ABSTRACT

Soybean oil used in processed foods is susceptible to lipid oxidation. Carotenes are utilized as colorants in processed foods containing soybean oil. Though carotenes are easily degraded during thermal processing, little is known regarding the effects of thermally degraded carotenes on the oxidative stability of soybean oil.

Thermally degraded β-carotene or lycopene solutions were added to soybean oil samples at concentrations of 0,25,45, and 50 ppm, respectively. Each sample, as well as controls containing 5, 25, and 50 ppm of unheated β-carotene or lycopene, contained 3 ppm of chlorophyll to allow photosensitized singlet oxygen oxidation to occur. The vial containing each sample was sealed airtight and stored either at 25°C in a light box (1650 lumens) or in a dark oven at 60°C. The oxidative stability of each soybean oil sample was determined by measuring (every 4 hours for 24 hours) peroxide value and headspace oxygen depletion by thermal conductivity gas chromatography. Oxidative stability was further identified by first utilizing solid phase microextraction (SPME) fibers to adsorb volatile oxidation products in the headspace of the samples, then quantifying and comparing the volatiles’ total peak area from their respective gas chromatogram.

Soybean oil samples containing 50 ppm degraded β-carotene displayed 11.5% higher peroxide values (under light) as well as higher headspace oxygen depletion values (in the

ii dark) when compared with controls (p<0.01). Lycopene degradation products (50 ppm) in soybean oil decreased peroxide values up to 10.5% under light, and significantly decreased headspace oxygen depletion of samples in the dark (p<0.05). Over all concentration ranges, headspace oxygen depletion values for samples stored under light containing either β-carotene or lycopene degradation products did not differ significantly from controls. After 30 days of storage in the dark at 60°C, samples containing 50 ppm degraded β-carotene displayed a significantly higher (p<0.05) SPME-GC total volatile peak area when compared with controls containing 50 ppm all-trans β-carotene. Under similar conditions, samples containing 50 ppm degraded lycopene displayed a significantly lower (p<0.05) SPME-GC total volatile peak area when compared with controls containing only oil.

These results indicate that thermally degraded β-carotene can act as a prooxidant in soybean oil exposed to elevated temperatures, which may cause a decrease in the oxidative stability of thermally processed foods containing soybean oil. Thermally degraded lycopene, however, may act as an antioxidant in soybean oil exposed to elevated temperatures and therefore may actually increase the oxidative stability of food

systems containing soybean oil.

iii

Once carotenoids have been solubilized in bulk lipid droplets in the stomach or intestine, bile salts and pancreatic lipases help to capture the carotenoids in micelles.

Duodenal mucosal cells absorb micelles containing the carotenoids by a passive diffusion mechanism similar to that of cholesterol and triglyceride lipolysis products. The carotenoids are then incorporated into chylomicrons and released into the lymphatics system (Stahl and Sies, 1996).

2.8.1.2 Tissue distribution

In order to reach the tissues of the body, carotenoids must first be transported by chylomicrons from the intestinal mucosa to the bloodstream via the lymphatic system.

The more non-polar carotenoids such as p-carotene and lycopene reside in the hydrophobic center ofthe chylomicron, while carotenoids with polar functional groups such as lutein and zeaxanthin are found closer to the outer surface. This differential orientation within the chylomicron molecule may affect the carotenoids' transfer to lipoproteins during circulation, as well as their uptake by extrahepatic tissues during hydrolysis ofchylomicron triglycerides.

In the bloodstream chylomicrons are broken down by lipolytic action due to lipoprotein lipase, which gives rise to chylomicron remnants. The liver clears the remnants from the blood and then can resecrete the carotenoids within lipoproteins for transport to other tissues. The distribution of p-carotene, a-carotene, and lycopene among the very low-density lipoproteins (VLDL), low-density lipoproteins (LDL), and high-density lipoproteins (HDL) are similar for all three carotenoids, with 58-73% in

LDL, 17-26% in HDL, and 10-16% in VLDL. The more polar dihydroxy carotenoids

70 lutein and zeaxanthin, however, are found predominantly in HOL (53%), with significantly lower proportions in LOL (31%) and VLOL (16%), respectively (Parker,

1996). Van Vliet (1996) actually determined that hepatic resecretion of carotenoids such as p-carotene occurs first by VLDL, upon which the carotenoids are then transferred to

LOL by delipidation. In short, the hydrocarbon carotenoids are transported primarily in

LOL, whereas the slightly polar carotenoids tend to be transported primarily in the more hydrophilic HDL lipoprotein fraction (Stahl and Sies, 1996). Significant differences have also been discovered when comparing distribution ofthe different isomeric forms of carotenoids in the human body. Though the amount of all-trans p-carotene greatly exceeds that of cis-p-carotene in the plasma, much increased proportions of cis-p­ carotene has been observed in peripheral tissues.

After transport throughout the body in the lipoproteins, carotenoids are primarily stored in adipose tissue in humans, although they have also been found in liver, lung, corpus luteum, and adrenal tissues (Rock et al., 1996). Stahl and Sies (1996) reported that the highest levels of p-carotene and lycopene have been found in liver, adrenal, and testes tissues, with lesser amounts in lung and kidney tissues (Table 6). In addition, lycopene concentrations exceeded those of p-carotene in every tissue except the ovary.

Prolonged over-consumption of vegetable juices has the capability of increasing carotenoid levels so high that the skin and liver can gain a yellow-orange discoloration

(Stahl and Sies, 1996).

71 Tissue p-carotene lycopene

liver 1.82 - 4.41 1.28 - 5.72 kidney 0.31- 0.55 0.15 - 0.62 adrenal 5.6 - 9.39 1.9 - 21.60 testes 2.68 -4.36 4.34 - 21.36 ovary 0.45 - 0.97 0.25 - 0.28 adipose 0.38 0.2 - 1.3 lung 0.12 - 0.35 0.22 - 0.57 colon 0.17 0.31 breast 0.71 0.78 skin 0.27 0.42

Table 6: Reported human tissue levels (nmol/g) of p-carotene and lycopene.

2.8.2 Vitamin A precursors

Perhaps the most important physiological function of carotenoids is to act as vitamin

A precursors in animals. Most animal species are capable of enzymatically converting carotenoids into the vitamin A compound retinol (see Figure 22). The provitamin A activity of a carotenoid depends on the presence ofat least one unsubstituted p-ring. As a result, p-carotene, with its two p-rings, is the carotenoid with the highest provitamin A activity. Other carotenoids containing one B-ring and exhibiting (to a lesser extent) provitamin A activity include: a.-carotene, y-carotene, 5,6- and 5,8-monepoxides of p- carotene, cryptoxanthin, and the p-apocarotenals (Table 7). Other factors contributing to

72 Carotenoid Activity (%)

all-frans- ~-carotene 100

9-cis-~-carotene 38

13-cis-~-carotene 53

all-trans-a-carotene 53

9-cis-a-carotene 13

13-cis-a-carotene 16

all-frans-cryptoxanthin 57

9-cis-cryptoxanthin 27

15-cis-cryptoxanthin 42

p-carotene 5,6-epoxide 21

p-carotene 5,8-epoxide (mutachrome) 50

y-carotene 42-50

p-zeacarotene 20-40

Table 7: Relative vitamin A activity ofsome carotenoids commonly found in vegetables.

provitamin A activity are state ofisomerization (cis vs. trans), gastrointestinal stability, and digestibility (O'Neil and Schwartz, 1992).

'Retinol equivalent' was introduced in 1974 by the NAS-Recommended Dietary

Allowances Panel to describe vitamin A content in terms of international unit activity, with 1 IV = 0.3 Jlg retinol. Therefore by definition:

retinol equivalent = 1 ug retinol

73 retinol equivalent = 6 ug p-carotene

= 12 ug other provitamin A

= 3.33 IV vitamin A activity from retinol 10 IV vitamin A activity from p-carotene

The recommended daily allowance for vitamin A is 1000 retinol equivalents. However, it is difficult to measure total carotenoid content (and therefore vitamin A content) in foods due to the sensitive nature ofcarotenoids to oxygen and solvents common in present analytical techniques. There are several specific nutritional roles for vitamin A in humans (Semba, 1998). The involvement of retinal, the vitamin A aldehyde, as the chromophore of the visual pigments in the eye, is crucial to human vision. In underdeveloped locations around the world where vitamin A deficiencies frequently occur, xerophthalmia, blindness, and premature death are all too common, especially among children. Vitamin A is also crucial in maintaining growth and reproductive efficiency, as well as maintenance of epithelial tissues and prevention of their keratinization. Recently it has even been shown to improve nonheme iron absorption from fortified rice, wheat, and com flours (Garcia-Casal, 1998).

