a review the chemistry and analysis of annatto food...
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The chemistry and analysis of annatto food colouring:a reviewM. Scotter aa DEFRA Food and Environment Research Agency , Sand Hutton, York YO61 1NW, UKPublished online: 20 Aug 2009.
To cite this article: M. Scotter (2009) The chemistry and analysis of annatto food colouring: a review, Food Additives &Contaminants: Part A, 26:8, 1123-1145, DOI: 10.1080/02652030902942873
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Food Additives and ContaminantsVol. 26, No. 8, August 2009, 1123–1145
The chemistry and analysis of annatto food colouring: a review
M. Scotter*
DEFRA Food and Environment Research Agency, Sand Hutton, York YO61 1NW, UK
(Received 9 March 2009; final version received 1 April 2009)
Annatto food colouring (E160b) has a long history of use in the food industry for the colouring of a wide rangeof food commodities. The principal colouring component of annatto is the oil-soluble diapo carotenoidbixin, which is the methyl ester of the dicarboxylic acid norbixin and soluble in aqueous alkali. Bixin andnorbixin, therefore, exhibit not only physicochemical properties normally associated with carotenoids, but alsocertain anomalous properties that have an impact on the stability, food colouring applications, and importantlythe analysis of annatto. This review summarizes the key aspects of the structural determination of bixin (andnorbixin) with special attention to cis-trans isomerization and how this links with its chemical structure,spectroscopic properties, and stability. The oxidative, thermal, and photo-stability of annatto and the subsequentimplications for its use in the colouring of foods, food processing, and the analysis of foods and beverages arediscussed along with important mechanistic, thermodynamic and kinetic aspects. The main analytical techniquesused for the chemical characterization of annatto, i.e. spectrophotometry, nuclear magnetic resonance (NMR),chromatography (particularly high-performance liquid chromatography (HPLC)) and mass spectrometry arereviewed in detail and other methods are discussed. This links in with a review of the methods available forthe detection and measurement of annatto in colour formulations and foods and beverages, which highlights theimportance of the need for a good understanding and knowledge of the chemistry of bixin and norbixin in orderto meet new analytical challenges.
Keywords: analysis – nuclear magnetic resonance (NMR); chromatography – gas chromatography-massspectrometry (GC/MS); chromatography – high-performance liquid chromatography (HPLC); liquid chromato-graphy-mass spectrometry (LC/MS); colours; process contaminants; volatiles; ingredients; processed foods
Overview
Annatto is a natural colouring agent obtained from
the outer coats of the seeds of the tropical shrub
Bixa orellana. Annatto and its extracts are designated
collectively as E160b and are permitted as a food
additive in the European Union and elsewhere, and
have widespread use in the food industry for the
colouring of many commodities including flour and
sugar confectionery, dairy and savoury products, soft
drinks and fish. The major colour principles of annatto
are the carotenoids bixin and norbixin. Though chem-
ically very similar, differences in their chemical proper-
ties present several challenges to the analytical chemist
with respect to stereochemistry, solubility, chromato-
graphic behaviour and stability. While current legisla-
tion on the extraction and use of annatto colours
and their applications in food are addressed briefly,
this review focuses on the chemistry, stability, and
analysis of annatto pertaining to its use as a permitted
food colouring.
Annatto in foods
Legislative aspects
The use of food colours in the European Union iscontrolled by European Community Directive 94/36/EC (European Commission 1994 as amended), whichcontains a list of permitted colours, a list of foodstuffsto which these colours may be added and, whereappropriate, maximum limits on the level of addition.The permitted uses of annatto and the maximum levelsof addition are given in Table 1. Annatto extracts arelisted amongst those colours that may be used singly orin combination in certain foods up to the maximumlevels specified (on a ready-for-consumption basis).Comprehensive on-line sources of information onpermitted food colour regulations and specificationsmay be found at the Nordic Food Additives Database(Nordic Working Group on Food Toxicology andRisk Assessment (NNT) 2008) and the Food Law siteof the Department of Food Biosciences, University ofReading (Jukes 2008).
*Email: [email protected]
ISSN 0265–203X print/ISSN 1464–5122 online
� 2009 Taylor & Francis
DOI: 10.1080/02652030902942873
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In July 2006, the European Commission publisheda set of four proposed Regulations intended toreplace the current system and provide a commonbasis for controls on food additives, food flavouringsand food enzymes. The proposals were publishedas separate Commission Documents on additives,flavourings, enzymes and a common authorizationprocedure (European Commission 2006). The proposalbrings together all the existing food additive regula-tions and plans to introduce comitology for additiveapprovals in place of the cumbersome co-decisionprocedure.
The specifications for food colours are laiddown in Commission Directive 95/45/EC (EuropeanCommission 1995) in which separate definitions andpurity criteria are prescribed for (1) solvent-extractedbixin and norbixin, (2) alkali-extracted annatto and(3) oil-extracted annatto. Solvent-extracted bixinand norbixin formulations are often referred toas indirectly extracted annatto formulations, whereasalkali- and oil-extracted annatto are termed directlyextracted. The purity specifications include a definitionof the source material(s) and the solvents permitted forextraction, the identification and minimum contentof the colouring material (measured by spectrophoto-metry), and the limits for residual solvents andheavy metals.
Use of annatto in foods
Annatto was reported to be the most commonlyconsumed natural colour additive in the UK (Ministryof Agriculture, Fisheries and Food (MAFF) 1989, 1993)where the per capita consumption was estimated to be0.065mgkg–1 body weight day–1 based on pure colour-ing component, representing some 12.5% of the accept-able daily intake (ADI). The chemistry and applicationsof oil- and water-soluble annatto colours in termsof their modes of applications to a wide range of foodproducts and the usage levels required to obtain thedesired colour shades have been reviewed (Collins 1992;Levy and Rivadeneira 2000). Crystalline bixin productsof 80–97% purity may be obtained by extraction ofannatto seed with certain permitted organic solventsand subsequent production of a solvent-free product,which is then processed to give a range of highpurity oil- and water-soluble annatto formulations.Oil-soluble bixin is generally used in fatty foodapplications, whereas norbixin, because of its abilityto bind strongly with protein, is especially suited forthe colouring of high protein content foods. Annattocolours are often formulated with other additivessuch as emulsifiers to produce forms of water-solubleannatto that are stable to the effects of, for example,acids, metal ions and salts. The applications andstability of spray-dried annatto formulations infruit and vegetable products have been studied(Satyanarayana et al. 2006).
Annatto intake
Bixin is reported to be rapidly absorbed in thebloodstream, comparable with other dietary carote-noids, with complete plasma clearance after 8 h and fornorbixin after 24 h (Levy 1997). While annatto intakeis an important issue within the regulatory context,intake estimates for it have in the past providedambiguous results largely due to the lack of reliabledata on the colour principals’ (bixin/norbixin) contentof annatto extracts (Levy and Rivadeneira 2000).In response to a request by the Joint FAO/WHOExpert Committee on Food Additives (JECFA) forinformation relating to the toxicity, intake and specifi-cations of annatto, the European annatto producersconsulted with the food industry to determine usagelevels of specific annatto extracts (JECFA 2002).The data obtained were combined with the levels ofbixin/norbixin in particular extracts to provide anestimate of their concentration in food. These datahave been combined with food consumption datausing various methods to estimate consumer intakes,which ranged from 1% to 163% of the ADI (Tennantand O’Callaghan 2005). The actual levels of annattoin foodstuffs were well below maximum limitsprescribed under European Union regulations and
Table 1. Permitted uses of annatto and maximum levels ofaddition (European Commission 1994).
Food commodityMaximum permitted
level (mg kg�1)a
Margarine, minarine, otherfat emulsions, and fats essentiallyfree from water
10
Decorations and coatings 20Fine bakery wares 10Edible ices 20Liqueurs, including fortified
beverages with less than15% alcohol by volume
10
Flavoured processed cheese 15Ripened orange, yellow and broken
white cheese; unflavouredprocessed cheese
15
Desserts 10‘Snacks’: dry, savoury potato, cereal
or starch-based products: extrudedor expanded savoury snackproducts
20
Other savoury snack products andsavoury coated nuts
10
Smoked fish 10Edible cheese rinds and edible casings 20Red Leicester cheese 50Mimolette cheese 35Extruded, puffed and/or fruit-
flavoured breakfast cereals25
Note: a100% bixin or norbixin.
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Codex standards, which had been confirmed by anearlier analytical study (Scotter et al. 2002).
Annatto chemistry
Elucidation of the chemical constitution of bixin(and thereafter norbixin) was first put forward byHeiduschka and Panzer (1917) who suggested thecorrect molecular formula for bixin (C25H30O4) as anunsymmetrical molecule. Herzig and Faltis (1923)recognized that bixin was the monomethyl ester of anunsaturated dicarboxylic acid. The results from theircatalytic hydrogenation studies led them to concludethat bixin contains nine carbon double bonds, which,evidenced by the intense red colour of the pigment,were conjugated. However, the unsymmetrical mole-cule hypothesis was abandoned when the now acceptedstructure was proposed (Kuhn and Winterstein 1932),later confirmed by Karrer et al. (1932). A new, highermelting form termed �-bixin was obtained during thecourse of pigment isolation (Herzig and Faltis 1923),which was later proposed as the trans-isomer and thatthe original form may be cis-isomer (Karrer et al.1929). A stable form of bixin identical to the �-form bytreatment of the ‘natural’ (or ‘labile’) form with iodinewas subsequently obtained (Kuhn and Winterstein1932). From the results of these investigations, it wasconcluded that bixin was the first known naturallyoccurring cis-polyene. The structural elucidation ofbixin was confirmed from various oxidation anddegradation experiments (Karrer and Jucker 1950).During investigations to determine the stereochemicalconfiguration of labile bixin, several stereoisomerswere isolated (Zechmeister 1960). The consequences
of cis-trans isomerism on the chemistry, stability and
analysis of annatto are significant and are discussed
below.The major colouring component of annatto is con-
firmed as the apo-carotenoid 90-cis-bixin (methyl hydro-
gen 90-cis-6,60-diapocarotene-6,60-dioate, C25H30O4), the
monomethyl ester of the dicarboxylic acid 90-cis-
norbixin, commonly referred to as cis-bixin (Figure 1).
