Keywords: Analysis -NMR, Chromatography -GC/MS, Chromatography -HPLC, LC/MS Additives/Contaminants: Colours, Process contaminants, Volatiles Food Types: Ingredients, Processed foods annatto, bixin, norbixin, food additives, chemistry, stability, 25

Annatto food colouring (E160b) has a long history of use in the food industry for the 4 colouring of a wide range of food commodities. The principle colouring components 5 of annatto is the oil-soluble diapo carotenoid bixin, which is the methyl ester of the 6 dicarboxylic acid norbixin, which is soluble in aqueous alkali. Bixin and norbixin 7 therefore exhibit not only physicochemical properties normally associated with 8 carotenoids but also certain anomalous properties that have an impact on the stability, 9 food colouring applications and importantly the analysis of annatto. This review 10 summarizes the key aspects of the structural determination of bixin (and norbixin) 11 with special attention to cis-trans isomerization and how this links with its chemical 12 structure, spectroscopic properties and stability. The oxidative, thermal and photo 13 stability of annatto and the subsequent implications for its use in the colouring of 14 foods, food processing, and the analysis of foods and beverages are discussed along 15 with important mechanistic, thermodynamic and kinetic aspects. The main analytical 16 techniques used for the chemical characterization of annatto i.e. spectrophotometry, 17 NMR, chromatography (particularly HPLC) and mass spectrometry are reviewed in 18 detail and other methods discussed. This links in with a review of the methods 19 available for the detection and measurement of annatto in colour formulations and 20 foods and beverages, which highlights the importance of the need for a good 21 understanding and knowledge of the chemistry of bixin and norbixin in order to meet 22 new analytical challenges.

Annatto is a natural colouring agent obtained from the outer coats of the seeds of the 28 tropical shrub Bixa orellana. Annatto and its extracts are designated collectively as 29

E160b and permitted as a food additive in the European Union and elsewhere, and 30 have widespread use in the food industry for the colouring of many commodities 31 including flour and sugar confectionery, dairy and savoury products, soft drinks and 32 fish. The major colour principles of annatto are the carotenoids bixin and norbixin. 33

Though chemically very similar, differences in their chemical properties present 34 several challenges to the analytical chemist with respect to stereochemistry, solubility, 35 chromatographic behaviour and stability. While current legislation on the extraction 36 and use of annatto colours and their applications in food are addressed briefly, this 37 review focuses on the chemistry, stability and analysis of annatto pertaining to its use 38 as a permitted food colouring. 39 40

Annatto in foods 41

Legislative aspects 42

The use of food colours in the European Union is controlled by European Community 43 Directive 94/36/EC (EC, 1994 as amended) which contains a list of permitted colours, 44 a list of foodstuffs to which these colours may be added, and where appropriate, 45 maximum limits on the level of addition. The permitted uses of annatto and the 46 maximum levels of addition are given in Table 1. Annatto extracts are listed amongst 47 those colours that may be used singly or in combination in certain foods up to the 48 maximum levels specified (on a ready-for-consumption basis). Comprehensive on-line 49 sources of information on permitted food colour regulations and specifications may be 50 the Joint FAO/WHO Expert Committee on Food Additives (JECFA) for information 100 relating to the toxicity, intake and specifications of annatto, the European annatto 101 producers consulted with the food industry to determine usage levels of specific 102 annatto extracts (JECFA, 2002). The data obtained were combined with the levels of 103 bixin/norbixin in particular extracts to provide an estimate of their concentration in 104 food. These data have been combined with food consumption data using various 105 methods to estimate consumer intakes, which ranged from 1 to 163% of the ADI 106 [START_REF] Tennant | Study of the carotenoid pigments of Bixa orellana L. seeds by 1606 thin layer chromatography[END_REF]. The actual levels of annatto in foodstuffs were well 107 below maximum limits prescribed under EU regulations and Codex standards, which 108 had been confirmed by an earlier analytical study [START_REF] Scotter | Method development and 1586 analysis of retail foods for annatto food colouring material[END_REF]. 109 110

Annatto chemistry 111

Elucidation of the chemical constitution of bixin (and thereafter norbixin) was first 112 put forward by [START_REF] Heiduschka | Zur Kenntnis des Bixins[END_REF] who suggested the correct molecular 113 formula for bixin (C 25 H 30 O 4 ) as an unsymmetrical molecule. Herzig and Faltis (1923) 114 recognised that bixin was the monomethyl ester of an unsaturated dicarboxylic acid. 115

The results from their catalytic hydrogenation studies led them to conclude that bixin 116 contains 9 carbon double bonds, which, evidenced by the intense red colour of the 117 pigment, were conjugated. However, the unsymmetrical molecule hypothesis was 118 abandoned when proposed the now accepted structure was proposed (Kuhn and 119 Winterstein, 1932), later confirmed by [START_REF] Karrer | Konstitution des 1361 Safranfarbstoffs Crocetin, Synthese des Perhydro-bixin-äthylesters und Perhydro-1362 norbixins. Vorläufige Mitteilung[END_REF]. A new, higher melting 120 form termed β-bixin was obtained during the course of pigment isolation (Herzig and 121 Faltis, 1923), which was later proposed as the trans-isomer that the original form may 122 be cis-isomer [START_REF] Karrer | Über Bixin. (XIII. 1358 Mitteilung über Pflanzenfarbstoffe)[END_REF]. A stable form of bixin identical to the β-form by 123 treatment of the 'natural' (or 'labile') form with iodine was subsequently obtained 124 [START_REF] Kuhn | Die dihydroverbindung der isomeren bixine und die 1384 elektronen-konfiguration der polyene[END_REF]. From the results of these investigations that it was 125 concluded that bixin was the first known naturally occurring cis-polyene. The 126 structural elucidation of bixin was confirmed from various oxidation and degradation 127 experiments [START_REF] Karrer | Chapter V. Cis-trans isomerism of carotenoids[END_REF]. During investigations to determine the 128 stereochemical configuration of labile bixin, several stereoisomers were isolated 129 [START_REF] Zechmeister | Cis-trans isomeric carotenoid pigments[END_REF]. The consequences of cis-trans isomerism on the chemistry, 130 stability and analysis of annatto are significant and are discussed below. 131 132

The major colouring component of annatto is confirmed as the apo-carotenoid 9'-cis-133 bixin (methyl hydrogen 9'-cis-6,6'-diapocarotene-6,6'-dioate, C 25 H 30 O 4 ), the 134 monomethyl ester of the dicarboxylic acid 9'-cis-norbixin, commonly referred to as 135 cis-bixin (Figure 1). 9'-Cis-bixin is soluble in most polar organic solvents to which it 136 imparts an orange colour but is largely insoluble in vegetable oil. It may be readily 137 converted to the all-trans isomer due to its instability in the isolated form in solution. 138

Trans-bixin is the more stable isomer and has similar properties to the cis-isomer but 139 exhibits a red colour in solution and is soluble in vegetable oil. Commercially,140 isomerisation is achieved by heating a suspension of the cis-isomer in oil to 130°C in 141 vacuo. The water-soluble analogue 9'-cis-norbixin (C 24 H 28 O 4 ) can be isolated from 142 annatto seeds by agitation in aqueous alkali at <70° C or formed by alkaline hydrolysis 143 of cis-bixin to give either the sodium or potassium salt. The dicarboxylic acid is 144 soluble in polar solvents to which it imparts an orange colour. 9'-Cis-norbixin is only 145 sparingly soluble in chloroform and 0.1M sodium hydroxide (Preston and Rickard, 146 1980). Under extraction conditions, 9'-cis-bixin undergoes isomerization to produce oil 147 solutions containing approximately 0.2 -0.5% of pigment comprising a mixture of all-148 While it is reported that 80% of the carotenoids in the annatto seed coat comprise bixin 154 [START_REF] Preston | Extraction and chemistry of annatto[END_REF][START_REF] Lauro | A primer on natural colours[END_REF], traces of bixin diesters may be found 155 (Mercadante et al., 1997b). The preparation and use of ethylbixin has been discussed 156 [START_REF] Levy | Annatto[END_REF]. 157 158The presence of other minor carotenoids in annatto has also been postulated, which 159 included β-carotene, cryptoxanthin, lutein, zeaxanthin and methyl bixin (Tirimanna, 160 1980). The presence of a range of lycopenoate analogues and other minor carotenoids 161 in annatto has been reported in a series of papers by Mercadante and co-workers (1996Mercadante and co-workers ( , 162 1997aMercadante and co-workers ( , 1997bMercadante and co-workers ( and 1999) ) and has been reviewed by [START_REF] Mercadante | Composition of carotenoids from annatto[END_REF] and 163 Satyanarayana, Prrabhakara Rao and Rao (2003). Bixin and three minor carotenoids 164 have been chemically synthesized using the Wittig reaction of the (Z)-terminus 165 followed by a Horner-Emmons reaction [START_REF] Haberli | Synthesis of bixin and three minor carotenoids from 1314 annatto (Bixa orellana)[END_REF]. 166 167

Molecular properties 168

It is the delocalisation of π-electrons along the polyene backbone that gives 169 carotenoids their characteristic electronic spectra and is largely responsible for the 170 photophysical and photochemical properties of these molecules, including cis-trans 171 photoisomerization. Detailed explanations of cis-trans isomerization may be found in 172 standard texts [START_REF] Karrer | Chapter V. Cis-trans isomerism of carotenoids[END_REF][START_REF] Lunde | Infrared spectra and cis-trans configurations of some 1415 carotenoid pigments[END_REF]Zechmeister, 173 Kolher, 1995). Only the basic properties of linear conjugated molecules will be 174 reviewed here along with a brief account of the simple concepts that apply to bixin 175 and norbixin, in order to provide a background for discussion on the UV-VIS 176 spectroscopy of these compounds and to show how UV-VIS spectra are affected by 177

isomerization. 178 179

The sufficiently large barriers to rotation about the formal double bonds in polyenes 180 or carotenoids allow double bond cis-and trans-isomers to be isolated as 181 independent, distinct compounds. Since the differences in excitation energies for cis-182 and trans-isomers of a given molecule are small compared to the change in excitation 183 energy that is associated with adding or subtracting a conjugated double bond, the 184 basic electronic structure is almost independent of isomeric form. The four single 185 bonds that surround a carbon-carbon double bond all lie in the same plane. In 186 consequence, each of the disubstituted and trisubstituted acyclic double bonds that 187 constitute the polyene chain of a carotenoid can exist in two forms i.e. geometric 188 isomers. Nomenclature of the cis-or trans-isomers is designated in accordance with 189 IUPAC rules (Weedon andMoss, 1995a, 1995b and disubstituted double bonds in the 15,15'-position, the effect is relatively small, as 199 it results from limited interference between two hydrogen atoms and hence these 200 isomers may be formed quite readily. With other disubstituted double bonds the 201 adoption of the cis-configuration results in major interference between a hydrogen 202 atom and a methyl group. This renders such molecules less stable than the 203 corresponding trans-form and hence less likely to be encountered (Karrer and Jucker, 204 1950;[START_REF] Zechmeister | Cis-trans isomeric carotenoid pigments[END_REF]Kolher, 1995;Weedon and Moss, 1995a). 205 206 Stereomutation studies, in which interconversion of geometrical isomers is 207 deliberately promoted, lend support to this theory. Interconversion generally produces 208 a 'set' of isomers that approximates to an equilibrium mixture of all possible geometric 209 forms proportional to their relative thermodynamic stabilities. The all trans-form 210 usually predominates, indicating that it is the most thermodynamically stable isomer. 211