2.8.3 Additional health effects

In addition to serving as precursors of vitamin A, other important biological functions have been shown to exist for carotenoids, including antioxidant activity (singlet oxygen quenching), and prevention ofdiseases such as cancer and cardiovascular disease.

74 2.8.3.1 Antioxidant protection

A biological antioxidant can be defined as "compounds that protect biological systems against the potentially harmful effects of processes or reactions that can cause excessive oxidations" (Palozza and Krinsky, 1992). The antioxidant activities of carotenoids have been demonstrated both in vitro and in vivo in several studies. Stahl et al. (1998) found that lycopene and lutein acted synergistically to increase the antioxidant effects of carotenoid mixtures containing tocopherols and carotene (a and P) in multilamellar liposomes. Martin et al. (1996) reported that carotenoid-loaded cells were partially or completely protected against oxidant-induced changes in lipid peroxidation, demonstrating that p-carotene and lutein (or their metabolites) protect HepG2 human liver cells in vitro against oxidant-induced damage independent of provitamin A activity.

However, Bast et al. (1996) found that p-carotene administered via the diet did not significantly influence liver microsomal lipid peroxidation in rats. p-carotene was shown to act synergistically with a-tocopherol as an effective radical-trapping antioxidant in liver microsome membranes (Palozza and Krinsky, 1992). In a similar study, p-carotene supplementation increased the induction period and decreased PC-OOH production in plasma during AAPH-induced lipid peroxidation. The findings suggested that dietary p-carotene may play an important role in the overall antioxidant defense system ofplasma (Meydani et al., 1994).

In vivo studies have shown similar results. Lepage et al. (1996) concluded that p­ carotene deficiencies in children with cystic fibrosis led to excessive lipid peroxidation.

Subsequent p-carotene supplementation reduced serum malonaldehyde concentrations,

75 indicating the carotenoid acted as an antioxidant in vivo. Ribaya-Mercado et al. (1995) found that when human skin was subjected to UV irradiation in vivo, skin lycopene and

~-carotene concentrations were lowered in a manner consistent with the consumption of free radicals through quenching processes. The researchers concluded that the antioxidant actions of lycopene and ~-carotene might be an important defense mechanism against the adverse effects of UV irradiation on the skin.

2.8.3.2 Cancer prevention

Studies have consistently shown that individuals with the highest intakes of carotenoid-rich fruits and vegetables have the lowest risks for cancers such as lung, oral cavity, stomach, and esophagus (Krinsky, 1994). Pool-Zobel et al, (1997) determined that men on a diet supplemented with tomato, carrot, or spinach products showed significantly lower endogenous levels of strand breaks in lymphocyte DNA. High serum levels of carotenoids have also been correlated with decreased risks for certain cancers

(Peto et aI., 1981; Bendich, 1993; van Poppel, 1996). ~-carotene has been shown to be particularly effective against smoking-related cervical intraepithelial neoplasia and cervical cancer (Charleux, 1996). Garewal (1995) found evidence of a chemopreventative role for the carotenoid ~-carotene against oral cavity cancer, the sixth most frequent cancer in the world. In another study, ~-carotene supplemented elderly men had significantly greater natural killer cell activity than elderly men receiving placebos. Because natural killer cells are crucial in the body's fight against tumor

76 growth, the increased activity can be correlated with decreased cancer risks (Santos et al.,

1996).

Lycopene has shown perhaps the most anti-cancer potential of all the carotenoids.

Giovannuci et al. (1995) found that among the carotenoids ~-carotene, a-carotene, lutein, lycopene, and ~-cryptoxanthin, only lycopene intake was related to a lower risk of non­ stage Al prostate cancer. Of the four vegetables or fruits that were found in the study to be significantly associated with lower prostate cancer risk, three (tomato sauce, tomato juice, and pizza) happened to be primary sources oflycopene. Dorgan et al. (1998) determined that the risk of developing breast cancer significantly decreased as serum concentrations oflycopene and lutein/zeaxanthin increased in women. Nagasawa et al.

(1995) examined the effects of chronic ingestion of lycopene on the development of spontaneous mammary tumors in SHN virgin mice. The lycopene treatment significantly suppressed the mammary tumor development compared with the non-lycopene-fed control group. Sharoni et al. (1995) stated that lycopene inhibited tumor growth both in vitro and in vivo. In vitro treatments oflycopene inhibited the growth of human skin fibroblasts, while lycopene treated rats developed fewer and significantly smaller mammary tumors in vivo when compared to the control or ~-carotene treated rats.

Lycopene delivered in cell culture medium from stock solutions in tetrahydrofuran more strongly inhibited proliferation of endometrial, mammary, and lung human cancer cells than did either a- or ~-carotene (Levy et al., 1995).

77 2.8.3.3 Cardiovascular disease

Carotenoids have been linked to reduced risk for the development of cardiovascular disease. Krinsky (1994) reported on findings showing a distinct inverse association between serum carotene levels and ischemic heart disease. A study of U.S. health professionals showed a similar association between dietary intake of p-carotene and heart disease risk. Men with the highest dietary p-carotene intakes had a 29% decrease in heart disease risk. Further analysis, however, determined that heart disease risk was reduced

70% in current smokers with high p-carotene intakes and 40% in former smokers, with lifelong nonsmokers actually having no significant correlation between p-carotene intake and risk of heart disease. In a parallel study of women nurses, those in the highest one­ fifth of the population in terms of p-carotene intake showed a 22% reduction in heart disease risk (Charleux, 1996).

2.9 Carotenoid oxidation/degradation

Due to their conjugated polyene backbone, most carotenoids are fairly unstable molecules and as a result are very sensitive to light, oxygen, and elevated temperatures.

The presence of these factors can cause oxidative degradation of carotenoids, resulting in destruction of the parent compound and formation of a variety of oxidized by-products.

2.9.1 Effects of oxidizing agents

EI-Tinay and Chichester (1970) used the radical initiator azo-bis-isobutyronitrile

(AlBN) to accelerate the formation p-carotene oxidation products. AIBN is capable of

78 thermal breakdown to a radical species (R·) that can rapidly react with oxygen to form peroxyl radicals (ROO·). The resulting initial oxidized product was reported as ~­ carotene-5,6- and 5,8-epoxides, with subsequent decomposition to other products such as

~-carotene-5,6-5' ,6' -diepoxide derivatives. Handelman et al. (1991) disputed the former claim, suggesting rather that the AIBN-initiated radical attack on ~-carotene occurred at multiple sites on the molecule, creating a 'series' ofapo-carotenals ofdifferent sizes such as retinal, ~-apo-14' -carotenal, ~-apo-12' -carotenal, and ~-apo-l 0' -carotenal.

Yamauchi et al. (1993) utilized a similar radical initiator, 2,2'-azobis(2,4­ dimethylvaleronitrile) (AMVN), to allow alkylperoxyl radical oxidation of ~-carotene.

The major resulting oxidized ~-carotene products included 12-formyl-ll-nor-~,~­ carotene, 15'-formyl-15-nor-~,~-carotene, 5,6-epoxy-5,6-dihydro-~,~-carotene, and 19­ oxomethyl-Iu-nor-Bfi-carotene. Using perphthalate as a chemical oxidizing agent, Seely and Meyer (1971) reported ~-carotene-5,6-monoepoxide as the principle product of oxidation. Copper stearate was also effective as a catalyst in the degradation of lycopene to smaller oxidized products (Cole and Kapur, 1957). Khachik et al. (1998) prepared the oxidative metabolites lycopene 1,2-epoxide and lycopene 5,6-epoxide by oxidizing all­ trans-lycopene with m-chloroperbenzoic acid (MCPBA), followed by acid hydrolysis.

Micro-Cel C, a common chromatographic adsorbent, was found to react with various carotenoids to yield hydroxides and epoxides when exposed in the presence of a nonpolar solvent. a-carotene was converted to 4-hydroxy-a-carotene, while ~-apo-8' -carotenal underwent hydroxylation at the allylic 4-position of the ~-ring. Lycopene was

79 completely oxidized by the Micro-Cel C, leaving lycopene 5,6-epoxide, 6'-apolycopenal, and lycopene-5,6-diol as degradation products (Ritacco et al., 1984; Ritacco et al., 1984).

Photosensitizers can also be effective initiators ofcarotenoid oxidation. Seely and

Meyer (1971) utilized hypericin, a powerful photodynamic agent, in the photosensitized oxidation of p-carotene to yield products such as mutatochrome and aurochrome. Lutein and zeaxanthin were found to undergo the photooxidative process more slowly than p­ carotene. By irradiating lycopene in an O2 atmosphere with the presence of methylene blue as a photosensitizer, Ukai et al. (1994) identified several oxidized products of lycopene, including 2-methyl-2-hepten-6-one and apo-6' -lycopenal (Figure 24).