90-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
exhibits a red colour in solution and is soluble in
vegetable oil. Commercially, isomerization is achieved
by heating a suspension of the cis-isomer in oil to 130�C
in vacuo. The water-soluble analogue 90-cis-norbixin
(C24H28O4) can be isolated from annatto seeds by
agitation in aqueous alkali at less than 70�C or formed
by alkaline hydrolysis of cis-bixin to give either the
sodium or potassium salt. The dicarboxylic acid is
soluble in polar solvents to which it imparts an orange
colour. 90-cis-norbixin is only sparingly soluble in
chloroform and 0.1M sodium hydroxide (Preston and
Rickard 1980). Under extraction conditions, 90-cis-bixin
undergoes isomerization to produce oil solutions
containing approximately 0.2–0.5% of pigment com-
prising amixture of all-trans- and 90-cis-bixin in variable
proportions and characteristic degradation products,
dependent upon extraction temperature and time
(see below).While it is reported that 80% of the carotenoids
in the annatto seed coat comprise bixin (Preston and
R1OOC
CH3CH3
CH3 CH3
COOR29’8′
7′
10′
11′
12′
13′
14′
15′
15
14
13
12
11
10
9
8
766′
R1OOC
CH3CH3
CH3
CH3
COOR2
9′
R1OOC
CH3CH3
CH3
CH3
COOR29′
13′9′,13′ - di-cis-
9′-cis-
trans-
Figure 1. Chemical structures of some bixin/norbixin isomers. R1¼H, R2¼H¼ norbixin; R1¼H, R2¼CH3¼ bixin.
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Rickard 1980; Lauro 1991), traces of bixin diesters maybe found (Mercadante et al. 1997b). The preparationand use of ethylbixin has been discussed (Levy andRivadeneira 2000).
The presence of other minor carotenoids inannatto has also been postulated, which included�-carotene, cryptoxanthin, lutein, zeaxanthin andmethyl bixin (Tirimanna 1980). The presence of arange of lycopenoate analogues and other minorcarotenoids in annatto has been reported in a seriesof papers by Mercadante et al. (1996, 1997a, 1997b,1999) and has been reviewed by Mercadante (2001)and Satyanarayana et al. (2003). Bixin and three minorcarotenoids have been chemically synthesized usingthe Wittig reaction of the (Z)-terminus followed by aHorner–Emmons reaction (Haberli and Pfander 1999).
Molecular properties
It is the delocalization of �-electrons along the polyenebackbone that gives carotenoids their characteristicelectronic spectra and is largely responsible for thephotophysical and photochemical properties of thesemolecules, including cis-trans photoisomerization.Detailed explanations of cis-trans isomerization maybe found in standard texts (Karrer and Jucker 1950;Lunde and Zechmeister 1954; Zechmeister 1960;Kohler 1995). Only the basic properties of linearconjugated molecules will be reviewed here along witha brief account of the simple concepts that apply tobixin and norbixin in order to provide a backgroundfor discussion on the UV-VIS spectroscopy of thesecompounds and to show how UV-VIS spectra areaffected by isomerization.
The sufficiently large barriers to rotation about theformal double bonds in polyenes or carotenoids allowdouble bond cis- and trans-isomers to be isolated asindependent, distinct compounds. Since the differencesin excitation energies for cis- and trans-isomers ofa given molecule are small compared with the changein excitation energy with which it is associated addingor subtracting a conjugated double bond, the basicelectronic structure is almost independent of isomericform. The four single bonds that surround a carbon-carbon double bond all lie in the same plane.In consequence, each of the disubstituted and tri-substituted acyclic double bonds that constitute thepolyene chain of a carotenoid can exist in two forms,i.e. geometric isomers. Nomenclature of the cis- ortrans-isomers is designated in accordance with IUPACrules (Weedon and Moss 1995). In recent years,however, these designations have been replaced largelyby Z and E, respectively.
Since each double bond in the polyene chain could,in principle, exist in one of two forms, a large numberof geometric isomers are theoretically possible for
any carotenoid. However, in practice few of theseisomers are encountered. An explanation for this isprovided by studying molecular models, which indicatethat the introduction of a cis-double bond normallyresults in steric hindrance thus rendering the cis-isomerless stable than the trans-form.With both tri-substituteddouble bonds and disubstituted double bonds in the15,150-position, the effect is relatively small, as itresults from limited interference between two hydro-gen atoms and hence these isomers may be formedquite readily. With other disubstituted double bondsthe adoption of the cis-configuration results in majorinterference between a hydrogen atom and a methylgroup. This renders such molecules less stable thanthe corresponding trans-form and hence less likely tobe encountered (Karrer and Jucker 1950; Zechmeister1960; Kohler 1995; Weedon and Moss 1995).
Stereo-mutation studies, in which interconversionof geometrical isomers is deliberately promoted,lend support to this theory. Interconversion generallyproduces a ‘set’ of isomers that approximate to anequilibrium mixture of all possible geometric formsproportional to their relative thermodynamic stabilities.The all trans-form usually predominates, indicatingthat it is the most thermodynamically stable isomer.A number of mono- and di-cis-isomers are usuallyalso present. However, those isomers with more thantwo cis-double bonds and/or those that aresterically hindered usually occur only in trace amounts,if at all. It is not surprising that most naturallyoccurring carotenoids are predominantly in the alltrans-form. However, bixin occurs predominantly asthe cis-isomer, which has a cis-configuration aboutthe 90-tri-substituted double bond. Since asymmetricbixin has nine alkene bonds (n¼ 9), theoretically512 (i.e. 2n¼ 29) geometric isomers are possible, whereassymmetric norbixin has only 272 (i.e. 2(n� 1/2)
�
(2(n� 1/2)þ 1) possible isomers. However, the presence
of stable cis-isomers at positions 7, 11, 120 and 80 arestearically hindered, hence the remaining five alkenebonds are capable of yielding 32 and 20 isomers forbixin and norbixin, respectively (Figure 1).
Provided that an adequate sample of the pureisomer is available or the selected analytical techniqueis adequately sensitive, spectroscopic analysis willnormally allow the unambiguous assignment of thegeometrical configuration of any carotenoid isomer.All linear polyenes, the carotenoids included, possesssimilar low-lying excited singlet (S1) states (Hudsonand Kohler 1974; Kohler 1977; Hudson et al. 1982).This is critically important since virtually all photoprocesses in linear polyenes originate in the lowest-energy singlet-excited state, the correct identificationand characterization of which is therefore alsoimportant. As might be anticipated from the simila-rities in electronic structure, the electronic absorptionspectrum of a given carotenoid closely resembles that
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of the unsubstituted polyene with the same numberof conjugated double bonds. There are, however, well-characterized principal differences due largely to thepresence of methyl substituents along the carotenoidskeleton, which affect the basic polyene electronicstructure:
. 10–30 nm shift of the lowest energy strongabsorption band to longer wavelength.
. Decreased vibrational fine structure.
Thus, cis- and trans-isomers may often be distin-guished on the basis of their UV-VIS absorptionspectra, but the most important differences observedbetween isomers are not related to excitation energiesbut to the relative intensities of high-energy absorptionbands, i.e. ‘cis-’ peaks (Dale 1954; Zechmeister 1960).It is well established that the lowest excited state oflinear polyenes (including carotenoids) is the 21Ag stateand that the origin of the main absorption band is thestrongly allowed 21Ag! 11Bu transition. The shapes ofelectronic absorption (and fluorescence) bands arederived from the vibrational levels that are associatedwith the initial and final electronic states. Thus, thetypical three-peaked shape of the main absorptionband of linear polyenes arises from transitions of thelowest vibrational level of the electronic ground stateto the lowest vibrational levels of the electronic excitedstates. A broadening of these peaks is observed becauseof rotational levels and inhomogeneity leading topeak overlap. This is particularly relevant for manycarotenoids measured as solutions at ambient tem-peratures (Kohler 1995).
The positions of the absorption maxima and theshape or fine structure of the UV-VIS spectrum ofcarotenoids are therefore characteristic. But while theUV-VIS spectrum gives information about the chro-mophores of the molecule, it yields nothing aboutfunctional groups apart from conjugated carbonylgroups that form part of the molecule (Scott 1964;Britton 1995a, 1995b). In the case of carotenoids, therelevant transition is the �!�* transition. For sucha conjugated system, in which the �-electrons arehighly delocalized, the excited state is of comparativelylow energy. The energy required to bring about thetransition is therefore relatively small and correspondsto light in the visible region. While the transitionresponsible for the main absorption band is strongly‘allowed’, transitions from the ground state to higherelectronic states are also possible, providing they obeythe symmetry selection rules. These high-energytransitions give rise to absorption bands in the UVregion which are usually weak, but are observedparticularly in the spectra of compounds with extendedchromophores.
When the symmetry properties of a carotenoidchange, absorption bands that are otherwise notdetected may become a significant feature of the
spectrum, as the transitions that produce them
become allowed (Britton 1995a). For trans-isomers,
the electronic structure has a centre of symmetry and the
ground state is a g state, so transitions to a higher g state
are forbidden. Transition to a higher excited g state is
only allowed when at least one double bond becomes
cis- and the original symmetry is lost. This gives rise to
an absorption band in the UV region, known as the
cis-band or cis-peak. The most important feature of
the absorption spectrum of a carotenoid is the main
absorption band in the visible region. Several important
pieces of information can be obtained from the
spectrum:
. The position of the main absorption band,
specified by lmax, provides structural infor-
mation because it is determined by the
chromophore of the molecule.. The intensity of the absorption at lmax (A)
is related both to the structure and to the
concentration of the carotenoid in the sample,
and provides the basis for quantitative
analysis.. The position or the intensity of the main
absorption band of a carotenoid can be
influenced by a number of factors such as a
change in the molecular environment of the
carotenoid, e.g. solvent.
Since the structure of the carotenoid chromophore
is related to the overall shape or fine structure of
the spectrum, the shape as well as the positions of
the absorption maxima may therefore be used as a
diagnostic tool, especially when comparing carotenoid
spectra (Britton 1995a). A numerical notation describ-
ing fine structure has proved convenient and removes
the requirement for presenting all spectra as diagrams
(Kohler 1995). In this notation, the baseline or zero
value is taken as the minimum between the two peaks,
the peak height of the longest wavelength absorption
band is designated as III, that of the middle absorption
band (usually lmax) as II (Figure 2). Spectral fine
structure is then expressed as the ratio of the peak
heights III/II, as a percentage.The annatto carotenoids bixin and norbixin are
unusual in that they contain two carbonyl (i.e. carboxyl)
groups, one at either end of the conjugated system and
in conjugation with it, which formally extends the
chromophore. This results in a spectral shift to longer
wavelength, usually accompanied by loss of spectral
fine structure, but the degree of effect is solvent
dependent. With dicarboxylic acids and their esters
such as bixin (and norbixin), the further extension of the
chromophore causes a large relative increase in lmax.
The spectral shift depends on the polarizability rather
than the polarity of the solvent, and the frequency
shifts to lower energy, i.e. a spectral shift to longer
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wavelength, as the refractive index of the solventincreases (Britton 1995a).