A number of mono-and di-cis-isomers are usually also present, however those 212 isomers with more than two cis-double bonds and/or those that are sterically hindered 213 usually occur only in trace amounts, if at all. It is not surprising that most naturally 214 occurring carotenoids are predominantly in the all trans-form. However, bixin occurs 215 predominantly as the cis-isomer, which has a cis-configuration about the 216 9'-trisubstituted double bond. Since asymmetric bixin has nine alkene bonds (n=9), 217 theoretically 512 (i.e. 2 n = 2 9 ) geometric isomers are possible, whereas symmetric 218 norbixin has only 272 (i.e. 2 (n-1/2) x (2 (n-1/2) + 1) possible isomers. However, the 219 presence of stable cis-isomers at positions 7, 11, 12' and 8' are stearically hindered, 220 hence the remaining 5 alkene bonds are capable of yielding 32 and 20 isomers for 221 bixin and norbixin respectively (Figure 1 Provided that an adequate sample of the pure isomer is available or the selected 224 analytical technique is adequately sensitive, spectroscopic analysis will normally 225 allow the unambiguous assignment of the geometrical configuration of any carotenoid 226 isomer. All linear polyenes, the carotenoids included, possess similar low-lying 227 excited singlet (S 1 ) states [START_REF] Hudson | Linear polyene electronic structure and spectroscopy[END_REF][START_REF] Kohler | Visual chromophore electronic structure[END_REF]Hudson, Kohler 228 and Schulten, 1982). This is critically important since virtually all photo processes in 229 linear polyenes originate in the lowest-energy singlet excited state, the correct 230 identification and characterization of which is therefore also important. As might be 231 anticipated from the similarities in electronic structure, the electronic absorption 232 spectrum of a given carotenoid closely resembles that of the unsubstituted polyene 233 with the same number of conjugated double bonds. There are however well 234 characterised principle differences due largely to the presence of methyl substituents 235 along the carotenoid skeleton, which affect the basic polyene electronic structure. 236

These are: 237

• a 10 to 30 nm shift of the lowest energy strong absorption band to 238 longer wavelength 239

• decreased vibrational fine structure 240 241 Thus, cis-and trans-isomers may often be distinguished on the basis of their UV-VIS 242 absorption spectra, but the most important differences observed between isomers are 243 not related to excitation energies but to the relative intensities of high-energy 244 absorption bands i.e. 'cis-' peaks [START_REF] Dale | Empirical relationships of the minor bands in the absorption spectra of 1245 polyenes[END_REF][START_REF] Zechmeister | Cis-trans isomeric carotenoid pigments[END_REF]. It is well 245 established that the lowest excited state of linear polyenes (including carotenoids) is 246 the 2 1 A g state and that the origin of the main absorption band is the strongly allowed 247 are derived from the vibrational levels that are associated with the initial and final 249 electronic states. Thus, the typical three-peaked shape of the main absorption band of 250 linear polyenes arises from transitions of the lowest vibrational level of the electronic 251 ground state to the lowest vibrational levels of the electronic excited states. 252

2 1 A g → 1 1 B u
Broadening of these peaks is observed because of rotational levels and inhomogeneity 253 leading to peak overlap. This is particularly relevant for many carotenoids measured 254 as solutions at ambient temperatures [START_REF] Kohler | Chapter 1: Electronic structure of carotenoids[END_REF]. 255 256The positions of the absorption maxima and the shape or fine structure of the UV-VIS 257 spectrum of carotenoids are therefore characteristic. But while the UV-VIS spectrum 258 gives information about the chromophores of the molecule, it yields nothing about 259 functional groups apart from conjugated carbonyl groups that form part of the 260 molecule [START_REF] Scott | Chapter 8: Application of spectral data to the investigation of gross 1557 molecular structure. Interpretation of the ultraviolet spectra of natural products[END_REF]Britton, 1995a and1995b). In the case of carotenoids, the 261 relevant transition is the π→ π* transition. For such a conjugated system, in which the 262 π-electrons are highly delocalized, the excited state is of comparatively low energy. 263

The energy required to bring about the transition is therefore relatively small and 264 corresponds to light in the visible region. While the transition responsible for the main 265 absorption band is strongly 'allowed', transitions from the ground state to higher 266 electronic states are also possible, providing they obey the symmetry selection rules. 267

These high energy transitions give rise to absorption bands in the UV region which 268 are usually weak, but are observed particularly in the spectra of compounds with 269 transitions that produce them become allowed (Britton, 1995a). For trans-isomers, 274 the electronic structure has a centre of symmetry and the ground state is a g state, so 275 transitions to a higher g state are forbidden. Transition to a higher excited g state only 276

becomes allowed when at least one double bond becomes cis-and the original 277 symmetry is lost. This gives rise to an absorption band in the UV region, known as the 278 cis-band or cis-peak. The most important feature of the absorption spectrum of a 279 carotenoid is the main absorption band in the visible region. Several important pieces 280 of information can be obtained from the spectrum: 281 Since the structure of the carotenoid chromophore is related to the overall shape or 295 fine structure of the spectrum, the shape as well as the positions of the absorption 296 maxima may therefore be used as a diagnostic tool, especially when comparing 297 carotenoid spectra (Britton, 1995a) proved convenient and removes the requirement for presenting all spectra as diagrams 299 [START_REF] Kohler | Chapter 1: Electronic structure of carotenoids[END_REF]. In this notation, the baseline or zero value is taken as the minimum 300 between the two peaks, the peak height of the longest wavelength absorption band is 301 designated as III, that of the middle absorption band (usually λ max ) as II (Figure 2). 302 Spectral fine structure is then expressed as the ratio of the peak heights III/II, as a 303 percentage. 304 305 [Insert Figure 2 about here] 306 307

The annatto carotenoids bixin and norbixin are unusual in that they contain two 308 carbonyl (i.e. carboxyl) groups, one at either end of the conjugated system and in 309 conjugation with it, which formally extends the chromophore. This results in a 310 spectral shift to longer wavelength, usually accompanied by loss of spectral fine 311 structure but the degree of effect is solvent dependent. With dicarboxylic acids and 312 their esters such as bixin (and norbixin), the further extension of the chromophore 313 causes a large relative increase in λ max . The spectral shift depends on the polarizability 314 rather than the polarity of the solvent, and the frequency shifts to lower energy, i.e. a 315 spectral shift to longer wavelength, as the refractive index of the solvent increases 316 Britton, 1995a). 317 318 While water can have a major effect on the spectra of carotenoids in water-miscible 319 solvents, both bixin and norbixin isomers are relatively polar carotenoid molecules, 320 which are significantly soluble in solvents with a high aqueous content and as such 321 similar solvent-related properties are not observed. Cis-norbixin is in fact highly 322 soluble in 0.1M sodium hydroxide solution, and both compounds are fairly soluble in 323 The differences that are observed consistently between the spectra of trans-and cis-327 isomers of carotenoids are therefore diagnostic for structural assignment. A small 328 hypsochromic shift in λ max of ca. 2 to 6 nm is usually observed for mono-cis isomers 329 along with a significant hypochromic effect and a reduction in vibrational fine 330 structure. A new absorption band appears at a characteristic position about 142 nm 331 below the longest wavelength absorption maximum, often referred to as the cis-peak. 332 333 For the all-trans form of bixin, the main absorption band (2 1 A g → 1 1 B u ) is very 334 intense. In the 9'-cis isomer), the intensity of this main band decreases as a weak cis-335 peak appears at 355 nm, corresponding to the transition to a higher energy level g 336 state. From their studies on β-carotene isomers, it has been shown that the 15-cis 337 isomer, in which the cis double bond is in the centre of the molecule, shows maximum 338 bending of the chromophore and a well developed cis-band, with corresponding 339 decrease in intensity of the main absorption band (Petterson and Jonsson, 1990). 340 341

The intensity of the cis-band is essentially greater as the cis double bond is nearer the 342 centre of the molecule and is therefore empirically diagnostic. In a symmetrical di-cis 343 carotenoid, the centre of symmetry may be restored so that the cis-band again 344 becomes a weak feature. For di-cis and poly-cis carotenoids, a larger hypsochromic 345 shift in the main absorption band may be seen e.g. 13 nm for di-cis norbixin (Figure 346 3) [START_REF] Scotter | Characterization 1562 of the principal colouring components of annatto using high performance liquid 1563 chromatography with photodiode array detection[END_REF] has been adapted to designate the relative intensity of the cis-peak [START_REF] Kohler | Chapter 1: Electronic structure of carotenoids[END_REF]. 352

The intensity or absorbance of the cis-peak is expressed as a percentage of the 353 absorbance of the middle main absorption band, which is usually the λ max . Scotter et 354 al. (1994) used this technique to study the spectra obtained by HPLC-photodiode 355 array analysis of geometrical isomers of bixin and norbixin. The absorption intensity 356 at λ max (Figure 4, III) for each isomer was normalised and a comparison of relative 357 intensity (REL (%)) made with the three other maxima at I, II and IV, to give REL(I), 358 REL(II) and REL(IV) respectively. In all cases, λ max (II) took the form of inflection 359 rather than a peak, which made exact location of the wavelength maximum difficult. 360 However, post-run analysis of the spectral data allowed first derivative spectra to be 361 taken which facilitated location of λ max (II), as shown in Figure 5. 362

Annatto stability 367

It is well known that the polyene chain in carotenoids is responsible for their 368 instability i.e. their susceptibility to oxidation by various agents such as oxygen and 369 peroxides, addition of electrophiles including H + and Lewis acids, and cis-/trans-370 isomerization due to various factors such as temperature and light as discussed above. 371

