Double Gloucester, low fat ND ND 0.2 ND 1.1 0.2
010 Edam, coloured ND ND 0.2 ND 0.7 0.1
011 Edam, coloured ND ND 0.4 ND 2.0 0.2
012 Blue Shropshire/Buxton ND ND 2.3 ND 1.1 0.2
014 Gouda, coloured ND ND 0.2 ND 0.4 0.1
025 Cheddar, light ND ND 0.1 ND 0.1 ND
026 Cheddar, dark ND ND 2.5 ND 3.4 0.3
027 Cheddar, dark ND ND 3.3 ND 2.7 0.2
028 Cheddar, dark ND ND 4.4 ND 3.6 0.3
029 Cheddar, dark ND ND 3.7 ND 4.4 0.5
031 Soft cheese with rind ND ND 1.3 ND 1.4 0.2
032 Coloured Cheshire ND ND 1.3 ND 1.3 0.2
034 Coloured Gouda ND ND 0.5 ND 0.9 0.2
Margarine, minarine, other fat emulsions, and fats essentially free from water (maximum permitted level 10 mg kg 1 ) variability of
resultsd ˆ 9%
015 Sun¯ower spread 2.4 0.9 ND 0.8 ND ND
016 Sun¯ower spread, low-fat 2.6 0.9 ND 0.9 ND ND
017 Butter substitute 2.9 0.9 ND 1.0 ND ND
018e Block 0.8 0.2 ND ND ND ND
019 Soft 1.7 0.8 ND 0.8 ND ND
020 Reduced fat spread 1.6 0.8 ND 0.8 ND ND
021 Soya spread 1.8 0.8 ND 0.8 ND ND
1
Smoked ®sh (maximum permitted level 10 mg kg ) variability of resultsd ˆ 4%
035 Kippers, with butter ND ND 1.5 ND 0.7 ND
036 Kippers, with butter ND ND 4.6 ND 1.6 0.2
037 Haddock ND ND 0.3 ND 0.1 ND
038 White ®sh ND ND 0.3 ND 0.1 ND
039 Haddock ND ND 0.3 ND 0.1 ND
040 Mackerel ND ND 6.2 ND 1.8 0.3
041e Haddock, with butter ND ND 0.2 ND ND ND
(continued )
 216 M. J. Scotter et al.

Table 4Ðcontinued
Data type

Quantitative (mg kg 1 ) Semi-quantitative (mg kg 1 †

Composite 9 0 -cis- trans- 9 0 -cis- Other bixin trans- Other norbixin

number Composite description Bixin Bixin Norbixin isomers Norbixin isomers

Edible ices (maximum permitted level 20 mg kg 1 ) variability of resultsd ˆ 5%

042 Vanilla ND ND 2.4 ND 1.2 0.3
043 Vanilla ND ND 2.5 ND 1.0 0.3
044 Vanilla, block ND ND 2.6 ND 1.1 0.3
045 Cornish ND ND 5.3 ND 2.9 0.5
048 Raspberry ripple ND ND 3.3 ND 1.0 0.3
050 Neapolitan ND ND 2.0 ND 0.7 0.2
056 ToŒee/Banana ND ND 2.5 ND 0.8 0.2
158 ToŒee/meringue roulade ND ND 1.0 ND 0.5 ND
049 Choc Ices ND ND 1.6 ND 1.1 0.2
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051 Vanilla ND ND 1.1 ND 0.6 0.2

