Bohn: Journal of AOAC International Vol. 102, No. x, 2019 1

SPECIAL GUEST EDITOR SECTION Determinants and Determination of Carotenoid Bioavailability from Infant Food Formulas and Adult Nutritionals Including Liquid Dairy Products

Torsten Bohn Luxembourg Institute of Health, Population Health Department, 1A-B, rue Thomas Edison, Strassen L-1445, Luxembourg

Carotenoids are typically tetraterpenoid and salmon (5), via natural accumulation or biofortification of phytochemicals that cannot be synthesized by feed rich in carotenoids. Another common source of carotenoids humans, some of which such as β-carotene can be is from fortified food items such as butter, as carotenoids can be metabolized into vitamin A. Sufficient carotenoid added as coloring agents (E160-E160f; 6), or directly through intake and tissue levels have been associated with supplements in which amounts of 5–50 mg per dosing (often a several health benefits including the reduction of capsule or caplet) are typically available on the market. cardiovascular diseases and some types of cancer The most common carotenoids in the diet vary according to and also the amelioration of age-related macular dietary habits but typically include β-carotene, lutein, lycopene, degeneration. Carotenoids and their metabolites β-cryptoxanthin, zeta-carotene, zeaxanthin, α-carotene, have also been related to reduced inflammation and phytoene, phytofluene, violaxanthin, and neoxanthin7 ( ). oxidative stress via interacting with transcription However, in many Asian diets, astaxanthin and fucoxanthin factors, such as NF-κB and Nrf-2, as well as with from algae are also predominant (8), whereas in societies that eat the nuclear receptors retinoic acid receptor/retinoid less tomatoes and processed tomato products such as ketchup, X receptor, implicated in immune functions and lycopene concentrations can be quite low (9). However, their cellular differentiation. Therefore, carotenoids dietary intake is generally reflected by blood plasma/serum are important for growth and development. They concentrations, except for the epoxycarotenoids violaxanthin could mark beneficial constituents in infant food and neoxanthin, which appear to be further metabolized prior formulas and adult nutritionals, the latter typically to their absorption, as their blood and tissue concentrations are constituting protein-rich liquid foods targeting typically low (10, 11). The xanthophylls can also be present meal replacements. Carotenoids may be present by as monoesters or, in some cases, diesters, which presumably nature (typically below 20 μg/100 mL) or following requires cleavage prior to their further absorption (12, 13). fortification (up to 200 μg/100 mL), such as for lutein Finally, in addition to the primary all-trans conformation and β-carotene. However, carotenoid bioavailability in plants, certain cis-isomers can be formed during food may be low and variable, especially in low-fat items. processing and following their absorption in vivo (14). Although most infant foods and adult nutritionals are Carotenoids in general, and also specific carotenoids, have rich in lipids and proteins, facilitating absorption and been related to several health benefits. One important aspect availability of carotenoids, unfortunately, very little of carotenoids is that they contribute to vitamin A intake; data is available. In addition, carotenoid detection several carotenoids (β-carotene, β-cryptoxanthin, α-carotene) for such lipid-rich matrices may be challenging as can be converted into vitamin A upon cleavage by β-carotene a result of low concentrations and matrix effects. oxygenase 1 (BCO1) in the gut and in other tissues (15, 16). This review aims to highlight considerations for Vitamin A is important for the optimal functioning of the carotenoid bioavailability from infant food formula immune system (17, 18), and it is involved in cell proliferation and adult nutritionals as well as summarize detection (19), among other functions. Many of these functions are methods for carotenoids from these items. conveyed by activating the nuclear receptors, retinoic acid receptor/retinoid X receptor (20, 21). For strict vegetarians/ vegans, carotenoids are their only natural dietary source of vitamin A. arotenoids are typically C40 tetraterpenoids of plant, In addition to providing vitamin A, dietary carotenoid intake bacterial, or fungal origin, but they could also comprise and blood plasma/serum concentrations have been related to C30 (1) or C50 (2) terpenoids. Although they cannot C several chronic diseases. For example, higher plasma β-carotene be produced by animals, including humans, they can also be concentrations in the elderly have been significantly associated found in animal sources, such as eggs (3), dairy products (4), with decreased all-cause mortality (22). Because of such findings, a carotenoid health index above 1 μM for total plasma carotenoid concentration has been proposed as a carotenoid target indicator (23). In addition to a potential direct antioxidant Guest edited as a special report on “Carotenoids: Absorption, Biological Activity, and Analysis” by Gregory L. Hostetler function, e.g., stabilizing cell membranes against lipid (per-) Corresponding author’s e-mail: [email protected] oxidation (24), carotenoids and metabolites have also been DOI: https://doi.org/10.5740/jaoacint.19-0015 shown to alter pathways of inflammation and oxidative stress 2 Bohn: Journal of AOAC International Vol. 102, No. x, 2019 via cellular transcription factors, such as NF-kB and Nrf2, Carotenoid Content in Infant Food Formulas and respectively (25). Adult Nutritionals Including Liquid Dairy Products However, bioavailability, i.e., the fraction of a compound that can be absorbed and used for its physiological function Infant Food Formulas and/or stored, for carotenoids is low (typically 5–40%; 26, 27) — especially for the more apolar, oxygen-free Infant food formula is generally composed of cow milk carotenes — and also variable, influenced by many dietary proteins such as whey and casein, to which vegetable oils such and host factors (7). For instance, it is well recognized that as rapeseed, corn, sunflower, or coconut oil may be added in dietary lipids can ameliorate the bioavailability of carotenoids addition to lactose and a mixture of minerals and vitamins. (28), whereas dietary fiber 29( ) and perhaps divalent minerals Also, skimmed milk, fructo-oligosaccharides, or fish oils may (30, 31) may reduce it. Regarding host factors, enzymes be added (44, 45). Carotenoids occur especially within milk and of digestion, cleavage enzymes (BCO1), and transporting vegetable oils. lipoproteins are known to influence bioavailability; however, The measurement of carotenoids in infant food formulas they have been more extensively reviewed elsewhere (7). has been met with increasing interest, for several reasons. Infant food formula includes formula for babies up First, because of the potential of several carotenoids (e.g., to the age of 12 months. It may be used to replace human α- and β-carotene and α- and β-cryptoxanthin) to yield vitamin milk, which is also a source of carotenoids for infants, A following cleavage by BCO1, and second, because of the containing approximately 80 nM (4.2 μg/100 g) β-carotene proposed health benefits of carotenoids. For example, lutein and 130 nM (7.4 μg/100 g) lutein, as reviewed recently (32). supplementation given to newborns on two occasions (2× Special European regulations exist for infant food formulas 0.28 mg) reduced circulating hydroperoxides and improved (33), and similar regulations are set by other governmental ferric reducing antioxidant power assay, which is a test for organizations such as the U.S. Food and Drug Administration antioxidant activity in blood plasma (46, 47). Similarly, (34, 35). In general, only very few additives for such food the supplementation of a mixture of lutein/β-carotene/ items are allowed. These include preformed vitamin A (up to lycopene in infant food formula at 210 μg/L reduced c-reactive 70–114 μg retinol equivalents/100 kcal; 36) and carotenoids, protein, a marker of systemic inflammation 39 ( ), following but only in the United States with maximum limits established approximately 40 weeks of intervention. Similarly, Capeding within generally recognized as safe (GRAS) notifications for et al. (48) fortified infant food formula with lutein at various food categories (35, 37), but not in the European 200 μg/L. After 16 weeks, no growth differences or bio­ Union (EU), where the use of food colors in infant foods is chemical blood parameters measured, such as glucose or prohibited (34). In light of their partial contribution to vitamin circulating proteins, differed between the two groups; however, A, and the contribution to antioxidant status as recently bioavailability and inflammatory markers were not reported. shown in infants (38, 39), carotenoids are considered valuable Because carotenoids are already naturally present in milk, micro-constituents for growing infants. For this reason, although at a low extent (Table 1), and, at least in some countries, β-carotene and lutein especially (and occasionally lycopene) can be added (e.g., in the United States but not in the EU), the have been supplemented to infant food formula, as the native carotenoid content and profile can vary considerably Table( 1), concentration of carotenoids (mostly β-carotene) is fairly low, depending on the individual ingredients and further fortification. typically below 20 μg/100 mL. In general, total carotenoid concentrations from 0 to about Likewise, carotenoids are frequently added to and are 40 μg/L have been reported in commercial products and up to present in adult nutritionals, typically beverages rich in proteins 200 μg/L in products used in infant trials, which is still low and intended to replace a meal, e.g., Boost, Ensure, Prosure, compared with the presence in some baby foods in which Slimfast, or similar products, often based on dairy and soy concentrations up to 48 mg/100 g have been reported (Table 1). products (40, 41). However, in Europe, carotenes and lutein So far, however, traditionally only β-carotene and, more recently, cannot be added directly to such beverages; the use of lycopene lutein, have been added to commercial products, such as in the for flavored drinks is only permitted up to 10 mg/L 42( ). The Similac brand of Abbott (Table 1). allowed amount in the United States is not always specified, such As carotenoids can be present in either cis or trans as for β-carotene, but should follow good manufacturing practice forms, a few researchers have examined the geometrical (35); whereas for other carotenoids, a maximum limit has been profiles of β-carotene, lutein, and lycopene49 ( , 50). These specified within GRAS notifications, i.e., suspended lutein in have included, in addition to the respective all-trans forms, infant food formula (250 μg/L) and 1 mg meso-zeaxanthin per 13-cis-lutein, 9-cis-lutein, 9′-cis-lutein, 13′-cis-lutein, 9-cis- meal for infant and toddler foods (37). β-carotene, 13-cis-β-carotene, 15-cis-β-carotene, 5-cis-lycopene, As carotenoids are lipo-soluble (with logP values between and additional nonidentified lycopene isomers, possibly 8 and 12; 43), it may be analytically challenging to extract including 13-cis-lycopene and 15-cis-lycopene. Although in and separate these compounds at low concentrations from a plants, the predominant form of carotenoids is the all-trans lipid-rich matrix, because of the large amount of coextracted form, a certain fraction of carotenoids may isomerize during lipids and the difficulty for further concentration and processing, i.e., heat application (51, 52) and upon ingestion purification. Normally, such matrices require a saponification in the enterocytes (53, 54). However, also in the final products step to cleave triglycerides and to also cleave potentially (infant food formulas and adult nutritionals), the predominant present xanthophyll (mostly lutein) esters. Because of these forms are the all-trans form (>2/3; 49, 50). The presence of challenges, and the importance of infant food formulas and various geometric forms is of interest because they also may adult nutritionals, this review aims to highlight aspects of differ in their bioavailability (see Bioavailability Aspects bioavailability from these products and analytical methods that of Carotenoids Regarding Infant Food Formulas, Adult have been developed to detect carotenoids in these matrices. Nutritionals, and Liquid Dairy Foods section). Bohn: Journal of AOAC International Vol. 102, No. x, 2019 3 ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) 64 70 70 65 62 67 49 70 70 70 70 70 49 70 55 144 144 144 145 144 146 146 147 147 147 148 148 149 ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( Ref. ( ( ( ( ( ( ( ( ( ( ( ( ( database b Ellis et al. Ellis et al. Jeon et al. Souci et al. Souci et al. Souci et al. Costa et al. Lipkie et al. Data source Hulshof et al. Hulshof et al. Mackey et al. Hanson et al. Hanson et al. Hanson et al. Hanson et al. Calderon et al. USDA database USDA database USDA database USDA database USDA database USDA database USDA database USDA USDA Sommerburg et al. Estimated from label Schweiggert and Carle 0 0 0 0 0 0 0 0.2 0.1 0.1 0.1 2.2 ND ND ND ND ND ND ND ND ND ND ND ND ND 0–13 0–135 0.1 (0.1) (μg/100 g) μg/100 mL μg/100 mL 0.3–0.4 (0.3) β-cryptoxanthin, ) ) ) 17 18 8 c 0 1 3 0 37 2.0 1.3 1.2 7.1 6.4 2.5 ND 0–7 144 0–20 5–100 28–46 0–11.9 1–13.3 6–9 ( 4.9 ± 7.6 0.2–21.0 18.7 ± 4.7 17.5 ± 7.4 (μg/100 g) μg/100 mL μg/100 mL 13–21 ( 10–20 ( β-carotene, 6.8–9.4 (7.9) 0–7156 (501) 14.7–19.1 (16.7) 0 0 0 0 0 0 0 0 0 0 0.2 0.6 8.0 ND ND ND ND ND ND ND ND ND ND ND ND 0-0.83 0–7.57 6.6 ± 5.6 (μg/100g) Lycopene, Lycopene, μg/100 mL μg/100 mL 0–2575 (92) (1.0) (0.8) c d d d 0 0 0 0 0 0 0 2.2 6.6 5.7 5.8 3.3 ND ND ND ND ND ND ND ND 7.9 1.5 4.0 ± 4.3 0.22-16.7 0.09–12.0 (μg/100 g) μg/100 mL μg/100 mL 2.3–2.4 (2.4) 0.8–1.4 0.5–0.8 0–45050 (452) Lutein/zeaxanthin,