Holman (1949) first investigated the oxidation of p-carotene in unsaturated oils, reporting that in the medium of an oxidizing unsaturated lipid, an intermediate product of fat oxidation stimulated p-carotene oxidation. Similar results were found in a later study that determined the addition of unsaturated oil resulted in a shorter induction period for the autooxidation of p-carotene and vitamin A in a paraffin solution. The prooxidant effect ofthe oil increased with an increasing iodine value and degree ofunsaturation

(Budowski and Bond, 1960). Carnevale et al. (1979) disputed these previous findings by stating that increased unsaturation of oil offers protection against autooxidation of carotenoids, because a higher degree of unsaturation in oil provides a substrate diversion away from the p-carotene molecule, resulting in a lower rate of carotenoid oxidation.

Oxygen has been used to initiate carotenoid oxidation. By passing a slow current ofpure oxygen through a solution of lycopene in hexane, Cole and Kapur (1957) claimed the oxidative products of lycopene to be acetone, methylheptenone, and laevulinic aldehyde.

80 lycopene

02, hv methylene blue

~o 2-methyl-2-hepten-6-one

apo-6' -lycopenal

Figure 24: Possible reaction pathway for photosensitized oxidation of lycopene.

Ben-Aziz et al. (1973) later isolated a series of epoxides and apo-lycopenals from tomatoes, including 1,2-epoxy-l ,2-dihydro-\j/,\j/-carotene and 5,6-epoxy-5,6-dihydro­

\j/,\j/-carotene, which may have been early products of the oxidative degradation of lycopene due to tissue senescence and!or physical injury. Teixeira Neto et al. (1981)

81 examined the kinetics of p-carotene oxidation in a model system of "nonfat" dry milk,

AVICEL microcrystalline cellulose, and crystalline p-carotene (simulating a dehydrated food product). Carotenoid oxidation could be accurately predicted by colorimetric assessment of the decoloration of p-carotene in the model system.

2.9.2 Effects of thermal processes

Carotenoid pigments possess an extended polyene structure that causes them to be very reactive in the presence of light and/or heat (Minguez-Mosquera and Jaren-Galan,

1995). Because carotenoids such as p-carotene and annatto (bixin) are widely used in industry as food colorants, thermal degradation (and resulting loss of color) during processing is of great concern to food manufacturers and nutritionists. Common degradation products from the heating of p-carotene can be seen in Figure 25.

Marty and Berset (1986) compared the degradation of all-trans-p-carotene during two thermal processes: heating in sealed glass tubes, and extrusion cooking. After heating the all-trans-p-carotene in sealed glass tubes for 2 hours at 180°C, the main degradation products were identified as p-carotene-5,6-epoxide, p-carotene-5,6,5',6' -diepoxide, and p-carotene-5,8-epoxide. Similar compounds were found after extrusion cooking of all­ trans-p-carotene, with p-carotene-5,6,5' ,8' -diepoxide also being present. Marty and

Berset (1988) found that after extrusion cooking, only 8% of the all-trans-p-carotene remained, with the other 92% of the p-carotene degrading into one of six main groups: 1) mono- or poly-cis stereoisomers, 2) a diepoxide derivative, 3) five apo-carotenals, 4) a polyene ketone, 5) a dihydroxide derivative, or 6) a monohydroxide diepoxide derivative.

82 mutatochrome

5,6,5',6'-diepoxy-p-carotene

0-----'

aurochrome

luteochrome

Figure 25: Nonvolatile compounds often formed during heating of p-carotene.

The authors concluded that the resistance ofall-trans-Bscarotene to high temperatures depends largely on the processing conditions. Different thermal treatments resulted in all-trans-p-carotene losses between 7.5% and 92%. Prolonged heating at I80aC caused

83 only limited breakdown of the carotenoid molecule, but the presence of other constituents such as starch and/or water combined with mechanical mixing favoring incorporation of additional oxygen lead to much higher losses of all-trans-~-carotene(Marty and Berset,

1990).

Ouyang et al. (1980) identified the main decomposition products of ~-carotene formed during a simulated commercial deodorization of palm oil to be ~-13-apo­ carotenone, ~-15-apo-carotenal, and ~-14' -apo-carotenal. When ~-carotene was heated in glycerol at 210°C for 5 min., 15 min., 1 hour, and 4 hours (to simulate the time/temperature combinations seen in deep fat frying and edible oil deodorization), respectively, over seventy nonvolatile compounds were observed by IRfMS (Onyewu et al., 1986).

A study examining the effect of microwave cooking on the stability of carotenoid pigments in sweet potato leaves demonstrated that the epoxy-containing carotenoids were more susceptible to heat loss than other carotenoids. Two lutein dehydration products were identified in the sweet potato leaves after microwave processing: 3,4-didehydro­

~,f:-caroten-3'-01, and 3,4-didehydro-~,~-caroten-3-01 (Chen and Chen, 1993). Khachik et al. (1992b) also found the epoxycarotenoids present in foods such as green vegetables and tomatoes to be more sensitive to the thermal processes involved in microwaving, boiling, steaming, and stewing than the hydrocarbon carotenoids such as neurosporene, u- and ~-carotene, lycopene, phytofluene, and phytoene. Godoy and Rodriguez-Amaya

(1987) examined the effects ofthennal processing on both mango slices and puree. The only significant change in the mango slices after heat treatments was an increase in the

84 measured luteoxanthin content. In the processed mango puree, processing at 80°C for 10 minutes resulted in a 13% decrease in ~-carotene content, 33% decrease in violaxanthin, and an increase in the auroxanthin content due to a 5,6- to 5,8-epoxide transformation.

Cole and Kapur (1957) were among the first to examine the extent oflycopene breakdown as a result of exposure to elevated temperatures. The authors reported lycopene losses of 15% and 25% in 3 hours of thermal treatment at 65°C and 100°C, respectively. A heat treatment of 97°C in water produced several novel ~-carotene thermal degradation products, including decanal, 4-ethylbenzaldehyde, and cetoisophorone. The compound 5,6..epoxy-Bcionone was also shown to be an important reaction intermediate, acting as a precursor for various volatile compounds such as ~­ ionone and 2-hydroxy-2,6,6-trimethylcyclohexanone (Kanasawud and Crouzet, 1990).

Using a similar thermal process, Kanasawud and Crouzet (1990) determined the resulting degradation products of lycopene by GC/MS analysis. The main characterized products included 2-methyl-2-hepten-6-one and citral, as well as the previously uncharacterized compounds 5-hexen-2-one, hexane-2,5-dione, 6-methyl-3,5-heptadien-2-one, and geranyl acetate (Figure 26). All-trans-Iycopene was also found to partially isomerize to the cis­ trans isomer as a result of the heat treatments. Henry et al. (1998) reported that in a safflower seed oil model system, the rates of thermal degradation for selected carotenoids between 75°C and 95°C were as follows: lycopene > all-trans-p-carotene = cis-~­ carotene> lutein.

85 all-trans-p-carotene

CS-C6 Cleavage CrCg Cleavage

o o 6-methyl-3,5-heptadien-2-one 2-methyl-2-hepten-6-one

geranial neral

Figure 26: Reaction sequence for the formation of volatile compounds during heat treatment of lycopene.

Chandler and Schwartz (1988) examined changes in the carotene content of sweet potatoes subjected to one of several different thermal processes. All-trans-p-carotene was found to be more susceptible to isomerization reactions resulting in the formation of cis isomers such as 13- and 15-cis-p-carotene than degradation reactions during most processing treatments. The extent of isomerization was largely related to the severity and

86 length ofthe individual heat treatment. Chen and Chen (1995) reported extensive isomerization in carrot carotenoids during canning (121°C, 30 min.) and HTST heating

(120°C, 30 sec.) thermal processes. The formation of several cis isomers of p-carotene, including 13-cis-p-carotene, 13-cis-Iutein, and 15-cis-a.-carotene may have been responsible for the carrot juice color change from orange to yellow during the intensive heat treatments. Ogunlesi and Lee (1979) reported a substantial increase in the concentration of cis isomers and a 25-35% decrease in all-trans isomers of p-carotene after retorting processed carrots, resulting in a 15% decrease in the vitamin A value.

