1044  Bohn: Journal of AOAC International Vol. 102, No.
4, 2019
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
phytochemicals that cannot be synthesized by
humans, some of which such as β-carotene can be
metabolized into vitamin A. Sufficient carotenoid
intake and tissue levels have been associated with
several health benefits including the reduction of
cardiovascular diseases and some types of cancer
and also the amelioration of age-related macular
degeneration. Carotenoids and their metabolites
have also been related to reduced inflammation and
oxidative stress via interacting with transcription
factors, such as NF-κB and Nrf-2, as well as with
the nuclear receptors retinoic acid receptor/retinoid
X receptor, implicated in immune functions and
cellular differentiation. Therefore, carotenoids
are important for growth and development. They
could mark beneficial constituents in infant food
formulas and adult nutritionals, the latter typically
constituting protein-rich liquid foods targeting
meal replacements. Carotenoids may be present by
nature (typically below 20 μg/100 mL) or following
fortification (up to 200 μg/100 mL), such as for lutein
and β-carotene. However, carotenoid bioavailability
may be low and variable, especially in low-fat items.
Although most infant foods and adult nutritionals are
rich in lipids and proteins, facilitating absorption and
availability of carotenoids, unfortunately, very little
data is available. In addition, carotenoid detection
for such lipid-rich matrices may be challenging as
a result of low concentrations and matrix effects.
This review aims to highlight considerations for
carotenoid bioavailability from infant food formula
and adult nutritionals as well as summarize detection
methods for carotenoids from these items.
C
arotenoids are typically C40 tetraterpenoids of plant,
bacterial, or fungal origin, but they could also comprise
C30 (1) or C50 (2) terpenoids. Although they cannot
be produced by animals, including humans, they can also be
found in animal sources, such as eggs (3), dairy products (4),
Guest edited as a special report on “Carotenoids: Absorption,
Biological Activity, and Analysis” by Gregory L. Hostetler.
Corresponding author’s e-mail: torsten.bohn@gmx.ch
DOI: https://doi.org/10.5740/jaoacint.19-0015
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and salmon (5), via natural accumulation or biofortification of
feed rich in carotenoids. Another common source of carotenoids
is from fortified food items such as butter, as carotenoids can be
added as coloring agents (E160-E160f; 6), or directly through
supplements in which amounts of 5–50 mg per dosing (often a
capsule or caplet) are typically available on the market.
The most common carotenoids in the diet vary according to
dietary habits but typically include β-carotene, lutein, lycopene,
β-cryptoxanthin, zeta-carotene, zeaxanthin, α-carotene,
phytoene, phytofluene, violaxanthin, and neoxanthin (7).
However, in many Asian diets, astaxanthin and fucoxanthin
from algae are also predominant (8), whereas in societies that eat
less tomatoes and processed tomato products such as ketchup,
lycopene concentrations can be quite low (9). However, their
dietary intake is generally reflected by blood plasma/serum
concentrations, except for the epoxycarotenoids violaxanthin
and neoxanthin, which appear to be further metabolized prior
to their absorption, as their blood and tissue concentrations are
typically low (10, 11). The xanthophylls can also be present
as monoesters or, in some cases, diesters, which presumably
requires cleavage prior to their further absorption (12, 13).
Finally, in addition to the primary all-trans conformation
in plants, certain cis-isomers can be formed during food
processing and following their absorption in vivo (14).
Carotenoids in general, and also specific carotenoids, have
been related to several health benefits. One important aspect
of carotenoids is that they contribute to vitamin A intake;
several carotenoids (β-carotene, β-cryptoxanthin, α-carotene)
can be converted into vitamin A upon cleavage by β-carotene
oxygenase 1 (BCO1) in the gut and in other tissues (15, 16).
Vitamin A is important for the optimal functioning of the
immune system (17, 18), and it is involved in cell proliferation
(19), among other functions. Many of these functions are
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.
In addition to providing vitamin A, dietary carotenoid intake
and blood plasma/serum concentrations have been related to
several chronic diseases. For example, higher plasma β-carotene
concentrations in the elderly have been significantly associated
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
function, e.g., stabilizing cell membranes against lipid (per-)
oxidation (24), carotenoids and metabolites have also been
shown to alter pathways of inflammation and oxidative stress
 Bohn: Journal of AOAC International Vol. 102, No. 4, 2019  1045
via cellular transcription factors, such as NF-kB and Nrf2,
respectively (25).
However, bioavailability, i.e., the fraction of a compound
that can be absorbed and used for its physiological function and/
or stored, for carotenoids is low (typically 5–40%; 26, 27) —
especially for the more apolar, oxygen-free carotenes — and also
variable, influenced by many dietary and host factors (7). For
instance, it is well recognized that dietary lipids can ameliorate
the bioavailability of carotenoids (28), whereas dietary fiber (29)
and perhaps divalent minerals (30, 31) may reduce it. Regarding
host factors, enzymes of digestion, cleavage enzymes (BCO1),
and transporting lipoproteins are known to influence bio­
availability; however, they have been more extensively reviewed
elsewhere (7).
Infant food formula includes formula for babies up
to the age of 12 months. It may be used to replace human
milk, which is also a source of carotenoids for infants,
containing approximately 80 nM (4.2 μg/100 g) β-carotene
and 130 nM (7.4 μg/100 g) lutein, as reviewed recently (32).
Special European regulations exist for infant food formulas
(33), and similar regulations are set by other governmental
organizations such as the U.S. Food and Drug Administration
(34, 35). In general, only very few additives for such food
items are allowed. These include preformed vitamin A (up to
70–114 μg retinol equivalents/100 kcal; 36) and carotenoids,
but only in the United States with maximum limits established
within generally recognized as safe (GRAS) notifications for
various food categories (35, 37), but not in the European
Union (EU), where the use of food colors in infant foods is
prohibited (34). In light of their partial contribution to vitamin
A, and the contribution to antioxidant status as recently
shown in infants (38, 39), carotenoids are considered valuable
micro-constituents for growing infants. For this reason,
β-carotene and lutein especially (and occasionally lycopene)
have been supplemented to infant food formula, as the native
concentration of carotenoids (mostly β-carotene) is fairly low,
typically below 20 μg/100 mL.
Likewise, carotenoids are frequently added to and are
present in adult nutritionals, typically beverages rich in proteins
and intended to replace a meal, e.g., Boost, Ensure, Prosure,
Slimfast, or similar products, often based on dairy and soy
products (40, 41). However, in Europe, carotenes and lutein
cannot be added directly to such beverages; the use of lycopene
for flavored drinks is only permitted up to 10 mg/L (42). The
allowed amount in the United States is not always specified, such
as for β-carotene, but should follow good manufacturing practice
(35); whereas for other carotenoids, a maximum limit has been
specified within GRAS notifications, i.e., suspended lutein in
infant food formula (250 μg/L) and 1 mg meso-zeaxanthin per
meal for infant and toddler foods (37).