) ) ) 17 18 8 a 0 37 2.8 3.5 ND ND ND ND ND ND ND ND ND ND 0–7 ND 10.1 14.0 12.9 16.4 18.5 20.9 10.4 6–9 ( 0.33–23.3 (μg/100 g) μg/100 mL μg/100 mL 0.22-29.43 13–21 ( 10–20 ( 0–47.500 (1113) Total carotenoids, Total f f Milk, raw Food item Breast milk Milk, organic Various items Various Milk, raw, organic Milk, raw, Milk, conventional Nestle, Boost Plus Full-fat milk, pasteurized Full-fat milk, pasteurized Several adult nutritionals = 8): (Beba 0, Humana 0, Beba Pre, Aptamil = 8): (Beba 0, Humana Beba Pre, Skimmed milk (nonfat milk) n Several infant food formulas Mead Johnson, ready-to-feed Full-fat milk, ultra-heat treated Aptamil 1, Nestle, ready-to-feed Semi-skimmed milk, pasteurized Semi-skimmed milk, pasteurized Kellogg’s Special K protein shake Kellogg’s Unilever, SlimFast, Shake Mix, powder Unilever, Ensure High Protein therapeutic nutrition Similar advance, Abbott Nutrition, ready to eat Similar advance, Abbott Ensure, Nutritional Shake, ready to use Preterm formula, Abbott Nutrition, ready-to-feed Preterm formula, PBM PRODUCTS, store brand, soy, ready-to-feed PBM PRODUCTS, store brand, soy, Transitional formula, Abbott Nutrition, ready-to-feed formula, Transitional Term standard formula, Abbott Nutrition, ready-to-feed standard formula, Term Pre, Pre Aletemil, PreAponti, Humana HA, Aletemil HA) Aletemil, PreAponti, Humana HA, Pre, Pre Mead Johnson, ENFAMIL, premium, infant, ready to use Mead Johnson, ENFAMIL, Similar Advance with OptiGRO, Abbott Nutrition, ready-to-feed Advance with OptiGRO, Similar = 7–9. Various products ( Various n e Carotenoids in infant food formula. Data are individual values or ranges (mean) when available e ND = Not determined/no data. Agriculture. = U.S. Department of USDA Only lutein. Group added for comparison. Fortified with respective carotenoid. Including powders,