2.9.3 Effects of storage

In addition to changes incurred as a result of processing, carotenoids are also capable of undergoing alterations in their composition and structure as a result of simple extended storage. During storage of mango slices in lacquered or plain tin-plate cans, no significant loss of p-carotene was observed after 10 months of storage. However, upon extended storage ofthe slices, a 50% reduction in total p-carotene was observed after 14 months, with continued degradation resulting in a p-carotene loss of 84% after 24 months. Other carotenoids such as violaxanthin and luteoxanthin also decreased in quantity during storage, while auroxanthin levels remained constant (Godoy and

Rodriguez-Amaya, 1987).

Kopas-Lane and Warthesen (1995) determined that light promoted carotenoid pigment losses in raw spinach, with 60% of the violaxanthin and 22% of the lutein present in the spinach being degraded after only 8 days of storage. Storage of raw

87 spinach in the dark did not affect spinach carotenoid levels, except for an 18% loss of all­ trans-~-carotene. For raw carrots, however, neither lighted nor dark cold storage affected the major carotenoids. In a similar study, degradation of lycopene in a vegetable juice model system was about one-fifth that ofa- and ~-carotene after an 8-day storage period

(Pesek and Warthesen, 1987). In addition, Wagner and Warthesen (1995) revealed that degradation of a- and ~-carotene during storage at 37°C occurred at the same rate.

2.9.4 Effects of encapsulation in minimizing degradation

Though the trend in the food industry is towards natural products as opposed to synthetic additives, carotenoids are limited by application problems. For example, creating acceptable water-soluble forms of carotenoids is extremely difficult due to the hydrophobicity ofpure carotene crystals. Encapsulation provides a method to transform liquids such as solubilized carotenes into stable free-flowing powders that can be easily incorporated into aqueous food systems.

Wagner and Warthesen (1995) determined that hydrolyzed starch of36.5 DE was more effective than 25, 15, and 4 DE in improving carotene retention during storage, with encapsulated carotenes enjoying a predicted half-life of450 days at 21°C, compared with

2 days for the spray-dried carrot juice control. In another study, the stability of ~­ carotene encapsulated in 25 DE maltodextrin by spray drying, freeze drying, and drum drying was evaluated. After 15 weeks ofstorage, drum drying gave the best ~-carotene preservation of all the encapsulation methods. Due to its smaller particle size and surface

88 carotenoid content, the spray-dried encapsulated p-carotene showed the fastest degradation, with 80% degraded after a seven-week storage period at 45°C.

2.10 Isolation and identification of carotenoids

Isolation of carotenoids from biological sources usually involves extraction, saponification, and separation processes. Once separated, identification of individual carotenoids is often based on a complex combination of spectrometric, chromatographic, and chemical tests. High performance liquid chromatography (HPLC), thin layer chromatography (TLC), Mass spectrometry (MS), Gas chromatography (GC), Nuclear magnetic resonance (NMR) spectroscopy, UltravioletlVisible (UVNis) spectroscopy, and circular dichroism (CD) are all routinely used for separation and/or structural elucidation of carotenoids and their oxidation/degradation products.

The analysis of carotenoids is further complicated due to their structural instability, tendency to stereomutate, photo- and thermo lability, and propensity towards oxidation.

As a result, all analytical experiments must be carried out in dim light with inert (under nitrogen or vacuum) atmospheric conditions. In addition, solvents must be purified, environmental temperatures must be no higher than 40°C, and samples must be dried and stored at -20°C under nitrogen (Schiedt and Liaaen-Jensen, 1995).

2.10.1 Extraction procedures

Extraction of carotenoids from biological materials must be done as rapidly as possible to minimize oxidative and/or enzymatic degradation. Often blanching ofplant

89 tissue and addition of calcium carbonate (CaC03) and antioxidants such as butylated hydroxytoluene (BHT) is included before extraction to minimize enzymatic reactions, acid hydrolysis, and oxidative degradation, respectively. To facilitate maximal extraction yields material should be ground into small pieces. The lipophilic carotenoids require organic solvents that are free of oxidizing compounds, acids, or halogens for efficient extraction to take place (Figure 27).

Usually extractions are carried out in a blender so that grinding and extraction can occur simultaneously. Hart and Scott (1995) prepared samples for carotenoid analysis by freezing them in liquid nitrogen and then grinding them under liquid nitrogen with a

Waring blender. Homogenates can be suction filtered through a Buchner funnel lined with filter paper coated with Celite (Silveira, Jr., and Evans, 1995). The extraction process can be repeated several times until all visible pigment is extracted from the source material (Khachik et aI., 1992b).

2.10.1.1 Solvents

Different solvents have been used with varying success in the extraction of carotenoids from biological sources. Hakala and Heinonen (1994) extracted 6 mg of lycopene from 10 grams of tomato puree using both petroleum ether and acetone.

Recovery and purity ofthe lycopene were both better in the sample extracted with petroleum ether compared to that extracted with acetone, due to a smaller portion ofpolar xanthophylls being extracted with the more nonpolar petroleum ether solvent.

Tetrahydrofuran (THF) was used as the solvent to extract carotenoids from several different raw and cooked vegetables for HPLC analyses. Complete extraction of the

90 WET BIOLOGICAL DEHYDRATED MATERIAL .....~--...... BIOLOGICAL MATERIAL

CRUDE EXTRACT

Extraction into ether/hexane Evaporation

DRY LIPID EXTRACT

Partition I hexane/aqueous L-. 85% methanol

HYPOPHASIC• EPIPHASIC NEUTRAL ACIDIC CAROTENOID CAROTENOID CAROTENOID CAROTENOID Partition + HYPOPHASIC• EPIPHASIC CAROTENOID CAROTENOID

Chromatography (CC, TLC, HPLC) + INDIVIDUAL CAROTENOIDS Rechromatography Crystallization Recrystallization

PURE CAROTENOID ~ Characterization

Figure 27: Sample procedure for isolation of carotenoids from a natural source.

91 A wrist-action shaker containing a solvent mix of hexane-acetone-ethanol (50:25:25) was utilized to extract carotenoids from tomato puree. Fifteen mLs of water was added to the mix to allow improved separation into distinct polar (clear aqueous) and nonpolar

(lycopene-containing red) layers.

Craft and Soares, Jr. et al. (1992) reported the relative solubility, stability, and absorptivity of several carotenoids in eighteen different organic solvents. Results showed that the solubility ofboth lutein and p-carotene was highest in tetrahydrofuran (THF), hexane exhibited the least solubility for lutein, and both methanol and acetonitrile exhibited the least solubility for p-carotene. Cyclohexanone caused the most degradation of carotenoid pigments after ten days of storage, with only 37% of lutein and 32% of p­ carotene absorbance remaining, respectively.

2.10.1.2 Supercritical fluids

The increasing demand for natural p-carotene has resulted in a growing interest in rapid, cost-effective methods of carotenoid extraction from plant sources. Most extraction methods presently involve the use of organic solvents, which are generally undesirable due to exposure to potentially toxic compounds, as well as environmental concerns. Vega et al. (1996) reported a maximum 99.5% extraction of p-carotene from carrot pulp using supercritical carbon dioxide extraction with 10% ethanol as a co­ solvent. Concentration of ethanol and temperature were determined to be the most important factors in determining extraction yield. High efficiency extractions of p­

92 carotene from natural sources such as carrot pulp were determined to be feasible by supercritical C02 + ethanol extraction methods.

2.10.2 Quantitation

Because carotenoids generally obey the Beer-Lambert law, their quantitative determination is often accomplished by spectrometric methods in which the absorbance ofa known volume of carotenoid solution is read at the wavelength of maximal absorption. Carotenoid content can therefore be determined in ug/g material by using the following equation:

ug carotenoid/g A x V x 106

A 1%",,,, X 100 x G

where V is the total volume (mL) containing G grams of sample, and A 1%lcm is the specific absorbance or extinction coefficient. Specifically, the extinction coefficient is defined as the theoretical absorbance of a 1% solution (w/v) in a 1 cm path-length cuvette. For colored carotenoids, extinction coefficient values are usually around 2500, so a solution with a carotenoid concentration of 1 ug/rnl, would give an absorbance (A) of approximately 0.25 (Davies, 1976).

2.10.2.1 UV/Vis Spectroscopy

The pursuit ofaccurate carotenoid quantitation is most commonly accomplished by using spectroscopy in the UV/Visible region ofthe spectrum. Kearsley and Rodriguez

(1981) utilized UV/Vis Spectroscopy to determine the content and stability of p-carotene in solution after exposure to thermal treatments, light, and changes in pH.

93 2.10.2.2 HPLC-PDA

High performance liquid chromatography-photodiode array detection (HPLC-PDA) has also more recently been used to quantitate carotenoids from various sources. The use of a photodiode array detector to quantitate carotenoids in biological extracts depends upon calibration with authentic source substances, or standards. Analysis precision is greatly increased by the addition of internal standards early in the analysis. Peak height

and peak area ratios ofthe compound of interest vs. the internal standard can be utilized

for quantitation. In the absence of standards only semiquantitative results can be compiled (De Leenheer and Nelis, 1992).