Other undesirable reactions may also be promoted by higher temperatures and the 372 light, and exposure to strong acids and alkalis should normally be avoided. 373 Annatto, especially norbixin, is susceptible to oxidation, particularly when applied in 376 powdered form due to the large surface area and when incorporated into foodstuffs; 377 although some foods can have a stabilizing effect [START_REF] Berset | Potential use of annatto in extrusion cooking[END_REF]Collins, 378 1992;[START_REF] Levy | Annatto[END_REF]. Spray-dried norbixin formulated with acaia gum 379 or maltodextrin as carriers have been reported to be particularly susceptible to 380 oxidation [START_REF] Henry | Natural food colours[END_REF]. The level of bixin prepared from annatto seeds and stored 381 for ca. 1 year at 30°C in packages comprising materials with different oxygen 382 transmission rates was reduced by ca. 10% during the first 2-3 weeks storage but 383 stabilized thereafter except for polyethylene film, which exhibited a degradation rate 384 of 0.04% per day, reflecting the permeability of the polyethylene (Carvalho et al., 385 1993). Several mechanisms have been put forward for the effect of water activity on 386 the reduction of bixin oxidation in microcrystalline cellulose-based model systems to 387 simulate dehydrated foods [START_REF] Gloria | Effect of water activity on the stability of 1305 bixin in annatto extract-microcrystalline cellulose model system[END_REF]. Bixin degradation followed first order 388 kinetics and the observed half-lives showed greater stability in systems of 389 intermediate and high water activity. It was postulated that this is because of the 390 ability of water to exclude oxygen from liposoluble materials by surface adsorption, 391 hydrogen bond with hydroperoxides, inactivate metal catalysts, reduce of free radicals 392 and lower the stability of singlet oxygen. Annatto has been shown to inhibit 393 hydroperoxide formation leading to triglyceride autoxidation by trapping peroxy 394 radicals [START_REF] Haila | Effects of lutein, lycopene, annatto and 1317 g-tocopherol on autoxidation of triglycerides[END_REF]. Annatto was among a number of mediterranean spices 395 whose antioxidant capacities were compared with permitted food antioxidants in lipid 396 peroxidation [START_REF] Martinez-Tomé | Antioxidant properties of Mediterranean spices compared with common food 1430 additives[END_REF]. Annatto was reported to have a greater 397 antioxidant capacity than either butylated hydroxylanisole (BHA) or butylated 398 prepared by oil extraction of seeds was found to be more stable than a powdered 402 formulation during storage over ca. 1 year. Samples were stored in glass bottles with a 403 3cm headspace of air. The greatest losses (60%) were observed for the powder at 404 ambient temperature in daylight, compared to ambient temperature in the dark (54%) 405 and at 5-8°C in the dark (23%). These results concur with the findings of Najar, 406 Bobbio and Bobbio, (1988) that light is the main degradation factor. Moreover, 407 photosensitized bixin is very reactive towards oxygen and thus may be considered as a 408 an oxygen quencher; the reaction of bixin with singlet oxygen is a related issue and is 409 discussed below. 410 411 Norbixin was the only carotenoid that inhibited the oxidative deterioration of lipids in 412 both olive oil and oil-in-water emulsions stored at 60°C and displayed a similar 413 activity to δ-tocopherol in stored oil (Kiokias and Gordon, 2003). In olive oil-in-water 414 emulsions, norbixin reduced hydroperoxide formation and a synergistic effect 415 between norbixin and ascorbic acid or ascorbyl palmitate was observed. 416 417 Bixin has been reported to be able to scavenge hydroxyl radicals generated by ferrous 418 ions (Fe 2+ ) and hydrogen peroxide (H 2 O 2 ) but no mechanism was suggested (Zhao et 419 al., 1998). Similarly, the behaviour of norbixin during in vivo plasmid DNA damage 420 induced by reactive oxygen species Fe 2+ , Sn 2+ and H 2 O 2 have been studied and it has 421 proposed that since norbixin contains two free carboxyl moieties, its protective action 422 may rely on the formation of complexes [START_REF] Kovary | I. Biochemical behaviour of norbixin during in vitro DNA 1381 damage induced by reactive oxygen species[END_REF]. Norbixin showed a 423 stronger affinity for Sn 2+ than for Fe 2+ but was readily displaced by EDTA. 424 425 During the isolation and analysis of carotenoids, the exclusion of atmospheric air by 426 inert gas or vacuum is strongly recommended in order to minimize the risk of 427 destruction or undesired reactions (Scheidt and Liaaen-Jensen, 1995) and annatto is 428 no different in this respect. The oxidation 'products' of bixin were identified 429 tentatively after transformation of bixin in corn oil at 125°C using spectrophotometry 430 and paper chromatography (McKeown and Mark, 1962). Evidence for the oxidative 431 decomposition of cis-bixin on TLC plates has been reported, where both powdered 432 colour formulations and chloroform solutions of cis-bixin exhibited decreased colour 433 content when stored in the dark in air at ambient temperature (Reith and Gielen, 434 1971). This was concluded to be due to the presence of oxygen, supported by 435 observing the relatively lower stability of cheese colour (norbixin in aqueous KOH) 436 compared to butter colour (bixin in vegetable oil), due to the presence of tocopherols 437 in the latter. The effects of light, air and pro-oxidants on the stability of annatto 438 extracts in chloroform over a 12 day period were monitored by spectrophotometry. 439

Air was much less effective at promoting loss of colour compared to light or to 440 benzoyl peroxide, a free-radical promoter [START_REF] Najar | Effects of light, air, anti-oxidants and pro-1483 oxidants on annatto extracts (Bixa orellana)[END_REF]. The 441 authors concluded that rapid loss of colour might occur whenever free radical 442 formation is promoted. 443 444

Reaction with singlet oxygen 445

Model studies on the photosensitized isomerization of cis-bixin show that while bixin 446 in the ground electronic state is stable to thermal isomerization, energy transfer via 447 which readily isomerizes to the trans-isomer [START_REF] Montenegro | 1476 Model studies on the photosensitized isomerisation of bixin[END_REF]. The rate of 449 isomerization is dependent on several factors which compete for deactivation of 450 3 BIX* e.g. ground state bixin and triplet oxygen ( 3 O 2 ). Primary reaction products are 451 only degraded in the presence of air and under prolonged illumination, which is due to 452 the formation of oxidation products by reaction with singlet oxygen ( 1 O 2 ). The 453 associated reaction mechanisms are discussed very elegantly by the authors. In a 454 similar study,), the 3 BIX* energy level was calculated used laser-induced 455 photoacoustic calorimetry of bixin in methanol; acetonitrile solution (Rios et al., 456 2007). The results of the study showed that bixin is a very efficient quencher of 1 O 2 in 457 fluid solutions due to an efficient energy-transfer process, and confirmed that that the 458 3 BIX* energy level is lower than that of 1 O 2 (18 ± 2 kcal/mol and 22.5 kcal/mol 459 respectively). 460 461

Thermal stability 462

While bixin and norbixin have good heat stability during food processing compared to 463 other carotenoids, 9'-cis-bixin undergoes a series of complex degradation reactions at 464 commercial extraction temperatures to produce a range of products coloured pale 465 yellow to orange [START_REF] Iversen | Uber den farbstoff in annatto-butterfarben[END_REF][START_REF] Levy | Annatto[END_REF]. Using paper 466 chromatography, the pigments in commercial annatto preparations were separated into 467 a series of bands that included a number of yellow bands comprising up to 40% of the 468 total pigments and including bright yellow fluorescent (sic) band [START_REF] Mckeown | Paper chromatography of bixin and related compounds[END_REF]. 469

This band was thought to be the pale yellow breakdown product of bixin identified 470 previously [START_REF] Iversen | Uber den farbstoff in annatto-butterfarben[END_REF]. The main thermal degradation product of 9'-cis-471 bixin has since been isolated and identified using paper chromatography and UV/VIS 472 (McKeown and Mark, 1962;474 McKeown, 1963 and[START_REF] Mckeown | Composition of oil-soluble annatto food colors. III. Structure of 1440 the yellow pigment formed by the thermal degradation of bixin[END_REF][START_REF] Preston | Extraction and chemistry of annatto[END_REF] The influence of heating time 475 on the thermal degradation of bixin in alkaline extracts of annatto showed that 476 pigment stability is related to the initial quantity of cis-and trans-bixin as well as to 477 the method used to obtain the extracts (Prentice-Hernández, Rusig and Carvalho, 478 1993). 479 480The C 17 product has since been confirmed to be predominantly the trans-isomer and 481 that cis-isomerisation of bixin was prerequisite to its formation [START_REF] Scotter | Characterisation of the coloured thermal degradation products of 1567 bixin from annatto and a revised mechanism for their formation[END_REF]. 482 However, this compound was shown to isomerise in solution to form small amounts 483 of cis-isomers and to be susceptible to hydrolysis thus forming a range of compounds 484 analogous to bixin and norbixin in terms of their chemical structures and 485 chromatographic properties. In the light of the results obtained, the mechanism of C 17 486 formation originally suggested [START_REF] Mckeown | Composition of oil-soluble food colours. II. Thermal 1436 degradation of bixin[END_REF] was postulated as a concerted 487 electrocyclic process followed by the elimination of m-xylene and, to a much lesser 488 extent toluene, toluic acid and toluic acid methyl ester, and the formation of C 17 which 489 can degrade further by a similar mechanism. 490

491

The analytical HPLC-photodiode array (PDA) method developed by Scotter et al. 492 (1994Scotter et al. 492 ( , 1995) ) provided superior qualitative and quantitative data compared with UV-493 VIS spectroscopic methods (McKeown and mark, 1962;Smith, Blake and Porter, 494 1983) for determining the colour content (as bixin and norbixin) in 21 commercial 495 annatto formulations, particularly with respect to the coloured thermal degradation 496 products [START_REF] Scotter | Analysis of annatto (Bixa 1571 orellana) food colouring formulations. 1. Determination of colouring components and 1572 coloured thermal degradation products by high-performance liquid chromatography 1573 with photodiode array detection[END_REF]. Moreover, the levels of the all-trans and di-cis-497 In a follow-up study, a method was developed which used ambient alkaline hydrolysis 510 followed by solvent extraction and gas chromatography (GC), to analyse annatto 511 colour formulations for the main aromatic hydrocarbon thermal degradation products 512 m-xylene and toluene [START_REF] Scotter | Analysis of annatto (Bixa 1577 orellana) food colouring formulations. 2. Determination of aromatic hydrocarbon 1578 thermal degradation products by gas chromatography[END_REF]. Of the 20 samples analysed, 15 contained 513 <5 mg/kg toluene Four samples contained between 5 and 10 mg/kg and one sample 514 contained 12 m/kg toluene but these levels were not indicative of significant toluene 515 formation via thermal degradation of annatto. In contrast, 6 samples comprising both 516 bixin and norbixin formulations contained m-xylene in the range 30 -200 mg/kg with 517 the highest level found in an oil-based bixin formulation. Moreover, the two norbixin 518 formulations of known production history analysed in the previous study (Scotter et 519 al., 1998) with HPLC-PDA and GC-MS to study this [START_REF] Scotter | Kinetics and yields for the formation of 1582 coloured and aromatic thermal degradation products of annatto in foods[END_REF]. Low levels (ca. 10 -530 15 ug/kg) of m-xylene were detected in the headspace of annatto-coloured retail 531 samples of custard powder, extruded snacks, margarine and breadcrumbs but not in 532 control samples. Much higher levels of m-xylene were detected in annatto-coloured 533 smoked herring (kippers) at ca. 150-200 ug/kg and m-xylene was observed in the 534 headspace of heated Red Leicester cheese (not quantified). The C 17 coloured annatto 535 degradation product was also detected, indicating that thermal degradation of the 536 principal annatto colouring agent 9'-cis-bixin in model systems and foods is facile. 537 However, the degradation is complicated many competing isomerisation reactions 538 which proceed at different rates towards equilibrium. This is further complicated by 539 the simultaneous and irreversible formation of C 17 associated with the production of 540 m-xylene and to a lesser extent, toluene. While norbixin was reported to degrade 541 similarly but more slowly, the levels of m-xylene formation were nonetheless 542 consistent with bixin / norbixin concentration in the food and occurred more rapidly at 543 higher temperatures. 544 545 In order to better understand the kinetics and yields for the formation of both the 546 coloured and aromatic hydrocarbon thermal degradation products of annatto, the 547 stability of bixin at the boiling point of three homologous alcohol solvents was 549 evaluated using HPLC-PDA to monitor the rate of loss of 9'-cis-bixin as well as the 550 appearance of a di-cis-and trans-isomer, and the C 17 degradation product. Loss of 551 linearity was observed at each temperature beyond 2 hours, suggesting that two or 552 more competing reactions were taking place at different rates. From the rate constants 553 calculated for the initial phase of the reaction, the Arrhenius activation energy for the 554 loss of 9'-cis-bixin in refluxing alcohol solvent was 35.7 kJ.mol -1 . Since the rate of 555 loss of 9'-cis-bixin was measured as a function of time regardless of reaction pathway 556 i.e. isomerisation vs. degradation), the authors concluded that the rate data represented 557 only the total (summed) values. Thus, several concurrent reaction pathways are 558 available hence deviation from first order kinetics at long observation times was not 559 unexpected as suggested in Figure 6. 560 561 [Insert Figure 6 about here] 562 563 [START_REF] Berset | Potential use of annatto in extrusion cooking[END_REF] had reported previously an activation energy of 125 kJ.mol -1 564 for the thermal degradation of annatto pigments in petroleum jelly using a simple 565 first-order kinetic model for the complete decay. This disparity in values therefore 566 suggests a controversy in the kinetic analysis or a misinterpretation of the 567 experimental data. Interestingly, bixin was reported to be easily transformed to the all-568 trans-isomer at ambient temperature in the presence of a photosensitizer and light, 569