While water can have amajor effect on the spectra ofcarotenoids in water-miscible solvents, both bixin andnorbixin isomers are relatively polar carotenoid mole-cules, which are significantly soluble in solvents witha high aqueous content and as such similar solvent-related properties are not observed. cis-norbixin is infact highly soluble in 0.1M sodium hydroxide solution,and both compounds are fairly soluble in mixturesof acetonitrile and dilute aqueous acetic acid, used asa mobile phase in HPLC analysis.
The differences that are observed consistentlybetween the spectra of trans- and cis-isomers ofcarotenoids are therefore diagnostic for structuralassignment. A small hypsochromic shift in lmax ofapproximately 2–6 nm is usually observed for mono-cisisomers along with a significant hypochromic effectand a reduction in vibrational fine structure. A newabsorption band appears at a characteristic positionabout 142 nm below the longest wavelength absorptionmaximum, often referred to as the cis-peak.
For the all-trans form of bixin, the main absorptionband (21Ag! 11Bu) is very intense. In the 90-cis isomer,the intensity of this main band decreases as a weak cis-peak appears at 355 nm, corresponding to the transi-tion to a higher energy level g state. From their studieson �-carotene isomers, it has been shown that the15-cis isomer, in which the cis double bond is in thecentre of the molecule, shows maximum bending ofthe chromophore and a well developed cis-band,with corresponding decrease in intensity of the mainabsorption band (Pettersson and Jonsson 1990).
The intensity of the cis-band is essentially greater asthe cis double bond is nearer the centre of the molecule
and is therefore empirically diagnostic. In a symmet-rical di-cis-carotenoid, the centre of symmetry may berestored so that the cis-band again becomes a weakfeature. For di-cis- and poly-cis-carotenoids, a largerhypsochromic shift in the main absorption band maybe seen, e.g. 13 nm for di-cis-norbixin (Figure 3)(Scotter et al. 1994).
A numerical notation similar to the percentage III/II notation to indicate spectral fine structure has beenadapted to designate the relative intensity of the cis-peak (Kohler 1995). The intensity or absorbance of thecis-peak is expressed as a percentage of the absorbanceof the middle main absorption band, which is usuallythe lmax. Scotter et al. (1994) used this technique tostudy the spectra obtained by HPLC-photodiode arrayanalysis of geometrical isomers of bixin and norbixin.The absorption intensity at lmax (Figure 4, III) for eachisomer was normalized and a comparison of relativeintensity (REL, %) made with the three other maximaat I, II and IV, to give REL(I), REL(II) and REL(IV),respectively. In all cases, lmax (II) took the form ofinflection rather than a peak, which made the exactlocation of the wavelength maximum difficult.However, post-run analysis of the spectral data
nm360 380 400 420 440 460 480 500 520
mAU
0
20
40
60
80
100
120
III
II
%III/II = III/II x 100
Figure 2. Spectral fine structure: calculation of percentageIII/II for a carotenoid (90-cis-norbixin).
(I)
(II)
(III)(IV)
300 400Wavelength (nm)
mA
U s
cale
d
500 600
Figure 4. HPLC-photodiode array spectra of bixin isomersshowing the locations of lmax (I)–(IV) (Scotter et al. 1994).
(a)(b)(c)
300 400Wavelength (nm)
mA
U s
cale
d
500 600
Figure 3. UV-VIS spectra of norbixin isomers (by HPLC-photodiode array): (a) 90-cis-norbixin, (b) di-cis-norbixin and(c) trans-norbixin (Scotter et al. 1994).
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allowed first-derivative spectra to be taken whichfacilitated location of lmax (II), as shown in Figure 5.
Annatto stability
It is well known that the polyene chain in carotenoidsis responsible for their instability, i.e. their susceptibil-ity to oxidation by various agents such as oxygen andperoxides, addition of electrophiles including Hþ andLewis acids, and cis-/trans-isomerization due to vari-ous factors such as temperature and light as discussedabove. Other undesirable reactions may also bepromoted by higher temperatures and light, andexposure to strong acids and alkalis should normallybe avoided.
Oxidative stability
Annatto, especially norbixin, is susceptible to oxida-tion, particularly when applied in powdered form dueto the large surface area and when incorporated intofoodstuffs, although some foods can have a stabilizingeffect (Berset and Marty 1986; Collins 1992; Levy andRivadeneira 2000). Spray-dried norbixin formulatedwith acaia gum or maltodextrin as carriers have beenreported to be particularly susceptible to oxidation(Henry 1992). The level of bixin prepared from annattoseeds and stored for approximately 1 year at 30�C inpackages comprising materials with different oxygentransmission rates was reduced by approximately 10%during the first 2–3 weeks’ storage but stabilizedthereafter except for polyethylene film, which exhibiteda degradation rate of 0.04% per day, reflecting thepermeability of the polyethylene (Carvalho et al. 1993).Several mechanisms have been put forward for theeffect of water activity on the reduction of bixinoxidation in microcrystalline cellulose-based modelsystems to simulate dehydrated foods (Gloria et al.1995). Bixin degradation followed first-order kineticsand the observed half-lives showed greater stability
in systems of intermediate and high water activity.It was postulated that this is because of the ability ofwater to exclude oxygen from liposoluble materialsby surface adsorption, hydrogen bond with hydroper-oxides, inactivate metal catalysts, reduce free radicalsand lower the stability of singlet oxygen. Annattohas been shown to inhibit hydroperoxide formationleading to triglyceride autoxidation by trappingperoxy radicals (Haila et al. 1996). Annatto wasamong a number of Mediterranean spices whoseantioxidant capacities were compared with permittedfood antioxidants in lipid peroxidation (Martinez-Tome et al. 2001). Annatto was reported to have agreater antioxidant capacity than either butylatedhydroxylanisole (BHA) or butylated hydroxytoluene(BHT) for preventing deoxyribose damage by hydroxylradicals. In aqueous media, annatto exhibited alower antioxidant activity than propyl gallate but wasmore effective at peroxide scavenging than BHA orBHT. Annatto oleoresin prepared by oil extractionof seeds was found to be more stable than a powderedformulation during storage over approximately 1 year.Samples were stored in glass bottles with a 3 cmheadspace of air. The greatest losses (60%) wereobserved for the powder at ambient temperature indaylight compared with ambient temperature in thedark (54%) and at 5–8�C in the dark (23%). Theseresults concur with the findings of Najar et al. (1988)that light is the main degradation factor. Moreover,photosensitized bixin is very reactive towards oxygenand thus may be considered as a an oxygen quencher;the reaction of bixin with singlet oxygen is a relatedissue and is discussed below.
Norbixin was the only carotenoid that inhibited theoxidative deterioration of lipids in both olive oil andoil-in-water emulsions stored at 60�C and it displayeda similar activity to �-tocopherol in stored oil (Kiokiasand Gordon 2003). In olive oil-in-water emulsions,norbixin reduced hydroperoxide formation and asynergistic effect between norbixin and ascorbic acidor ascorbyl palmitate was observed.
Bixin has been reported to be able to scavengehydroxyl radicals generated by ferrous ions (Fe2þ) andhydrogen peroxide (H2O2), but no mechanism wassuggested (Zhao et al. 1998). Similarly, the behaviourof norbixin during in vivo plasmid DNA damageinduced by reactive oxygen species Fe2þ, Sn2þ andH2O2 have been studied and it has proposed thatsince norbixin contains two free carboxyl moieties,its protective action may rely on the formation ofcomplexes (Kovary et al. 2001). Norbixin showeda stronger affinity for Sn2þ than for Fe2þ but wasreadily displaced by ethylenediamine tetra-acetic acid(EDTA).
During the isolation and analysis of carotenoids,the exclusion of atmospheric air by inert gas or vacuumis strongly recommended in order to minimize the risk
λmax(II)
300 400Wavelength (nm)
mA
U s
cale
d
500 600
Figure 5. HPLC-photodiode array spectrum of 90-cis-bixin(broken line) and its first derivative spectrum (solid line)highlighting the inflection at lmax (II) (Scotter et al. 1994).
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of destruction or undesired reactions (Scheidt andLiaaen-Jensen 1995) and annatto is no different inthis respect. The oxidation ‘products’ of bixin wereidentified tentatively after transformation of bixinin corn oil at 125�C using spectrophotometry andpaper chromatography (McKeown and Mark 1962).Evidence for the oxidative decomposition of cis-bixinon thin-layer chromatography (TLC) plates has beenreported, where both powdered colour formulationsand chloroform solutions of cis-bixin exhibiteddecreased colour content when stored in the dark inair at ambient temperature (Reith and Gielen 1971).This was concluded to be due to the presence ofoxygen, supported by observing the relatively lowerstability of cheese colour (norbixin in aqueous KOH)compared with butter colour (bixin in vegetable oil),due to the presence of tocopherols in the latter. Theeffects of light, air and pro-oxidants on the stabilityof annatto extracts in chloroform over a 12-day periodwere monitored by spectrophotometry. Air was muchless effective at promoting loss of colour comparedwith light or to benzoyl peroxide, a free-radicalpromoter (Najar et al. 1988). The authors concludedthat rapid loss of colour might occur whenever freeradical formation is promoted.
Reaction with singlet oxygen
Model studies on the photosensitized isomerizationof cis-bixin show that while bixin in the groundelectronic state is stable to thermal isomerization,energy transfer via photosensitization gives rise to thehigher energy triplet state (3BIX*) precursor, whichreadily isomerizes to the trans-isomer (Montenegroet al. 2004). The rate of isomerization is dependenton several factors that compete for deactivation of3BIX*, e.g. ground state bixin and triplet oxygen (3O2).Primary reaction products are only degraded in thepresence of air and under prolonged illumination,which is due to the formation of oxidation productsby reaction with singlet oxygen (1O2). The associatedreaction mechanisms are discussed very elegantly bythe authors. In a similar study, the 3BIX* energy levelwas calculated used laser-induced photo-acousticcalorimetry of bixin in methanol: acetonitrile solution(Rios et al. 2007). The results of the study showedthat bixin is a very efficient quencher of 1O2 in fluidsolutions due to an efficient energy-transfer process,and it confirmed that that the 3BIX* energy levelis lower than that of 1O2 (18� 2 kcalmol�1 and22.5 kcalmol�1, respectively).