052 Mixed Splits ND ND 0.5 ND 0.5 0.2
053 Cones and wafers ND ND 0.6 ND 0.4 0.2
054 Fruit lollies ND ND 8.3 ND 1.8 1.0
055 Sponge Rolls ND ND 2.8 ND 1.3 0.3
046 Choc-Ices ND ND 0.7 ND 0.5 0.2
047 Choc-Ices ND ND 1.1 ND 0.6 0.2
057 Banana and toŒee bars ND ND 2.3 ND 0.8 0.2
058 Iced lollies ND ND 1.3 ND 0.4 0.4
059 Chocolate ND ND 0.5 ND 0.4 0.2
067 Mixed fruit splits ND ND 1.0 ND 0.7 0.2
Snacks; extruded or expanded savoury snack products (maximum permitted level 20 mg kg 1 ) variability of resultsd ˆ 6%
061 Extruded snack, cheese ¯avoured 3.4 1.8 1.1 0.5 1.5 0.9
062 Extruded snack, cheese ¯avoured 3.2 2.5 0.5 0.6 0.5 ND
063 Extruded snack, prawn ¯avoured ND ND 0.6 ND 1.4 0.5
064 Extruded snack, prawn ¯avoured ND ND 0.6 ND 1.5 0.5
068 Extruded snack, cheese ¯avoured 3.4 7.6 1.0 2.5 1.3 1.0
092 Extruded snack, mixed ¯avours ND ND 0.3 ND 0.3 0.3
Snacks; other savoury snack products and savoury coated nuts (maximum permitted level 10 mg kg 1 ) variability of resultsd ˆ 6%
091 Popcorn ND ND 0.8 ND 0.8 0.7
093 Tortilla chips/French fries ND ND 0.6 ND 0.9 0.4
095 Oriental rice crackers ND ND ND ND ND ND
Extruded, puŒed and/or fruit-¯avoured breakfast cereals (maximum permitted level 25 mg kg 1 ) variability of resultsd ˆ 6%
094 Extruded cereals ND ND 4.1 ND 4.3 0.9
120 Breakfast cereal, toastable with jam 0.5 0.7 2.2 1.2 1.1 0.2
Fine bakery wares (maximum permitted level 10 mg kg 1 ) Variability of resultsd ˆ 5%
065 Swiss rolls ND ND 0.1 ND ND ND
066 Swiss rolls ND ND 0.3 ND 0.2 ND
069 Scones ND ND 0.1 ND 0.1 ND
060 Swiss rolls ND ND 0.5 ND 0.2 0.1
072 Gateau ND ND 0.3 ND 0.2 ND
073 Gateau ND ND 0.1 ND 0.2 ND
074 Cake, saŒron/cinnamon 0.1 0.1 0.3 ND 0.8 0.1
075 Cake, Battenburg 0.2 0.3 ND ND ND 0.1
076 Cake, lemon/orange ®lling ND ND 2.4 ND 1.6 0.1
077 Cake, sponge ND ND 0.1 ND 0.6 ND
078 Cake, Swiss rolls 0.1 0.2 0.2 ND 0.3 0.1
079 Jam tarts, apricot ND ND 0.2 ND 0.2 0.1
080 Cakes, iced ND ND 0.1 ND 0.2 ND
 Method development and analysis of retail foods for annatto food colouring material 217

Table 4Ðcontinued
081 Cakes, mixed Danish, twists ND ND ND ND ND ND
and splits
082 Cakes, with custard ND ND 0.1 ND ND ND
083 Biscuits, shortbread ND ND 0.3 ND 0.3 ND
084 Cakes, cream ND ND ND ND ND ND
085 Cakes, tarts ND ND ND ND ND ND
086 Biscuits, cream 0.5 0.2 0.3 ND 0.3 ND
087 Biscuits, cream 0.2 0.2 0.1 ND ND ND
088 Cocktail snacks ND ND 1.8 ND 1.8 0.6
089 Wafers ND ND 2.3 ND 0.9 0.3
090 Brandy snap ND ND 0.1 ND ND ND
096 Fruit cake N/A
128 Gateaux, orange/lemon ND ND 0.5 ND 0.3 ND
151 Cakes, chilled/frozen ND ND 0.4 ND 0.3 ND
152 Cakes, chilled/frozen 0.2 0.2 1.3 ND 0.7 ND
153 Cakes, chilled/frozen ND ND 0.2 ND 0.3 ND
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154 Cakes ND ND 0.1 ND 0.2 ND