Table 1. Table Food category Infant food formula Baby food, general Breast milk Adult nutritionals Milk products a b c d e f 4 Bohn: Journal of AOAC International Vol. 102, No. x, 2019

Adult Nutritionals Including Liquid Dairy Products formula (Similac Advance, Abbott; Table 1) and human milk. Although the amount of all carotenoids increased with the higher Carotenoid concentration of full-fat milk varies according doses of infant food formulas, final plasma concentrations were to the diet of cows and has been reported to be typically not significantly higher compared with human breast milk–fed in the range of 6–21 μg/100 mL, with mostly β-carotene infants, which is typically of lower carotenoid concentration being reported (Table 1). Schweiggert and Carle stated that (Table 1). over 90% of carotenoids in milk are β-carotene (55). Lutein These results are in line with another human trial (63), in which (0.5–2.4 μg/100 mL; Table 1), zeaxanthin (0.1–0.5 μg/100 mL; lutein bioavailability (expressed as serum concentration) of human Table 1), and β-cryptoxanthin (0.3–0.4 μg/100 mL) have been milk and infant food formula were compared. More specifically, occasionally detected. Because most adult nutritionals are in a double blinded study, human milk was 4 times more efficient produced on a milk base and are further diluted as additional in delivering lutein than the infant food formulas, i.e., the authors ingredients (which normally contain less carotenoids, except concluded that 4 times greater concentrations of lutein in formula perhaps certain vegetable oils) are added, the content of would be required to achieve plasma concentrations comparable nonfortified carotenoids is expected to decrease. The same is with human milk. Also, in line with this finding is a study true for products based on semi-skimmed or skimmed milk, investigating carotenoid status by plasma levels and skin status as the lower concentration of fat correlates with a decreased using noninvasive Raman spectroscopy (58), and both measures concentration of carotenoids (Table 1). were well correlated (R = 0.44, P = 0.01). In this study, preterm Only little data are available on the carotenoid content infants were fed either infant food formula or human milk with in adult nutritionals. Basal levels as low as 1–3 μg/100 μL unspecified amounts of carotenoids for 2 weeks. Although both β-carotene have been found. For certain β-carotene–fortified serum and skin carotenoids tended to decline with the formula products, concentrations of up to 144 μg/100 mL have been intervention, concentrations tended to increase upon human reported (Table 1). milk intake, although it is likely that the higher concentrations of In conclusion, unfortified concentrations of carotenoids are carotenoids to be expected in breast milk versus the nonfortified quite low in infant food formulas and in adult nutritionals; only infant food formula (Table 1) contributed to the findings. β-carotene appears to be present in measurable, although in low Additional carotenoids were monitored by Sommerburg et al. (64), concentrations, whereas many other carotenoids are virtually who followed newborns receiving either human milk or infant absent. Concentrations following fortification has resulted in food formula for up to 14 days, finding that, compared with birth, levels of 100 μg/mL or even higher. formula feeding resulted in decreased concentrations of β-carotene, β-cryptoxanthin, lycopene, and α-carotene, with the latter two becoming no longer detectable in blood plasma, whereas breast Bioavailability Aspects of Carotenoids Regarding feeding enhanced slightly, although not significantly, carotenoid Infant Food Formulas, Adult Nutritionals, and Liquid concentrations compared with levels at the time of birth. Dairy Foods The reasons for the relatively low bioavailability of carotenoids from infant food formula are unclear and can Infant Food Formulas be either because of poorer release and micellization from the The bioavailability of carotenoids can be assessed by several matrix (a prerequisite for later absorption), cellular uptake methods, including measuring the area under the time curve of and transport, or further absorption and biodistribution. newly absorbed carotenoids in the plasma–triacylglycerol-rich Interestingly, bioaccessibility, i.e., the fraction of a compound lipoprotein fraction, which is possibly the most accepted and that is liberated during digestion and available for absorption, conducted method (56, 57), especially for postprandial trials, has been reported to not differ significantly between infant food although for ethical reasons, it cannot be applied to infants formula and mother’s milk. In a study by Lipkie et al. (65), the because of the several blood draws required. In addition, baseline bioaccessibility of lutein was 29 ± 2% from human milk versus subtracted plasma measurements can be carried out, which may 36 ± 4% from fortified infant food formula, both containing be the method of choice following long-term feeding trials and comparable amounts of lutein. In contrast, fractional cellular for infants. It is also frequently used for postprandial trials, uptake was 4.5 times greater by Caco-2 cells from human although in this latter case, it also requires several blood draws milk than from the infant food formula, suggesting that either over several hours (31), 24 h in an optimal case. Other methods absorptive processes do differ between the two sources or the include measuring skin color after several weeks of feeding by composition and/or size of the mixed micelles differed between Raman spectrometry (58). When determining the effect of lutein/ the two sources, influencing absorption capability. zeaxanthin supplementation, which is selectively accumulated In an earlier in vitro study, it was already shown that adding in the macula of the human eye, macula-pigment optical synthetic carotenoids in an isolated form to milk resulted in good density measurement may also constitute a valid bioavailability solubilization but apparently poorer uptake into small micelles, indicator (59). Isotopically labeled carotenoids may also be as filtration largely reduced β-carotene from the present larger followed in the blood over time, but these methods are generally droplets (66). The presence of β-carotene in milk, i.e., in milk more expensive (60) and used rather in compartment modeling fat globules, has been well described by Schweiggert and (61) to study further transport and metabolism. Carle (55), in which carotenoids are well dissolved in unipolar When comparing bioavailability across food sources, it has lipids followed by a surrounding monolayer of polar lipids, been estimated that the bioavailability of carotenoids from cytoplasm, and then another bilayer of polar lipids (Figure 1). infant food formulas is lower compared with that of mother’s This is presumably much different than merely added synthetic milk (62). In the latter study, two infant food formulas fortified carotenoids to milk. with β-carotene (54, 93 μg/L), lutein (33, 53 μg/L) and lycopene Higher carotenoid intake and serum concentrations are likely (43, 81 μg/L) were given for 56 days versus a control infant food related to higher target tissue concentrations. For example, Bohn: Journal of AOAC International Vol. 102, No. x, 2019 5