Thermal stability
While bixin and norbixin have good heat stabilityduring food processing compared with other
carotenoids, 90-cis-bixin undergoes a series of complexdegradation reactions at commercial extraction tem-peratures to produce a range of products colouredpale yellow to orange (Iversen and Lam 1953; Levy andRivadeneira 2000). Using paper chromatography,the pigments in commercial annatto preparations wereseparated into a series of bands that included a numberof yellow bands comprising up to 40% of the totalpigments and including a bright yellow fluorescent band(McKeown 1961). This band was thought to be the paleyellow breakdown product of bixin identified pre-viously (Iversen and Lam 1953). The main thermaldegradation product of 90-cis-bixin has since beenisolated and identified using paper chromatographyand UV/VIS spectrophotometry as the yellow-coloured17-carbon polyene 4,8,dimethyl-tetradecahexaenediocacid monomethyl ester ‘C17’ (McKeown and Mark1962; McKeown 1963, 1965; Preston and Rickard1980). The influence of heating time on the thermaldegradation of bixin in alkaline extracts of annattoshowed that pigment stability is related to the initialquantity of cis- and trans-bixin as well as to themethod used to obtain the extracts (Prentice-Hernandez et al. 1993).
The C17 product has since been confirmed tobe predominantly the trans-isomer and that cis-isomerization of bixin was prerequisite to its formation(Scotter 1995). However, this compound was shownto isomerize in solution to form small amounts ofcis-isomers and to be susceptible to hydrolysis, thusforming a range of compounds analogous to bixin andnorbixin in terms of their chemical structures andchromatographic properties. In the light of the resultsobtained, the mechanism of C17 formation originallysuggested (McKeown 1963) was postulated as aconcerted electrocyclic process followed by the elimi-nation of m-xylene and, to a much lesser extent,toluene, toluic acid and toluic acid methyl ester, andthe formation of C17 which can degrade further bya similar mechanism.
The analytical HPLC-photodiode array (PDA)method developed by Scotter et al. (1994) andScotter (1995) provided superior qualitative and quan-titative data compared with UV-VIS spectroscopicmethods (McKeown and Mark 1962; Smith et al. 1983)for determining the colour content (as bixin andnorbixin) in 21 commercial annatto formulations,particularly with respect to the coloured thermaldegradation products (Scotter et al. 1998). Moreover,the levels of the all-trans and di-cis-isomers of norbixindetermined from chromatographic profiles of twodifferent norbixin formulations were found to beconsistent with their known production history, i.e.indicative of the degree of thermal treatment. Theformulation obtained by direct aqueous alkalineextraction contained higher levels of these isomerscompared with solvent pre-extracted bixin followed by
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alkaline hydrolysis obtained using lower temperatures.However, the authors pointed out that while theisomer profiles obtained by HPLC-PDA analysissupport this, the different extraction proceduresmight also give rise to different isomer profiles due todifferential solubilities and stabilities in the extractionmedium. The effects of light and oxygen may furthercomplicate this during extraction and handling, and bythe nature of the source material.
In a follow-up study, a method was developed thatused ambient alkaline hydrolysis followed by solventextraction and gas chromatography (GC) to analyseannatto colour formulations for the main aromatichydrocarbon thermal degradation products m-xyleneand toluene (Scotter et al. 2000). Of the 20 samplesanalysed, 15 contained less than 5mgkg�1 toluene,four samples contained between 5 and 10mgkg�1, andone sample contained 12mgkg�1 toluene, but theselevels were not indicative of significant toluene forma-tion via thermal degradation of annatto. In contrast,six samples comprising both bixin and norbixinformulations contained m-xylene in the range30–200mgkg�1, with the highest level found in anoil-based bixin formulation. Moreover, the two nor-bixin formulations of known production historyanalysed in the previous study (Scotter et al. 1998)differed markedly in m-xylene content, which appearedto be consistent with the degree of thermal treatment.
For comparison with the alkaline hydrolysis-solvent extraction procedure, seven of the annattoformulations were submitted for headspace (HS)-GCanalysis for toluene and m-xylene in order to monitorthe effects of heating in a closed controlled environ-ment (90�C for 20min). An increase in m-xylene wasobserved, with the bixin in oil formulations showingthe highest rise in m-xylene concentration on heating.The authors anticipated that HS-GC could be used tomonitor the thermal degradation of annatto in foodsystems and thus conducted a number of experimentsin combination with HPLC-PDA and GC-MS to studythis (Scotter et al. 2001). Low levels (approximately10–15 mg kg�1) of m-xylene were detected in the head-space of annatto-coloured retail samples of custardpowder, extruded snacks, margarine and breadcrumbsbut not in control samples. Much higher levels ofm-xylene were detected in annatto-coloured smokedherring (kippers) at approximately 150–200 mg kg�1
and m-xylene was observed in the headspace ofheated Red Leicester cheese (not quantified). The C17
coloured annatto degradation product was alsodetected, indicating that thermal degradation of theprincipal annatto colouring agent 90-cis-bixin in modelsystems and foods is facile. However, the degradationis complicated by many competing isomerizationreactions which proceed at different rates towardsequilibrium. This is further complicated by the simul-taneous and irreversible formation of C17 associated
with the production of m-xylene and to a lesser extenttoluene. While norbixin was reported to degrade
similarly but more slowly, the levels of m-xylene
formation were nonetheless consistent with bixin/
norbixin concentration in the food and occurred
more rapidly at higher temperatures.In order to understand better the kinetics and yields
for the formation of both the coloured and aromatic
hydrocarbon thermal degradation products of annatto,
the authors carried out a number of experiments in
model systems (Scotter et al. 2001). The thermal
stability of bixin at the boiling point of three homol-ogous alcohol solvents was evaluated using HPLC-
PDA to monitor the rate of loss of 90-cis-bixin as well
as the appearance of a di-cis- and trans-isomer, and the
C17 degradation product. Loss of linearity was
observed at each temperature beyond 2 h, suggestingthat two or more competing reactions were taking
place at different rates. From the rate constants
calculated for the initial phase of the reaction, the
Arrhenius activation energy for the loss of 90-cis-bixin
in refluxing alcohol solvent was 35.7 kJmol�1. Sincethe rate of loss of 90-cis-bixin was measured as a
function of time regardless of reaction pathway, i.e.
isomerization versus degradation), the authors con-
cluded that the rate data represented only the total
(summed) values. Thus, several concurrent reaction
pathways are available, hence deviation from first-order kinetics at long observation times was not
unexpected as suggested in Figure 6.Berset and Marty (1986) had reported previously an
activation energy of 125 kJmol�1 for the thermal
degradation of annatto pigments in petroleum jellyusing a simple first-order kinetic model for the complete
decay. This disparity in values therefore suggests a
controversy in the kinetic analysis or a misinterpreta-
tion of the experimental data. Interestingly, bixin was
reported to be easily transformed to the all-trans-isomerat ambient temperature in the presence of a photo-
sensitizer and light, where the activation energy
for the excitation of bixin to an excited triplet state
was approximately 25 kJmol�1 as discussed above
(Montenegro et al. 2004), which suggests strongly that
a greater energy barrier may be anticipated for thethermal isomerization of 90-cis-bixin to trans-bixin.
Trans-isomer 9′-cis-isomer Di-cis-isomers
Other mono-cis-isomers
?
Poly-cis-isomers
Degradation products
Figure 6. Suggested reaction pathways for the thermaldegradation of 90-cis-bixin (Scotter et al. 2001).
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A more detailed kinetic study on the thermaldegradation of bixin in an aqueous model systemcomprising water:ethanol (8:2) as a function of tem-perature has been described, where HPLC was used tomonitor the decay of 90-cis-bixin and the formationof the di-cis- and trans-isomers, as well as C17 (Rioset al. 2005). The reactions were found not to followfirst-order rate characteristics but rather fitted wellto a bi-exponential model. The rate constants forthe formation of the primary products of bixin andthe energy barriers for each step were calculated.Di-cis-isomers were formed immediately (energy bar-rier approximately 63 kJmol�1) followed by a slowconsumption (with the associated decay of 90-cis-bixin), indicating their role as reaction intermediates.The di-cis-isomers can either revert readily to 90-cis-bixin (approximately 13 kJmol�1) or yield the primaryC17 degradation product with a higher energy require-ment of approximately 27 kJmol�1). However, theisomerization of 90-cis-bixin to trans-bixin requiresapproximately 100 kJmol�1, thereby explaining itsrelatively slow formation. The Arrhenius plot obtainedfrom the initial decay component for 90-cis-bixinyielded an activation energy of approximately33 kJmol�1, which concurs with earlier data (Scotteret al. 2001). In conclusion, while the activation energyobtained for the 90-cis-! trans-isomerization of bixinis very similar to that reported for �-carotene(Mınguez-Mosquera and Jaren-Galan 1995), thevalue of approximately 155 kJmol�1 for the summedisomerization steps of bixin is much higher than thosereported for the thermal isomerization of C40 carote-noids (approximately 105 kJmol�1). Thus, the reactionscheme suggested by Scotter et al. (2001) and thegreater relative stability of bixin, especially during itsisolation and manipulation were confirmed (Figure 7).
Thermogravimetric analysis has been used toinvestigate the thermal degradation of bixin derivedfrom annatto seeds at different heating rates overthe 25–900�C range (Silva et al. 2005). The resultsindicated that the decomposition of solid 90-cis-bixinoccurs in the liquid phase and that four decompositionstages are evident over the range 205–545�C, withisomerization to the trans-isomer occurring between200 and 240�C. The calculated activation energy wasdependent upon heating rate (i.e. 5, 10 or 15Kmin�1)at approximately 108, 147 and 128 kJmol�1 respec-tively compared with the value of approximately100 kJmol�1 reported by Rios et al. (2005) obtainedin solution. In a similar follow-up study, cis-norbixinwas heated at rates of 5, 10 and 20�Cmin�1 over therange 25–900�C, where the thermal decompositionreactions occurred in the solid-phase (Silva et al. 2007).Using the Coats-Redfern model, the calculated acti-vation energy was dependent upon heating rate atapproximately 154, 131 and 99 kJmol�1 at 5, 10 and20�Cmin�1 respectively for the first-order process.
Heating solid non-purified extracts of annattoseeds as a thin film deposited on a silicon waferin vacuo and monitoring using time of flight (ToF)secondary ion mass spectrometry (SIMS) does not givethe same results as heating in solution (Bittencourtet al. 2005). Principal component analysis revealed thatthe thermal degradation of the annatto extracts underthese conditions occurs in three distinct temperatureranges: below 70�C, the extracts remain thermallystable, but above this temperature dimerization reac-tions occur and the signals attributed to bixin decrease.Near to 100�C, the bixin molecules begin to degrade,leading to fragmentation with extensive degradationof bixin above 120�C. However, the nature of thedegradation mechanism described is not fully under-stood since there was no evidence for the formation ofC17 or related fragments from solid bixin.