2.10.3 Separation

Chromatography is perhaps the most important single technique in the separation of

carotenoid pigments. The separation process of chromatography is based upon two phases known as the stationary phase and the mobile phase. A mixture of compounds is

added to the mobile phase that is subsequently carried through the chromatographic

system. As the mobile phase passes through the stationary phase, each compound in the

mixture reaches an equilibrium distribution at a specific point between the two phases,

resulting in differential migration rates through the system (Pfander, 1995).

The compounds to be separated can interact with the two phases in two specific ways,

partition and adsorption. Partitioning occurs ifthe sample mixture diffuses into the

interior ofa liquid stationary phase. The latter term is applicable ifthe sample mixture is

attracted to the surface ofa solid stationary phase. Many adsorbents are utilized in

carotenoid chromatography, among them starch, CaC03, MgC03, Ah03, and silica gels.

94 However, these various absorbents can achieve separation ofcompounds in different ways. Materials such as CaC03 and CaC03 have affinities for double-bond systems, so separation of carotenoids is determined by the number and type ofdouble bonds in the molecule. Adsorbents such as alumina or silica, on the other hand, separate carotenoid groups ofdiffering polarity such as hydrocarbons, monohydroxy carotenoids, and polyhydroxy carotenoids according to polarity, with the most polar carotenoids being most strongly adsorbed.

Silica and alumina are now the most widely used stationary phases for the separation ofcarotenoids, and are usually used in bonded-phase chromatography. Bonded phases are prepared by modifying the reactive groups on the surface ofthe stationary phase

(silica or alumina). To do this, the silanol groups (SiOH) are reacted with an alkylating agent such as dimethyloctadecylchlorosilane (ODS) to form a CIS bonded phase.

Trimethylchlorosilane can also be reacted with the silanol groups in a second alkylation process, known as endcapping, which minimizes the number ofremaining silanol groups

(Figure 28).

2.10.3.1 HPLC

High-performance liquid chromatography (HPLC) is by far the method ofchoice for carotenoid analysis for many reasons. Its ability to distinctly separate many compounds in a relatively short time proves its efficiency. HPLC analyses can be highly sensitive, with the detection ofsmall amounts ofimpurities, trace carotenoids, and/or geometrical isomers being possible. Finally, HPLC analyses with a photodiode array detector and computer-aided data processor can yield much information about the sample being

95 Residual silanol group

CH3

Si---O--~i--(CH2)17CH3 Bonded ODS group I CH3 CH3

Si---o--L--CH3 Endcapped silanol I

Figure 28: Main features ofa CI8 bonded-phase silica material.

analyzed. High-performance liquid chromatography, which has also been referred to as

'high-pressure liquid chromatography', includes liquid chromatographic methods that used stationary phases ofparticle size not> 10 urn and pressures> 20 bar (300 psi).

An HPLC system is comprised ofseveral important components that can be viewed schematically in Figure 29. The pump is responsible for producing the pressures necessary to force mobile phase through the stationary phase particles. A sintered metal frit is often placed after the solvent reservoir to prevent solid impurities from reaching the

