Study of Annatto from Bixa Orellana Seeds: an Application of Time-Of-Flight Secondary Ion Mass Spectrometry

VOL. 17 NO. 2 (2005) ARTICLE

Study of annatto from Bixa orellana seeds: an application of time-of-flight secondary ion mass spectrometry

Carla Bittencourt, Marcella P. Felicissimo, Jean-Jacques Pireaux and Laurent Houssiau Laboratoire Interdisciplinaire de Spectroscopie Electronique (LISE), Facultés Universitaires Notre-Dame de la Paix, University of Namur, 61 rue de Bruxelles, B-5000 Namur, Belgium

Introduction A trend in the analysis of natural prod- Pulsed operation of the primary beam As colour is often a key consumer percep- ucts is the search for new techniques allows insulating surfaces to be neutral- tion for food preference and acceptability, that can be applied to their characterisa- ised between pulses using a low energy alimentary colourants play a key factor in tion without unduly interfering with their electron beam. Among the advantages the food industry. Natural and synthetic structure. During the analyses of natural attributed to the ToF-SIMS, one can cite colourants have been extensively used products, some of the following preven- the possibility to directly analyse the to colour food, drugs and cosmet- tive measures may be essential: reduced samples, without the need to extract ics. Since the first synthetic colourants time of analysis, elimination of oxygen, their components; in some cases detec- were introduced into the market at the protection from light, temperature control tion of a concentration lower than 1 ppm end of the eighteenth century, a set of and use of high-purity solvents. In this can be achieved, the spatial distribution prohibition rules have been established study we applied time-of-flight second- of elements and molecules with 100 nm by governments worldwide as some of ary ion mass spectrometry (ToF-SIMS) lateral distribution may be analysed and these molecules proved to be harm- in order to study annatto, a natural food insulating materials may be analysed. ful to human health. Even so the use colourant. ToF-SIMS, a technique already Moreover, measurements are performed of synthetic colourants is still conceived well developed to analyse organic and under high vacuum avoiding reaction as undesirable and harmful. In contrast, inorganic samples, can become a power- with undesired chemical elements. Large natural colourants, being nature-derived ful tool for elucidating the chemical and molecular ions and molecular fragmen- products, are considered by the consum- molecular compositions, spatial distribu- tation patterns (or “fingerprints”) are ers as healthy and of good quality, so tion of elements and degradation charac- characteristic of the sample. A review of the demand for natural colorants has teristics of natural products. ToF-SIMS is a surface analysis with ToF-SIMS can be increased. Hence better understanding surface analytical technique that uses an found in Reference 1. of their chemistry and biochemistry is ion beam to remove atoms or molecules Among the naturally occurring colour- required; in the food industry, the safety from the outermost layer of a surface. A ants, an important one is annatto of colourants is indeed a very important short pulse of primary ions strikes the (E160b). It is a carotenoid-based dye, issue. sample surface, and the secondary ions extracted from the surface of the seed of Compared with their synthetic coun- produced during the impact are extracted a tropical tree called Bixa orellana L. (achi- terparts, some natural colourants can be from the sample surface into a time-of- ote in Spanish, urucum in Portuguese); more sensitive to heat, pH or light; i.e. flight mass spectrometer. These second- named after the 16th century Spanish their colouring agent can degrade more ary ions are dispersed in time according scientist and explorer of South America, easily. The degradation of the colouring to their velocities, which are proportional Franscisco de Orellana. This tree is native agent can be used to tailor some prop- to (m/z)–1/2, where m/z is their mass/ to the Central and South American rain erties of the colourant. However, no charge ratio. Discrete packets of ions of forest; however, through Spanish influ- matter whether it is of natural or synthetic differing mass are detected as a function ence it became grown in South-East origin, the degradation of the colourant of time at the end of the flight tube. ToF- Asia. Annato was classified by the Food may generate undesirable products. SIMS is capable of detecting ions over a and Drug Administration in the USA Consequently, careful analysis of their large mass range, up to 10,000 atomic as a “color additive exempt of certifi- production and storage is important in mass units (Daltons), at a mass reso- cation”.2 Its principal colouring agent is order to establish safety specifications. lution (m/∆m) of 8000 at mass 29. bixin (C25H30O4), a carotenoid having

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+ C5H9 pressure during the experiment was better than 10–9 mbar. The mass reso- lution (m/∆m) near mass 29 was typi- cally 8000. Electron flood-gun charge HOOC compensation was necessary during the measurements recorded directly on the