Light stability
The effect of light at 900 lux intensity on the 30-daystability of a microencapsulated water-miscible extractof bixin compared with that of a purified bixin extractwas studied by measuring the loss of spectrophoto-metric absorbance at 470 nm with time (Prentice-Hernandez and Rusig 1999). The degradation rate ofbixin in the microencapsulated extract was approxi-mately 0.05% compared with 0.11% per day for thepurified extract.
Ferreira et al. (1999) submitted commercial water-soluble annatto (norbixin) solutions to different timeand temperature treatments to investigate colourstability. The colour change was measured by spectro-photometry using the Hunter Lab System and theresults presented in terms of changes in the norbixinconcentration and L, a, and b colour parameters.Data were analysed for reaction order and thetemperature dependence was explained by theArrhenius model, with activation energy valuesbetween 46 and 105 kJmol�1. The changes in colourshowed an increase in lightness and yellow colour anda decrease in red colour. Norbixin degradation reac-tion followed second-order kinetics whereas for othercolour parameters first-order kinetics were followed.
The light stability of spray-dried bixin encapsulatedwith gum arabic or maltodextrin plus Tween 80surfactant has been reported, where the kineticbehaviour of bixin photodegradation in all systemswas characterized by two first-order decays due to the
Trans -isomer 9′-cis-isomer Di-cis-isomers C17k4
k1
k2 k3
Figure 7. Coupled reaction scheme proposed for the degra-dation of bixin and the formation of its primary products(Rios et al. 2005).
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presence of bixin both inside and outside the micro-capsules (Barbosa et al. 2005). Unsurprisingly, approx-imately two orders of magnitude greater stability wasobserved for bixin in the dark compared with illumi-nated conditions and in the absence of light, and bixinstability in encapsulated solutions was approximatelyten times greater than in non-encapsulated systems. Theeffect of processing conditions used for the preparationof traditional Indian foods on bixin stability includingbaking, frying, microwave cooking and pressure cook-ing have been monitored by following losses usingspectrophotometry (Rao et al. 2005). The losses of bixinunder model processing conditions were compared withthe preparation of cakes, chegodis, biscuits and friedrice. The greatest losses of bixin were observed in directexposure to oven baking (54% loss) and deep-fat frying(47%), whereas microwave cooking did not affect thecolour during direct exposure or in food products. Themaximum loss of bixin (65%) was observed for deep-fried snack due largely to leaching of the dye into the oil.Pressure cooking produced losses of between 25% and33%. In cakes, the loss was 30%, but negligible losseswere found for biscuits (1.5%).
Similarly, the combined effects of light and temper-ature on annatto extract under different storageconditions were evaluated spectrophotometrically at470 nm in chloroform over a period of 360 days(Balaswamy et al. 2006). Annatto oleoresin was gener-ally more stable during storage with respect to bixincontent than annatto powder obtained by solventextraction of annatto seeds. The bixin loss in oleoresinstored under cold (5–8�C), dark conditions was minor(11%) throughout the study, whereas considerablelosses were observed for the powdered dye (23%).Likewise, the bixin lost after storage at ambienttemperature in the dark were 8% and 54% for oleoresinand powder, respectively. Under diffused daylightambient and temperature the losses were 14% and60%, respectively, whereas bixin seed stored in jutesacks showed a loss of only 15%. As expected, the rateconstants for bixin degradation were much higher inpowder compared with oleoresin and were reported tofollow second-order kinetics. It was proposed that thecolour is protected from exposure to oxygen and lightby the oleoresin compared with the dry powder, whichhas a large surface area.
Bixin complexed with �-cyclodextrin is alsoreported to be more resistant to the damage caused bylight and air (Lyng et al. 2005).
Analytical methods for annatto
Spectrophotometry
Historically, chloroform has been used as a solvent forthe spectrophotometric analysis of bixin and dilutesodium hydroxide (approximately 0.1M) for norbixin.
Absorbance measurements at the two most intensespectral peaks (III and IV in Figure 4) are used forquantitative analysis, where peak IV is preferredbecause it is less prone to interference from yellowdecomposition products. This interference was cor-rected by using a factor related to the absorbances atlmax and at 404 nm in determining the total pigmentcontent of annatto formulations (McKeown andMark 1962). In practice, the spectrophotometricdetermination of annatto (as bixin or norbixin) issomewhat confused by the use of conflicting extinctioncoefficients. This has been discussed in detail and thepublished (E1 %
1cm) extinction coefficients for norbixinand bixin summarized and compared with highlightdisparities (Levy and Rivadeneira 2000). Depending onthe extinction coefficient used, large errors might beincurred and a practical conversion factor is proposedto correlate the relative absorbances at the two peakmaxima. This is based on the increase in absorbanceobserved upon hydrolysis of bixin to norbixin atconstant concentration – thus proving that the extinc-tion value for norbixin must be higher than that forbixin, which was also reported (Smith et al. 1983).Furthermore, from data recorded by the authors frommore than 1000 spectrophotometric measurements ofdifferent samples of bixin before and after hydrolysis,the difference between the extinction values of bixin andnorbixin was reported to be in the order of 6%. Whencompared with a value of E1 %
1cm ¼ 3208 reported for purenorbixin, this equates to an extinction coefficient forbixin of E1 %
1cm ¼ 3016, which concurs with the valuesreported for purified bixin in chloroform (Scotter et al.1994).
However, these extinction values to not agree withthose adopted for colour purity specifications by theEuropean Union (European Commission 1995) or theFAO/WHO (1996), largely due to misassumptionsmade regarding solvent effects. The discrepancy inpublished extinction values might be traced back to the‘erroneous’ coefficient reported by Reith and Gielen(1971) that has been used subsequently as a referencevalue by various other workers. Serious doubt isexpressed over the validity of the extinction values fornorbixin in aqueous alkaline solution at 453 nm (2850)and 482 nm (2550). Moreover, the same reservationswere expressed over the value of 3473 at 453 nmreported by the FAO/WHO (1981) specification.
An interesting and important aspect of the spectro-photometric analysis of bixin in chloroform is its rapidrate of degradation when contained in a quartz cuvette,which unlike glass cuvettes allows the transmission ofultraviolet light (i.e.5300 nm) (Levy and Rivadeneira2000).
Planar chromatography
Before 1961 there were few references in the literatureto paper and adsorption chromatography, which dealt
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mainly with the gross separation of different carote-noids, and from chlorophylls. The first paper chro-matographic method for the direct separation ofannatto colouring components used Whatman 3MMpaper impregnated with 50% N,N-dimethylformamide(DMF) in acetone and which developed withcyclohexane:chloroform:DMF and acetic acid(85 : 10 : 3 : 2) (McKeown 1961). This method wasused in a number of subsequent studies on annattoand its main thermal degradation product C17
(McKeown and Mark 1962; McKeown 1963). Thefirst thin-layer method for the separation of annattocolour and other fat-soluble dyes shortly thereafteremployed silica gel G, plaster of Paris and silicic acidmedia with amyl acetate mobile phase (Ramamurthyand Bhalerao 1964). However, of the 30 solventsystems studied, bixin was reported to migrate fromthe baseline only when acetic acid was present (Francis1965). The findings suggested that the amyl acetatesolvent used by Ramamurthy and Bhalerao (1964)must have contained acetic acid as an impurity, whichwas proven by subsequent experimentation. Latermethods used silica gel with various solvent systemscontaining acetic acid for the separation of bixin andnorbixin in colour formulations (Dendy 1966) andcheese colour, i.e. norbixin (Reith and Gielen 1971),who also employed cellulose media for the analysis ofbutter colour, i.e. bixin. Other methods include those
developed by Preston and Rickard (1980) and Corradiand Micheli (1981). Chao et al. (1991) used reverse-phase (C18) plates with methanol:water mobile phaseto separate annatto pigments from supercritical CO2
extractions of annatto seeds. More recently, TLChas been used for the detection of bixin and otherfood colour carotenoids derived from red pepper(Mınguez-Mosquera et al. 1995) and for the isolationand identification of new (trace) apocarotenoids fromannatto seeds (Mercadante et al. 1997b) and in thebioautographic detection of antimicrobial compoundsin water-soluble annatto extracts (Galindo-Cuspineraand Rankin 2005). The various methods are summar-ized in Table 2.
HPLC
As discussed above, developments in HPLC techniqueshave enabled more detailed studies of other bixin andnorbixin isomers as well as their degradation productscompared with TLC methods and have been utilized togain a greater understanding of the stability of annattoand which in turn have been applied to the detectionand measurement of annatto colour in foodstuffs(see below).
Literature references on the application of HPLCto the separation of annatto colouring components aresparse. Early methods include the HPLC analysis of
Table 2. Summary of planar chromatographic methods for annatto colours.
Sample type Adsorbent Mobile phase Reference
Bixin, norbixin, C17 Paper CHX :CHCl3 :DMF :HOAc(85 : 10 : 3 : 2)
McKeown (1961, 1963)
Annatto and otherfat-soluble dyes
Silica gel G Amyl acetate Ramamurthy and Bhalerao(1964)
Annatto Silica gel G 1% HOAc in amyl acetate Francis (1965)Bixin Silica gel CHCl3 :ACE :HOAc
(50 : 50 : 1)Dendy (1966)
(1) Bixin (1) Cellulose CHX :CHCl3 :HOAc(65 : 5 : 1)
Reith and Gielen (1971)
(2) Norbixin (2) Silica gel CHCl3 : EtOH :HOAc(68 : 2 : 1)
Annatto and otherpigments
Silica gel G(two-dimensional)
CHCl3 : EtOAc (4 : 1)Et2O
Tirimanna (1980)
Bixin and norbixincommercialformulations
Silica gel GF PE : Et2O :HOAc (85 :15 : 2.5) Preston and Rickard (1980)
Ether extracts offoods
Silica gel CHCl3 :HOAc (9 : 1) Corradi and Micheli (1981)Et2O : IPA (9 : 1)
Annatto seeds KC18 reverse phase MeOH :H2O (70 : 30) Chao et al. (1991)Bixin and othercarotenoids
Silica gel GF HEX :ACE (10 : 9) Mınguez-Mosquera et al.(1995)DCM :Et2O (9 : 1)
PE : BZ (1 : 1)PE
Annato seeds Silica gel MgO/Kieselguhr
HEX : t-BME (90 : 10) Mercadante et al. (1997b)HEX :ACE (85 : 15)
Annattoformulations
Silica gel GF CHCl3 :HOAc :ACN :ACE(8 : 1 : 0.5 : 0.5)
Galindo-Cuspinera andRankin (2005)
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annatto extract (Nishizawa et al. 1983) and Smith et al.(1983), who reported the use of an isocratic reverse-phase system employing an ODS column and metha-nol/aqueous acetic acid mobile phase. Using thissystem the cis- and trans-isomers of both bixin andnorbixin were separated within 10min. However, thecis- and trans-bixin peaks were not fully resolved andthe peak shapes were generally very poor. A methodfor the reverse-phase separation of bixin, norbixin andthree curcuminoids using both isocratic and gradientelution systems, comprising a Zorbax ODS columnand water/THF mobile phase, was later developedand gave improved chromatographic separation(Rouseff 1988). However, only separation of the‘main’ annatto-colouring components were reportedand no reference to stereoisomer separation wasgiven. Other approaches have been reported for theanalysis of cheese extracts (Luf and Brandl 1988) andof foods after protease digestion (Chatani and Adachi1988). A procedure similar to that reported by Smithet al. (1983) has been developed and applied to thedetermination of annatto in selected foodstuffs withreasonable success (Lancaster and Lawrence 1995).