As carotenoids are lipo-soluble (with logP values between
8 and 12; 43), it may be analytically challenging to extract
and separate these compounds at low concentrations from a
lipid-rich matrix, because of the large amount of coextracted
lipids and the difficulty for further concentration and
purification. Normally, such matrices require a saponification
step to cleave triglycerides and to also cleave potentially
present xanthophyll (mostly lutein) esters. Because of these
challenges, and the importance of infant food formulas and
adult nutritionals, this review aims to highlight aspects of
bioavailability from these products and analytical methods that
have been developed to detect carotenoids in these matrices.
Carotenoid Content in Infant Food Formulas and
Adult Nutritionals Including Liquid Dairy Products
Infant Food Formulas
Infant food formula is generally composed of cow milk
proteins such as whey and casein, to which vegetable oils such as
rapeseed, corn, sunflower, or coconut oil may be added in addition
to lactose and a mixture of minerals and vitamins. Also, skimmed
milk, fructo-oligosaccharides, or fish oils may be added (44, 45).
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Carotenoids occur especially within milk and vegetable oils.
The measurement of carotenoids in infant food formulas
has been met with increasing interest, for several reasons.
First, because of the potential of several carotenoids (e.g.,
α- and β-carotene and α- and β-cryptoxanthin) to yield
vitamin A following cleavage by BCO1, and second,
because of the proposed health benefits of carotenoids. For
example, lutein supplementation given to newborns on two
occasions (2×0.28 mg) reduced circulating hydroperoxides
and improved ferric reducing antioxidant power assay,
which is a test for antioxidant activity in blood plasma
(46, 47). Similarly, the supplementation of a mixture of lutein/
β-carotene/lycopene in infant food formula at 210 μg/L
reduced c-reactive protein, a marker of systemic inflammation
(39), following approximately 40 weeks of intervention.
Similarly, Capeding et al. (48) fortified infant food formula
with lutein at 200 μg/L. After 16 weeks, no growth
differences or bio­chemical blood parameters measured, such
as glucose or circulating proteins, differed between the two
groups; however, bioavailability and inflammatory markers
were not reported.
Because carotenoids are already naturally present in milk,
although at a low extent (Table 1), and, at least in some countries,
can be added (e.g., in the United States but not in the EU), the
carotenoid content and profile can vary considerably (Table 1),
depending on the individual ingredients and further fortification.
In general, total carotenoid concentrations from 0 to about
40 μg/L have been reported in commercial products and up to
200 μg/L in products used in infant trials, which is still low
compared with the presence in some baby foods in which
concentrations up to 48 mg/100 g have been reported (Table 1).
So far, however, traditionally only β-carotene and, more recently,
lutein, have been added to commercial products, such as in the
Similac brand of Abbott (Table 1).
As carotenoids can be present in either cis or trans
forms, a few researchers have examined the geometrical
profiles of β-carotene, lutein, and lycopene (49, 50). These
have included, in addition to the respective all-trans forms,
13-cis-lutein, 9-cis-lutein, 9′-cis-lutein, 13′-cis-lutein, 9-cis-
β-carotene, 13-cis-β-carotene, 15-cis-β-carotene, 5-cis-lycopene,
and additional nonidentified lycopene isomers, possibly
including 13-cis-lycopene and 15-cis-lycopene. Although in
plants, the predominant form of carotenoids is the all-trans
form, a certain fraction of carotenoids may isomerize during
processing, i.e., heat application (51, 52) and upon ingestion
in the enterocytes (53, 54). However, also in the final products
(infant food formulas and adult nutritionals), the predominant
forms are the all-trans form (>2/3; 49, 50). The presence of
various geometric forms is of interest because they also may
differ in their bioavailability (see Bioavailability Aspects
of Carotenoids Regarding Infant Food Formulas, Adult
Nutritionals, and Liquid Dairy Foods section).
Table 1.  Carotenoids in infant food formula. Data are individual values or ranges (mean) when available

Total carotenoids, Lutein/zeaxanthin, Lycopene, β-carotene, β-cryptoxanthin,

μg/100 mL μg/100 mL μg/100 mL μg/100 mL μg/100 mL
Food category Food item (μg/100 g) (μg/100 g) (μg/100g) (μg/100 g) (μg/100 g) Data source Ref.
a
Infant food formula Various products (n = 8): (Beba 0, Humana 0, Beba Pre, Aptamil ND ND 0 0–20 0–13 Sommerburg et al. (64)
Pre, Pre Aletemil, PreAponti, Humana HA, Aletemil HA)
b
PBM PRODUCTS, store brand, soy, ready-to-feed 37 0 0 37 0 USDA database (70)
Mead Johnson, ENFAMIL, premium, infant, ready to use 0 0 0 0 0 USDA database (70)
Mead Johnson, ready-to-feed 10.1 7.9c 0 2.0 0.2 Lipkie et al. (65)
Similar advance, Abbott Nutrition, ready to eat 2.8 1.5d 0 1.3 ND Mackey et al. (62)
Similar Advance with OptiGRO, Abbott Nutrition, ready-to-feed 3.5 2.2 ND 1.2 ND Jeon et al. (67)
Preterm formula, Abbott Nutrition, ready-to-feed 14.0 6.6 0.2 7.1 0.1 Hanson et al. (144)
Transitional formula, Abbott Nutrition, ready-to-feed 12.9 5.7 0.6 6.4 0.1 Hanson et al. (144)
Term standard formula, Abbott Nutrition, ready-to-feed 16.4 5.8 8.0 2.5 0.1 Hanson et al. (144)
Aptamil 1, Nestle, ready-to-feed ND 3.3 ND ND ND Costa et al. (145)
Several infant food formulasf 0.33–23.3 0.09–12.0 0–7.57 0.2–21.0 ND (49)
e
Baby food, general Various items 0–47.500 (1113) 0–45050 (452) 0–2575 (92) 0–7156 (501) 0–135 USDA database (70)
Breast milke Breast milk 18.5 4.0 ± 4.3 6.6 ± 5.6 4.9 ± 7.6 2.2 Hanson et al. (144)
Adult nutritionals Nestle, Boost Plus ND 0 0 144c 0 USDA database (70)
Unilever, SlimFast, Shake Mix, powder ND 0 0 1 0 USDA database (70)
Kellogg’s Special K protein shake ND 0 0 3 0 USDA database (70)
Abbott Ensure, Nutritional Shake, ready to use ND 0 0 0 0 USDA database (70)
1046  Bohn: Journal of AOAC International Vol. 102, No. 4, 2019

Ensure High Protein therapeutic nutrition ND ND ND 5–100 ND Estimated from label

Several adult nutritionalsf 0.22-29.43 0.22-16.7 0-0.83 0–11.9 ND (49)
Milk products Full-fat milk, pasteurized 20.9 0.8–1.4d (1.0) ND 14.7–19.1 (16.7) 0.3–0.4 (0.3) Hulshof et al. (146)
Skimmed milk (nonfat milk) 0–7 0 0 0–7 0 USDA database (70)
Semi-skimmed milk, pasteurized 10.4 0.5–0.8d (0.8) ND 6.8–9.4 (7.9) 0.1 (0.1) Hulshof et al. (146)
Full-fat milk, pasteurized 13–21 (17) ND ND 13–21 (17) ND Souci et al. (147)
Semi-skimmed milk, pasteurized 6–9 (8) ND ND 6–9 (8) ND Souci et al. (147)
Full-fat milk, ultra-heat treated 10–20 (18) ND ND 10–20 (18) ND Souci et al. (147)
Milk, organic ND ND ND 18.7 ± 4.7 ND Ellis et al. (148)
Milk, conventional ND ND ND 17.5 ± 7.4 ND Ellis et al. (148)
Milk, raw, organic ND 2.3–2.4 (2.4) ND 1–13.3 ND Calderon et al. (149)
Milk, raw ND ND ND 28–46 ND Schweiggert and Carle (55)
a
  ND = Not determined/no data.