COOCH3 seeds. For thermal degradation study, the samples were heated in situ by a regu- Figure 1. Chemical structure of bixin. lated resistive heater. a carboxylic acid and a methyl ester as ents of Bixa orellana seeds, its extract and Result and discussion its end groups (see the chemical struc- its degradation products. Annatto seed and extract ture in Figure 1). The average annual analysis production of annatto seeds is near 104 Sample preparation In order to determine the spatial localisa- tons, 60% of which comes from Latin Four sets of samples were analysed. The tion of the colourant, ToF-SIMS analyses America, 27% from Africa and 12% from first one was composed of Bixa orellana were performed on sectioned seeds in Asia. Among natural colourants, annatto seeds that were purchased from a local which the endosperm and the aril were ranks second in economic importance;3 retail outlet in São Carlos (State of São analysed (see the cross-section from the it is a substitute of Tartrazine, a synthetic Paulo, Brazil). A second set of samples B. orellana seed in Figure 2). Organic colourant that has been prohibited in was composed of the organic extracts fragments dominate typical spectra several countries.4 obtained by washing seeds of Bixa orel- recorded from the endosperm; further- The use of annatto as a food colour- lana with a (1 : 3) solution of methanol more, it is important to note the pres- ant can be traced back to the ancient and dichloromethane at room tempera- ence of a peak at m/z 55.93 (Figure 3), Aztecs, whose original chocolate drink is ture and which were analysed just after which indicates the presence of Fe inside reported to have contained annatto seeds. the extraction. To perform the analysis, the seeds. Bixin and its related fragments Given annatto’s high fat content and its the extracts were dripped onto a sili- are found to be present only at the seed’s red colour that could have been associ- con substrate and blown dry under a aril. Figure 4 shows the typical positive ated with blood (which of course had reli- flow of nitrogen. Finally, to study possi- ToF-SIMS spectrum recorded at the seed gious connotations in Aztec society) its use ble degradation, the previous sample set aril: this spectrum should be consid- is entirely probable. Until the 17th century, was divided into two sub-sets: one was ered as the colourant fingerprint, since the use of annatto in Europe to deepen analysed after being heated in vacuo, no extraction treatment, which could the colour of chocolate was also common. in situ in the ToF-SIMS spectrometer to interfere with its structure, was used. Nowadays, annatto is primarily used for different temperatures ranging from The peak at m/z 396 is assigned to the colouring cheese (50%), other dairy prod- room temperature to 150°C; the other bixin molecular ion plus two hydrogen + ucts (butter, margarine, snacks etc.) and set was analysed after exposure in air atoms (C25H32O4 or M + 2). The peaks body care products. The colouring agent to ambient light during three months at higher masses (m/z 397 and 398) are bixin is extracted by heating in vacuo a at room temperature. A colour change (M + 2) molecular ions containing one or suspension of the seeds in vegetal oil to was observed, which is an indication of two 13C isotopes (the natural abundance a maximum temperature of 130°C. At this degradation. of 13C is 1.1%). The peak at m/z 410 is extraction temperature, bixin undergoes a attributed to methyl bixin, a minor carote- series of degradation reactions to produce Experimental a range of products coloured from pale The mass spectra of the samples were yellow to orange. The principal thermal recorded on a ToF-SIMS IV instrument Aril degradation product is the yellow coloured from Ion-Tof GmbH, Germany. The

C17O4H20 molecule, the formation of which sample was bombarded with a pulsed is accompanied by release of m-xylene, Gallium ion beam. The secondary ions toluene, toluic acid and toluic acid methyl generated were extracted with a 2 keV Endosperm ester, all undesirable molecules in prepara- voltage and their time-of-flight, from the tions intended for food use. In considering sample to the detector, was measured the use of annatto as a colouring agent, it in a reflectron mass spectrometer. Typical is necessary to develop suitable methods analysis conditions for this work were of analysis to study directly the extracted 25 keV pulsed Ga+ beam at a 45° inci- annatto preparations and relate them to dence, 2 pA pulsed current rastered over the characteristics of the seeds and the a 130 × 130 µm area. The ion fluence –12 –2 extraction mechanism. In this work we was kept below 3 × 10 ions cm to Figure 2. Cross-section from a B. orellana applied ToF-SIMS to determine constitu- ensure static conditions. The background seed.

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Table 1. Bixin and some of its fragments.