where the activation energy for the excitation of bixin to an excited triplet state was 570 ca. 25 kJ.mol -1 as discussed above [START_REF] Montenegro | 1476 Model studies on the photosensitized isomerisation of bixin[END_REF], which suggests strongly 571 desribed, where HPLC was used to monitor the decay of 9'-cis-bixin and the 577 formation of the di-cis-and trans-isomers, as well as C 17 [START_REF] Rios | Thermal degradation kinetics of 1533 bixin in an aqueous model system[END_REF]. The 578 reactions were found not to follow first order rate characteristics but rather fitted well 579 to a biexponential model. The rate constants for the formation of the primary products 580 of bixin and the energy barriers for each step were calculated. Di-cis-isomers were 581 formed immediately (energy barrier ca. 63 kJ.mol -1 ) followed by a slow consumption 582 (with the associated decay of 9'-cis-bixin), indicating their role as reaction 583 intermediates. The di-cis-isomers can either revert readily to 9'-cis-bixin (ca. 13 584 kJ.mol -1 ) or yield the primary C 17 degradation product with a higher energy 585 requirement of ca. 27 kJ.mol -1 ). However, the isomerisation of 9'-cis-bixin to trans-586 bixin requires ca. 100 kJ.mol -1 , thereby explaining its relatively slow formation. The 587 Arrhenius plot obtained from the initial decay component for 9'-cis-bixin yielded an 588 activation energy of ca. 33 kJ.mol -1 , which concurs with earlier data (Scotter et al., 589 2001). In conclusion, while the activation energy obtained for the 9'-cis-→ trans-590 isomerisation of bixin is very similar to that reported for β-carotene, the value of ca. 591 155 kJ.mol -1 for the summed isomerisation steps of bixin is much higher than those 592 reported for the thermal isomerisation of C 40 carotenoids (ca. 105 kJ.mol -1 ). Thus the 593 reaction scheme suggested by [START_REF] Scotter | Kinetics and yields for the formation of 1582 coloured and aromatic thermal degradation products of annatto in foods[END_REF] and the greater relative stability of 594 bixin, especially during its isolation and manipulation were confirmed (Figure 7). Thermogravimetric analysis has been used to investigate the thermal degradation of 599 bixin derived from annatto seeds at different heating rates over the 25 -900°C 600 temperature range [START_REF] Silva | Thermogravimetric investigations on the thermal degradation 1591 of bixin, derived from the seeds of annatto (Bixa orellana L.)[END_REF]. The results indicated that the decomposition of 601 solid 9'-cis-bixin occurs in the liquid phase and that four decomposition stages are 602 evident over the temperature range 205-545°C, with isomerisation to the trans-isomer 603 occurring between 200 and 240°C. The calculated activation energy was dependent 604 upon heating rate (i.e. 5, 10 or 15 K.min -1 ) at ca. 108, 147 and 128 kJ.mol -1 605 respectively compared to the value of ca. 100 kJ.mol -1 reported by [START_REF] Rios | Thermal degradation kinetics of 1533 bixin in an aqueous model system[END_REF] 606 obtained in solution. In a similar follow up study, cis-norbixin was heated at rates of 607 5, 10 and 20°C.min -1 over the temperature range 25 -900°C, where the thermal 608 decomposition reactions occurred in the solid phase [START_REF] Silva | Kinetics study of 1595 norbixin's first stage thermal decomposition, using dynamic method[END_REF]. Using the 609 Coats-Redfern model, the calculated activation energy was dependent upon heating 610 rate at ca. 154, 131 and 99 kJ.mol -1 at 5, 10 and 20°C.min -1 respectively for the first-611 order process. 612 613 Heating solid non-purified extracts of annatto seeds as a thin film deposited on a 614 silicon wafer in vacuo and monitored using time of flight (ToF) secondary ion mass 615 spectrometry (SIMS), does not give the same results as heating in solution 616 [START_REF] Bittencourt | ToF-SIMS 1206 characterization of thermal modifications of bixin from Bixa orellana fruit[END_REF]. Principal component analysis revealed that the thermal 617 degradation of the annatto extracts under these conditions occurs in three distinct 618 temperature ranges; below 70°C, the extracts remain thermally stable but above this 619 temperature dimerization reactions occur and the signals attributed to bixin decrease. 620 Near to 100°C, the bixin molecules begin to degrade, leading to fragmentation with 621 extensive degradation of bixin above 120°C. However, the nature of the degradation 622 mechanism described is not fully understood since there was no evidence for the 623 formation of C 17 or related fragments from solid bixin. 624 625

Light stability 626

The effect of light at 900 lux intensity on the 30-day stability of a microencapsulated 627 water-miscible extract of bixin compared to that of a purified bixin extract have been 628

studied by measuring the loss of spectrophotometric absorbance at 470nm with time 629 [START_REF] Prentice-Hernández | Effect of light on the stability of a 1508 microencapsulated extract obtained from annatto (Bixa orellana L.)[END_REF]. The degradation rate of bixin in the 630 microencapsulated extract was ca. 0.05% compared to 0.11% per day for the purified 631 extract. 632 633 [START_REF] Ferreira | Kinetics of 1290 colour degradation of water soluble commercial annatto solutions under thermal 1291 treatments[END_REF] submitted commercial water-soluble annatto (norbixin) 634 solutions to different time and temperature treatments to investigate colour stability. 635

The colour change was measured by spectrophotometry using the Hunter Lab System 636 and the results presented in terms of changes in the norbixin concentration and L, a, b 637 colour parameters. Data were analysed for reaction order and the temperature 638 dependence was explained by the Arrhenius model, with activation energy values 639 between 46 and 105 kJ.mol -1 The changes in colour showed an increase in lightness 640 and yellow colour and a decrease in red colour. Norbixin degradation reaction 641 followed second order kinetics whereas for other colour parameters, first order 642 kinetics was followed. 643

644

The light stability of spray-dried bixin encapsulated with gum Arabic or maltodextrin 645 plus Tween 80 surfactant has been reported, where the kinetic behaviour of bixin 646 photo degradation in all systems was characterized by two first-order decays due to 647 the presence of bixin both inside and outside the microcapsules [START_REF] Barbosa | The light stability of spray-1191 fried bixin encapsulated with different edible polysaccharide preparations[END_REF]. 648

Unsurprisingly, approximately two orders of magnitude greater stability was observed 649 for bixin in the dark compared to illuminated conditions and in the absence of light, 650 and bixin stability in encapsulated solutions was ca. ten times greater than in non-651 encapsulated systems. The effect of processing conditions used for the preparation of 652 traditional Indian foods on bixin stability including baking, frying, microwave 653 cooking and pressure cooking have been monitored by following losses using 654 spectrophotometry [START_REF] Rao | Effect of 1522 processing conditions on the stability of annatto (Bixa orellana L.) dye incorporated 1523 into some foods[END_REF]. The losses of bixin under model processing 655 conditions was compared to the preparation of cakes, chegodis, biscuits and fried rice. 656

The greatest losses of bixin were observed in direct exposure to oven baking (54% 657 loss ) and deep fat frying (47%), whereas microwave cooking did not affect the colour 658 during direct exposure or in food products. The maximum loss of bixin (65%) was 659 observed for deep fried snack due largely to leaching of the dye into the oil. Pressure 660 cooking produced losses of between 25% and 33%. In cakes, the loss was 30% but 661 negligible losses were found for biscuits (1.5%). 662 663 Similarly, the combined effects of light and temperature on annatto extract under 664 different storage conditions were evaluated spectrophotometrically at 470nm in 665 chloroform over a period of 360 days (Balaswamy, Prabhakara Rao, Satyanarayana 666 and Rao, 2006). Annatto oleoresin was generally more stable during storage with 667 respect to bixin content than annatto powder obtained by solvent extraction of annatto 668 seeds. The bixin lost in oleoresin stored under cold (5-8°C), dark conditions was 669 minor (11%) throughout the study, whereas considerable losses were observed for the 670 powdered dye (23%). Likewise, the bixin lost after storage at ambient temperature in 671 the dark were 8% and 54% for oleoresin and powder respectively. Under diffused 672 daylight ambient and temperature the losses were 14% and 60% respectively, whereas 673 bixin seed stored in jute sacks showed a loss of only 15%. As expected, the rate 674 constants for bixin degradation were much higher in powder compared to oleoresin 675 and were reported to follow second order kinetics. It was proposed that the colour is 676 protected from exposure to oxygen and light by the oleoresin compared to the dry 677 powder, which has a large surface area. 678 679 Bixin complexed with α-cyclodextrin is also reported to be more resistant to the 680 damage caused by light and air [START_REF] Lyng | Bixin and alpha-cyclodextrin inclusion 1418 complex and stability tests[END_REF]. 681 682