Carotenoid Membrane associated vitamin Cholestero l Unpolarvitamin (A, E) Inner monolayer of polar lipid s Membrane associated carotenoid Protein

Cytoplasm Cholestero l

Outerlipid bilayer

OH

HO

0.2–15 µm

Figure 1. The presence of carotenoids in milk fat globules. Based on reports by Schweiggert et al. (55). in a study on rhesus macaques by Jeon et al. (67), carotenoids higher bioaccessibility from the chicken/vegetable dishes administered to offspring in breast milk was compared with (31–78% versus 3–68% for β-carotene, β-cryptoxanthin, infant food formula either fortified (with lutein, zeaxanthin, lutein, and lycopene) and a generally higher bioaccessibility β-carotene, and lycopene at 13.5, 1.1, 4.0, and 18.1 μg/100 mL, for xanthophylls versus carotenes, likely because of the higher respectively), or unfortified infant formula (2.2, 0.1, 1.2, and lipophilicity of the latter and poorer micellization. 0 μg/100 mL, respectively). Also in this study, serum concent­ Also, the geometric form of carotenoids can influence their rations were higher following breast feeding, and β-carotene, bioaccessibility and bioavailability. cis-Isomers are of shorter lutein, and zeaxanthin accumulation in the brain was higher apparent structure as they appear more bended and tend less compared with the formula-fed animals, indicating that not only toward crystallization (14). Consequently, higher micellization blood, but also tissue distribution, is likely influenced by the of cis-isomers as compared to their all-trans form has been different mode of feeding, although human data on this is scant. reported in several studies (72, 73). However, this does not Another factor that may potentially limit carotenoid appear to result in improved bioavailability, at least not for all bioavailability is the addition of mineral mixtures containing carotenoids. Whereas a higher bioavailability for cis-isomers divalent minerals, e.g., calcium and magnesium. Several in of lycopene has been clearly demonstrated in several studies vitro studies (30, 68) and also in vivo studies on carotenoids (74, 75), i.e., up to 8.5-fold, the bioavailability of β-carotene from plant matrices, although the latter with mixed results appears superior in the all-trans form (76). However, as (31, 57), have suggested that higher concentrations of divalent β-carotene appears to be reisomerized to the all-trans form minerals, with intake amounts of approximately 250 mg of in the enterocyte, the absorption of the cis-forms may be calcium or magnesium for adults, could compromise carotenoid underestimated, as reviewed previously (14). bioaccessibility. This is because divalent minerals can bind to free fatty acids and bile salts during digestion, reducing the Adult Nutritionals Including Liquid Dairy Products micellization efficiency of carotenoids. However, whether the concentration of divalent minerals in infant food formulas (up To the author’s knowledge, no published reports exist on to 7.5 g/kg as the sum of calcium and magnesium; 69, 70) does aspects of the bioaccessibility or bioavailability of carotenoids have any negative effects remains to be elucidated. Given that from adult nutritionals, possibly also because the nonfortified up to 750 mL liquid formula is consumed per day, containing beverages only contain very low concentrations of carotenoids, approximately up to 200 g solids, this would translate to up which would be difficult to detect in vivo. to 150 mg of divalent minerals, a range at which negative The bioavailability from unfortified milk has also never been interactions may occur. reported and would be low in absolute terms because of the Although in vitro methods such as simulated gastrointestinal rather low content of carotenoids in milk (Table 1). The only digestion methods are comparatively easy to conduct, affordable, estimates can be obtained either from human milk (see Infant and not restricted by ethical concerns, only one study has been Food Formulas section) or milk blended with carotenoid-rich conducted with infant food formula (65). Infant food formula food items, although then, the physical state and presence of bioaccessibility was 36, 51, 31, and 27% for lutein, zeaxanthin, carotenoids in such a mixture becomes uncertain. β-cryptoxanthin, and β-carotene, respectively. When comparing The bioaccessibility of milk- and soy-based fruit beverages bioaccessibility with other products, such as for other baby have been investigated by Cilla et al. (77). In their study, partially foods, infant food formula does appear to compare reasonably temperature-treated and high-pressure processed beverages well. The bioaccessibility of infant foods based on chicken were digested gastrointestinally in an in vitro system. The following and vegetables as well as berries and deserts were investigated beverages were tested: whole-milk fruit beverages (75%, v/v by Jiwan et al. (71). For both types, the bioaccessibility was fruit juice and 16.5% of milk), skimmed-milk fruit beverages comparatively high, ranging from 3 to 100%, with a generally (mixture as with whole milk beverages), and soy-milk fruit 6 Bohn: Journal of AOAC International Vol. 102, No. x, 2019 beverages (50%, v/v fruit juice and 41.5% of soy milk), containing However, again, the micelles formed may be larger than the approximately 137–220 and 122–158 μg/100 mL carotenoids normally formed mixed micelles containing bile salts and may for the milk, respectively, and 24–58 μg/100 mL carotenoids not contribute to the same extent to bioaccessibility compared for the soy-based beverages. Corresponding bioaccessibilities with typical mixed micelles (66). of total carotenoids were 39–99% and 13–47% for the whole- Therefore, in contrast to the positive effect of lipids, not and skimmed-milk–based beverages, respectively, and 18–73% much is known on the effect of proteins regarding carotenoid for the soy-based beverages, indicating that the lower amount bioaccessibility or bioavailability. As peptides produced during of fat in the skimmed milk products reduced bioaccessibility. digestion may have emulsifying properties and may protect Regarding individual carotenoid bioaccessibility, no consistent carotenoids from oxidation via chelating iron or forming a strong differences were encountered between β-carotene, surface film around lipid droplets, it could be speculated that they β-cryptoxanthin, lutein, zeaxanthin, and violaxanthin/neox­ aid in carotenoid micellization and bioaccessibility, as reviewed anthin, which is of interest as typically, the bioaccessibility of previously (90). On the other hand, they may also reduce the more apolar carotenoids (β-carotene, lycopene) is lower than enzymatic access to lipid droplets and reduce the transition that of the more polar xanthophylls such as lutein. Both heat of lipid droplets to mixed micelles, hampering micellization. treatments resulted in decreased fractional bioaccessibility; the Thus, the effects of proteins may be more mixed, depending on reasons for this are not quite clear, but it could have included digestive conditions (91) and require more detailed investigation. enhanced crystallinity of carotenoids or negative interactions A summary of factors influencing carotenoid bioavailability with fiber, which is not uncommon, for example, in high-pressure from infant food formulas and adult nutritionals is given processing, which may enhance the stability of the fiber in Table 2. In short, the bioavailability of carotenoids from network (78) in vegetable-containing beverages. milk products appears to be good or even high, at least when The positive influence of dietary lipids on carotenoid present in native form, i.e., in nonfortified products, because of bioavailabilty has been well described (26, 27). As mixed the embedding of carotenoids in liquid form in a lipid matrix micelles require emulsifying constituents, the digestion of that ensures their protection and a good micellization, owing triacylglycerides, producing free fatty acids, monoglycerides, to the presence of emulsifying constituents supporting micelle and diacylglycerides, which can act as emulsifiers, aided in their formation, including lipid digestion products, phospholipids, bioaccessibility (79). For example, adding whole milk versus and proteins. However, fortified products appear to show skimmed milk to a mixture of persimmon enriched milk (as a much lower bioavailability, even although the bioaccessibiltiy carotenoid source) drastically improved the bioaccessibility of typically measured following in vitro digestion and filtration various carotenoids by up to 4-fold (80). Similarly, addition (200 nm filters) may not be compromised, for reasons that of full-fat milk to wolfberries enhanced the bioavailability of may be related to different types and sizes of micelles formed; zeaxanthin in a human trial 3-fold, compared with a wolfberry– however, this remains hypothetical. skimmed milk formulation (81). Also, it has been assumed that additional lipids can foster chylomicron formation and thus Analytical Methods for Carotenoid Isolation and result in the further transport and absorption from carotenoids Quantification already present in the enterocytes (82). Although the generally positive influence of lipids on the Analytical methods may be separated into the isolation/ bioaccessibility and bioavailablity are undisputed, it remains purification/concentration steps and the actual quantification. less clear whether short-chain fatty acids such as in milk fat Major challenges for carotenoid determination in infant food really aid in the micellization of apolar microconstitutents. formula and adult nutritionals include the following: Several studies have suggested that rather the more long- (1) The relatively low concentration of carotenoids, i.e., below chain fatty acids may enhance carotenoid micellization and 20 μg/100 mL for nonfortified items, and up to approximately dietary uptake, as contrary to short-chain fatty acids (which are 200 μg/100 mL for fortified products. absorbed via the portal vein and are more water soluble), they (2) The relatively high protein and fat content of the food require also micellization and uptake via chylomicrons (83, 84), products, which may perturb the extraction of the lipophilic fostering their formation. However, other studies have reported carotenoids because of the coextraction of large quantities of higher bioaccessibility of carotenoids from butter fat than olive lipids and difficulties in their purification, unless the lipids are oil and peanut oil, as the shorter fatty acids and monoglycerides saponified beforehand, which bears the risk of carotenoid losses. and diglycerides appeared to contribute to a higher absolute In the following, the main isolation/purification/concentration zeta-potential, stabilizing the micelles (85, 86). Thus, there may and identification/quantification methods (that can be) used for be more specific matrix–lipid type interactions than previously the analysis of carotenoids in infant food formulas and adult anticipated which determines bioavailability. nutritionals are described. In another study, the addition of fat-free milk to fruit juice showed a significant positive effect on the bioaccessibility of Analytical Methods – Extraction and Purification lutein, zeaxanthin and β-cryptoxanthin (although not significant of Carotenoids for β-carotene) following simulated gastrointestinal digestion, also highlighting that positive effects of the addition of milk Liquid–Liquid Extraction may be because of additional factors other than lipids (87). For example, it was suggested that casein-phospholipids may chelate The most common methods of extraction for carotenoids iron, which could otherwise result in oxidative breakdown of include liquid–liquid extraction (LLE) and solid-phase carotenoids (88), and the presence of casein micelles per se extraction (SPE). LLE typically includes a fairly strong apolar may contribute to enhanced carotenoid bioaccessibility (89). and nonwater miscible solvent of reasonable low boiling point Bohn: Journal of AOAC International Vol. 102, No. x, 2019 7