b
  USDA = U.S. Department of Agriculture.
c
  Fortified with respective carotenoid.
d
  Only lutein.
e
  Group added for comparison.
f
  Including powders, n = 7–9.

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 Bohn: Journal of AOAC International Vol. 102, No. 4, 2019  1047
Adult Nutritionals Including Liquid Dairy Products
Carotenoid concentration of full-fat milk varies according
to the diet of cows and has been reported to be typically
in the range of 6–21 μg/100 mL, with mostly β-carotene
being reported (Table 1). Schweiggert and Carle stated that
over 90% of carotenoids in milk are β-carotene (55). Lutein
(0.5–2.4 μg/100 mL; Table 1), zeaxanthin (0.1–0.5 μg/100 mL;
Table 1), and β-cryptoxanthin (0.3–0.4 μg/100 mL) have been
occasionally detected. Because most adult nutritionals are
produced on a milk base and are further diluted as additional
ingredients (which normally contain less carotenoids, except
perhaps certain vegetable oils) are added, the content of
nonfortified carotenoids is expected to decrease. The same is
true for products based on semi-skimmed or skimmed milk,
as the lower concentration of fat correlates with a decreased
concentration of carotenoids (Table 1).
Only little data are available on the carotenoid content
in adult nutritionals. Basal levels as low as 1–3 μg/100 μL
β-carotene have been found. For certain β-carotene–fortified
products, concentrations of up to 144 μg/100 mL have been
reported (Table 1).
In conclusion, unfortified concentrations of carotenoids are
quite low in infant food formulas and in adult nutritionals; only
β-carotene appears to be present in measurable, although in low
concentrations, whereas many other carotenoids are virtually
absent. Concentrations following fortification has resulted in
levels of 100 μg/mL or even higher.
Bioavailability Aspects of Carotenoids Regarding
Infant Food Formulas, Adult Nutritionals, and Liquid
Dairy Foods
Infant Food Formulas
The bioavailability of carotenoids can be assessed by several
methods, including measuring the area under the time curve of
newly absorbed carotenoids in the plasma–triacylglycerol-rich
lipoprotein fraction, which is possibly the most accepted and
conducted method (56, 57), especially for postprandial trials,
although for ethical reasons, it cannot be applied to infants
because of the several blood draws required. In addition, baseline
subtracted plasma measurements can be carried out, which may
be the method of choice following long-term feeding trials and
for infants. It is also frequently used for postprandial trials,
although in this latter case, it also requires several blood draws
over several hours (31), 24 h in an optimal case. Other methods
include measuring skin color after several weeks of feeding by
Raman spectrometry (58). When determining the effect of lutein/
zeaxanthin supplementation, which is selectively accumulated
in the macula of the human eye, macula-pigment optical
density measurement may also constitute a valid bioavailability
indicator (59). Isotopically labeled carotenoids may also be
followed in the blood over time, but these methods are generally
more expensive (60) and used rather in compartment modeling
(61) to study further transport and metabolism.
When comparing bioavailability across food sources, it has
been estimated that the bioavailability of carotenoids from
infant food formulas is lower compared with that of mother’s
milk (62). In the latter study, two infant food formulas fortified
with β-carotene (54, 93 μg/L), lutein (33, 53 μg/L) and lycopene
(43, 81 μg/L) were given for 56 days versus a control infant food
formula (Similac Advance, Abbott; Table 1) and human milk.
Although the amount of all carotenoids increased with the higher
doses of infant food formulas, final plasma concentrations were
not significantly higher compared with human breast milk–fed
infants, which is typically of lower carotenoid concentration
(Table 1).
These results are in line with another human trial (63), in which
lutein bioavailability (expressed as serum concentration) of human
milk and infant food formula were compared. More specifically,
in a double blinded study, human milk was 4 times more efficient
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in delivering lutein than the infant food formulas, i.e., the authors
concluded that 4 times greater concentrations of lutein in formula
would be required to achieve plasma concentrations comparable
with human milk. Also, in line with this finding is a study
investigating carotenoid status by plasma levels and skin status
using noninvasive Raman spectroscopy (58), and both measures
were well correlated (R = 0.44, P = 0.01). In this study, preterm
infants were fed either infant food formula or human milk with
unspecified amounts of carotenoids for 2 weeks. Although both
serum and skin carotenoids tended to decline with the formula
intervention, concentrations tended to increase upon human
milk intake, although it is likely that the higher concentrations of
carotenoids to be expected in breast milk versus the nonfortified
infant food formula (Table 1) contributed to the findings.
Additional carotenoids were monitored by Sommerburg et al. (64),
who followed newborns receiving either human milk or infant
food formula for up to 14 days, finding that, compared with birth,
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
feeding enhanced slightly, although not significantly, carotenoid
concentrations compared with levels at the time of birth.