The method developed by Scotter et al. (1994) hasplayed a key role in the advancement of HPLCcapabilities for the separation and characterization ofnorbixin and bixin isomers, and has been refined andadapted for the study of annatto stability and for thedetermination of annatto colouring components incolour formulations, foodstuffs and human plasma.These are summarized along with other publishedmethods in Table 3.
While the development of column stationaryphases been vital in allowing separation of geometricalisomers of bixin and norbixin, C17 analogues and otherfood components, it is the power of the detectionsystems that has enabled the development of highlyuseful qualitative and quantitative analyses. Manydeveloped methods utilize detection with fixed wave-length UV-visible (UV-VIS) detectors at wavelengthsspecific to bixin/norbixin isomer absorption maximaquite successfully. However, photodiode-array (PDA)technology offers combined sensitivity and specificitycoupled to real-time qualitative (spectral) confirmatoryanalysis, thereby enabling powerful isomer identifi-cation and measurement. PDA allows isomer peakswith different lmax wavelengths to be monitoredusing a spectral bandwidth that encompasses them.A reference wavelength can also be used to subtractbackground absorbances and to allow for baselinedrift, which is usually set outside of the absorbancerange of the main analyte and interfering peaks,e.g. at 600 nm� 4 nm bandwidth. The lack of avail-ability of authenticated reference standards is themain limiting factor in the HPLC analysis of annattocolouring components but methods are available forthe isolation, purification and characterization of the
main bixin and norbixin isomers (Scotter et al. 1994)and for C17 analogues (Scotter 1995). Other workershave exploited the use of PDA detection for theidentification of trace levels of other apocarotenoidsin annatto seeds very successfully (Mercadante et al.1997b). Figure 8 shows the HPLC separation of bixinand norbixin isomers (Scotter et al. 1994).
Mass spectrometry (MS)
A comprehensive review on the use of mass spectro-metry in the study of carotenoids in general maybe found elsewhere (Enzell and Back 1990). This workcites earlier reviews and studies that consolidate theimportance of the technique not only for elucidationof structure but also for analytical research, not leastthose carried out by Vetter et al. (1971), Budzikiewicz(1974) and Enzell and Wahlberg (1980). The 1990review covers in detail ionization techniques, tandemMS, combined chromatographic-MS techniques, andelimination reactions of in-chain units and terminalgroups. The first method for electrospray liquidchromatography-mass spectrometry (LC-ES-MS) ofcarotenoids employed gradient reversed-phase HPLCwith PDA and MS detection in tandem (Van Breemen1995). Molecular ions, M(.þ), without evidence of anyfragmentation, were observed in the ES mass spectraof both xanthophylls and carotenes but neither bixinnor norbixin were studied.
In common with other carotenoids, the MS spectraof bixin and norbixin are characterized by fragmenta-tion leading to losses of toluene and xylene fromthe polyene chain and the structural significance of theintensity ratio of the [M – 92]þ. and [M – 106]þ. ions(and to a lesser extent the [M – 158]þ. ion), which isrelated to the number of conjugated double bonds.It is the apo-configuration that gives rise to anomalousMS properties of bixin and norbixin that have diag-nostic value, i.e. the -CH2-CH¼CH-CH2-COOH end-group gives characteristic fragments at [M – 44]þ. and[M – 99]þ, whereas the -CH2-CH¼CH-CH2-COOCH3
end-group gives characteristic fragments at [M – 31]þ,[M – 59]þ. and [M – 113]þ. Solid probe electronionization (EIþ) was used to confirm the structures ofisolated and purified bixin and norbixin isomers(Scotter et al. 1994). Both the 90-cis- and trans-isomersgave a molecular ion at m/z 394 (bixin) and m/z 380(norbixin), with major fragment ions at m/z [M – 106],106 (xylene), 105 (methyl tropylium) and 91. Usingthermospray analysis, [MþH]þ was identified as thebase peak along with the presence of sodium and(possibly) water adducts, and fragment ions corre-sponding to [M – H2O]þ and [M – CH3OH]þ. In a laterstudy, similar analytical conditions were used tocharacterize the 17-carbon major thermal degradationproduct of annatto (Scotter 1995). Solid-probe EIrevealed the molecular ion at m/z 288 along with
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Table
3.Summary
ofhigh-perform
ance
liquid
chromatography(H
PLC)methodsusedfortheanalysisofannatto.
Sample
matrix
Analyte(s)
HPLC
conditions
Column
Mobilephase
Detector
Reference
Annattocolour
Bixin
andnorbixin
isomers
HRPB
C8/C
18250�4.6mm,
5mm
ACN:2%
HOAc(65:35)
isocratic
1mlm
in�135�C
UV-V
ISPDA
452,460nm
Scotter
etal.(1994)
Annattocolour
Bixin
andnorbixin
isomersandC17
isomers
HRPB
C8/C
18250�4.6mm,
5mm
ACN:0.4%
HOAc(65:35)
isocratic
1mlm
in�135�C
UV-V
ISPDA
435�60nm
Scotter
(1995);Scotter
etal.(1998,2001)
Foods
Cis/transbixin
and
norbixin
Supelco
LC-18250�4.6mm,
5mm
MeO
H:2%
HOAc(9:1)
iscocratic
1mlm
in�1
UV-V
IS500nm
Lancaster
andLawrence
(1995)
Foods
Bixin,norbixin
and
carm
inic
acid
Supelco
LC-18250�4.6mm,
5mm
MeO
H:6%
HOAcgradient
1mlm
in�1
UV-V
IS493nm
Lancaster
andLawrence
(1996)
Plasm
aBixin
andnorbixin
isomers
S5ODS1
ACN:2%
HOAcisocratic
1.5mlm
in�1
UV-V
ISPDA
460nm
Levyet
al.(1997)
DNA
Bixin
andnorbixin
Supelco
LC-8
250�4.6mm,
10mm
ACN:0.08%
CF3CO
2H
(85:15)
isocratic
1mlm
in�1
UV-V
IS470nm
Kovary
etal.(2001)
Foods
Bixin
andnorbixin
isomers
HRPB
C8/C
18250�4.6mm,
5mm
ACN:0.4%
HOAc(65:35)
isocratic
1mlm
in�135�C
UV-V
ISPDA
455�10nm
Scotter
etal.(2002)
Cheese
Cis/transbixin
and
norbixin
ODS2C18250�4mm,5mm
ACN:2%
HOAc(75:25)
isocratic
1mlm
in�1
UV-V
IS460nm
Bareth
etal.(2002)
Corn
snacks
Norbixin
ODS2C18150�4mm,3mm
ACN:2%
HOAc(65:35)
isocratic
1mlm
in�129�C
UV-V
ISPDA
450nm
RiosandMercadante
(2004)
Food
Bixin,norbixin
andother
carotenoids
YMC
C30250�4.6mm,5mm
A:MeO
H:H
2O:TEA
(90:10:0.1)
B:MTBE:MeO
H:H
2O:TEA
(90:6:4
:0.1)gradient
1mlm
in�135�C
UV-V
ISPDA
450�4nm
þLC-M
S
Breithaupt(2004)
Bixin
Photodegradation
products
VydacC18250�4.6mm,5mm
ODS2C18150�4.6,3mm
ACN:2%
HOAc:
DCM
(65:35:2)isocratic
1mlm
in�125�C
UV-V
ISPDA
450nm
Montenegro
etal.(2004)
Aqueous
model
system
Bixin
thermaldegradation
products
ODS2C18150�4mm,3mm
ACN:2%
HOAc(65:35)or
ACN:2%
HOAc:
DCM
(65:35:2)isocratic
1mlm
in�129�C
UV-V
ISPDA
450nm
Rioset
al.(2005)
Watersoluble
annatto
Cis/transnorbixin
BeckmanC18250�4.6mm,
5mm
ACN:0.4%
HOAcþ5%
ACN
isocraticandgradient
1mlm
in�1
UV-V
ISPDA
250-600nm
þLC-M
S
Galindo-C
uspineraand
Rankin
(2005)
Notes:ACN,acetonitrile;DCM,dichloromethane;
HOAc,
aceticacid;MeO
H,methanol;MTBE,methyltertiary
butylether;TEA,triethylamine.
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fragment ions at m/z [M – 106], 106, 105 and 91, andthermospray analysis identified the base peak as[MþH]þ as well as sodium adducts at m/z 311([MþNa]þ) and 333 [M – Hþ2Na]þ.
Complementary to other analytical techniques,EIþ and fast-atom bombardment (FAB) MS wasused to determine the structure of the bixin family ofapocarotenoids (Kelly et al. 1996). Both cis- and trans-bixin isomers gave EIþ molecular ion abundancesequivalent to approximately 30% of the base peaks atm/z 59 or 91, and the [Mþ1] and [Mþ2] ion intensitieswere consistent with predictions based upon calculated13C isotope patterns. As expected, loss of xylene as aneutral group was most pronounced for cis-bixin butno loss of neutral toluene was observed although them/z 91 peak was prominent. FABþ spectra of cis- andtrans-bixin gave the molecular ion as the base peak butthe abundance of the [Mþ1] peak exceeded thecalculated isotopic abundance by 55–75%, indicatinga small contribution from [MþH]. Small amounts ofsodium adducts were observed but ions due to elim-ination of toluene were not. However, significantamounts of m/z 105, 115 and 165 were observed.These observations were consistent with other FABspectra of carotenoids where odd electron molecularions are frequently observed due presumably to theirlower ionization potentials (Vetter and Meister 1985).