156 Cakes, dessert 0.1 0.1 ND ND ND ND
Desserts (maximum permitted level 10 mg kg 1 ) variability of resultsd ˆ 5%
070 Cheesecake ND ND 1.7 ND 0.6 0.1
071 Cheesecake N/A
097 Tri¯e, strawberry ND ND 0.8 ND 0.4 0.1
098 Tri¯e, raspberry ND ND 0.8 ND 0.4 0.1
099 Tri¯e ND ND 0.3 ND 0.3 0.1
100# Tri¯e mix ND ND 12.6 ND 3.0 0.7
101 Jelly, strawberry ND ND 1.3 ND 0.2 0.1
102# Jelly crystals ND ND 67.2 ND 10.8 1.6
103 Jelly ND ND 0.8 ND 0.12 ND
104# Custard powder ND ND 48.4 ND 7.6 0.9
105# Custard, instant mix ND ND 6.6 ND 2.4 0.4
106 Custard, ready-to-serve ND ND 0.7 ND 1.5 0.2
107 Creme caramel ND ND 0.4 ND 0.8 0.2
108# Instant dessert mix ND ND 6 ND 1.6 ND
109# Instant dessert mix ND ND 4.8 ND 0.8 ND
110# Instant dessert mix ND ND 23.4 ND 4.6 0.5
111# Custard powder ND ND 25.3 ND 6.6 1.0
112# Instant dessert mix ND ND 19.6 ND 3.7 0.8
113 Custard style yoghurt ND ND 0.4 ND 0.2 0.2
114 Custard style yoghurt ND ND 0.3 ND 0.1 0.3
115 Fool, apricot ND ND 0.3 ND 0.5 0.1
116 Yoghurt ND ND 0.2 ND 0.1 ND
117 Yoghurt ND ND 2.2 ND 1.4 0.2
118 Yoghurt ND ND 0.2 ND 0.2 0.1
119 Yoghurt, peach ND ND 0.4 ND 0.2 0.1
135 Yoghurt, orange ND ND 2.7 ND 0.8 0.2
136 Yoghurt, apricot ND ND 0.5 ND 0.3 ND
137 Yoghurt, fruit ND ND 0.4 ND 0.3 0.2
138 Yoghurt, tropical ND ND 0.3 ND 0.2 ND
139 Yoghurt, peach ND ND 0.3 ND 0.2 ND
140 Cheesecake, individual ND ND 0.4 ND 0.2 0.1
141 Cheesecake ND ND 1.0 ND 1.3 0.4
150 Gateaux, meringue 0.1 0.1 0.1 ND ND ND
155 Sponge pudding with custard ND ND 0.1 ND 0.4 ND
157 Mousse, raspberry ripple ND ND 0.3 ND 0.8 0.2
159 Yoghurt, fruit low-fat ND ND 0.1 ND 0.1 ND
160 ToŒee/fudge/banana ¯avour ND ND 0.1 ND 0.3 0.1
161 Jelly/mousse deserts ND ND 0.5 ND 0.5 0.1
(continued )
 218 M. J. Scotter et al.

Table 4Ðconcluded
Data type
1
Quantitative (mg kg ) Semi-quantitative (mg kg 1 †

Composite 9 0 -cis- trans- 9 0 -cis- Other bixin trans- Other norbixin

number Composite description Bixin Bixin Norbixin isomers Norbixin isomers

162 Tri¯es, mixed fruit ND ND 0.2 ND 0.3 0.1

163 Chocolate custard/mousse ND ND 0.1 ND 0.1 ND
165# Dessert/cake mixes ND ND 2.6 ND 1.2 0.1
166# Dessert mixes ND ND 8.4 ND 1.8 0.2
167# Custard, instant mix ND ND 4.2 ND 3.0 0.4
168 Mousse, orange/apricot ND ND 0.6 ND 0.3 ND
170 Milk shake ND ND 0.4 ND 1.0 0.3
Compound foods/ready meals. Variability of resultsd ˆ 9%
121 Pasta ready meal ND ND 0.2 0.2 ND
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122 Pasta ready meal ND ND ND ND ND ND

123 Pasta ready meal ND ND ND ND ND ND
124 Pasta ready meal ND ND ND ND ND ND
125 Pizza ND ND 0.2 ND 0.2 ND
126 Pizza ND ND 0.2 ND 0.2 ND
127 Pizza ND ND 0.2 ND 0.2 ND
129 Pasta ready meal ND ND 0.1 ND 0.2 0.1
130 Pasta ready meal ND ND ND ND ND ND
131 Lattice pie ND 0.1 ND ND ND ND
132 Fish pie ND ND 0.2 ND 0.2 0.1
133 Garlic bread with cheese ND ND 0.2 ND 0.2 ND
134 Garlic bread ND ND ND ND ND ND
142 Ready meals ND ND ND ND ND ND
143 Fish product N/A ND
144 Ready meals ND ND 0.1 0.1 ND
145 Breaded ®sh ND ND 0.1 ND 0.4 ND
146 Smoked haddock ready meal ND ND 0.2 ND 0.3 ND
147 Ready meals ND ND ND ND ND ND
148 Ready meals ND ND 0.2 ND 0.2 ND
149 Ready meals ND ND 0.1 ND 0.3 ND
Decorations and coatings (maximum permitted level 20 mg kg 1 ) variability of resultsd ˆ 5%
169 Sugar strands/jelly slices 1.5 0.4 0.8 ND 0.2 ND
a
Limit of quanti®cation (LOQ) 0.1 mg kg 1 ; b results not corrected for recovery; c results reported on an `as consumed’ basis by
applying appropriate dilution factor where necessary; d results variability estimated from relative standard deviation (RSD%)
obtained from repeat analysis of spiked samples. ND, not detected i.e. <LOQ; N/A, not analysed (samples 071, 096 and 143
were replicate samples of composites already analysed). e Single samples, not composites. # Sample analysed as received and not
as prepared according to manufacturer’s instructions before analysis but result not reported on `as consumed’ basis (see c ).