Analytical methods for annatto 683

Spectrophotometry 684 Historically, chloroform has been used as solvent for the spectrophotometric analysis 685 of bixin and dilute sodium hydroxide (ca. 0.1M) for norbixin. Absorbance 686 measurements at the two most intense spectral peaks (III and IV in Figure 4) are used 687 for quantitative analysis, where peak IV is preferred because it is less prone to 688 interference from yellow decomposition products. This interference was corrected by 689 using a factor related to the absorbances at λ max and at 404nm in determining the total 690 pigment content of annatto formulations (McKeown and Mark, 1962). In practice, the 691 spectrophotometric determination of annatto (as bixin or norbixin) is somewhat 692 confused by the use of conflicting extinction coefficients. This has been discussed in 693 detail and the published ( E 1cm 1% ) extinction coefficients for norbixin and bixin 694 summarized and compared to highlight disparities [START_REF] Levy | Annatto[END_REF]. 695

Depending upon the extinction coefficient used, large errors might be incurred and 696 propose a practical conversion factor to correlate the relative absorbances at the two 697 peak maxima. This is based upon the increase in absorbance observed upon hydrolysis 698 of bixin to norbixin at constant concentration -thus proving that the extinction value 699 for norbixin must be higher than that for bixin, which was also reported (Smith et al., 700 1983). Furthermore, from data recorded by the authors from more than 1000 701 spectrophotometric measurements of different samples of bixin before and after 702 hydrolysis, the difference between the extinction values of bixin and norbixin was 703 reported to be of the order of 6%. When compared with a value of E 1cm 1% = 3208 704 reported for pure norbixin, this equates to an extinction coefficient for bixin of E 1cm 1% = 705 3016, which concurs with the values reported for purified bixin in chloroform (Scotter 706 et al., 1994). 707 708 However, these extinction values to not agree with those adopted for colour purity 709 specifications by the European Union (EC, 1995) or the FAO/WHO (1996), largely 710 due to misassumptions made regarding solvent effects. The discrepancy in published 711 extinction values might be traced back to the 'erroneous' coefficient reported by Reith 712 and Gielen (1971) that has been used subsequently as a reference value by various 713 other workers. Serious doubt is expressed over the validity of the extinction values for 714 norbixin in aqueous alkaline solution at 453nm (2850) and 482nm (2550). Moreover, 715 the same reservations were expressed over the value of 3473 at 453nm reported by the 716 FAO/WHO specification (FAO/ WHO, 1981). 717 718 An interesting and important aspect of the spectrophotometric analysis of bixin in 719 chloroform is its rapid rate of degradation when contained in a quartz cuvette, which 720 [START_REF] Mckeown | Paper chromatography of bixin and related compounds[END_REF]. This method was used in a number of subsequent studies on 731 annatto and its main thermal degradation product, C 17 (McKeown and Mark, 1962;732 McKeown, 1963). The first thin layer method for separation of annatto colour and 733 other fat-soluble dyes shortly thereafter, employed silica gel G, plaster of Paris and 734 silicic acid media with amyl acetate mobile phase (Ramamurthy and Bhalerao, 1964). 735 However, of the 30 solvent systems studied, bixin was reported to migrate from the 736 base line only when acetic acid was present (Francis, 1965). The findings suggested 737 that the amyl acetate solvent used by Ramamurthy and Bhalerao (1964) must have 738 contained acetic acid as an impurity, which was proven by subsequent 739 experimentation. Later methods used silica gel with various solvent systems 740 containing acetic acid for the separation of bixin and norbixin in colour formulations 741 (Dendy, 1966) and cheese colour i.e. norbixin [START_REF] Reith | Properties of bixin and norbixin and the composition of 1526 annatto extracts[END_REF], who also 742 employed cellulose media for the analysis of butter colour i.e. bixin. Other methods 743 include those developed by [START_REF] Preston | Extraction and chemistry of annatto[END_REF] and Corradi and Micheli 744 (1981). [START_REF] Chao | Supercritical 1228 CO 2 extraction of annatto (Bixa orellana) pigments and some characteristics of the 1229 color extracts[END_REF] (Mínguez-Mosquera, Hornero-Méndez 748 and Garrido-Fernández, 1995) and for the isolation and identification of new (trace) 749 apocarotenoids from annatto seeds (Mercadante, Steck and Pfander, 1997b) As discussed above, developments in HPLC techniques have enabled more detailed 758 studies of other bixin and norbixin isomers as well as their degradation products 759 compared to TLC methods and have been utilized to gain a greater understanding of 760 the stability of annatto and which in turn have been applied to the detection and 761 measurement of annatto colour in foodstuffs (below). 762 763 Literature references on the application of HPLC to the separation of annatto colouring 764 components are sparse. Early methods include the HPLC analysis of annatto extract 765 [START_REF] Nishizawa | Studies on the analysis of natural 1489 dyes: Analysis of annatto extract and gardenia yellow dye in foods and natural dye 1490 preparations[END_REF] and [START_REF] Smith | Determination of added natural colours in 1599 foods[END_REF], who reported the use of an isocratic 766 reverse-phase system employing an ODS column and methanol/aqueous acetic acid 767 mobile phase. Using this system the cis-and trans-isomers of both bixin and norbixin 768 were separated within 10 minutes. However, the cis-and trans-bixin peaks were not 769 fully resolved and the peak shapes were generally very poor. A method for the reverse-770 phase separation of bixin, norbixin and three curcuminoids using both isocratic and 771 gradient elution systems, comprising a Zorbax ODS column and water/THF mobile 772 phase was later developed that gave improved chromatographic separation (Rouseff, 773 1988) developed. However, only separation of the 'main' annatto colouring 774 components were reported and no reference to stereoisomer separation was given. 775

Other approaches have been reported for the analysis of cheese extracts (Luf and 776 Brandl, 1988) and of foods after protease digestion [START_REF] Chatani | Determination of norbixin in foods by HPLC after 1232 protease digestion[END_REF]. A 777 procedure similar to that reported by [START_REF] Smith | Determination of added natural colours in 1599 foods[END_REF] has been developed and 778 appled to the determination of annatto in selected foodstuffs with reasonable success 779 [START_REF] Lancaster | Determination of annatto in high-fat dairy 1391 products, margarine and hard candy by solvent extraction followed by high-1392 performance liquid chromatography[END_REF]. 780 781The method developed by [START_REF] Scotter | Characterization 1562 of the principal colouring components of annatto using high performance liquid 1563 chromatography with photodiode array detection[END_REF] has played a key role in the 782 advancement of HPLC capabilities for the separation and characterization of norbixin 783 and bixin isomers, and has been refined and adapted for the study of annatto stability 784 and for the determination of annatto colouring components in colour formulations, 785 foodstuffs and human plasma. These are summarized along with other published 786 methods in Table 3. 787 788 [Insert Table 3 about with different λ max wavelengths to be monitored using a spectral bandwidth that 800 encompasses them. A reference wavelength can also be used to subtract background 801 absorbances and to allow for baseline drift, which is usually set outside of the 802 absorbance range of the main analyte and interfering peaks e.g. at 600nm x 4nm 803 bandwidth. The lack of availability of authenticated reference standards is the main 804 limiting factor in the HPLC analysis of annatto colouring components but methods are 805 available for the isolation, purification and characterization of the main bixin and 806 norbixin isomers [START_REF] Scotter | Characterization 1562 of the principal colouring components of annatto using high performance liquid 1563 chromatography with photodiode array detection[END_REF] and for C 17 analogues [START_REF] Scotter | Characterisation of the coloured thermal degradation products of 1567 bixin from annatto and a revised mechanism for their formation[END_REF]. Other 807 workers have exploited the use of PDA detection for the identification of trace levels 808 of other apocarotenoids in annatto seeds very successfully (Mercadante, Steck and 809 Pfander, 1997b). Figure 8 shows the HPLC separation of bixin and norbixin isomers 810 [START_REF] Scotter | Characterization 1562 of the principal colouring components of annatto using high performance liquid 1563 chromatography with photodiode array detection[END_REF]. 811 812 [Insert Figure 8 about here] 813 814

Mass spectrometry(MS) 815

A comprehensive review on the use of mass spectrometry in the study of carotenoids 816 in general may be found elsewhere (Enzell and Back, 1990). This work cites earlier 817 reviews and studies that consolidate the importance of the technique not only for 818 elucidation of structure but also for analytical research, not least those carried out by 819 [START_REF] Vetter | Spectroscopic methods[END_REF][START_REF] Budzikiewicz | Mass spectra of carotenoids -labelling studies[END_REF] and [START_REF] Enzell | Specifications for identity and purity of food additives (carrier 1278 solvents, emulsifiers and stabilizers, enzyme preparations, flavouring agents, food 1279 colours, sweetening agents, and other food additives)[END_REF]. The 820 was used to confirm the structures of isolated and purified bixin and norbixin isomers 838 [START_REF] Scotter | Characterization 1562 of the principal colouring components of annatto using high performance liquid 1563 chromatography with photodiode array detection[END_REF]. Both the 9´-cis-and trans-isomers gave a molecular ion at m/z 839 394 (bixin) and m/z 380 (norbixin), with major fragment ions at m/z [M-106], 106 840 (xylene), 105 (methyl tropylium) and 91. Using thermospray analysis, [M+H] + was 841 identified as the base peak along with the presence of sodium and (possibly) water 842 adducts, and fragment ions corresponding to [M-H 2 O] + and [M-CH 3 OH] + . In a later 843 study, similar analytical conditions were used to characterize the 17-carbon major 844 thermal degradation product of annatto [START_REF] Scotter | Characterisation of the coloured thermal degradation products of 1567 bixin from annatto and a revised mechanism for their formation[END_REF]. Solid probe EI revealed the 845 (FAB) MS was used to determine the structure of the bixin family of apocarotenoids 851 [START_REF] Kelly | NMR, MS and X-ray crystal structure 1366 determination of the bixin family of apocarotenoids[END_REF]. Both cis-and trans-bixin isomers gave EI+ molecular ion 852 abundancies equivalent to ca. 30% of the base peaks at m/z 59 or 91, and the [M+1] 853 and [M+2] ion intensities were consistent with predictions based upon calculated 13 C 854 isotope patterns. As expected, loss of xylene as a neutral group was most pronounced 855 for cis-bixin but no loss of neutral toluene was observed although the m/z 91 peak was 856 prominent. Fast-atom bombardment (FAB+) spectra of cis-and trans-bixin gave the 857 molecular ion as the base peak but the abundance of the [M+1] peak exceeded the 858 calculated isotopic abundance by 55-75%, indicating a small contribution from 859 [M+H]. Small amounts of sodium adducts were observed but ions due to elimination 860 of toluene were not. However, significant amounts of m/z 105, 115 and 165 were 861 observed. These observations were consistent with other FAB spectra of carotenoids 862 where odd electron molecular ions are frequently observed due presumably to their 863 lower ionization potentials (Vetter and Meister, 1985). 864 865 Bixin was among the polyenes studied using electrospray ionization (EI) and high 866 resolution (HR) matrix-assisted laser desorption ionization (MALDI) time-of-flight 867 (TOF) mass spectrometry (Guaratini et al., 2004). In this study, the ability of neutral 868 organic molecules to give up an electron for oxidation was exploited, which is 869 governed by the energy of their highest occupied molecular orbital (HOMO) and can 870 from ESI analysis of bixin, whereas M .+ was detected but only in the specific capillary 877 voltage range of 0.1 to 0.7 kV . The accurate mass measurement afforded by the HR-878 MALDI-TOF analysis showed M .+ for bixin at an observed mass of 394.2147 with 879 40% ion intensity, but [M+H] + was not observed. 880