96 .•.­ 9 8 7 10 2:J 4 0

~~~'-. 5 ffi1

Figure 29: Schematic diagram ofa typical HPLC system. 1. solvent reservoir, 2. sintered metal frit, 3. high-pressure pump, 4. pulse damper, 5. drain valve, 6. pressure gauge, 7. pre-column, 8. injection syringe, 9. injection valve, 10. column, 11. thermostatted oven (optional), 12. detector, 13. recorder/integrator/plotter, 14. fraction collector.

pump and damaging it. A pulse damper can lessen or even remove the pulsations that

result from pump action. A pre-column is most commonly inserted between the sample

injector and the separation column to protect the latter from impurities in the sample or

mobile phase. The separation column itself is usually constructed from 316-grade

stainless steel, which is resistant to the high HPLC pressures as well as most chemical

corrosion. The detectors utilized in HPLC analyses are usually UVNis due to their

sensitivity and ease in operation (Johnson and Stevenson, 1978). The development of

photodiode-array (PDA) detectors has been very helpful in the analysis of carotenoids

due to their rapid data acquisition and ability to store entire spectra for later comparison

(De Leenheer and Nelis, 1992; Pfander and Riesen, 1995). Most recently,

electrochemical detection (ECD) has been shown to be a particularly useful alternative to

97 UVNis detection methods for LC analyses requiring extremely high (fmol) sensitivity

(Ferruzzi et al., 1998).

Both UVNis and PDA detectors generate chromatograms that consist of a curved peak for each separated compound from the sample. The chromatogram provides the following information to the researcher: 1) the retention time of the compound(s), and 2) the area and/or height of the peak(s). Retention times ofunknown compounds and standards can be compared for tentative identification purposes. The arealheight of a peak can be used, with the help of a calibration curve, to estimate the relative amounts of each compound present in the sample.

Carotenoid separations can be accomplished by either normal-phase or reverse-phase

HPLC. Normal-phase employs adsorptive phases such as silica and alumina, as well as polar bonded phases such as alkylamine or alkylnitrile in combination with nonpolar mobile phases. In this type ofHPLC, polar sites on the carotenoid compete for adsorptive sites on the stationary phase, resulting in the least polar carotenoids

(carotenes) eluting first, while the more polar oxygenated carotenoids (xanthophylls) are retained in the column longer. Reverse-phase HPLC includes nonpolar bonded phases

(Cs and CIS) and polymer phases in conjunction with polar mobile phases. During reverse-phase HPLC, xanthophylls are more induced to stay in the polar mobile phase and therefore elute first, while the carotenes partition preferentially into the stationary phase and elute later. Both normal-phase and reverse-phase HPLC can be used with the same mobile phase solvent compositions throughout the analysis (isocratic), or the solvent compositions can change during the analysis (gradient) (Craft, 1992).

98 The investigation of carotenoids by HPLC has been ongoing for almost 30 years, with a wide variety of papers being published on the subject during that time. Handelman et aI. (1991) utilized a mobile phase of85% acetonitrilel15% methanol through a CI8 column in a gradient elution process in order to separate the degradation products of p­ carotene. Ammonium acetate (0.01%) was added to the initial mobile phase to help increase HPLC recovery of carotenoids. Several apocarotenoids (autooxidized products of p-carotene) were recorded on chromatograms at a wavelength of 350 nm. In a similar study, epoxide products of p-carotene antioxidant reactions were separated by reverse­ phase HPLC using a mobile phase of methanol-hexane (85: 15 v/v) with a flow rate of 1.5 mLimin. Further resolution of epoxide products was successfully accomplished through the use of a cyano-column (Liebler and Kennedy, 1992). Marty and Berset (1990)

separated thermally oxidized p-carotene compounds by using a HPLC elution system of n-hexane/diethyl ether (95:5 v/v) with a flow rate of 1 mLimin.

HPLC has been employed as an effective technique to separate and quantify various carotenoids present in human plasma samples. Eighteen different carotenoids, including

vitamin A, were separated from extracts ofhuman plasma by HPLC on reversed-phase

CI8 silica-based nitrile-bonded columns (Khachik et al., 1992a). Epler et al. (1993)

developed a HPLC method for quantitative measurement ofthe six major carotenoids

found in human serum. The mobile phase consisted of a mixture of acetonitrile,

methanol, and ethyl acetate, each containing 0.05% triethylamine (TEA) to increase

carotenoid recovery. In addition, ammonium acetate (0.05 M) was also added to the

methanol to minimize carotenoid losses on the column. Stahl et al. (1993) separated five

99 geometrical isomers of p-carotene and seven of lycopene in human serum and tissues using improved reverse-phase HPLC methods. Mobile phases consisted of methanol/acetonitrile/2-propanol (54/44/2), or methanol/acetonitrile/2-propanol/H20

(l0/40/40/1 0) with a flow rate of 1.0 mLlmin and detection at 460 nrn.

The carotenoid content of human serum was first examined using a non-aqueous reverse-phase (NARP) HPLC method developed by Nelis and De Leenheer (1983).

NARP mobile phases used included mixtures of acetonitrile, dichloromethane, methanol, tetrahydrofuran (THF), and ethyl acetate. The NARP method showed superior sample solubility of nonpolar carotenoid components when compared with conventional aqueous reverse-phase chromatography, mainly due to the fact that carotenoids typically are only sparingly soluble in partially aqueous solvents.

Several HPLC methods have been developed to determine the carotenoid contents of fresh or processed fruits and vegetables both qualitatively and quantitatively. The major carotenoid constituents of extracts from several raw and cooked green vegetables

(broccoli, green beans, and spinach) (Khachik et al., 1992b), as well as fresh tomatoes and tomato paste (Khachik et al., 1992b; Tonucci et al., 1995) have been separated by

HPLC on a Cis-reverse phase column. An isocratic mixture of acetonitrile (85%), methanol (10%), dichloromethane (2.5%) and hexane (2.5%) was used for the mobile phase, with chromatographic analyses simultaneously being monitored at several different wavelengths (Khachik et al., 1992b). Tan (1988) also identified the carotenoids present in tomato paste by using isocratic reverse-phase HPLC with several different

NARP solutions. Carotenoids from apricot, peach, cantaloupe, and pink grapefruit extracts were also separated and quantitated on a Cis-reverse phase column (Khachik et

100 al., 1989). Kopas-Lane and Warthesen (1995) developed a reverse-phase gradient HPLC method on a CIS column for the separation ofxanthophylls, carotenes, and cis f3-carotene isomers from raw spinach and carrots. Initial conditions for the mobile phase included

90% acetonitrile/5% water/5% methanol, with a linear gradient increasing the methanol content to 100% in 15 minutes. An HPLC solvent system of acetonitrile/methanollchloroform/hexane (75:12.5:7.5:7.5 v/v/v/v) pumped at a flow rate of 1.0 mLlmin was employed to determine the stability of carotenoids in sweet potato

leaves to microwave cooking. Eluate was monitored at 440 nm and comparing retention times of separated peaks with those of the reference standards (Chen and Chen, 1993) identified carotenoids.

More recently novel polymeric C30 stationary phase columns have been shown to be

effective in HPLC separation of carotenoid compounds and even geometric isomers

(Sander et al., 1994). Using such a column, Emenhiser et al. (1995) was able to obtain

superior resolution of several isomers of the asymmetrical carotenoids lutein, a-carotene,

and f3-cryptoxanthin, as well as the symmetrical carotenoids zeaxanthin and f3-carotene.

A mobile phase of methyl-tert.-butyl-ether (MTBE) in methanol was used isocratically to

achieve separations on the polymeric C30 stationary phase. The same C30 column was

later used by Emenhiser et al. (1996) to distinguish the different geometrical carotenoid

isomers present in human serum, carrots, algae extract, and a poultry feed supplement.

Again a methanol-MTBE mobile phase was utilized for chromatographic analysis.

Addition oftriethylamine (0.1% v/v) to the mobile phase was found to increase the

recovery of f3-carotene from the C30 column from 52% to 88%.

101 Another proposal to increase HPLC resolution between carotenoid isomers was made by Schmitz et al. (1995). The use of a calcium hydroxide stationary phase in HPLC analyses was advocated for the specific purpose of increasing the resolution between the various members ofthe acyclic and cyclic geometric carotenes containing 5, 7, or 11 aliphatic double bonds.

Depending on how HPLC methods are used in the separation and identification of carotenoids, occasionally unanticipated results have been known to occur. Piretti et al.

(1996) found that when using a normal-phase cyano-amino HPLC column, lycopene samples and standards both exhibited differing numbers of peaks depending on the solvent used to prepare the sample for injection. Scott et al. (1992) reported that in addition to reactions between carotenoids, injection solvents, and the mobile phase, metal surfaces such as the stainless steel of metal frits in HPLC systems may be damaging to carotenoid compounds. In addition, changes in ambient temperature during HPLC analyses of carotenoids were reported to cause dramatic differences in the data collected.

A reduction of 1 minute in elution time for every 1°C rise in temperature was described, with optimum resolution occurring at 20-22.5°C (Scott and Hart, 1993).

2.10.3.2 TLC

Thin-layer chromatography (TLC) first gained popularity in the 1950's as a replacement for paper chromatography. Though more recently TLC has tended to be superseded by the more efficient and sensitive HPLC systems, TLC is still a simple and inexpensive method that is often utilized for pilot screenings of carotenoid mixtures of

102 unknown composition. The results ofTLC screenings often direct the choice of conditions for subsequent preparative chromatography and/or HPLC (Schiedt, 1995).

TLC is generally applied on a micro- or semimicro scale, which permits rapid and sharp separation ofthe various compounds present in the sample as well as detection of substances at the trace level. Usually silica gels are used as the adsorbent; basic oxides or carbonates such as MgO are also commonly used. Tentative identification ofa compound by TLC is based on comparison ofits Rrvalue (the distance moved by the solute divided by the distance moved by the mobile front) with that of an authentic standard.