Table 2. Dietary and technological factors likely influencing bioavailability from infant food formula and adult nutritionals

Factor Positive or negative influence Type of influence Ref.

Synthetically added carotenoids ↓a compared with natively Higher crystallinity, presence not in milk fat globules, (55, 65, 66) present carotenoids different micelle structure, lower cellular uptake. Presence of lipids ↑b Improved micellization with higher levels of fat due to (14, 26, 28) formation of emulsifying compounds (mono-diglycerides), higher bioavailability. Milk fat ↑↓c Negative effects because of the presence of short-chain fatty (83, 84) acids not absorbed via chylomicrons in some studies; Other studies suggest positive effects via smaller mixed (85, 86) micelles rich in milk fats. Phospholipids ↑ Enhanced emulsifying capacity. (150, 151) High-pressure homogenization ↓↑ Negative effects in vegetable matrices possible, although (152) no negative effects in matrices with structural barriers (chromoplasts, cell clusters), effects on milk products unclear. Proteins ↓↑ May stabilize lipid droplets, casein micelles may aid (88, 90) in carotenoid emulsification, casein-phospholipids may chelate iron, but also prevention of enzymatic lipid-droplet degradation possible. Presence of mineral mixes ↓ Higher concentrations of divalent lipids may reduce carotenoid (30, 31, 57) absorption, although effect only shown for Ca at high physiological concentrations and not in all studies. a ↓ = Negative effects. b ↑ = Positive effects. c ↑↓ = Mixed effects. with good extractability for carotenoids (Figure 2). As for infant followed by hexane (50) and hexane–acetone (1+1, v/v; 49), food formula and adult nutritionals, these carotenoids include with both methods yielding a high recovery (Table 3). Spiking β-carotene (logP 11.12), lutein (logP 8.55), and, to a lesser was done with externally added carotenoids and may not fully extent, lycopene (logP 11.92) (43). Typical solvents have been represent the extractability of the native carotenoids from their including hexane (30) petroleum ether (92), diethyl ether (68), matrix. However, following LLE, which is typically repeated tetrahydrofuran (THF; 49), methyl-tert-butyl-ether (MTBE; 49) for a total number of three extractions, the combined phases are and chloroform/dichloroethane or ethyl acetate, as reviewed evaporated to dryness, typically under a stream of nitrogen, or, earlier (93). When THF is used, because of the possible formation alternatively, with a rotary evaporator under reduced pressure, of peroxides, an antioxidant such as butylated hydroxytoluene especially for larger volumes. (BHT) should be added (93). Also, the use of an internal As xanthophylls may be present in the form of esters, standard to account for losses during extraction, such as including those present in dairy-based products, a prior β-apo-8′-carotenal, may be recommended. However, as the saponification with potassium hydroxide is recommended to miscibility of these solvents with water is limited, a solvent simplify later quantification. In addition, this will saponify carrier such as acetone (30) is typically added in order to triglycerides (and also phospholipids), which would otherwise foster the transition of carotenoids into the apolar phase. The be coextracted in the following LLE, perturbing further application of a binary mixture is also an advantage for a concentration and purification of the extracts. Saponification mixture of xanthophylls such as lutein and carotenes such as has also been used during the extraction of carotenoids from β-carotene, as the prior are better dissolvable in more polar plant matrices, without coextracting chlorophylls that could organic solutions such as MTBE, diethyl ether, and acetone, potentially interfere during chromatographic analysis (94). In whereas carotenes are better dissolvable in more apolar solvents this case, the addition of BHT as an antioxidant (0.02–0.1%) such as hexane. Extraction with diethyl ether requires further or ascorbic acid (e.g., 3–5%, w/v) may be advised (93). Within drying with sodium sulfate because diethyl ether tends to bind a typical saponification procedure, dried powder (infant food, small quantities of water. adult nutritional, about 1 g) is taken up in a volume of, e.g., As carotenoids have a long chain-like structure, it is assumed 15 mL methanol–water (2+1, v/v), containing a final concen­ that the solubility and extractability in hexane or petroleum tration of 3% KOH, and the mixture heated, for 15 min at ether is usually good. Indeed, in a previous study on their 60°C (49); however, different concentrations, times, and tempe­ extraction from rapeseed, among the tested solvents (acetone, ratures have been applied, with higher lipid content typically petroleum ether, methanol, chloroform, and petroleum requiring stronger conditions, as reviewed earlier (94). Shorter ether–acetone; 1+1), the latter combination had the strongest times but higher concentrations of KOH have also been applied, extraction capacity (92). Applying ultrasonication, heat (50°C) using, for example, 10 mL 5% KOH to 2 mL formula for 1 min. and a high solvent:material ratio (40:1) further improved Typically, conditions ranging from exposure times of extractability. The solvents that were used in a validated method 30 min at room temperature (RT) to 1 h at 80°C have been to detect carotenoids in infant formula and adult nutritionals applied. Choosing more drastic conditions increases the risk included extraction with a mixture of MTBE–THF (1+1, v/v) of carotenoid degradation. Biehler et al. (95) reported losses 8 Bohn: Journal of AOAC International Vol. 102, No. x, 2019