881

The major carotenoid composition of Bixa orellana seeds has been ascertained using 882 TOF-MS with X-ray photoelectron spectroscopy [START_REF] Felicissimo | Time-of-flight 1286 secondary ion mass spectrometry and x-ray photoelectron spectroscopy analyses of 1287 Bixa orellana seeds[END_REF] The confirmation of twelve different carotenoids used as food colorants was achieved 907 utsing positive atmospheric pressure chemical ionization (APcI)+ LC-MS (Breithaupt, 908 (2004). The [M+H]+ ions were monitored for norbixin and bixin at m/z 381 and 395 909 respectively. Based on the presence of at least one carboxyl group, APcI 910 measurements in the negative mode were also carried out on bixin and norbixin but no 911 significant enhancement in sensitivity was observed. A similar approach has been 912 used for the analysis of water-soluble annatto extracts in both positive and negative 913 electrospray detection modes [START_REF] Galindo-Cuspinera | Key: ACE = Acetone; ACN = Acetonitrile; BZ = Benzene; CHX = Cyclohexane, CHCl 3 = Chloroform; DMF = N,N-Dimethylformamide; EtOAc = Ethyl acetate; EtOH = Ethanol; Et 2 O = Diethyl ether; HEX = Hexane; HOAc = Acetic acid; IPA = isopropyl alcohol; PE = Petroleum ether; t-BME = tertiary butylmethyl ether[END_REF]. ES-detection 914 mode showed a major peak at m/z 379 corresponding to [M-H] -for norbixin, whereas 915 the major peak at m/z 381 was found using ES+ mode. An ion at m/z 117 was 916 identified in in the ES-spectrum of 9´-cis-norbixin but not in the spectrum of the 917 trans-isomer. Conversely, the trans-isomer showed an ion at m/z 111.1 in ES+ mode 918 that was not present on the spectrum of 9´-cis-isomer. This was thought to be due to 919 differences in fragmentation patterns determined by stereochemical configuration. 920 elelctrospray mass spectrometric confirmatory analysis can be used to identify and 922 measure norbixin and bixin in meat products using precursor ions at m/z 379 and 395 923 respectively and monitoring characteristic product ions at m/z 253,291,310 and 335 924 (norbixin) and m/z 317, 335, 345, 363 and 377 (bixin) [START_REF] Noppe | Determination of bixin and norbixin in meat using liquid 1497 chromatography and photodiode array detection[END_REF]. 925 926

Nuclear magnetic resonance (NMR) spectroscopy 927

A comprehensive review on the use of NMR spectroscopy in the study of carotenoids 928 in general is given by [START_REF] Englert | NMR spectroscopy[END_REF], in which a detailed treatise on the experimental 929 aspects, chemical shifts of end groups, chemical shifts and spin couplings, 930 stereoisomerization , and simple and multidimensional experiments are given for 1 H 931 and 13 C nuclei. 932

933

The earliest published use of NMR in the study of bixin stereochemistry used low 934 resolution (40 MHz) instrumentation to assign 1 H frequencies and deduce that the cis-935 bond of the methyl analogue of 'natural or α-' bixin was in the 9´-(equivalent) 936 position [START_REF] Barber | Studies in nuclear 1187 magnetic resonance. Part IV. Stereochemistry of the bixins[END_REF]. The high frequency shift of the proton assigned to H-8´ 937 was attributed to deshielding by the 11´-12´ alkene bond when compared to the trans-938 (or β-) isomer, which was confirmed via synthesis and more detailed structural 939 assignments [START_REF] Pattenden | Carotenoids and related compounds. Part 1501 XXVI. Synthesis of methyl natural bixin[END_REF]. Fourier transform (FT) NMR was 940 used later to assign the 13 C spectra of methyl cis-and trans-bixin using deuterated 941 compounds, however no experimental details were given and assignments were partly 942 derived from spectra of carotenoids with similar structural characteristics (Moss, 943 1976). The 1 H FT-NMR spectrum of cis-bixin and cis-methyl bixin at 250Mhz has 944 been reported but is limited to assignment of the terminal acrylate moieties (Jondiko 945 (1989). Proton NMR at 250MHz was used to confirm the structures of 946 purified trans-and 9´-cis-bixin, where the chemical shifts and coupling constants 947 associated with the change in stereochemistry were consistent with those reported 948 previously [START_REF] Barber | Studies in nuclear 1187 magnetic resonance. Part IV. Stereochemistry of the bixins[END_REF] but afforded much higher resolution (Scotter et al., 949 1994). A similar approach was used to confirm the structure of the principal thermal 950 degradation product of bixin as trans-4,8-dimethyltetradeca -hexaenedioc acid 951 monomethyl ester or C 17 [START_REF] Scotter | Characterisation of the coloured thermal degradation products of 1567 bixin from annatto and a revised mechanism for their formation[END_REF]. The structure of a minor apocarotenoid 952 isolated from Bixa orellana was confirmed as methyl 9´Z-apo-6´-lycopenate using 953 proton NMR at 500 MHz [START_REF] Mercadante | Isolation 1462 of methyl 9'Z-apo-6'-lycopenoate from Bixa orellana[END_REF] and a similar approach used to 954 identify apocarotenoids not previousy found in annatto (Mercadante, Steck and 955 Pfander, 1997b;1999). NMR (300 MHz 1 H) was used alongside TLC and HPLC in 956 the bioautographic detection of antimicrobial compounds in water-soluble annatto 957 extracts where peak assignments were reported to be consistent with previous reports 958 [START_REF] Galindo-Cuspinera | Key: ACE = Acetone; ACN = Acetonitrile; BZ = Benzene; CHX = Cyclohexane, CHCl 3 = Chloroform; DMF = N,N-Dimethylformamide; EtOAc = Ethyl acetate; EtOH = Ethanol; Et 2 O = Diethyl ether; HEX = Hexane; HOAc = Acetic acid; IPA = isopropyl alcohol; PE = Petroleum ether; t-BME = tertiary butylmethyl ether[END_REF]. 959 960

The most comprehensive study to date on the determination of the structure of the 961 bixin family of apocarotenoids is by [START_REF] Kelly | NMR, MS and X-ray crystal structure 1366 determination of the bixin family of apocarotenoids[END_REF], who utilised a combination of 962 1D and 2D NMR techniques in conjunction with mass spectrometry and X-ray 963 diffraction analysis. Chemical shift, coupling constants and 1 H correlation data were 964 examined alongside the ion abundances and intensity ratios from standard electron 965 impact (EI+) and fast atom bombardment (FAB+) MS spectra, and bond 966 measurement, cell dimension and degree of hydrogen bonding from X-ray diffraction 967 data to elucidate and compare the crystal structures of the cis-and trans-isomers of 968 bixin and methyl bixin. Notwithstanding where specific techniques have been discussed elsewhere in this 972 review, there are several less widely known techniques that have been used in the 973 study of annatto either alone or in conjunction with complementary techniques. These 974 include infra-red spectroscopy, where the characteristic strong absorption due to the 975 C=O stretching frequency between 1740 and 1700 cm -1 and the complex bands in the 976 1300-1050 cm-1 region due to C-O single bond characteristic of esters and carboxylic 977 acids has been used [START_REF] Lunde | Infrared spectra and cis-trans configurations of some 1415 carotenoid pigments[END_REF][START_REF] Reith | Properties of bixin and norbixin and the composition of 1526 annatto extracts[END_REF]Chao et 978 al., 1991;Bernard and Grosjean, 1995). Photoacoustic spectrometry in the UV, VIS 979 and IR regions has been used for the qualitative and quantitative analysis of annatto in 980 commercial seasoning products (Haas and Vinha, 1995) and more recently in the 981 determination of the triplet state energy of bixin [START_REF] Rios | Triplet state energy of the 1537 carotenoid bixin determined by photoacoustic calorimetry[END_REF]. X-ray 982 photoelectron spectroscopy was used by [START_REF] Felicissimo | Time-of-flight 1286 secondary ion mass spectrometry and x-ray photoelectron spectroscopy analyses of 1287 Bixa orellana seeds[END_REF] to ascertain the 983 major carotenoid composition of Bixa orellana seeds and X-ray diffraction in 984 conjunction with NMR and mass spectrometry has been used to determine of the 985 structure of the bixin family of apocarotenoids [START_REF] Kelly | NMR, MS and X-ray crystal structure 1366 determination of the bixin family of apocarotenoids[END_REF]. 986 987

Analysis of foods 988

Prior to 1970, there were very few published methods for the extraction of annatto 989 from foods. The qualitative and quantitative analytical aspects of annatto extraction 990 methods published prior to 1976 have been reviewed briefly (Aparnathi and Sharma, 991 1991). These relatively simple methods generally involve extraction with solvent (e.g. 992 chloroform, benzene, petroleum spirit or ether) with or without some form of sample 993 pre-treatment such as protein precipitation, washing and adsorption onto an inert 994 which reflects the relatively narrow scope of annatto usage at that time. 996 997 Annatto has been extracted from whey solids with dilute ammonium hydroxide where 998 proteins were precipitated by the addition of ethanol and phosphate buffer 999 (Hammond, Chang and Reinhold, 1973), and from meats [START_REF] Mcneal | Qualitative tests for added colouring matter in meat products[END_REF]. Annatto 1000 may be analysed in milk and ice-cream by precipitation with boiling acetic acid and 1001 extraction of the whey with diethyl ether, and the colour extracted from macaroni and 1002 noodles with 80% ethanol followed by back-extraction into diethyl ether under 1003 alkaline conditions (AOAC, 1980). 1004 1005 Rapid methods for the extraction of annatto from foods have been described where 1006 drinks and syrups were dissolved in water, acidified with acetic acid and annatto was 1007 partitioned into diethyl ether [START_REF] Corradi | A rapid research and identification method for the natural 1241 colorant E160b bixin, norbixin (Oriana, annatto) in food products. Industrie 1242 Alimentari[END_REF]. Products with a high fat 1008 content e.g. butter and margarine, were dissolved in petroleum spirit and annatto was 1009 β-apo-8´-carotenal but other food colours and emulsifiers did not affect the analysis. 1070