The reasons for the relatively low bioavailability of
carotenoids from infant food formula are unclear and can
be either because of poorer release and micellization from the
matrix (a prerequisite for later absorption), cellular uptake
and transport, or further absorption and biodistribution.
Interestingly, bioaccessibility, i.e., the fraction of a compound
that is liberated during digestion and available for absorption,
has been reported to not differ significantly between infant food
formula and mother’s milk. In a study by Lipkie et al. (65), the
bioaccessibility of lutein was 29 ± 2% from human milk versus
36 ± 4% from fortified infant food formula, both containing
comparable amounts of lutein. In contrast, fractional cellular
uptake was 4.5 times greater by Caco-2 cells from human
milk than from the infant food formula, suggesting that either
absorptive processes do differ between the two sources or the
composition and/or size of the mixed micelles differed between
the two sources, influencing absorption capability.
In an earlier in vitro study, it was already shown that adding
synthetic carotenoids in an isolated form to milk resulted in good
solubilization but apparently poorer uptake into small micelles,
as filtration largely reduced β-carotene from the present larger
droplets (66). The presence of β-carotene in milk, i.e., in milk
fat globules, has been well described by Schweiggert and
Carle (55), in which carotenoids are well dissolved in unipolar
lipids followed by a surrounding monolayer of polar lipids,
cytoplasm, and then another bilayer of polar lipids (Figure 1).
This is presumably much different than merely added synthetic
carotenoids to milk.
Higher carotenoid intake and serum concentrations are likely
related to higher target tissue concentrations. For example,
1048  Bohn: Journal of AOAC International Vol. 102, No. 4, 2019
Carotenoid Membrane associated
Cholesterol vitamin
Unpolarvitamin (A, E)
O
H
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
administered to offspring in breast milk was compared with
infant food formula either fortified (with lutein, zeaxanthin,
β-carotene, and lycopene at 13.5, 1.1, 4.0, and 18.1 μg/100 mL,
respectively), or unfortified infant formula (2.2, 0.1, 1.2, and
0 μg/100 mL, respectively). Also in this study, serum concent­
rations were higher following breast feeding, and β-carotene,
lutein, and zeaxanthin accumulation in the brain was higher
compared with the formula-fed animals, indicating that not only
blood, but also tissue distribution, is likely influenced by the
different mode of feeding, although human data on this is scant.
Another factor that may potentially limit carotenoid
bioavailability is the addition of mineral mixtures containing
divalent minerals, e.g., calcium and magnesium. Several in
vitro studies (30, 68) and also in vivo studies on carotenoids
from plant matrices, although the latter with mixed results
(31, 57), have suggested that higher concentrations of divalent
minerals, with intake amounts of approximately 250 mg of
calcium or magnesium for adults, could compromise carotenoid
bioaccessibility. This is because divalent minerals can bind to
free fatty acids and bile salts during digestion, reducing the
micellization efficiency of carotenoids. However, whether the
concentration of divalent minerals in infant food formulas (up
to 7.5 g/kg as the sum of calcium and magnesium; 69, 70) does
have any negative effects remains to be elucidated. Given that
up to 750 mL liquid formula is consumed per day, containing
approximately up to 200 g solids, this would translate to up
to 150 mg of divalent minerals, a range at which negative
interactions may occur.
Although in vitro methods such as simulated gastrointestinal
digestion methods are comparatively easy to conduct, affordable,
and not restricted by ethical concerns, only one study has been
conducted with infant food formula (65). Infant food formula
bioaccessibility was 36, 51, 31, and 27% for lutein, zeaxanthin,
β-cryptoxanthin, and β-carotene, respectively. When comparing
bioaccessibility with other products, such as for other baby
foods, infant food formula does appear to compare reasonably
well. The bioaccessibility of infant foods based on chicken
and vegetables as well as berries and deserts were investigated
by Jiwan et al. (71). For both types, the bioaccessibility was
comparatively high, ranging from 3 to 100%, with a generally
Inner monolayer of polar lipids
Membrane associated
carotenoid
Protein
Cytoplasm
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Cholesterol
Outer lipid bilayer
higher bioaccessibility from the chicken/vegetable dishes
(31–78% versus 3–68% for β-carotene, β-cryptoxanthin,
lutein, and lycopene) and a generally higher bioaccessibility
for xanthophylls versus carotenes, likely because of the higher
lipophilicity of the latter and poorer micellization.
Also, the geometric form of carotenoids can influence their
bioaccessibility and bioavailability. cis-Isomers are of shorter
apparent structure as they appear more bended and tend less
toward crystallization (14). Consequently, higher micellization
of cis-isomers as compared to their all-trans form has been
reported in several studies (72, 73). However, this does not
appear to result in improved bioavailability, at least not for all
carotenoids. Whereas a higher bioavailability for cis-isomers
of lycopene has been clearly demonstrated in several studies
(74, 75), i.e., up to 8.5-fold, the bioavailability of β-carotene
appears superior in the all-trans form (76). However, as
β-carotene appears to be reisomerized to the all-trans form
in the enterocyte, the absorption of the cis-forms may be
underestimated, as reviewed previously (14).
Adult Nutritionals Including Liquid Dairy Products
To the author’s knowledge, no published reports exist on
aspects of the bioaccessibility or bioavailability of carotenoids
from adult nutritionals, possibly also because the nonfortified
beverages only contain very low concentrations of carotenoids,
which would be difficult to detect in vivo.
The bioavailability from unfortified milk has also never been
reported and would be low in absolute terms because of the
rather low content of carotenoids in milk (Table 1). The only
estimates can be obtained either from human milk (see Infant
Food Formulas section) or milk blended with carotenoid-rich
food items, although then, the physical state and presence of
carotenoids in such a mixture becomes uncertain.
The bioaccessibility of milk- and soy-based fruit beverages
have been investigated by Cilla et al. (77). In their study, partially
temperature-treated and high-pressure processed beverages
were digested gastrointestinally in an in vitro system. The following
beverages were tested: whole-milk fruit beverages (75%, v/v
fruit juice and 16.5% of milk), skimmed-milk fruit beverages
(mixture as with whole milk beverages), and soy-milk fruit
 Bohn: Journal of AOAC International Vol. 102, No. 4, 2019  1049
beverages (50%, v/v fruit juice and 41.5% of soy milk), containing
approximately 137–220 and 122–158 μg/100 mL carotenoids
for the milk, respectively, and 24–58 μg/100 mL carotenoids
for the soy-based beverages. Corresponding bioaccessibilities
of total carotenoids were 39–99% and 13–47% for the whole-
and skimmed-milk–based beverages, respectively, and 18–73%
for the soy-based beverages, indicating that the lower amount
of fat in the skimmed milk products reduced bioaccessibility.