Figure 2. Typical separation possibilities for carotenoids from infant formula and adult nutritionals. Given volumes are an example based on previous publications (49, 50). AA = Ascorbic acid; MTBE = methyl-tert-butyl-ether; RT = room temperature.

of carotenoids from plant matrices between 7 and 16%, when highest retentions. However, protein removal was needed using 3.75% KOH for 15 mins at RT, although without the beforehand by precipitation, and both matrices were low in addition of any added antioxidant. lipids (96). In another study, an Oasis hydrophilic-lipophilic balance (HLB; a water-wettable, RP polymer), Abselut Nexus (a polymeric sorbent of high porosity and surface area), and SPE Lichrolut C18 phases were compared for the extraction of lutein and β-carotene from cereals (97), concluding that the Instead of LLE, SPE with silica or, more commonly, Oasis HLB followed by dichloromethane as an eluent worked reversed-phase (RP) material with C18 alkyl-chains can be used best. Additionally, proteins had to be removed by ethanol to purify carotenoids. Shen et al. (96) compared the retention precipitation first (Figure 2), and better results were obtained by of C18, C30, diol, and silica sorbents for extracting β-carotene a preceding saponification. However, following elution, further and lutein from human plasma and breast milk, followed by concentration by evaporation under nitrogen gas or by means of eluting with acetone. They concluded that C18 and C30 had a rotary evaporator is usually carried out. Bohn: Journal of AOAC International Vol. 102, No. x, 2019 9

Table 3. Validated method parameters for the detection of carotenoids from infant food formulas and adult nutritionals, according to Schimpf et al. (49) and Hostetler (50). The methods are based on saponification with KOH, LLE, and HPLC-C30-UV-Vis detection

Parameter investigated Corresponding performance method (49) Corresponding performance method (50) Remarks

LLE Hexane–MTBE (75+25, v/v) MTBE–THF (1+1, v/v), hexane Carotenoid identification All-trans-lutein, 13-cis lutein, 13′-cis-lutein, zeaxanthin, 13-cis lutein, 13′-cis lutein, zeaxanthin, all-trans β-carotene, 9-cis β-carotene, 13-cis β-apo-8′-carotenal, 13-cis β-carotene, β-carotene, all-trans-lycopene, unknown-cis-lycopene 15-cis β-carotene, α-carotene, all-trans 1a, unknown-cis-lycopene 2a, 5-cis-lycopene β-carotene, 9-cis β-carotene, all-trans-lycopene Repeatability, % 1.9–18.7 all-trans-lutein, 1.9–10.1 all-trans β-carotene, 0.1–6.2 all-trans-lutein and 0.4–47.4 all- High variation with 3.1–6.3 all-trans-lycopene trans β-carotene high-fat product Intermediate precision 1.9–23.9 all-trans-lutein, 3.5–54.4 all-trans β-carotene, 1.6–5.9 or all-trans-lutein and 0.7–4.6 all- (reproducibility), % 4.8–10.7 all-trans-lycopene trans β-carotene Recovery, % 90.3–95.3 lutein, 89.3–108 β-carotene, 97.3–109 92.3–105.5 lutein and 100.1–107.5 lycopeneb β-caroteneb Linearity (μg/L) 0.99991 lutein (10–250), 0.99993 β-carotene 1.0000 for β-apo-8′-carotenal, β-carotene, (25–200), 0.99980 lycopene (5–100) lutein LOQ, μg/100 g 0.4 lutein, 0.1 β-carotene, and 0.3 lycopene 0.27 lutein and β-carotene a Tentatively identified. b Unless otherwise specified, it is referred to the all-trans form.

Supercritical Fluid Extraction giving an additional possibility for their identification (102). Most carotenoids of relevance for infant food formula and In addition to the more classical LLE and SPE, supercritical adult nutritionals such as lutein, β-carotene, and lycopene absorb fluid extraction with CO2 has been used as a rapid method of in the area of 450–470 nm, depending slightly on the solvent, carotenoid isolation from the matrix, making use of the lipophilic with extinction maxima of 445 nm for lutein (144 900 L × –1 –1 character of the carotenoids. A further advantage of this method mol × cm in acetone; 95), 452 nm for β-carotene (140 663 –1 –1 is the potential for upscaling and being compatible with food L × mol × cm in acetone; 95), and 470 nm for lycopene –1 –1 production facilities as it is solvent free; a drawback remains (182 000 L × mol × cm in hexane; 103). the still-high operating costs of this method. For example, The major disadvantage of simple spectrophotometric extracting carotenoid from algae was reported to be highest at methods without prior separation of carotenoids by chromato­ 200 bar and 60°C (98), similar to other reported algae extraction graphic methods is the impossibility of quantification of methods (300 bar, 50°C; 99). This method has also been used individual carotenoids in a mixture because of their general for carotenoid extraction from other matrices such as pumpkin over­lap of absorption spectra. However, they are fast and quite rich in α- and β-carotene, β-cryptoxanthin, and lutein (35 bars, affordable. Although methods have been described to estimate 50–70°C), with a preceding vacuum drying resulting in the the total amounts of carotenoids in mixtures, based on pro­ highest extraction capability enhancing tissue disaggregation posing a typical mean molecular extinction coefficient and a (100). A further enhancement of the extraction can be achieved wavelength suitable for most carotenoids, this method allows by the addition of small quantities of other solvents, such as water only for a reasonable estimate. For example, Biehler et al. (95) or ethanol, to CO2. Indeed, such entrainers (a combination of proposed a method for determining carotenoids in a number of 10% water or ethanol and 10% olive oil) enhanced the extraction fruits and vegetables following saponification, using a mean of –1 –1 yield of carotenoids from pumpkin (100). Supercritical CO2 has 135 310 L × mol × cm at 450 nm, with results being very also been used for extracting β-carotene from infant food close to the HPLC method (on average, 1.4% difference but formulas (101), with recoveries of 70% compared with with larger deviations for individual food items) and worked conventional extraction, although a higher sample throughput reasonable well for vegetables rich in both β-carotene and lutein, and smaller sample volume required were emphasized. Further which may imply that a crude estimation of carotenoids in infant research in this area to improve extraction conditions for food formula and adult nutritionals is possible. Other methods, in complex matrices is warranted. part for more specific applications, such as for paprika 104( ) and for chlorophyll-rich pigments (105) have also been published. However, such methods can only be used as a first screening tool. Methods of Detection Liquid Chromatography Spectrophotometric Methods Spectrophotometric methods making use of the high General aspects – stationary phase and eluent.—As molecular extinction coefficients of most carotenoids (between carotenoids are relatively large molecules which are heat 110 000–180 000 L × mol–1 × cm–1) because of the delocalized sensitive and prone to degradation, LC methods, that is HPLC π-electron system are generally very sensitive and the most and ultra-HPLC (UHPLC), have been the method of choice applied method for carotenoid detection. In addition, most for separating individual carotenoids and their isomers. carotenoids have a typical and relatively specific spectrum, Several reviews exist in general on this topic, summarizing 10 Bohn: Journal of AOAC International Vol. 102, No. x, 2019