The recovery of annatto colouring spiked into cheese, processed cheese, butter and 1071 ice-cream ranged between 80 and 100%. Nine samples of cheese were analysed in 1072 which norbixin was found in the range <0.15 to 11.89 mg.kg -1 , whereas no bixin was 1073 detected (>0.15 mg.kg -1 ). 1074 1075The methods described by [START_REF] Scotter | Characterization 1562 of the principal colouring components of annatto using high performance liquid 1563 chromatography with photodiode array detection[END_REF]1998), [START_REF] Lancaster | Determination of annatto in high-fat dairy 1391 products, margarine and hard candy by solvent extraction followed by high-1392 performance liquid chromatography[END_REF]Lawrence 1076 (1995) and Navaz [START_REF] Diaz | Fluorimetric determination of curcumin in 1486 yogurt and mustard[END_REF] were further developed and consolidated 1077 to encompass a wide range of food commodities [START_REF] Scotter | Method development and 1586 analysis of retail foods for annatto food colouring material[END_REF] With the exception of regime 5, samples were extracted essentially using 1085 ethanol:water:ammonia solution with or without a hexane partition to remove excess 1086 lipid. After centrifugation in the presence of Celite filter aid, the annatto colour was 1087 partitioned into chloroform:acetic acid solution, centrifuged and the solvent removed 1088 using vacuum-assisted rotary evaporation. To minimise analyte losses via oxidation, a 1089 0.1% solution of butylated hydroxyl toluene (BHT) was added. For regime 5 matrices, 1090 samples were mixed with Celite in the presence of dilute hydrochloric acid and 1091 extracted using a biphasic solvent system comprising hexane (to remove excess lipid) 1092 and 2 single food samples covering a wide range of foods at levels above the 1097 analytical reporting limit of 0.1 mg.kg -1 were obtained. Quantitative results were 1098 given for those annatto colouring components for which reference standards were 1099 available (9´-cis-bixin, trans-bixin and 9´-cis-norbixin), whereas semi-quantitative 1100 results were given for other bixin and norbixin isomers. The method was single-1101 laboratory validated by the repeat (n = 4 to 9) analysis of 12 different sample types of 1102 food commodity covering the permitted range of annatto content, spiked with annatto 1103 at levels of between 1.7 and 27.7 mg.kg -1 and by the analysis of in-house reference 1104 matrices. Mean recoveries of between 61 and 96% were obtained from foods spiked 1105 with annatto. 1106 1107 Using response surface methodology to establish optimum conditions, a method for 1108 the determination of annatto colour in extruded corn snack products has been 1109 developed that exhibits improved accuracy and precision compared to the method 1110 described by [START_REF] Scotter | Method development and 1586 analysis of retail foods for annatto food colouring material[END_REF] [START_REF] Rios | Novel method for the determination of added 1529 annatto colour in extruded corn snack products[END_REF]. However, pre-1111 treatment of the samples with α-amylase was necessary to remove starch and a total 1112 of 8 solvent extractions with ethyl acetate were required for complete extraction of the 1113 annatto colour. Lipids were removed using alkaline saponification therefore all of the 1114 bixin present was hydrolysed to norbixin and determined as such by HPLC. [START_REF] Scotter | Characterization 1562 of the principal colouring components of annatto using high performance liquid 1563 chromatography with photodiode array detection[END_REF]. [START_REF] Scotter | Characterization 1562 of the principal colouring components of annatto using high performance liquid 1563 chromatography with photodiode array detection[END_REF].

Figure 5. HPLC-photodiode array spectrum of 9'-cis-bixin (broken line) and its first derivative spectrum (solid line) highlighting the inflection at λ max (II) [START_REF] Scotter | Characterization 1562 of the principal colouring components of annatto using high performance liquid 1563 chromatography with photodiode array detection[END_REF].

Figure 6. Suggested reaction pathways for the thermal degradation of 9'-cis-bixin [START_REF] Scotter | Kinetics and yields for the formation of 1582 coloured and aromatic thermal degradation products of annatto in foods[END_REF].

Figure 7. Coupled reaction scheme proposed for the degradation of bixin and the formation of its primary products [START_REF] Rios | Thermal degradation kinetics of 1533 bixin in an aqueous model system[END_REF]. Assignment of peaks: 1. Trans-norbixin. 2. Di-cis-norbixin. 3. 9'-cis-norbixin. 4.

Trans-bixin. 5 and 9. Di-cis-bixin isomers. 6. 9'-cis-bixin. Table 4. Summary of extraction regimes used for annatto in foods [START_REF] Scotter | Method development and 1586 analysis of retail foods for annatto food colouring material[END_REF].

Regime

  9'-cis-bixin in variable proportions and characteristic degradation products, 149 dependent upon extraction temperature and time (see below)

  transition. The shapes of electronic absorption (and fluorescence) bands 248

  properties of a carotenoid change, absorption bands that are 272 otherwise not detected may become a significant feature of the spectrum, as the 273

282• 286 ••

 286 The position of the main absorption band, specified by λ max , provides 283 structural information because it is determined by the chromophore of the 284 molecule 285 The intensity of the absorption at λ max , (A) is related both to the structure and 287 to the concentration of the carotenoid in the sample, and provides the basis for 288 quantitative analysis 289 290 The position or the intensity of the main absorption band of a carotenoid can 291 be influenced by a number of factors such as a change in the molecular 292 environment of the carotenoid e.g. solvent 293 294

  annatto exhibited a lower antioxidant activity than propyl gallate but 400 was more effective at peroxide scavenging than BHA or BHT. Annatto oleoresin 401

  photosensitization gives rise to the higher energy triplet state ( 3 BIX*) precursor, 448

  yellow coloured 17-carbon polyene 4,8,dimethyl-473 tetradecahexaenedioc acid monomethyl ester "C 17 "

  found to be consistent with their known production 499 history i.e. indicative of the degree of thermal treatment. The formulation obtained by 500 direct aqueous alkaline extraction contained higher levels of these isomers compared 501 to solvent pre-extracted bixin followed by alkaline hydrolysis obtained using lower 502 temperatures. However, the authors pointed out that while the isomer profiles 503 obtained by HPLC-PDA analysis support this, the different extraction procedures 504 might also give rise to different isomer profiles due differential solubilities and 505 stabilities in the extraction medium. The effects of light and oxygen may further 506 complicate this during extraction and handling, and by the nature of the source 507 material. 508 509

  differed markedly in m-xylene content, which appeared to be consistent 520 with the degree of thermal treatment. the alkaline hydrolysis-solvent extraction procedure, 7 of the 523 annatto formulations were submitted for headspace (HS) GC analysis for toluene and 524 m-xylene in order to monitor the effects of heating in a closed controlled environment 525 (90°C for 20 minutes). An increase in m-xylene was observed, with the bixin in oil 526 formulations showing the highest rise in m-xylene concentration on heating. The 527 authors anticipated that HS-GC could be used to monitor the thermal degradation of 528 annatto in food systems and thus conducted a number of experiments in combination 529

  a number of experiments in model systems (ibid.). The thermal 548

  energy barrier may be anticipated for the thermal isomerisation of 9'-cis-572 bixin to trans-bixin. 573574A more detailed kinetic study on the thermal degradation of bixin in an aqueous 575 model system comprising water: ethanol (8:2) as a function of temperature has been 576

  , there were few references in the literature to paper and adsorption 725 chromatography, which dealt mainly with the gross separation of different 726 carotenoids, and from chlorophylls. The first paper chromatographic method for the 727 direct separation of annatto colouring components used Whatman 3MM paper 728 impregnated with 50% N,N-dimethylformamide (DMF) in acetone and which 729 developed with cyclohexane: chloroform: DMF and acetic acid (85:10:3:2) 730

  of column stationary phases been vital in allowing separation 791 of geometrical isomers of bixin and norbixin, C 17 analogues and other food 792 components, it is the power of the detection systems that have enabled the 793 development of highly useful qualitative and quantitative analyses. Many developed 794 methods utilise detection with fixed wavelength UV-visible (UV-VIS) detectors at 795 bixin/norbixin isomer absorption maxima quite successfully. 796 However, photodiode-array (PDA) technology offers combined sensitivity and 797 specificity coupled to real-time qualitative (spectral) confirmatory analysis, thereby 798 enabling powerful isomer identification and measurement. PDA allows isomer peaks 799

  in detail ionization techniques, tandem MS, combined 821 chromatographic-MS techniques and elimination reactions of in-chain units and 822 terminal groups. The first method for electrospray liquid chromatography-mass 823 spectrometry (LC-ES-MS) of carotenoids employed gradient reversed-phase HPLC 824 with PDA and MS detection in tandem (van Breemen, 1995). Molecular ions, M (.+) , 825 without evidence of any fragmentation, were observed in the ES mass spectra of both 826 xanthophylls and carotenes but neither bixin nor norbixin were studied. 827 828 In common with other carotenoids, the MS spectra of bixin and norbixin are 829 characterized by fragmentation leading to losses of toluene and xylene from the 830 polyene chain and the structural significance of the intensity ratio of the [M-92] +. and 831 [M-106] +. ions (and to a lesser extent the [M-158] +. ion), which is related to the 832 number of conjugated double bonds. It is the apo-configuration that gives rise to 833 anomalous MS properties of bixin and norbixin that have diagnostic value i.e. the -834 CH 2 -CH=CH-CH 2 -COOH end group gives characteristic fragments at [M-44] +. and 835 [M-99] +. , whereas the -CH 2 -CH=CH-CH 2 -COOCH 3 end group gives characteristic 836 fragments at [M-31] +. , [M-59] +. and [M-113] +. . Solid probe electron ionization (EI+) 837

  m/z 288 along with fragment ions at m/z [M-106], 106, 105 and 91, 846 and thermospray analysis identified the base peak as [M+H] + as well as sodium 847 adducts at m/z 311 ([M+Na] + ) and 333 [M-H+2Na] + . 848 849 Complementary to other analytical techniques, EI+ and fast atom bombardment 850

  for an ionization process that produces the molecular ion M .+ in 872 ESI and HR-MALDI MS of polyenes, and the correlation of the observed ions to the 873 oxidation potential. The formation of M .+ and [M+H] + species was shown to be 874 dependent upon energetic variations and the presence of water or another protic 875 solvent. Niether the [M+H] + nor the [M+H-H2O] + were detected as the major ions 876