Carotenoid thermal degradation products have often been separated by TLC methods.

Kanasawud and Crouzet (1990) used aluminum oxide TLC plates of 1.5 mm thickness with an acetone-petroleum ether (4:95 v/v) elution solvent to separate the various non­ volatile thermal degradation products of p-carotene. In a similar study, alumina TLC plates separated five nonvolatile compounds produced by heat treatment of lycopene.

Separation by TLC was followed by determination of absorption spectra by UVNis spectroscopy (Kanasawud and Crouzet, 1990). The degradation products of p-carotene from heating in sealed glass tubes and extrusion cooking were fractionated on an alumina

TLC plate using 10% diethyl ether in n-hexane as a solvent. Each colored band was recovered in methylene chloride and rechromatographed twice to ensure purity (Marty and Berset, 1986). Onyewu et al. (1986) also utilized TLC plates with 10% diethyl ether in n-hexane to fractionate p-carotene products degraded by heating in glycerol.

Vegetable carotenoid pigment separation has also been achieved by using column chromatography in conjunction with TLC. Tomato carotenoids were separated on an

103 alumina column, then each fraction rechromatographed on TLC plates consisting of silica gel and MgO-kieselguhr (1:1) (Ben-Aziz et al., 1973). Ritacco et al. (1984) reported the use of a MgO TLC plate with an eluting solvent of 20% acetone in petroleum ether for the efficient separation of Micro-Cel C carotenoid artifacts.

2.10.4 Structure elucidation

In order to either identify a known naturally occurring carotenoid or elucidate the structure of a previously unknown carotenoid, the application of a variety of physical and chemical methods is usually required. The assignment ofstereochemistry in terms ofnot only chirality, but also geometrical configuration, usually is the source of most ofthe complexity in carotenoid analyses. As a result, structural elucidation involves various spectrometric methods as well as chemical derivatization (Britton et al., 1995).

Before a carotenoid can be characterized, chromatographic purification is necessary.

Chromatography also provides information regarding the polarity ofthe carotenoid in question so that tentative assignment as a hydrophobic carotene, monool, or more polar carotenoid can be made (Liaaen-Jensen, 1995). After chromatographic analyses are completed, spectrometric methods are usually applied next.

2.10.4.1 UVNis Spectroscopy

The UVNis spectrum of a carotenoid is usually examined first to provide information regarding the chromophore present. Specifically, examination of the spectral fine structure and position ofthe Amax ofthe main absorption band can help determine the specific number of conjugated C=C double bonds present in an aliphatic, monocyclic, or

104 dicyclic carotenoid (Britton, 1995). Values for Amax for some ofthe most commonly encountered carotenoids can be seen in Table 8.

Yamauchi et aI. (1993) determined the main UVNis spectral peaks of several oxidized products of ~-carotene in order to help characterize their structures. For example, three Amax peaks at 405,426, and 452 nm helped to define one of the oxidized products as 13,15'-epoxyvinyleno-13,15'-dihydro-~,~-carotene. In a similar study EI­

Tinay and Chichester (1970) also utilized UVNis spectroscopy to help determine the products formed upon oxidation of ~-carotene. Lycopene epoxides present in fresh tomatoes were partially characterized through the use ofUVNis spectroscopy. Fractions were dissolved in petroleum and Amax values were recorded for comparison with known standards (Ben-Aziz et aI., 1973). Britton and Goodwin (1969) also identified a lycopene oxidation product (phytoene 1,2-oxide) in ripe tomato fruits by comparing Amax values with those ofphytoene.

Jensen et al. (1982) examined the fine structure ofUVNis chromatograms of photoisomerized all-trans-~-carotene in order to identify exactly where and when cis­ trans isomerization occurred. Findings showed a significant hypochromic shift and a reduction in vibrational fine structure in both the 9- and IS-cis isomers of ~-carotene in hexane when compared with the all-trans isomer.

2.10.4.2 IR Spectroscopy

IR spectra are most useful in carotenoid structural elucidation for their ability to reveal the presence or absence of particular functional groups in the molecular structure.

105 Carotenoid Amax(nm) Solvent

Astaxanthin 480 A 485 B,C 478 EtOH 468 P

p-carotene 429 452 478 A 435 462 487 B 435 461 485 C 450 476 EtOH 425 450 477 H, P

Lutein 432 458 487 B 435 458 485 C 422 445 474 EtOH 421 445 474 P

Lycopene 448 474 505 A 455 487 522 B 458 484 518 C 446 472 503 EtOH 444 470 502 P

Phytoene 276 286 297 H, P

Zeaxanthin 430 452 479 A 440 463 491 B 433 462 493 C 424 449 476 P

Table 8: UVNis spectroscopic data for several common carotenoids. Solvents: A, acetone; B, benzene; C, chloroform; EtOH, ethanol; H, hexane; P, light petroleum.

106 Infrared (IR) radiation emitted from an IR spectrophotometer enhances the vibration of atoms in a molecule so that stretching ofthe bonds and variation in specific bond angles occurs. Energy is absorbed (and an absorption band formed) when the frequency of the

IR radiation matches the frequency of a particular vibrational mode. Therefore, compounds with many different atoms and functional groups will give rise to many different absorption bands in the IR spectrum (Figure 30). It is the specific shape, size, and position ofthe absorption band that lends information as to the type offunctional group that it represents. The positions ofthe absorption bands are typically indicated by the frequency in wavenurnbers (the reciprocal value of the wavelength).

Unknown purified compounds can be identified by a direct comparison of its IR spectra with that of a standard (Onyewu et al., 1986). IR spectroscopy is advantageous as a method for structural elucidation due to its simplicity and rapidity at relatively low cost.

The recent advent of Fourier-transform (FTIR) instruments has greatly improved both the

r I I -.IlI.. 1 l .'. • ~ ~I ~ ~ -'­ •• II."'A" II ~ ... , - " I ,.. -­ I I 1\ hr , \ rl'1 I 1\ ~ . II I I "

. ~ 1 1· I I .­ . ,- . - 1 1 1 1: .1 . . ..

Figure 30: Typical IR spectrum for lycopene.

107 sensitivity and accuracy of traditional IR spectroscopy. FTIR spectrometers can measure an IR spectrum with 10 to 100x the sensitivity and in l/OOOth the time required to obtain a similar spectrum with a traditional instrument. The main disadvantage to IR spectroscopy, however, is the limited information given, especially for the hydrocarbon carotenes which lack any type offunctional groups (Bernhard and Grosjean, 1995).

The methylene and methyl groups present in carotenoids and their derivatives typically give rise to bands at 2950-2850 em", while the alkene groups present in their polyene structure gives rise to weak bands between 1650 and 1550 em" and a strong band between 990 and 960 em-I (Yamauchi et aI., 1993). Onyewu et aI. (1986) determined that an unknown carotenoid compound with absorption bands only at the methyl C-H (2950-2850 em") and polyene C=C (1650 em", 990 em") wavenumbers must be a hydrocarbon carotene with no oxygenated groups present.

The presence ofa Z (cis) double bond in a carotenoid compound results in the appearance of strong absorption in the region around 750-800 em". Mangoon and

Zechmeister (1957) utilized this information in showing the stepwise process in which prolycopene was converted into all-trans-Iycopene. As the cis double bonds in prolycopene were converted to trans, the absorption band at approximately 750-800 cm-I became smaller until it disappeared in the IR spectrum of all-trans-Iycopene.

Alcohol groups often show a very distinct broad absorption band in the 3300-3400

em-I range due to the stretching of the O-H·· ..O bonds, while aldehyde and ketone

groups have a C=O stretching band near 1715 em-I. Lu et al. (1995) declared that due to

the absence ofabsorption bands in the OH and C=O regions, an oxygenated carotenoid

compound they were examining must have an ether linkage. In this study, the

108 information obtained from the IR spectra helped to identify the structure of a novel carotenoid with an epoxyiridane skeleton. Ouyang et al. (1980) reported that many ofthe structures of l3-carotene degradation products from palm oil deodorization contained aldehyde end groups, due to the presence of strong absorption bands in the 1700 cm-I region. Ukai et al. (1994) reported similar absorption bands in the region of 1678 em" for an oxidized lycopene product containing an aldehyde group (apo-6'-lyocopenal),

2.10.4.3 Mass spectrometry

The largest contribution mass spectrometry (MS) provides for structural elucidation of carotenoids is identification of the molecular mass of the molecule. In addition, characteristic fragmentation seen in a mass spectrum lends additional information as to the functional groups present in the compound, i.e., 18 mass units (water) from alcohols,

32 mass units (methanol) from methyl ethers, 80 mass units for 5,6- and 5,8-epoxides, etc

(Figure 31). To obtain a mass spectrum only a few ug of pure sample are required, though high precision instruments may require slightly more (Enzell and Back, 1995).

MS employs several different ionization techniques for compound identification. The first, and still most popular, technique is electron impact (EI) mass spectrometry. In this method, electrons are accelerated and directed at the sample, with the resulting impact causing the generation of the positively charged molecular ion M+. The particular intensity of the ions produced by EI mass spectrometry is highly dependent upon specific conditions such as temperature and ionizing voltage. Marty and Berset (1988) used EI mass spectroscopy to help identify the degradation products of trans-l3-carotene produced during extrusion cooking. The EI mass spectrum presented fragments specific for

109 M-138 M-191 o .-----_. .-----_. M-56 : -H : -H I I I I I 92 I I I I I I I I I

I ~ ~ I I I I I I I I I I I I I I I :L. M-203• I------_.I o -H -H

Figure 31: Main fragmentations ofthe molecular ion ofcanthaxanthin by FAB MS.

carotenoids with 1 or 2 cyclohexenyl rings, as well as two fragments characteristic of 5,6­ epoxide or 5,8-epoxide functions.

A comparison by Ouyang et al. (1980) of sample spectra with the EI mass spectra of pure 9-cis-retinal showed identical relative abundance and fragmentation patterns, allowing positive identification ofthe sample p-carotene degradation product as 9-cis­ retinal. Khachik et al. (1992a) elucidated the structures of eighteen different carotenoids and their oxidation products in human serum samples with the help ofEI mass spectroscopy. EI MS has been used by several authors to help determine the molecular structures ofproducts formed during the oxidation/degradation of p-carotene