LC methods for carotenoid detection (106–110). Although thin conjunction with a binary mixture of 20 mM ammonium acetate layer chromatography methods have also been reported and in methanol–water (98+2, v/v) and 100% MTBE in the method reviewed (111), these are typically rather low in resolution and of Hostetler (50) to separate carotenoids within 32 min, whereas sensitivity compared with HPLC/UHPLC; however, they are in the method of Schimpf et al. (49), carotenoids were separated more affordable, do not require expensive equipment, and can within 30 min, using a C30 column and a binary gradient be used as a semi-quantitative screening tool. with methanol–MTBE (85+15, v/v) and pure MTBE. Both Few reports exist on separation/detection methods focusing methods allowed for a good separation of all-trans-β-carotene, specifically on infant food formulas and adult nutritionals, lutein, and lycopene, as well as several of their more common although validated and sufficient sensitive methods developed cis-isomers (Table 3). for other matrices may be equally well used. Regarding HPLC/ Several earlier reports have been published investigating UHPLC, a first distinction regarding normal phase versus RP carotenoids in infant food formulas. In the study by Yuhas can be made, the latter one allowing the injection of water et al. (120), lutein was detected after a C30 column separation, containing mixtures, which may be an asset for detecting a following an unspecified saponification and the extraction wide range of carotenoids. For this reason, and for the more with ethanol, with a binary system of MTBE and methanol, recent development of RP-C30 columns, RP chromatographic but the method was only validated down to a concentration of methods are dominating. The C30 phase consists of a longer 25 μg/L of lutein. In a study by Jewell et al. (121), samples brush type of alkyl chains, which interacts well with the were saponified by combining 200 μL milk/formula with carotenoid backbone. Consequently, geometrical isomers as 150 μL 50% aqueous KOH and 150 μL ethanol and 100 μL well as the often-difficult separation of lutein and zeaxanthin internal standard echinenone to account for losses during sapon­ can be achieved better in general compared with C18 RP ifi­cation, followed by extraction with hexane and separation columns, as previously reviewed (112). on a C30 column, with a binary mixture of methanol–MTBE Regarding the elution of carotenoids, both isocratic (113) and (89+11, v/v) and methanol–MTBE (62+38, v/v), allowing for the nonisocratic gradient methods (82, 114) have been developed, detection of zeaxanthin, lutein, β-cryptoxanthin, α-cryptoxanthin, with nonisocratic methods generally allowing the separation of β-carotene, and α-carotene. Concentrations as low as 0.2 nmol/g a wider range of carotenoids in a shorter period of time, possibly fat (approximately 1 μg/g fat) could be detected, suggesting a minimizing diffusion. However, seven or more carotenoids detection limit lower than 50 μg/100 g, although this was not have been resolved with an earlier isocratic method using clearly specified. A similar method was reported by Johnson 5% THF and 95% methanol (113). Although gradient methods et al. (122) to separate isomers of β-carotene from breast have been typically used to optimize RP chromatographic milk, reporting a LOD of approximately 0.2 pmol (approximately methods, normal phase, typically silica-based methods, have 0.1 ng), and measured concentrations of 0.4–2.7 μM β-carotene instead used isocratic methods (115), in part as when these and 0.05 and 0.4 μM for 9-cis β-carotene. methods were developed, pumps allowing running binary Some methods do further detect additional apolar compounds. eluents were less common. Regarding solvents used today for For example, Woolard et al. present an HPLC normal column RP chromatography, binary mixtures are most commonly used method for measuring β-carotene in infant food formula, (e.g., methanol–water–based systems versus those being more also detecting vitamin E and vitamin A, using all-E-β-apo- apolar such as containing acetonitrile or MTBE). For example, a 8′-carotenoic acid ethyl ester as the internal standard (123), by mixture of methanol–water–ammonium acetate–MTBE (88+5+ means of UV-Vis detection for carotenoids and fluorescence for 2+5, v/v) and MTBE–methanol–water–ammonium acetate vitamin A/E. (79+16+3+2, v/v) was used by Biehler et al. (95) to separate Liquid chromatography with alternative detection methods.— 10 carotenoids within 40 min. It is thought that the ammonium In addition to coupling HPLC to UV-Vis, electrochemical acetate reduces the tailing of carotenoid peaks. detection such as with Coularray-detectors have been used for A variation of LC that is not commonly available because of quantifying carotenoids (82, 124). This method is generally the more sophisticated equipment required is supercritical fluid more sensitive than UV-Vis, although possibly not by much for chromatography. For example, a mixture of eight carotenoids the carotenoids with a very high molecular extinction coefficient extracted from microalgae were separated on two columns in around 100 000–150 000 L × mol–1 × cm–1. There was an series (C18 combined with a normal-phase column) within improvement by a factor of 10–100 for lycopene detection 16 min, using liquid CO2 and methanol as the mobile phase in with this method, detecting as low as 50 fmol (approximately a gradient method, increasing methanol concentration over time, 0.025 ng) with a C30 RP method during 50 min (125). However, with a flow rate at 5 mL/min and a pressure of 120 bar at 32°C. a typical detector is also several times more expensive compared The advantages of this method include low solvent consumption with UV-Vis. As the possibility to measure UV-Vis absorption and a possible fast separation time. Further information about this spectra is lost, the possibility to study different oxidation method is available elsewhere (116–118). No report on using this potentials is gained, and sometimes coeluting peaks can be method for infant food formula or adult nutritionals is available; resolved electrochemically, thus this method may have some however, this method has been used to detect other lipophilic complementarity to UV-Vis. constituents including retinol from infant formulas (119). Finally, in addition to UV-Vis and electrochemical detection, fluorescence has been used for detecting individual carotenoids, Published Methods Investigating Carotenoids in Infant especially phytofluene, which is less sensitive for UV-Vis Food Formulas and Adult Nutritionals detection than other carotenoids because of its lower molecular extinction coefficient (102) and its sufficient fluorescence In the two AOAC methods validated specifically for infant intensity when excited at 346 nm, measuring emission intensity food formula and adult nutritionals, C30 columns were used in at 520 nm as reviewed previously (126). For β-carotene, Bohn: Journal of AOAC International Vol. 102, No. x, 2019 11 fluorescence detection has also been reported, with 480 and matrices, has been carried out by Raman spectroscopy. This 560 nm of excitation and emission, respectively (127), but technique measures vibrational, rotational, and other low- fluorescence strongly dependeds on the solvent. Fluorescence frequency modes of selected compounds induced by inelastic has also been applied to study algae carotenoids (predominantly scattering from a laser source. The strength of this method rests β-carotene) by microscopic methods, with excitation in the detection of carotenoids in the original matrix