Regarding individual carotenoid bioaccessibility, no consistent
strong differences were encountered between β-carotene,
β-cryptoxanthin, lutein, zeaxanthin, and violaxanthin/neox­
anthin, which is of interest as typically, the bioaccessibility of the
more apolar carotenoids (β-carotene, lycopene) is lower than that
of the more polar xanthophylls such as lutein. Both heat
treatments resulted in decreased fractional bioaccessibility; the
reasons for this are not quite clear, but it could have included
enhanced crystallinity of carotenoids or negative interactions
with fiber, which is not uncommon, for example, in high-pressure
processing, which may enhance the stability of the fiber
network (78) in vegetable-containing beverages.
The positive influence of dietary lipids on carotenoid
bioavailabilty has been well described (26, 27). As mixed
micelles require emulsifying constituents, the digestion of
triacylglycerides, producing free fatty acids, monoglycerides,
and diacylglycerides, which can act as emulsifiers, aided in their
bioaccessibility (79). For example, adding whole milk versus
skimmed milk to a mixture of persimmon enriched milk (as a
carotenoid source) drastically improved the bioaccessibility of
various carotenoids by up to 4-fold (80). Similarly, addition
of full-fat milk to wolfberries enhanced the bioavailability of
zeaxanthin in a human trial 3-fold, compared with a wolfberry–
skimmed milk formulation (81). Also, it has been assumed that
additional lipids can foster chylomicron formation and thus
result in the further transport and absorption from carotenoids
already present in the enterocytes (82).
Although the generally positive influence of lipids on the
bioaccessibility and bioavailablity are undisputed, it remains
less clear whether short-chain fatty acids such as in milk fat
really aid in the micellization of apolar microconstitutents.
Several studies have suggested that rather the more long-
chain fatty acids may enhance carotenoid micellization and
dietary uptake, as contrary to short-chain fatty acids (which are
absorbed via the portal vein and are more water soluble), they
require also micellization and uptake via chylomicrons (83, 84),
fostering their formation. However, other studies have reported
higher bioaccessibility of carotenoids from butter fat than olive
oil and peanut oil, as the shorter fatty acids and monoglycerides
and diglycerides appeared to contribute to a higher absolute
zeta-potential, stabilizing the micelles (85, 86). Thus, there may
be more specific matrix–lipid type interactions than previously
anticipated which determines bioavailability.
In another study, the addition of fat-free milk to fruit juice
showed a significant positive effect on the bioaccessibility of
lutein, zeaxanthin and β-cryptoxanthin (although not significant
for β-carotene) following simulated gastrointestinal digestion,
also highlighting that positive effects of the addition of milk
may be because of additional factors other than lipids (87). For
example, it was suggested that casein-phospholipids may chelate
iron, which could otherwise result in oxidative breakdown of
carotenoids (88), and the presence of casein micelles per se
may contribute to enhanced carotenoid bioaccessibility (89).
However, again, the micelles formed may be larger than the
normally formed mixed micelles containing bile salts and may
not contribute to the same extent to bioaccessibility compared
with typical mixed micelles (66).
Therefore, in contrast to the positive effect of lipids, not
much is known on the effect of proteins regarding carotenoid
bioaccessibility or bioavailability. As peptides produced during
digestion may have emulsifying properties and may protect
carotenoids from oxidation via chelating iron or forming a
surface film around lipid droplets, it could be speculated that they
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aid in carotenoid micellization and bioaccessibility, as reviewed
previously (90). On the other hand, they may also reduce
enzymatic access to lipid droplets and reduce the transition
of lipid droplets to mixed micelles, hampering micellization.
Thus, the effects of proteins may be more mixed, depending on
digestive conditions (91) and require more detailed investigation.
A summary of factors influencing carotenoid bioavailability
from infant food formulas and adult nutritionals is given in
Table 2. In short, the bioavailability of carotenoids from milk
products appears to be good or even high, at least when present
in native form, i.e., in nonfortified products, because of the
embedding of carotenoids in liquid form in a lipid matrix that
ensures their protection and a good micellization, owing to
the presence of emulsifying constituents supporting micelle
formation, including lipid digestion products, phospholipids,
and proteins. However, fortified products appear to show
much lower bioavailability, even although the bioaccessibiltiy
typically measured following in vitro digestion and filtration
(200 nm filters) may not be compromised, for reasons that
may be related to different types and sizes of micelles formed;
however, this remains hypothetical.
Analytical Methods for Carotenoid Isolation and
Quantification
Analytical methods may be separated into the isolation/
purification/concentration steps and the actual quantification.
Major challenges for carotenoid determination in infant food
formula and adult nutritionals include the following:
(1) The relatively low concentration of carotenoids, i.e., below
20 μg/100 mL for nonfortified items, and up to approximately
200 μg/100 mL for fortified products.
(2) The relatively high protein and fat content of the food
products, which may perturb the extraction of the lipophilic
carotenoids because of the coextraction of large quantities of
lipids and difficulties in their purification, unless the lipids are
saponified beforehand, which bears the risk of carotenoid losses.
In the following, the main isolation/purification/concentration
and identification/quantification methods (that can be) used for
the analysis of carotenoids in infant food formulas and adult
nutritionals are described.
Analytical Methods – Extraction and Purification
of Carotenoids
Liquid–Liquid Extraction
The most common methods of extraction for carotenoids
include liquid–liquid extraction (LLE) and solid-phase
extraction (SPE). LLE typically includes a fairly strong apolar
and nonwater miscible solvent of reasonable low boiling point
1050  Bohn: Journal of AOAC International Vol. 102, No. 4, 2019
Table 2.  Dietary and technological factors likely influencing bioavailability from infant food formula and adult nutritionals
Factor Positive or negative influence
a
Synthetically added carotenoids ↓ compared with natively
present carotenoids
b
Presence of lipids ↑ Improved micellization with higher levels of fat due to
c
Milk fat ↑↓
Phospholipids ↑ Enhanced emulsifying capacity.
High-pressure homogenization ↓↑
(chromoplasts, cell clusters), effects on milk products unclear.