(Kanasawud and Crouzet, 1990; Handelman et al., 1991) and lycopene (Kanasawud and

Crouzet, 1990).

Chemical ionization (CI) generates mass spectra through ionic reactions. In this method a reagent gas such as ammonia or methane is introduced under pressure to the ion source, and then the gas is bombarded with electrons to yield ions and neutral molecules.

110 The reagent gas molecules then react with the vaporized sample molecules, resulting in a less fragmented and simpler spectra compared with EI mass spectrometry. Marty and

Berset (1988) utilized CIMS with NH3 as the reagent gas to help identify fourteen different ~-carotene thermal degradation products. In a similar study CI mass spectrometry was used to confirm the parent ion (and therefore the molecular weight) of several ~-carotene thermal degradation products (Onyewu et al., 1986).

Fast atom bombardment (FAB) mass spectrometry is a 'soft' ionization technique normally used for structure elucidation ofeither nonvolatile or thermally labile compounds. In this technique high-energy atoms such as xenon or cesium bombard a solution ofthe sample dissolved in an involatile matrix such as glycerol. Volatilization and ionization ofthe sample compound occurs by way ofdissipating kinetic energy from the bombarding atom beam, resulting in the formation ofeven-electron protonated molecular ion(s). Delocalization ofthe charge in the carotenoid extended polyene system prevents the usual protonization ofthe carotenoid sample from occurring, however, and results in the formation ofodd-electron molecular ions. Subjecting carotenoids to FAB mass spectrometry typically yields only the radical ion M+ in the molecular-ion region of the spectrum. An advantage of FAB MS over EI or CI techniques is the lack ofsample vaporization prior to volatilization, which reduces the likelihood ofcarotenoid degradation during MS analysis itself. Schmitz et al. (1992) described interfacing FAB mass spectroscopy with HPLC to provide a powerful tool in the analysis ofcarotenoids.

A mixture of3-nitrobenzyl alcohol (NAB) and glycerol (80:20 v/v) was described as an effective FAB matrix for analysis of the more nonpolar carotenoids due to NAB's ability to interact with and disperse the carotenoid throughout the matrix. Using FAB-LC/MS,

III van Breemen et al. (1993) determined the limits of detection to be as low as 5 ng and 15 ng for lutein and a-carotene, respectively. Lu et al. (1995) used FAB-MS to identify a new hydrogen peroxide oxidation product from lycopene. Van Breemen (1996) reported that though FAB mass-spectroscopy linked with HPLC can produce highly sensitive and unambiguous molecular weight confirmations for both oxygenated xanthophylls and hydrocarbon carotenes, it is limited by NAB fouling of the FAB probe that results in lower sample throughput and decreased sensitivity.

Matrix-assisted laser desorption ionization (MALDl) mass spectrometry is one more type ofMS which has been recently applied to the analysis ofcarotenoids. MALDl was originally introduced as an extremely sensitive method for mass analysis of very large molecules such as proteins up to 300 kD in size. While other MS methods have difficulty analyzing carotenoids due to their lack of volatility and thermal lability (El and CI) or lack of solubility and surface activity in common liquid matrices (FAB), MALDl mass spectroscopy methods have the potential to overcome most of these drawbacks

(Kaufmann et al., 1996).

2.10.4.4 NMR Spectroscopy

Nuclear magnetic resonance (NMR) spectroscopy is commonly referred to as the most powerful technique for overall structural elucidation. The reason for this is simple: proton eH) NMR is capable of identifying the structural surroundings of every hydrogen atom in a carotenoid, while carbon-13 (l3C) NMR identifies the degree of saturation for each carbon atom (Sp3, Sp2, sp) and its structural surroundings. Detailed lH and l3C NMR analyses alone can identify a carotenoid structure as well as its stereochemistry and

112 geometry of carbon-carbon double bonds. In addition, a typical IH-NMR spectrum requires only 100-200 Ilg of sample. As the magnetic field strengths ofNMR instruments have increased to as high as 750 MHz, the dispersion of chemical shifts in samples has increased to the point that interpretation of I H-NMR spectra has been greatly simplified. In turn, the amount of structural information that can be deduced from the spectra has greatly increased as well (Figure 32).

= 250 MtU

;,4 :0':: 1D" -i.I ~,

,..

:t' :$ 'I Ili: ~ r ~t.q.~ I~"""'- ~ )~Jla·, Jj __,.I' • •.JWJI\1¥J I :o.e ~! ill fU iz :lFfI1

Figure 32: Olefinic section ofthe IH-NMR spectrum of a typical carotenoid measured at 250 MHz (top), 400 MHz (middle), and 600 MHz (bottom).

113 Mordi et aI. (1991) identified several oxidized products of j3-carotene (5,6-epoxy-j3­ carotene, retinal, j3-ionone, j3-apo-14'-carotenal) using IH-NMR spectroscopy. In a similar study, 'H-NMR was performed at 270 MHz, the 'H-'H chemical shift-correlated

(COSY) NMR technique was employed to assign 'H shifts and couplings, and 13C-NMR was run at 70 MHz with proton decoupling in order to identify the products formed by the peroxyl radical oxidation of j3-carotene (Yamauchi et aI., 1993).

A 400 MHz 'H-NMR and a 270 MHz 13C-NMRspectrophotometer were utilized in an analysis ofthe photosensitized oxidation products oflycopene from tomato puree

(Ukai et aI., 1994). Lu et ai. (1995) used '3C-NMR, 'H-NMR, and COSY two­ dimensional NMR experiments to help identify lycopene-oxidized products. With the application of 'H-NMR spectroscopy at 270 MHz and the help of 13C-NMR spectroscopy, Englert et al. (1979) was first able to report the complete assignment of stereochemistry to the carotene prolycopene as 7,9,7',9' -tetra-czs-w,\V-carotene.

114 CHAPTER 3

MATERIALS AND METHODS

3.1 Materials

~-carotene, lycopene, and chlorophyll standards were obtained from Sigma Chemical

Company (St. Louis, MO). Lycopene (>98% pure) was generously donated by LycoRed

Natural Products Industries (Beer-Sheva, Israel). HPLC grade methanol, methyl-tert­ butyl ether, acetonitrile, and chloroform were purchased from Fisher Scientific

(Pittsburgh, PA). Helium for both gas chromatography and HPLC sparging, compressed air for gas chromatography and HPLC autosampler function, hydrogen for gas chromatography, and nitrogen for gas chromatography, sample drying, and storage were obtained from the Chemical Store at The Ohio State University. Analytical reagent grade methanol, acetone, and hexane (Fisher Scientific, Pittsburgh, PA) were used for lycopene extraction. All solid phase microextraction (SPME) fibers [polydimethylsiloxane

(PDMS), 100 urn; polydimethylsiloxane/divinylbenzene (PDMS/DVB), 65 urn; polydimethylsiloxane/carboxen (PDMS/carboxen), 75 urn; carbowax/divinylbenzene

(CW/DVB), 65 urn; polyacrylate, 85 urn] and fiber assembly holders used in soybean oil volatile analyses were purchased from Supe1co, Inc. (Bellefonte, PA). Volatile standards

115 t,t-2,4-decadienal, 2-heptenal, t,t-2,4-heptadienal, n-hexanal, and n-pentane were procured from Aldrich (Milwaukee, WI). Refined, bleached, and deodorized (RBD) soybean oil was obtained from Abitec Corp. (Columbus, OH).

3.2 Lycopene extraction

To obtain 85-90% pure crude lycopene extract for experimental procedures, lycopene was extracted from supermarket-bought (Kroger brand) canned tomato paste. Under dark conditions, 109 of tomato paste were mixed with 25 mL of methanol and 4 g of Celite, with the mixture being tissumized for approximately 2-3 minutes. The homogenized sample was then filtered through #1 and #45 Whatman filter papers and the filtrant placed in a beaker with 50 mL ofacetonelhexane (1:1). Again the mixture was tissumized for approximately 2-3 minutes and then filtered through the same #1 and #45 Whatman filter papers. The filtrate was poured into a separatory funnel, with the filtrant being discarded.

The filtrate was washed with 10 mL ofdistilled water 3 times in the funnel, each time with removal of the bottom layer (waste) and retention of the top hexane layer. The top hexane layer was then transferred to 4 mL amber vials and dried under nitrogen gas until the lycopene extract was in a solid crystalline state. The vials were sealed, wrapped in aluminum foil to minimize light exposure, and stored at -4°C until needed.

3.3 Thermal treatment ofcarotenes

3.3.1 J3-carotene and lycopene

A solution of 500 ppm J3-carotene or lycopene solubilized in acetone was added to a glass vial that was sealed airtight with a Teflon-faced rubber septum and aluminum cap

116 (Supelco, Inc., Bellefonte, PA). A portion of the unheated solution was saved. The remaining portion in the glass vial was wrapped in aluminum foil to minimize light exposure, then placed in a water bath set at 90°C to allow a specific amount of thermal degradation of the carotene to occur. After the ~-carotene or lycopene solution received the proper length of thermal treatment (Figures 33 and 34, respectively), the sealed glass vial was removed from the water bath and allowed to cool to room temperature. Out of the cooled glass vial one of three heated solutions containing either 250 ppm ~-carotene +

250 ppm degraded products, 50 ppm ~-carotene + 450 ppm degraded products, or 0 ppm

~-carotene + 500 ppm degraded products was collected (Figure 35), contents verified by

RP-HPLC, and stored in a freezer at -4°C for further use, respectively.

3.3.2 Crude lycopene extract

A 40 mL solution of 500 ppm lycopene solubilized in methyl-tert-butyl-ether

(MTBE) was added to a 100 mL glass vial that was sealed airtight with a Teflon-faced rubber septum and aluminum cap. A 13 mL portion of the unheated solution was saved.

The remaining portion in the glass vial was placed in a water bath at 90°C to allow thermal degradation of the carotene to occur. After receiving the proper length of thermal treatment, the sealed glass vial was removed from the water bath and allowed to cool to room temperature. Out of the cooled glass vial, a solution containing either 250 ppm lycopene + 250 ppm degraded products, 50 ppm lycopene + 450 ppm degraded products, or 0 ppm lycopene + 500 ppm degraded products was collected, contents verified with HPLC, then placed in a sealed glass vial and stored at _4°C.

117 2.00E+08

y = -504997x + 2E+08 1.50E+08 2 R = 0.9469 -s~ ~ .~ 1.00E+08 til ..::.:: tU ~ ~ 5.00E+07

O.OOE+OO 0 50 100 150 200 250 300 350 Time (minutes at 90 deg C)

Figure 33: Increased degradation of p-carotene (decreased p-carotene peak size) with increased thermal processing at 90°C.

2.0E+08

1.6E+08 y = -208486x + 2E+08 2 -~ R = 0.9983 ~ 1.2E+08 ~ .~ til ..::.:: 8.0E+07 tU ~ ~ 4.0E+07

O.OE+OO 0 200 400 600 800 1000 Time (minutes at 90 deg C)

Figure 34: Increased degradation oflycopene (decreased lycopene peak size) with increased thermal processing at 90°C.

118