Bixin was among the polyenes studied using EI andhigh resolution (HR) matrix-assisted laser desorptionionization (MALDI) time-of-flight (TOF) mass spec-trometry (Guaratini et al. 2004). In this study, theability of neutral organic molecules to give up anelectron for oxidation was exploited, which is governed
by the energy of their highest occupied molecularorbital (HOMO) and can be estimated bymeasurementof the half-wave potential for solution oxidation. Strongevidence was reported for an ionization process thatproduces the molecular ion M.þ in ESI and HR-MALDI MS of polyenes, and the correlation of theobserved ions to the oxidation potential. The formationofM.þ and [MþH]þ species was shown to be dependentupon energetic variations and the presence of water oranother protic solvent. Neither the [MþH]þ nor the[MþH-H2O]þwere detected as the major ions fromESIanalysis of bixin, whereas M.þ was detected but only inthe specific capillary voltage range of 0.1–0.7 kV. Theaccurate mass measurement afforded by the HR-MALDI-TOF analysis showed M.þ for bixin at anobserved mass of 394.2147 with 40% ion intensity, but[MþH]þ was not observed.
The major carotenoid composition of Bixa orellanaseeds has been ascertained using TOF-MS with X-rayphotoelectron spectroscopy (Felicissimo et al. 2004).The presence of bixin was revealed in the seed arilwithout any sample pretreatment from the detectionof ions attributable to [Mþ2H] at m/z 396 withassociated 13C isotope analogues at m/z 397 and 398.The presence of characteristic fragments at m/z 337was attributed to C23H29O
þ2 obtained from the previ-
ous molecular ion with loss of a COOCH3 ester group,and at m/z 281, a fragment compatible with lossof a C6O2H8 end-group plus a hydrogen atom, i.e.C19H21O
þ2 . The characteristic presence of xylene was
confirmed via the detection of the C8Hþ9 ion at m/z
105. Analysis of the coloured interior of the seedsfollowing cutting did not show any fragments consis-tent with bixin. A methanol:chloroform extract of theseeds was analysed immediately after preparationby blow-drying under nitrogen onto a silver substrate,and then after exposure to ambient light for 3 months.TOF-MS analysis of the fresh extract was dominatedby the molecular peak at m/z 396 along with all othercharacteristic fragments. As expected after 3 months’exposure to light, the colour of the extract hadlightened to a more yellow shade with an associatedfive-fold decrease in the intensity of the [Mþ2]þ ionand with a concomitant two-fold increase in theintensity of the C8H
þ9 ion, indicating the formation
of xylene via degradation. In a related study,Bittencourt et al. (2005) analysed extracts of Bixaorellana using TOF-MS as a means of characterizingthermal effects. The spectrum was characterized by alarge number of peaks generated by the principal ionsand their multiple fragmentation patterns but also,more notably, by the presence of ions at m/z 790([C50H62O8]
þ¼ 2Mþ2H), 804 ([C51H64O8]
þ¼ 2Mþ
2HþCH2) and 818 ([C52H66O8]þ¼ 2Mþ2Hþ2CH2)
attributed to the presence of dimers.The confirmation of twelve different carotenoids
used as food colorants was achieved using positive
min10 15 20 25 30 35
mAU
0
10
20
30
40
50
60
70
80
90
12
3
4
5
6
7 8 9
Figure 8. HPLC separation of bixin and norbixin isomers.Conditions: column HRPB C8/C18 250� 4.6mm, 5 mm;mobile phase acetonitrole: 0.4% acetic acid (65:35) isocraticelution at 1mlmin�1 35�C; detection photodiode arrayat 455� 10 nm. Assignment of peaks: 1, trans-norbixin;2, di-cis-norbixin; 3, 90-cis-norbixin; 4, trans-bixin; 5 and 9,di-cis-bixin isomers; 6, 90-cis-bixin; 7, 15-cis-bixin (tentative);and 8, 130-cis-bixin (tentative).
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atmospheric pressure chemical ionization (APcI)þLC-MS (Breithaupt 2004). The [MþH]þ ions weremonitored for norbixin and bixin at m/z 381 and 395,respectively. Based on the presence of at least onecarboxyl group, APcI measurements in the negativemode were also carried out on bixin and norbixin, butno significant enhancement in sensitivity was observed.A similar approach has been used for the analysisof water-soluble annatto extracts in both positiveand negative electrospray detection modes (Galindo-Cuspinera and Rankin 2005). ES-detection modeshowed a major peak at m/z 379 corresponding to[M – H]� for norbixin, whereas the major peak at m/z381 was found using ESþmode. An ion at m/z 117 wasidentified in the ES-spectrum of 90-cis-norbixin butnot in the spectrum of the trans-isomer. Conversely,the trans-isomer showed an ion at m/z 111.1 in ESþmode that was not present on the spectrum of 90-cis-isomer. This was thought to be due to differences infragmentation patterns determined by stereochemicalconfiguration. More recently, it has been shown thatHPLC-PDA in combination with ion-trap electrospraymass spectrometric confirmatory analysis can be usedto identify and measure norbixin and bixin in meatproducts using precursor ions at m/z 379 and 395respectively and monitoring characteristic product ionsat m/z 253, 291, 310 and 335 (norbixin) and m/z 317,335, 345, 363 and 377 (bixin) (Noppe et al. 2009).
NMR spectroscopy
A comprehensive review on the use of NMR spectros-copy in the study of carotenoids in general is given byEnglert (1995), in which a detailed treatise on theexperimental aspects, chemical shifts of end-groups,chemical shifts and spin couplings, stereoisomeriza-tion, and simple and multidimensional experiments aregiven for 1H and 13C nuclei.
The earliest published use of NMR in the study ofbixin stereochemistry used low-resolution (40MHz)instrumentation to assign 1H frequencies and deducethat the cis-bond of the methyl analogue of ‘natural or�-’ bixin was in the 90- (equivalent) position (Barberet al. 1961). The high frequency shift of the protonassigned to H-80 was attributed to deshielding by the110-120 alkene bond when compared with the trans-(or �-) isomer, which was confirmed via synthesis andmore detailed structural assignments (Pattenden et al.1970). Fourier transform (FT) NMR was used laterto assign the 13C spectra of methyl cis- and trans-bixinusing deuterated compounds, however no experimentaldetails were given and assignments were partly derivedfrom spectra of carotenoids with similar structuralcharacteristics (Moss 1976). The 1H FT-NMR spec-trum of cis-bixin and cis-methyl bixin at 250MHz hasbeen reported but is limited to assignment of the
terminal acrylate moieties (Jondiko and Pattenden(1989). Proton NMR at 250MHz was used to confirmthe structures of purified trans- and 90-cis-bixin, wherethe chemical shifts and coupling constants associatedwith the change in stereochemistry were consistent withthose reported previously (Barber et al. 1961) butafforded much higher resolution (Scotter et al. 1994).A similar approach was used to confirm the structureof the principal thermal degradation product ofbixin as trans-4,8-dimethyltetradeca-hexaenedioc acidmonomethyl ester or C17 (Scotter 1995). The structureof a minor apocarotenoid isolated from Bixa orellanawas confirmed as methyl 90Z-apo-60-lycopenate usingproton NMR at 500MHz (Mercadante et al. 1996) anda similar approach used to identify apocarotenoidsnot previously found in annatto (Mercadante et al.1997b, 1999). NMR (300MHz 1H) was used alongsideTLC and HPLC in the bioautographic detection ofantimicrobial compounds in water-soluble annattoextracts where peak assignments were reported to beconsistent with previous reports (Galindo-Cuspineraand Rankin 2005).
The most comprehensive study to date on thedetermination of the structure of the bixin family ofapocarotenoids is by Kelly et al. (1996), who utilizeda combination of one-dimensional (1D) and 2D-NMRtechniques in conjunction with mass spectrometry andX-ray diffraction analysis. Chemical shift, couplingconstants and 1H correlation data were examinedalongside the ion abundances and intensity ratios fromstandard electron impact (EIþ) and FABþ MSspectra, and bond measurement, cell dimension anddegree of hydrogen bonding from X-ray diffractiondata to elucidate and compare the crystal structures ofthe cis- and trans-isomers of bixin and methyl bixin.
Other analytical techniques
Notwithstanding where specific techniques have beendiscussed elsewhere in this review, there are several lesswidely known techniques that have been used in thestudy of annatto either alone or in conjunction withcomplementary techniques. These include infraredspectroscopy, where the characteristic strong absorp-tion due to the C¼O stretching frequency between1740 and 1700 cm�1 and the complex bands in the1300–1050 cm�1 region due to C-O single bond char-acteristic of esters and carboxylic acids has been used(Lunde and Zechmeister 1954; Reith and Gielen 1971;Chao et al. 1991; Bernard and Grosjean 1995).Photoacoustic spectrometry in the UV, VIS and IRregions has been used for the qualitative and quanti-tative analysis of annatto in commercial seasoningproducts (Haas and Vinha 1995) and more recently inthe determination of the triplet state energy of bixin(Rios et al. 2007). X-ray photoelectron spectroscopy
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was used by Felicissimo et al. (2004) to ascertain themajor carotenoid composition of Bixa orellana seedsand X-ray diffraction in conjunction with NMR andmass spectrometry has been used to determine of thestructure of the bixin family of apocarotenoids (Kellyet al. 1996).
Analysis of foods
Before 1970 there were very few published methodsfor the extraction of annatto from foods. The quali-tative and quantitative analytical aspects of annattoextraction methods published before 1976 have beenreviewed briefly (Aparnathi and Sharma 1991). Theserelatively simple methods generally involve extractionwith solvent (e.g. chloroform, benzene, petroleumspirit or ether) with or without some form of samplepre-treatment such as protein precipitation, washingand adsorption onto an inert substance. The foodstuffsanalysed by these methods largely comprise dairyproducts, which reflects the relatively narrow scopeof annatto usage at that time.
Annatto has been extracted from whey solidswith dilute ammonium hydroxide where proteinswere precipitated by the addition of ethanol andphosphate buffer (Hammond et al. 1975), and frommeats (McNeal 1976). Annatto may be analysed inmilk and ice-cream by precipitation with boiling aceticacid and extraction of the whey with diethyl ether, andthe colour extracted from macaroni and noodles with80% ethanol followed by back-extraction into diethylether under alkaline conditions (Association of OfficialAnalytical Chemists (AOAC) 1980).
Rapid methods for the extraction of annatto fromfoods have been described where drinks and syrupswere dissolved in water, acidified with acetic acid andannatto was partitioned into diethyl ether (Corradiand Micheli 1981). Products with a high fat content,e.g. butter and margarine, were dissolved in petroleumspirit and annatto was partitioned into aqueousammonaical ethanol. Three extractions were requiredfor quantitative extraction of the colour. The aqueousextracts were acidified with acetic acid and back-extracted with diethyl ether. For foods containing fatand protein, e.g. yoghurt, cheese and pastries, sampleswere ground with sand and aqueous ammonaicalethanol. The mixture was transferred to a centrifugetube and the fat was removed by agitation withpetroleum spirit, centrifugation and siphoning off thepetroleum spirit phase. The aqueous ammonaicalphase was retained, acidified with acetic acid and theannatto partitioned into diethyl ether.