without wavelengths of 450/488 and emission being highest at 680 nm the need of extraction/purification or separation. The potential (128). However, as fluorescence is not generally applicable to of Raman spectroscopy for measuring carotenoids, although in carotenoids at high sensitivity and because of the higher costs of microorganisms, has been previously reviewed (140). Various the detector, this method is not commonly applied. wavelengths for excitation (514, 488, 844, and 1064 nm) may be used, and spectra between 100 and 800 cm-1 are typically recorded. In general, the measurement of β-carotene (140), Mass Spectrometry zeaxanthin (140), and lycopene (141) have been reported. Limitations of this method include that not all carotenoids MS, especially tandem mass spectrometry (LC-MS or may be discriminated between and that the sensitivity is lower LC-MS/MS), such as with triple quadrupole instruments, compared with HPLC-UV-Vis. For infant food formula and has been conducted for detecting carotenoids from various adult nutritionals, although lutein and β-carotene have been matrices including cashew apples (129), tomato fruits (130), simultaneously detected by Raman spectroscopy before (142), and human samples such as plasma (131). The ionization mode and sensitivity (nanograms per gram range) may be sufficient, of choice, being crucial for sensitivity, appears to constitute this method has not accurately discriminated between more atmospheric pressure ionization (APCI), resulting in better complex mixtures of carotenoids. ionization efficiency compared with the generally more often Whithall et al. used Raman spectroscopy to measure used electrospray ionization, although there are some reports carotenoids in tomatoes, carrots, and red peppers in situ (143), that used this latter technique on carotenoids, e.g., in methods detecting the major carotenoids α- and β-carotene, capsanthin, detecting several classes of liposoluble constituents (132). and capsorubin, although a more detailed analysis was not The advantage for using MS methods is that at least coeluting possible, and lycopene appeared not clearly detectable. No compounds can be differentiated as long as their masses differ, validated Raman method exists for carotenoid detection in which, however, is not the case for several more difficult infant formulas or adult nutritionals. separations such as lutein/zeaxanthin or the separation of In conclusion, spectrophotometric methods based on UV- geometrical isomers. However, the strength of LC-MS/MS rests Vis, especially following prior separation by HPLC/UHPLC in the possibility to detect less UV-active constituents such as are the most commonly used methods and are generally phytoene/phytofluene (61, 133), and carotenoid metabolites sensitive enough for the analysis of infant formulas and adult such as apo-carotenoids (134), with higher sensitivity. Typical nutritionals. Following saponification and extraction, most carotenoid fragments formed by APCI have been reviewed and often by LLE, although SPE or even supercritical extraction can serve as an aid in identification by Rivera et al. 135( ). with CO are applied, separation has most often been achieved Regarding the different types of MS, ion-trap (136), 2 for infant food formulas by C30 RP column chromatography, quadrupole/triple quadrupole (131), orbitrap (133), and allowing the separation of cis-isomers of especially β-carotene quadrupole time-off-flight 61 ( ) have been used. Although and lycopene with alcohol/aqueous phase compatible solvents, orbritrap allows for high-resolution investigation (i.e., exact such as methanol and MTBE. Coupling to MS-based methods mass determination), the triple-MS has typically been described may allow for a slightly more sensitive analysis, but are often as the more sensitive method (137). not available and have been used instead for the identification Kopec et al. compared the sensitivity of an APCI–LC-MS/MS of metabolites present at lower concentrations, whereas method (using a triple quadrupole instrument) of several spectrophotometric methods alone may allow for a decent carotenoid standards and extracts from chylomicron rich estimate of total carotenoids only. A general alternative to fractions from humans. Compounds eluted within 18 min, UV-Vis is electrochemical detection, but this technique is less highlighting that for lycopene and α- and β-carotene, the MS available because of the considerable higher price of the detector. method was up to 37 times more sensitive than LC coupled to Measurements in situ, i.e., without extracting carotenoids from UV-Vis (131), whereas for lutein, the LC-UV-Vis method was the test matrix, such as by Raman spectrometry, may become 8 times more sensitive. The sensitivity for lycopene was about more available in the future, but presently, discrimination equal, with an on-column sensitivity of 25 fmol. between various carotenoids is not feasible. Nimalaratne et al. (138) validated an APCI–LC-MS/MS method to detect lutein, zeaxanthin, and β-carotene together Conclusions with other lipophilic constituents such as vitamin D and retinol from infant food formulas and dietary supplements, with LODs Because of their reputation as health beneficial phytochemicals of 10, 10, and 9 ng/mL (pg/L) formula preparation for lutein, or micronutrients (for pro-vitamin A carotenoids such as β-carotene, and zeaxanthin, respectively, making this method a β-carotene), carotenoids may be more and more frequently very sensitive, and with 15 min separation time, a very fast method. encountered in a broad array of products, including adult nutritionals, and in part also infant food formula, given that Methods of Detection – Other the legislative situation allows for their fortification, as their native concentration in dairy-based products is low, usually An interesting and noninvasive way of quantifying under 20 μg/100 mL. Beneficial health effects may include carotenoids such as in the skin (139), but also in various improved antioxidant and reduced inflammatory status, as well 12 Bohn: Journal of AOAC International Vol. 102, No. x, 2019 as prevention of age-related macular degeneration in the elderly. (14) Desmarchelier, C., & Borel, P. (2017) Trends Food Sci. Technol. Dairy products may constitute a good vehicle for these lipophilic 69, 270–280. doi:10.1016/j.tifs.2017.03.002 compounds, as bioavailability as a result of the presence of lipids, (15) dela Seña, C., Narayanasamy, S., Riedl, K.M., Curley, R.W., Jr, phospholipids, and possibly proteins is reasonable or good. Schwartz, S.J., & Harrison, E.H. (2013) J. Biol. Chem. 288, Whether additional carotenoids, i.e., other than lutein, β-carotene, 37094–37103. doi:10.1074/jbc.M113.507160 (16) Palczewski, G., Amengual, J., Hoppel, C.L., & von Lintig, J. and lycopene used at present, will be employed to fortify these (2014) FASEB J. 28, 4457–4469. doi:10.1096/fj.14-252411 food items remains to be seen, but it is possible. This and the fact (17) Erkelens, M.N., & Mebius, R.E. (2017) Trends Immunol. 38, that processing increases the risk of trans-cis isomerization, and 168–180. doi:10.1016/j.it.2016.12.006 possibly also the formation of carotenoid degradation products/ (18) Larange, A., & Cheroutre, H. (2016) Annu. Rev. Immunol. 34, metabolites such as apo-carotenoids, requires the presence of 369–394. doi:10.1146/annurev-immunol-041015-055427 sensitive and accurate methods to measure carotenoids in such (19) Moreb, J.S., Ucar-Bilyeu, D.A., & Khan, A. (2017) Cancer matrices. At the present, extraction with an apolar solvent, in Chemother. 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