Proteins ↓↑
Presence of mineral mixes ↓ Higher concentrations of divalent lipids may reduce carotenoid
a
  ↓ = Negative effects.
b
  ↑ = Positive effects.
c
  ↑↓ = Mixed effects.
with good extractability for carotenoids (Figure 2). As for infant
food formula and adult nutritionals, these carotenoids include
β-carotene (logP 11.12), lutein (logP 8.55), and, to a lesser
extent, lycopene (logP 11.92) (43). Typical solvents have been
including hexane (30) petroleum ether (92), diethyl ether (68),
tetrahydrofuran (THF; 49), methyl-tert-butyl-ether (MTBE; 49)
and chloroform/dichloroethane or ethyl acetate, as reviewed
earlier (93). When THF is used, because of the possible formation
of peroxides, an antioxidant such as butylated hydroxytoluene
(BHT) should be added (93). Also, the use of an internal
standard to account for losses during extraction, such as
β-apo-8′-carotenal, may be recommended. However, as the
miscibility of these solvents with water is limited, a solvent
carrier such as acetone (30) is typically added in order to
foster the transition of carotenoids into the apolar phase. The
application of a binary mixture is also an advantage for a
mixture of xanthophylls such as lutein and carotenes such as
β-carotene, as the prior are better dissolvable in more polar
organic solutions such as MTBE, diethyl ether, and acetone,
whereas carotenes are better dissolvable in more apolar solvents
such as hexane. Extraction with diethyl ether requires further
drying with sodium sulfate because diethyl ether tends to bind
small quantities of water.
As carotenoids have a long chain-like structure, it is assumed
that the solubility and extractability in hexane or petroleum
ether is usually good. Indeed, in a previous study on their
extraction from rapeseed, among the tested solvents (acetone,
petroleum ether, methanol, chloroform, and petroleum
ether–acetone; 1+1), the latter combination had the strongest
extraction capacity (92). Applying ultrasonication, heat (50°C)
and a high solvent:material ratio (40:1) further improved
extractability. The solvents that were used in a validated method
to detect carotenoids in infant formula and adult nutritionals
included extraction with a mixture of MTBE–THF (1+1, v/v)
Type of influence Ref.
Higher crystallinity, presence not in milk fat globules, (55, 65, 66)
different micelle structure, lower cellular uptake.
(14, 26, 28)
formation of emulsifying compounds (mono-diglycerides),
higher bioavailability.
Negative effects because of the presence of short-chain fatty (83, 84)
acids not absorbed via chylomicrons in some studies;
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Other studies suggest positive effects via smaller mixed (85, 86)
micelles rich in milk fats.
(150, 151)
Negative effects in vegetable matrices possible, although (152)
no negative effects in matrices with structural barriers
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.
(30, 31, 57)
absorption, although effect only shown for Ca at high
physiological concentrations and not in all studies.
followed by hexane (50) and hexane–acetone (1+1, v/v; 49),
with both methods yielding a high recovery (Table 3). Spiking
was done with externally added carotenoids and may not fully
represent the extractability of the native carotenoids from their
matrix. However, following LLE, which is typically repeated
for a total number of three extractions, the combined phases are
evaporated to dryness, typically under a stream of nitrogen, or,
alternatively, with a rotary evaporator under reduced pressure,
especially for larger volumes.
As xanthophylls may be present in the form of esters,
including those present in dairy-based products, a prior
saponification with potassium hydroxide is recommended to
simplify later quantification. In addition, this will saponify
triglycerides (and also phospholipids), which would otherwise
be coextracted in the following LLE, perturbing further
concentration and purification of the extracts. Saponification
has also been used during the extraction of carotenoids from
plant matrices, without coextracting chlorophylls that could
potentially interfere during chromatographic analysis (94). In
this case, the addition of BHT as an antioxidant (0.02–0.1%)
or ascorbic acid (e.g., 3–5%, w/v) may be advised (93). Within
a typical saponification procedure, dried powder (infant food,
adult nutritional, about 1 g) is taken up in a volume of, e.g.,
15 mL methanol–water (2+1, v/v), containing a final concen­
tration of 3% KOH, and the mixture heated, for 15 min at
60°C (49); however, different concentrations, times, and tempe­
ratures have been applied, with higher lipid content typically
requiring stronger conditions, as reviewed earlier (94). Shorter
times but higher concentrations of KOH have also been applied,
using, for example, 10 mL 5% KOH to 2 mL formula for 1 min.
Typically, conditions ranging from exposure times of
30 min at room temperature (RT) to 1 h at 80°C have been
applied. Choosing more drastic conditions increases the risk
of carotenoid degradation. Biehler et al. (95) reported losses
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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
using 3.75% KOH for 15 mins at RT, although without the
addition of any added antioxidant.
SPE
Instead of LLE, SPE with silica or, more commonly,
reversed-phase (RP) material with C18 alkyl-chains can be used
to purify carotenoids. Shen et al. (96) compared the retention
of C18, C30, diol, and silica sorbents for extracting β-carotene
and lutein from human plasma and breast milk, followed by
eluting with acetone. They concluded that C18 and C30 had
highest retentions. However, protein removal was needed
beforehand by precipitation, and both matrices were low in
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
Lichrolut C18 phases were compared for the extraction of
lutein and β-carotene from cereals (97), concluding that the
Oasis HLB followed by dichloromethane as an eluent worked
best. Additionally, proteins had to be removed by ethanol
precipitation first (Figure 2), and better results were obtained by
a preceding saponification. However, following elution, further
concentration by evaporation under nitrogen gas or by means of
a rotary evaporator is usually carried out.
1052  Bohn: Journal of AOAC International Vol. 102, No. 4, 2019
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)
LLE Hexane–MTBE (75+25, v/v)
Carotenoid identification All-trans-lutein, 13-cis lutein, 13′-cis-lutein, zeaxanthin,
all-trans β-carotene, 9-cis β-carotene, 13-cis
β-carotene, all-trans-lycopene, unknown-cis-lycopene
a a
1 , unknown-cis-lycopene 2 , 5-cis-lycopene
Repeatability, % 1.9–18.7 all-trans-lutein, 1.9–10.1 all-trans β-carotene,
3.1–6.3 all-trans-lycopene
Intermediate precision 1.9–23.9 all-trans-lutein, 3.5–54.4 all-trans β-carotene,
(reproducibility), % 4.8–10.7 all-trans-lycopene
Recovery, % 90.3–95.3 lutein, 89.3–108 β-carotene, 97.3–109
b b
lycopene
Linearity (μg/L) 0.99991 lutein (10–250), 0.99993 β-carotene
(25–200), 0.99980 lycopene (5–100)
LOQ, μg/100 g 0.4 lutein, 0.1 β-carotene, and 0.3 lycopene
a
  Tentatively identified.
b
  Unless otherwise specified, it is referred to the all-trans form.