Methods for the extraction and determination ofannatto in margarine, cheese and boiled sweets havebeen investigated using techniques similar to those
described previously, with modifications to enablemeasurement by spectrophotometry and HPLC(Smith et al. 1983). Margarine samples were saponifiedto separate fat and to convert any bixin to norbixin,thereby facilitating its extraction into aqueous mediaand subsequent purification. However the reportedHPLC conditions gave poor peak shapes and insuffi-cient resolution. A method for the determination ofannatto in cheese in which a simple acetone extractionwas used, followed by concentration by rotary evap-oration has been described (Luf and Brandl 1988).Spectrophotometric (derivative) and HPLC techniqueswere used to quantify annatto in the presence of othercarotenoids, based on the procedure described for theanalysis of certain baked goods. However, the cis- andtrans-isomers of bixin and norbixin were not identifiedseparately under the stated conditions.
More recently, other workers have developedrefined methods for the extraction of annatto fromhigh-fat foods, dairy products and candy utilizingsolvent pre-extraction of fat and extraction of annattointo ethanolic aqueous ammonia (Lancaster andLawrence 1995) and to separate mixtures of bixinand norbixin from carminic acid in fruit beverages,yoghurt and candies (Lancaster and Lawrence 1996).HPLC was used to measure both the cis- and trans-isomers of bixin and norbixin but no significantimprovements in peak resolution were demonstratedcompared with those reported previously (Smith et al.1983), and impure reference materials were usedfor calibration. Recovery of norbixin from spikedcheese samples was reported to average 93% overthe range 1–110mgkg�1, and the recovery of bixinfrom spiked wafers also averaged 93% over therange 0.1–445mgkg�1. The recovery of norbixinfrom laboratory-prepared hard candies averaged 88%.
TLC and HPLC were used to determine bixin andother carotenoid colours in products derived from redpepper (Mınguez-Mosquera et al. 1995). A simpleacetone extraction was used followed by partition withether and sodium chloride solution and alkalinesaponification. Back extraction with ether followingacidification of the saponifying medium was necessaryto recover the annatto colour (as norbixin). Whilegood chromatographic separation of the carotenoidswas obtained, no distinction between norbixin isomerswas made. However, the method demonstrated thecapability of detecting of colours added fraudulently tointensify the natural colour of paprika paste.
Whilst remaining an uncommon analytical tech-nique in food laboratories, photoacoustic spectrometry(PAS) has been used for the analysis of annattoproducts (Haas and Vinha 1995). The method is limitedto semi quantitative (�1% ‘annatto content’) andqualitative analysis of commercial seasonings compris-ing mixtures of corn meal and powdered annatto seeds
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or annatto extract known as ‘Colorifico du Urucum’.The particle size of the samples has a strong influence onthe amplitude of the PAS signal and therefore requiresclose control.
Based on the methods described previously (Scotteret al. 1994; Lancaster and Lawrence 1995), HPLC andspectrophotometric methods have been developedfor the simple and rapid determination of annatto incheese and milk products (Bareth et al. 2002). Solid-phase extraction (SPE) on amino phase was used toseparate annatto components from fat and �-carotene.The choice of end method was determined by thepresence of other colouring materials, i.e. curcuminor �-apo-80-carotenal but other food colours andemulsifiers did not affect the analysis. The recoveryof annatto colouring spiked into cheese, processedcheese, butter and ice-cream ranged between 80 and100%. Nine samples of cheese were analysed in whichnorbixin was found in the range50.15–11.89mg kg�1,whereas no bixin was detected (40.15mg kg�1).
The methods described by Scotter et al. (1994,1998), Lancaster and Lawrence (1995) and Navaz Diazand Ramos Peinado (1992) were further developed andconsolidated to encompass a wide range of foodcommodities (Scotter et al. 2002). Specific solventextraction regimes were developed for specific samplematrices, with HPLC-PDA used for spectral confir-mation and measurement of the main isomers ofbixin and norbixin. The different extraction regimesare summarized in Table 4.
With the exception of regime 5, samples wereextracted essentially using ethanol:water:ammoniasolution with or without a hexane partition toremove excess lipid. After centrifugation in the pres-ence of Celite filter aid, the annatto colour waspartitioned into chloroform:acetic acid solution, cen-trifuged and the solvent removed using vacuum-assisted rotary evaporation. To minimize analytelosses via oxidation, a 0.1% solution of butylatedhydroxyl toluene (BHT) was added. For regime fivematrices, samples were mixed with Celite in the
presence of dilute hydrochloric acid and extractedusing a biphasic solvent system comprising hexane(to remove excess lipid) and acetonitrile, whichwas then concentrated using vacuum-assisted rotaryevaporation.
Using this method, comprehensive quantitativeand qualitative data on 165 composite and two singlefood samples covering a wide range of foods at levelsabove the analytical reporting limit of 0.1mg kg�1 wereobtained. Quantitative results were given for thoseannatto colouring components for which referencestandards were available (90-cis-bixin, trans-bixinand 90-cis-norbixin), whereas semi-quantitative resultswere given for other bixin and norbixin isomers.The method was single-laboratory validated by therepeat (n¼ 4 – 9) analysis of twelve different sampletypes of food commodity covering the permitted rangeof annatto content, spiked with annatto at levels ofbetween 1.7 and 27.7mgkg�1 and by the analysisof in-house reference matrices. Mean recoveries ofbetween 61% and 96% were obtained from foodsspiked with annatto.
Using response surface methodology to establishoptimum conditions, a method for the determinationof annatto colour in extruded corn snack products hasbeen developed that exhibits improved accuracy andprecision compared with the methods described byScotter et al. (2002) and Rios and Mercadante (2004).However, pre-treatment of the samples with �-amylasewas necessary to remove starch and a total of eightsolvent extractions with ethyl acetate were required forcomplete extraction of the annatto colour. Lipids wereremoved using alkaline saponification therefore allof the bixin present was hydrolysed to norbixin anddetermined as such by HPLC.
Accelerated solvent extraction has been comparedwith manual solvent extraction to determine severalfood colouring carotenoids including bixin and nor-bixin in processed foods (Breithaupt 2004). Reverse-phase HPLC with a C30 column successfully separatedbixin and norbixin from 7 other carotenoids butthe cis- and trans-isomers were not distinguishable.Due to its ostensibly higher polarity, lower recoveriesof norbixin were reported for accelerated extraction(67� 1.0mg kg�1) compared with manual extraction(88.7 � 6.2mg kg�1). However, a similar difference inrecoveries was reported for less polar bixin (91.0� 2.7and 98.0� 1.7mgkg�1, respectively) although bixinrecovery was higher than norbixin with improvedprecision. The limit of quantitation for bixin andnorbixin was in the range 0.53–0.79mg kg�1 forpudding mix and cereals. More recently, a methodfor the determination of norbixin and bixin in meatproducts using HPLC-PDA and LC-MSn that givesrecoveries of between 99% and 102% and a limit ofquantitation of 0.5mg kg�1 has been reported (Noppeet al. 2009).
Table 4. Summary of extraction regimes used for annatto infoods (Scotter et al. 2002).
Regime Matrices
1 Cheese, cheese products and cheese-basedcompound foods
2 Custard powder and low-fat dessert dry mixes3 Desserts, cake decorations, fine bakery wares,
extruded snacks and breakfast cereals4 Margarine, fat-based emulsions and spreads,
butter and fat-based compound foods5 Fish, ice cream and ice cream-based
confectionery, yoghurt and other dairydesserts
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Annatto as an illegal food dye
The illegal use of annatto to colour milk goes back asfar as the early 20th century where it was reported byUK Public Analyst laboratories (Ellis Richards 1923;Collingwood Williams 1925). Amongst other specificfood commodities, annatto is currently permittedin the European Union for the colouring of certainmargarines and cheeses, but is not permitted forthe colouring of milk cream or butter (EuropeanCommission 1994 as amended). Moreover, whileannatto is permitted for use in food commoditiessuch as savoury snack products, coated nuts, extrudedproducts and flavoured breakfast cereals, it is notpermitted for use in spices. However, amongst othernon-permitted dyes bixin was detected in 18 of 893samples of spices, sauces and oils by UK enforcementlaboratories during 2005–2006 as part of the UKImported Food Programme (Food Standards Agency(FSA) 2006). This has led directly to a need foranalytical methods capable of detecting very low levelsof annatto in food ingredients and commodities inwhich it is not permitted, driven not only by theenforcement of regulations on a national scale(disseminated through the European Union RapidAlert System; European Union 2008), but also by theneed for the food manufacturing industry to ensurecompliance, especially in a proactive manner andthrough the adoption of a ‘zero tolerance’ approachas applied to the monitoring of illegal dyes such as theSudan Red group. Established HPLC methods capableof detecting bixin or norbixin at approximately0.1mgkg�1 in samples using UV-VIS or diode-arraytechnology are not sufficiently sensitive. LC-MS/MSmethodology is the obvious candidate but sufficientlydetailed methods in peer-reviewed publications havenot been forthcoming to date. Nevertheless, it isgenerally considered amongst analytical chemists work-ing in this area that LC-MS/MS is capable of detectingbixin at approximately 0.01mgkg�1 in certain com-modities, but this is heavily dependent upon the degreeof signal suppression caused by matrix effects. This cangive rise to false negative results using a screeningapproach, which in turn identifies a need for suitableextract clean up regimes, and guarding against ionsuppression by using the method of standard addition.
Future aspects
There is a clear requirement in the future for thedevelopment and validation of highly sensitive meth-ods of analysis for annatto in food commodities andother food ingredients which is driven by the needto ensure compliance with food quality regulationsand especially in the light of the pursuit of suitablealternatives to synthetic food colours. An in-depthunderstanding of the chemistry and stability of annatto
is therefore requisite and brings clear benefits to theproduction of annatto, and to the formulation andapplication of food colouring to a wide range of foodcommodities. Greater understanding of the processesof degradation may also benefit studies in the areasof food safety, particularly in risk assessment, andbiomarkers of exposure such as circulating (plasma)levels of norbixin. Here, complementary analyticaltechniques such as HPLC-PDA, LC-MS/MS andNMR will play a vital role in the detection, confirma-tion, and measurement of comparatively low levels ofbixin and norbixin isomers.
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