custard powders and dry dessert mixes, to allow for
preparation as per the commodity package instruc-
tions. The determined estimated levels found in com-
modities selected for analysis in this project are
summarized by commodity in table 5. These data
show at a glance the estimated range of results
obtained for each commodity and compares the
®gures with the appropriate maximum permitted
levels of annatto. Examples of chromatograms ob-
tained from extracts of smoked ®sh, cake decoration,
extruded snack, mousse dessert and Red Leicester
cheese are given in ®gures 3±7.
Validated extraction methods utilizing modern HPLC
analysis techniques coupled with spectral con®rma-
tion have been developed. These have provided accu-
rate and precise qualitative and quantitative data on
the annatto content of a selected range of foods
currently on sale in the UK. Isomer pro®les of the
major bixin and norbixin colour principles have been
 Method development and analysis of retail foods for annatto food colouring material 219

Table 5. Ranges of annatto content of retail foods.

Maximum permitted Number of Esimated range
Food commodity type level (mg kg 1 ) composites analysed (mg kg 1 )a

Margarine, minarine, other fat emulsions, etc. 10 7 1.0±4.8

Decorations and coatings 20 1 2.9
Fine bakery wares 10 29 ND±4.2
Edible ices 20 20 1.1±11.1
Red Leicester cheese 50 4 23.7±37.5
Flavoured processed cheese 15 8 0.2.±21.4
Ripened orange, yellow and broken white, cheese etc. 15 16 0.2±9.6
Desserts 10 44 0.2±5.7
Dry, savoury potato, cereal snacks, etc. 20 6 0.9±16.8
Other savoury snack products and nuts 10 3 ND±2.3
Smoked ®sh 10 7 0.3±8.3
Extrude, puŒed, fruit ¯avoured breakfast cereals 25 2 4.7±9.3
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Compound foods and ready meals N/A 20 ND±0.5

a
Results are derived from combined quantitative and qualitative data and are, therefore, to be regarded as semiquantitative
estimates only. Figures are indicative of likely ranges present and should not be interpreted as actual values. N/A, not
applicable; ND, not determined above LOQ (0.1 mg kg 1 ).

mAU

3
4

1 2

Retention time (min)

5 10 15 20 25

Figure 3. HPLC-PDA chromatogram of smoked ®sh extract. For peak assignments refer to ®gure 1.
 220 M. J. Scotter et al.
Norm.

3
10

4
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1
2
4
2 5

0
Retention time (min)

5 10 15 20 25 30

Figure 4. HPLC-PDA chromatogra m of cake decoration extract. For peak assignments refer to ®gure 1.

N orm.

6
5

4
1
2

1
3 5

2 U 8

0
Retention time (min)

5 10 15 20 25 30

Figure 5. HPLC-PD chromatogra m of extruded snack extract. For peak assignments refer to ®gure 1. U ˆ Unknown,
tentative identi®cation 13 0 -cis-norbixin.
 Method development and analysis of retail foods for annatto food colouring material 221

mAU

20
3

17.5

15

12.5

10

1
7.5
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2.5

4
0

Retention time (min)

5 10 15 20 25

Figure 6. HPLC-PD chromatogra m of dessert (mousse) extract. For peak assignments refer to ®gure 1.

mAU

1
40

m AU
1 .5 U
35 3 1
0 .5
0
30
40 0 50 0 nm

m AU
25 3
7 .5
5
2 .5
20
0
40 0 50 0 nm

15 m AU
1
15
10
10 5
0
U 40 0 50 0 nm
5 2

0
Retention time (min)

5 10 15 20 25

Figure 7. HPLC-PD chromatogra m of Red Leicester cheese extract with corresponding diode-array spectra. For peak
assignments refer to ®gure 1. U = Unknown, tentative identi®cation 13 0 -cis-norbixin.
 222 M. J. Scotter et al.
obtained which, with the use of prepared reference
materials, has enabled their quantitative determina-
tion at levels well below the maximum levels speci®ed
in the Council Directive on colours for use in
foodstuŒs (EC 1994). This is particularly relevant
to the ADI, which is based on pure colouring
component.
Acknowledgement
Financial support for the work was provided by the
UK Ministry of Agriculture Fisheries and Food,
Joint Food Safety and Standards Group, which in
Downloaded by [New York University] at 05:32 23 June 2015
April 2000 became part of the new UK Food
Standards Agency.
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