Supercritical Fluid Extraction
In addition to the more classical LLE and SPE, supercritical
fluid extraction with CO2 has been used as a rapid method of
carotenoid isolation from the matrix, making use of the lipophilic
character of the carotenoids. A further advantage of this method
is the potential for upscaling and being compatible with food
production facilities as it is solvent free; a drawback remains
the still-high operating costs of this method. For example,
extracting carotenoid from algae was reported to be highest at
200 bar and 60°C (98), similar to other reported algae extraction
methods (300 bar, 50°C; 99). This method has also been used
for carotenoid extraction from other matrices such as pumpkin
rich in α- and β-carotene, β-cryptoxanthin, and lutein (35 bars,
50–70°C), with a preceding vacuum drying resulting in the
highest extraction capability enhancing tissue disaggregation
(100). A further enhancement of the extraction can be achieved
by the addition of small quantities of other solvents, such as water
or ethanol, to CO2. Indeed, such entrainers (a combination of
10% water or ethanol and 10% olive oil) enhanced the extraction
yield of carotenoids from pumpkin (100). Supercritical CO2 has
also been used for extracting β-carotene from infant food
formulas (101), with recoveries of 70% compared with
conventional extraction, although a higher sample throughput
and smaller sample volume required were emphasized. Further
research in this area to improve extraction conditions for
complex matrices is warranted.
Methods of Detection
Spectrophotometric Methods
Spectrophotometric methods making use of the high molecular
extinction coefficients of most carotenoids (between 110 000–
180 000 L × mol–1 × cm–1) because of the delocalized π-electron
system are generally very sensitive and the most applied method for
carotenoid detection. In addition, most carotenoids have a typical
and relatively specific spectrum, giving an additional possibility for
Corresponding performance method (50) Remarks
MTBE–THF (1+1, v/v), hexane
13-cis lutein, 13′-cis lutein, zeaxanthin,
β-apo-8′-carotenal, 13-cis β-carotene,
15-cis β-carotene, α-carotene, all-trans
β-carotene, 9-cis β-carotene,
all-trans-lycopene
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0.1–6.2 all-trans-lutein and 0.4–47.4 all- High variation with
trans β-carotene high-fat product
1.6–5.9 or all-trans-lutein and 0.7–4.6 all-
trans β-carotene
92.3–105.5 lutein and 100.1–107.5
β-carotene
1.0000 for β-apo-8′-carotenal, β-carotene,
lutein
0.27 lutein and β-carotene
their identification (102). Most carotenoids of relevance for infant
food formula and adult nutritionals such as lutein, β-carotene,
and lycopene absorb in the area of 450–470 nm, depending slightly
on the solvent, with extinction maxima of 445 nm for lutein
(144 900 L × mol–1 × cm–1 in acetone; 95), 452 nm for β-carotene
(140 663 L × mol–1 × cm–1 in acetone; 95), and 470 nm for
lycopene (182 000 L × mol–1 × cm–1 in hexane; 103).
The major disadvantage of simple spectrophotometric
methods without prior separation of carotenoids by chromato­
graphic methods is the impossibility of quantification of
individual carotenoids in a mixture because of their general
over­lap of absorption spectra. However, they are fast and quite
affordable. Although methods have been described to estimate
the total amounts of carotenoids in mixtures, based on pro­
posing a typical mean molecular extinction coefficient and a
wavelength suitable for most carotenoids, this method allows
only for a reasonable estimate. For example, Biehler et al. (95)
proposed a method for determining carotenoids in a number of
fruits and vegetables following saponification, using a mean of
135 310 L × mol–1 × cm–1 at 450 nm, with results being very
close to the HPLC method (on average, 1.4% difference but
with larger deviations for individual food items) and worked
reasonable well for vegetables rich in both β-carotene and
lutein, which may imply that a crude estimation of carotenoids
in infant food formula and adult nutritionals is possible. Other
methods, in 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.
Liquid Chromatography
General aspects – stationary phase and eluent.—As
carotenoids are relatively large molecules which are heat
sensitive and prone to degradation, LC methods, that is HPLC
and ultra-HPLC (UHPLC), have been the method of choice
for separating individual carotenoids and their isomers.
Several reviews exist in general on this topic, summarizing
 Bohn: Journal of AOAC International Vol. 102, No. 4, 2019  1053
LC methods for carotenoid detection (106–110). Although thin
layer chromatography methods have also been reported and
reviewed (111), these are typically rather low in resolution and
sensitivity compared with HPLC/UHPLC; however, they are
more affordable, do not require expensive equipment, and can
be used as a semi-quantitative screening tool.
Few reports exist on separation/detection methods focusing
specifically on infant food formulas and adult nutritionals,
although validated and sufficient sensitive methods developed
for other matrices may be equally well used. Regarding HPLC/
UHPLC, a first distinction regarding normal phase versus RP
can be made, the latter one allowing the injection of water
containing mixtures, which may be an asset for detecting a
wide range of carotenoids. For this reason, and for the more
recent development of RP-C30 columns, RP chromatographic
methods are dominating. The C30 phase consists of a longer
brush type of alkyl chains, which interacts well with the
carotenoid backbone. Consequently, geometrical isomers as
well as the often-difficult separation of lutein and zeaxanthin
can be achieved better in general compared with C18 RP
columns, as previously reviewed (112).
Regarding the elution of carotenoids, both isocratic (113) and
nonisocratic gradient methods (82, 114) have been developed,
with nonisocratic methods generally allowing the separation of
a wider range of carotenoids in a shorter period of time, possibly
minimizing diffusion. However, seven or more carotenoids
have been resolved with an earlier isocratic method using
5% THF and 95% methanol (113). Although gradient methods
have been typically used to optimize RP chromatographic
methods, normal phase, typically silica-based methods, have
instead used isocratic methods (115), in part as when these
methods were developed, pumps allowing running binary
eluents were less common. Regarding solvents used today for
RP chromatography, binary mixtures are most commonly used
(e.g., methanol–water–based systems versus those being more
apolar such as containing acetonitrile or MTBE). For example, a
mixture of methanol–water–ammonium acetate–MTBE (88+5+
2+5, v/v) and MTBE–methanol–water–ammonium acetate
(79+16+3+2, v/v) was used by Biehler et al. (95) to separate
10 carotenoids within 40 min. It is thought that the ammonium
acetate reduces the tailing of carotenoid peaks.
A variation of LC that is not commonly available because of
the more sophisticated equipment required is supercritical fluid
chromatography. For example, a mixture of eight carotenoids
extracted from microalgae were separated on two columns in
series (C18 combined with a normal-phase column) within
16 min, using liquid CO2 and methanol as the mobile phase in
a gradient method, increasing methanol concentration over time,
with a flow rate at 5 mL/min and a pressure of 120 bar at 32°C.