  . The 883 presence of bixin was revealed in the seed aril without any sample pretreatment from 884 the detection of ions attributable to [M+2H] at m/z 396 with associated 13 C isotope 885 analogues at m/z 397 and 398. The presence of characteristic fragments at m/z 337 886 was attributed to C 23 H 29 O 2 + obtained from the previous molecular ion with loss of a 887 COOCH 3 ester group, and at m/z 281, a fragment compatible with loss of a C 6 O 2 H 8 888 end group plus a hydrogen atom i.e. C 19 H 21 O 2 + . The characteristic presence of xylene 889 was confirmed via the detection of the C 8 H 9 + ion at m/z 105. Analysis of the coloured 890 interior of the seeds following cutting did not show any fragments consistent with 891 bixin. A methanol:chloroform extract of the seeds was analysed immediately after 892 preparation by blow-drying under nitrogen onto a silver subtstrate, and then after 893 exposure to ambient light for 3 months. TOF-MS analysis of the fresh extract was 894 dominated by the molecular peak at m/z 396 along with all other characteristic 895 expected after 3 months exposure to light, the colour of the extract had 896 lightened to a more yellow shade with an associated 5-fold decrease in the intensity of 897 the [M+2] + ion and with a concomitant 2-fold increase in the intensity of the C 8 H 9 + 898 ion, indicating the formation of xylene via degradation. In a related study, Bittencourt 899 et al. (2005) analysed extracts of Bixa orellana using TOF-MS as a means of 900 characterising thermal effects. The spectrum was characterised by a large number of 901 peaks generated by the principal ions and their multiple fragmentation patterns but 902 also, more notably, by the presence of ions at m/z 790 ([C 50 H 62 O 8 ] + = 2M+2H), 804 903 ([C 51 H 64 O 8 ] + = 2M+2H+CH 2 ) and 818 ([C 52 H 66 O 8 ] + = 2M+2H+2CH 2 ) attributed to 904 the presence of dimers. 905 906

  it has been shown that HPLC-PDA in combination with ion-trap 921

  foodstuffs analysed by these methods largely comprise dairy products, 995

  partitioned into aqueous ammonaical ethanol. Three extractions were required for 1010 quantitative extraction of the colour. The aqueous extracts were acidified with acetic 1011 acid and back-extracted with diethyl ether. For foods containing fat and protein e.g. 1012 yoghurt, cheese and pastries, samples were ground with sand and aqueous 1013 ammonaical ethanol. The mixture was transferred to a centrifuge tube and the fat was 1014 removed by agitation with petroleum spirit, centrifugation and siphoning off the 1015 petroleum spirit phase. The aqueous ammonaical phase was retained, acidified with 1016 acetic acid and the annatto partitioned into diethyl ether. annatto components from fat and β-carotene. The choice of end 1068 method was determined by the presence of other colouring materials i.e. curcumin or 1069

  , comprehensive quantitative and qualitative data on 165 composite 1096

Figure 1 .

 1 Figure 1. Chemical structures of some bixin/norbixin isomers. R 1 =H, R 2 =H = norbixin;

Figure 2 .

 2 Figure 2. Spectral fine structure. Calculation of %III/II for a carotenoid (9'-cis-

Figure 3 .

 3 Figure 3. UV-VIS spectra of norbixin isomers (by HPLC-photodiode array): (a) 9'-cis-

Figure 4 .

 4 Figure 4. HPLC-photodiode array spectra of bixin isomers showing the locations of

Figure 8 .

 8 Figure 8. HPLC separation of bixin and norbixin isomers. Conditions: Column HRPB

[

  Key: ACN = Acetonitrile; DCM = Dichloromethane; MeOH = Methanol; MTBE = Methyl tertiary butyl ether; TEA = Triethylamine]

  -based emulsions and spreads, butter and fat-based compound foods 5 Fish, ice cream and ice cream-based confectionery, yoghurt and other dairy desserts

  used reverse phase (C 18 ) plates with methanol:water mobile 745 More recently, TLC has been used for the detection of bixin and other food 747 colour carotenoids derived from red pepper

	746	phase to separate annatto pigments from supercritical CO 2 extractions of annatto
		seeds.
		F o r
		P
		e
		e r
		R
		e
		v i e
		w
		O n l
		y
		32

uk Food Additives and Contaminants

	Page 75 of 83			Food Additives and Contaminants	Page 74 of 83
	mAU trans-isomer		9'-cis-isomer		k 1	di-cis-isomers
	R 1 OOC 100 other mono-cis-isomers CH 3 10 8 %III/II = III/II x 100 12 13 CH 3 14 11 9 7 6 120 II trans-isomer 9'-cis-isomer 15' 15 ? k 4	14'	CH 3 13' 12' poly-cis-isomers CH 3 7' 11' III 9' 8' 10' di-cis-isomers k 2	COOR 2 6' C 17 k 3
						trans-
	80
	R 1 OOC 60 40	F o r P F o r P F o r P F o r P F o r P F o r P F o r P F o r P F o r P F o r P F o r P F o r P F o r P F o r P F o r P F o r P F o r P F o r P F o r P F o r P F o r P F o r P F o r P CH 3 F o r F o r F o r F o r F o r F o r CH 3 P P P P P P	CH 3 9'-cis-CH 3		CH 3	CH 3 degradation products 9' COOR 2
	R 1 OOC 20 0		e e r e e r e e r e e r e e r e e r e e r e e r e e r e e r e e r e e r e e r e e r e e r e e r e e r e e r e e r e e r e e r e e r e e r e e e e e e 9',13'-di-cis-e r e r e r e r e r e r	CH 3 13'
		360	380	400	420	R e R e R e R e R e R e R e R e R e R e R e R e R e R e R e R e R e R e R e R e R e R e R e R 440 460 R R R R R e e e e e e	9' CH 3 480	500	COOR 2 520	nm
						v i e v i e v i e v i e v i e v i e v i e v i e v i e v i e v i e v i e v i e v i e v i e v i e v i e v i e v i e v i e v i e v i e v i e v i e v i e v i e v i e v i e v i e
								w w w w w w w w w w w w w w w w w w w w w w w w w w w w w
									O n l O n l O n l O n l O n l O n l O n l O n l O n l O n l O n l O n l O n l O n l O n l O n l O n l O n l O n l O n l O n l O n l O n l O n l O n l O n l O n l O n l O n l
										y y y y y y y y y y y y y y y y y y y y y y y y y y y y y
	1115
	1116

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Table 1 .

 1 7. 15-cis-bixin*. 8. 13'-cisbixin*. (*tentative). Permitted uses of annatto and maximum levels of addition(EC, 1994).

	Food Commodity type		Maximum permitted
				level (mg/kg)*
	Margarine, minarine, other fat emulsions, and fats	10
	F essentially free from water o r Decorations and coatings		20
	Fine bakery wares	P		10
	Edible ices	e		20
	15% alcohol by volume Liqueurs, including fortified beverages with less than r e	10
	Flavoured processed cheese	R	15
	e Ripened orange, yellow and broken white cheese; v unflavoured processed cheese i e Desserts w 'Snacks': dry, savoury potato, cereal or starch-based	15 10 20
	products: Extruded or expanded savoury snack products O Other savoury snack products and savoury coated nuts 10 n l y Smoked fish 10
	Edible cheese rinds and edible casings	20
	Red Leicester cheese		50
	Mimolette cheese			35
	Extruded, puffed and/or fruit-flavoured breakfast cereals 25
	[* Refers to 100% bixin or norbixin]

Table 2 .

 2 Summary of planar chromatographic methods for annatto colours.

	Sample	Adsorbent	Mobile phase	Reference(s)
	type
	Bixin,	Paper	CHX:CHCl 3 :DMF:HOAc	McKeown,
	norbixin,		(85:10:3:2)	1961, 1963
	C 17
	Annatto and	Silica gel G	Amyl acetate	Ramamurthy
	other fat			and Bhalerao,
	soluble dyes			1964
	Annatto	Silica gel G	1% HOAc in amyl acetate	Francis, 1965
	Bixin	Silica gel	CHCl 3 :ACE:HOAc (50:50:1) Dendy, 1966
	(1) Bixin	(1) Cellulose	(1) CHX:CHCl 3 :HOAc	Reith and
	(2) Norbixin	(2) Silica gel	(65:5:1)	Gielen, 1971
			(2) CHCl 3 :EtOH:HOAc
			(68:2:1)
	Annatto and	Silica gel G	(1) CHCl 3 :EtOAc (4:1)	Tirimanna,
	other	(2-dimensional)	(2) Et 2 O	1980
	pigments
	Bixin and	Silica gel GF	PE:Et 2 O:HOAc (85:15:2.5)	Preston and
	norbixin			Rickard,
	commercial			1980
	formulations
	Ether	Silica gel	(1) CHCl 3 : HOAc (9:1)	Corradi and
	extracts of		(2) Et 2 O: IPA (9:1)	Micheli, 1981
	foods
	Annatto
	seeds

Table 3 .

 3 Summary of HPLC methods used for analysis of annatto.

	Page 81 of 83		Food Additives and Contaminants
	Sample	Analyte(s)		HPLC conditions		Reference(s)
	matrix
			Column	Mobile phase	Detector
	Annatto colour	Bixin and norbixin isomers	1 ml.min -1 35°C HRPB C 8 /C 18 250 x 4.6mm, 5um ACN: 2% HOAc (65:35) isocratic	452, 460nm UV-VIS PDA	Scotter et al., 1994
	Annatto colour	Bixin and norbixin isomers	HRPB C 8 /C 18 250 x 4.6mm, 5um ACN: 0.4% HOAc (65:35) isocratic 1 ml.min -1 35°C	UV-VIS PDA 435 x 60nm	Scotter, 1995; Scotter et al, 1998,
		and C 17 isomers				2001
	Foods	Cis/trans bixin	Supelco LC-18 250 x 4.6mm,	MeOH: 2%HOAc (9:1) iscocratic	UV-VIS	Lancaster and
		and norbixin	5um	1 ml.min -1	500nm	Lawrence, 1995
	Foods	Bixin, norbixin	Supelco LC-18 250 x 4.6mm,	MeOH: 6%HOAc gradient	UV-VIS	Lancaster and
		and carminic	5um	1 ml.min -1	493nm	Lawrence, 1996
		acid
	Plasma	Bixin and	S5ODS1	ACN: 2% HOAc isocratic	UV-VIS PDA	Levy et al., 1997
		norbixin isomers		1.5 ml.min -1	460nm
	DNA	Bixin and norbixin	Supelco LC-8 250 x 4.6mm, 10um	ACN: 0.08% CF 3 CO 2 H (85:15) isocratic 1 ml.min -1	UV-VIS 470nm	Kovary et al., 2001
	Foods	Bixin and norbixin isomers	1 ml.min -1 35°C HRPB C 8 /C 18 250 x 4.6mm, 5um ACN: 0.4% HOAc (65:35) isocratic	455 x 10nm UV-VIS PDA	Scotter et al., 2002
	Cheese	Cis/trans bixin and norbixin	ODS2 C 18 250 x 4mm, 5um	ACN: 2%HOAc (75:25) isocratic 1 ml.min -1	UV-VIS 460nm	Bareth, Strohmar and Kitzelmann,
						2002
	Corn snacks	Norbixin	ODS2 C 18 150 x 4mm, 3um	ACN: 2%HOAc (65:35) isocratic 1 ml.min -1 29°C	UV-VIS PDA 450nm	O Rios and Mercadante, 2004
	Food	Bixin, norbixin	YMC C30 250 x 4.6mm, 5um	A: MeOH: H 2 O: TEA (90:10:0.1)	UV-VIS PDA	Breithaupt, 2004
		and other		B: MTBE: MeOH: H 2 O:TEA	450 x 4nm

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