m/z Fragments Label 1 H 1

15 CH3 2

27 C2H3 3

29 C2H5 4

41 C3H5 5

55 C4H7 6

69 C5H9 7 Figure 3. Typical spectrum recorded from the endosperm of the B. orellana seed. 81 C6H9 8

105 C8H9 9

121 C9H13 10

137 C8H9O2 11

151 C9H11O2 12

177 C11H13O2 13

191 C12H15O2 14

396 C25H32O4 15

Thermal and light degradation In order to quantify the degrada- tion induced by light, the extract was reanalysed after three months of expo- sure to ambient light. By visually inspect- ing the appearance of the extract, it was easy to observe that the initially orange pigment had turned to light yellow (Figure 6). The change in the extract colouration is due to the known degrada- tion of the colourant agent.5 The spectra obtained after light degradation exhibit a severe decrease (by a factor of five) in the intensity of the peak associated to the [M + 2]+ molecular ion, indicat- ing degradation of the bixin, along with + a two-fold increase of the C8H9 signal, attributed to xylene, an aromatic degra- dation product not welcome in food! For the thermal degradation study of bixin, a set of analyses was performed Figure 4. Typical spectrum recorded from the aril of the B. orellana seed. Labels (1–15) are after heating in situ the extract to differ- described in Table 1. ent temperatures. Figure 7 shows the evolution of the normalised intensity noid which is known to coexist with bixin ular peak at m/z 396. Moreover, simi- to the total spectral intensity of the in the Annato seeds. lar characteristic fragments peaks were peaks related to the bixin m/z = 396 The positive ToF-SIMS spectrum obtained both from the seed’s aril and and the fragments m/z = 137 and 177. recorded on the extract just after it had on the extract. The principal fragments Three temperature ranges can be distin- been prepared is similar to the one are shown in Table 1 and the possible guished: the first, shows a plateau from recorded on the aril of the B. orellana fragmentation sites in the bixin molecule 20 to 80°C ; near 80°C, a sharp inten- seed. It is dominated by the bixin molec- are shown in Figure 5. sity decrease is observed, accompanied

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191 Concluding remarks 151 The experimental study described in this article shows that ToF-SIMS can be used HOOC to analyse natural samples. This tech- 137 nique allows preventive measures, such as protection from light, temperature 177 COOCH3 control, inert atmosphere and no solvent use to be achieved during the analysis. Figure 5. Main degradation sites of bixin. It was shown that the technique allows the direct measurement on the seed; this opens a new possibility to be consid- 5 12

10 ered for the analysis of natural samples. 4 C H 5 9 By analysing the seed’s aril, it was shown 8 that bixin, one of the main colouring 3 6 constituents of the annatto seed, could 4 2 C2H5 Intensity (arb. un.) (arb. Intensity be easily detected by ToF-SIMS, without CH3 a) C H a) b) 2 3 2 any sample treatment. After prolonged 1 Figure 6. Photograph showing a) fresh 0 exposure to light, a significant degrada- 20 40 60 80 100 120 140 160 annatto extract and b) light degraded Temperature (°C) tion of bixin is observed. It was shown annatto extract. that degradation of the colourant agent Figure 8. Low mass fragments increase as occurs by heating near 80°C. The 0.9 temperature increases. systematic identification of the products 0.8 m/z 137 0.7 m/z 177 of degradation opens a new possibility m/z 396 0.6 for ToF-SIMS application. The molecular 0.5 sensitivity of ToF-SIMS has been shown 0.4 to 120°C for CH3, C2H3 and C2H5 and a to allow the straightforward detection of 0.3 slight decrease for C H This suggests the main constituents present in annatto Intensity (arb. un.) Intensity 0.2 5 9.

0.1 that the thermal degradation of bixin seeds, with potential applications in food 0.0 above 100°C leads to the creation of industry. Results also support some tradi- 20 40 60 80 100 120 140 160 smaller species, explaining the enhance- tional cooking practice. Temperature (°C) ment of the intensities arising from small Figure 7. Temperature effect on bixin and fragments, but in which the “repetition Acknowledgement some high mass fragments. unit”, C5H9, remains (Figure 1). The ToF-SIMS instrument was acquired During the heating experiment the thanks to the contribution of the FRFC formation of aromatic degradation prod- (Fonds de la Recherche Fondamentale by another plateau from 100 to 120°C; ucts, as inferred during the degradation Collective). finally, another sharp intensity drop by light, is expected. However, these appears at 120°C, until another plateau products were not observed during the References is achieved at 130°C. One can deduce heating experiment, which is probably 1. A. Benninghoven, Angew. Chem., Int from this figure that the colourant agent due to desorption of volatile compounds Ed. Engl. 33, 1023 (1994). begins to degrade near 80°C. By visual caused by heating. It has to be noted 2. J.B. Hallagan, D.C. Allen and J.F. inspection, it was possible to observe a that during “real” cooking situations it Borzelleca, Food Chem. Toxicol. 33, change in the colourant colour near this is reasonably feasible that these vola- 515 (1995). temperature. This observation supports tile compounds will suffer various fates, 3. A.Z. Mercadante, A. Steck and H. the important traditional cooking practice depending upon the food compo- Pfander, J. Agric. Food Chem. 45, of adding food colourant and some green nents, the heating conditions employed 1050–1054 (1997). herbs that need to keep their colour just and where the degradation occurs. For 4. JECFA, Evaluation of Certain Food at the end of the food preparation, in instance, aromatics formed at the surface Additives and Contaminants; Twenty- order to avoid their colour degradation, of the food are more likely to be vola- Sixth Report of the Joint FAP/WHO i.e. to grow pale. tilised and effectively removed, whereas Expert Committee on Food Additives; Figure 8 shows the evolution of the formation of aromatics within the inte- Technical Report Series No. 683. normalised intensity to the total spectral rior of the food may be trapped.5 The WHO, Geneva (1982). intensity of the peaks related to some traditional cooking practice of stirring 5. M.J. Scotter, L. Castle and G.P. fragments of lower mass for increasing the food while in the pan may in certain Appleton, Food Chem. 74, 365–375 temperatures. One can observe a gradual cases helps to avoid the imprisonment (2001). increase of the intensity in the range 100 of undesired volatile products.

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