The advantages of this method include low solvent consumption
and a possible fast separation time. Further information about this
method is available elsewhere (116–118). No report on using this
method for infant food formula or adult nutritionals is available;
however, this method has been used to detect other lipophilic
constituents including retinol from infant formulas (119).
Published Methods Investigating Carotenoids in Infant
Food Formulas and Adult Nutritionals
In the two AOAC methods validated specifically for infant
food formula and adult nutritionals, C30 columns were used in
conjunction with a binary mixture of 20 mM ammonium acetate
in methanol–water (98+2, v/v) and 100% MTBE in the method
of Hostetler (50) to separate carotenoids within 32 min, whereas
in the method of Schimpf et al. (49), carotenoids were separated
within 30 min, using a C30 column and a binary gradient
with methanol–MTBE (85+15, v/v) and pure MTBE. Both
methods allowed for a good separation of all-trans-β-carotene,
lutein, and lycopene, as well as several of their more common
cis-isomers (Table 3).
Several earlier reports have been published investigating
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carotenoids in infant food formulas. In the study by Yuhas
et al. (120), lutein was detected after a C30 column separation,
following an unspecified saponification and the extraction
with ethanol, with a binary system of MTBE and methanol,
but the method was only validated down to a concentration of
25 μg/L of lutein. In a study by Jewell et al. (121), samples
were saponified by combining 200 μL milk/formula with
150 μL 50% aqueous KOH and 150 μL ethanol and 100 μL
internal standard echinenone to account for losses during sapon­
ifi­cation, followed by extraction with hexane and separation
on a C30 column, with a binary mixture of methanol–MTBE
(89+11, v/v) and methanol–MTBE (62+38, v/v), allowing for the
detection of zeaxanthin, lutein, β-cryptoxanthin, α-cryptoxanthin,
β-carotene, and α-carotene. Concentrations as low as 0.2 nmol/g
fat (approximately 1 μg/g fat) could be detected, suggesting a
detection limit lower than 50 μg/100 g, although this was not
clearly specified. A similar method was reported by Johnson
et al. (122) to separate isomers of β-carotene from breast
milk, reporting a LOD of approximately 0.2 pmol (approximately
0.1 ng), and measured concentrations of 0.4–2.7 μM β-carotene
and 0.05 and 0.4 μM for 9-cis β-carotene.
Some methods do further detect additional apolar compounds.
For example, Woolard et al. present an HPLC normal column
method for measuring β-carotene in infant food formula,
also detecting vitamin E and vitamin A, using all-E-β-apo-
8′-carotenoic acid ethyl ester as the internal standard (123), by
means of UV-Vis detection for carotenoids and fluorescence for
vitamin A/E.
Liquid chromatography with alternative detection methods.—
In addition to coupling HPLC to UV-Vis, electrochemical
detection such as with Coularray-detectors have been used for
quantifying carotenoids (82, 124). This method is generally
more sensitive than UV-Vis, although possibly not by much for
the carotenoids with a very high molecular extinction coefficient
around 100 000–150 000 L × mol–1 × cm–1. There was an
improvement by a factor of 10–100 for lycopene detection
with this method, detecting as low as 50 fmol (approximately
0.025 ng) with a C30 RP method during 50 min (125). However,
a typical detector is also several times more expensive compared
with UV-Vis. As the possibility to measure UV-Vis absorption
spectra is lost, the possibility to study different oxidation
potentials is gained, and sometimes coeluting peaks can be
resolved electrochemically, thus this method may have some
complementarity to UV-Vis.
Finally, in addition to UV-Vis and electrochemical detection,
fluorescence has been used for detecting individual carotenoids,
especially phytofluene, which is less sensitive for UV-Vis
detection than other carotenoids because of its lower molecular
extinction coefficient (102) and its sufficient fluorescence
intensity when excited at 346 nm, measuring emission intensity
at 520 nm as reviewed previously (126). For β-carotene,
1054  Bohn: Journal of AOAC International Vol. 102, No. 4, 2019

fluorescence detection has also been reported, with 480 and
560 nm of excitation and emission, respectively (127), but
fluorescence strongly dependeds on the solvent. Fluorescence
has also been applied to study algae carotenoids (predominantly
β-carotene) by microscopic methods, with excitation
wavelengths of 450/488 and emission being highest at 680 nm
(128). However, as fluorescence is not generally applicable to
carotenoids at high sensitivity and because of the higher costs of
the detector, this method is not commonly applied.
matrices, has been carried out by Raman spectroscopy. This
technique measures vibrational, rotational, and other low-
frequency modes of selected compounds induced by inelastic
scattering from a laser source. The strength of this method rests
in the detection of carotenoids in the original matrix without
the need of extraction/purification or separation. The potential
of Raman spectroscopy for measuring carotenoids, although in
microorganisms, has been previously reviewed (140). Various
wavelengths for excitation (514, 488, 844, and 1064 nm) may
be used, and spectra between 100 and 800 cm-1 are typically

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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
geometrical isomers. However, the strength of LC-MS/MS
UV-Vis, especially following prior separation by HPLC/
rests in the possibility to detect less UV-active constituents such
UHPLC are the most commonly used methods and are generally
as 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 CO2 are applied, separation has most often been achieved
Regarding the different types of MS, ion-trap (136),
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
 Bohn: Journal of AOAC International Vol. 102, No. 4, 2019  1055
as prevention of age-related macular degeneration in the elderly.  
Dairy products may constitute a good vehicle for these lipophilic
compounds, as bioavailability as a result of the presence of lipids,  
phospholipids, and possibly proteins is reasonable or good.
Whether additional carotenoids, i.e., other than lutein, β-carotene,
 
and lycopene used at present, will be employed to fortify these
food items remains to be seen, but it is possible. This and the fact  
that processing increases the risk of trans-cis isomerization, and
possibly also the formation of carotenoid degradation products/  
metabolites such as apo-carotenoids, requires the presence of
sensitive and accurate methods to measure carotenoids in such  
matrices. At the present, extraction with an apolar solvent, in
conjunction with saponification (KOH) to liberate xanthophyll
esters and to avoid coextraction of dietary lipids, together with  
an antioxidant, followed by C30-based LC appears state of the
art. Improvements such as faster separation based on UHPLC and  
methods allowing the detection of further isomers and degradation
products/metabolites are expected in the near future. Also, the
development of improved in situ techniques, perhaps via Raman  
or other spectrometric methods, may be achievable and would be
welcome.
 
Acknowledgments
 
The insights obtained in the EU COST Actions  
EUROCAROTEN (CA 15136) and POSITIVE (FA 1403) are
much appreciated.  
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