Trends in Analytical Chemistry 56 (2014) 49–73

Contents lists available at ScienceDirect

Trends in Analytical Chemistry

journal homepage: www.elsevier.com/locate/trac

Review

Review of methods for analysis of carotenoids q

K.T. Amorim-Carrilho, A. Cepeda, C. Fente, P. Regal ⇑
Laboratorio de Higiene e Inspección de Alimentos (LHICA), Department of Analytical Chemistry, Nutrition and Bromatology, Faculty of Veterinary Medicine,
University of Santiago de Compostela, Lugo, Spain

a r t i c l e i n f o a b s t r a c t

Keywords: This review covers current analytical techniques, instruments and methodologies used in the analysis of
Analytical method carotenoids in foods and human samples. We also cover the importance of carotenoids for human health,
Bioavailability
carotenoid content in foods, bioavailability of carotenoids and evaluation of human intake of carotenoids.
Carotenoid
There are a wide variety of extraction methods and analytical techniques for determination of
Extraction
Food analysis
carotenoids. Recent advances in analytical instruments and the discovery of unknown metabolites of
Food composition carotenoids widened the scope of carotenoid studies, especially through the application of metabolomics
Foodomics tools. Omics instruments and statistical methods perform untargeted and targeted profiling of carotenoids
Human sample in foods and human samples, thus advancing knowledge of the composition of food containing
Metabolomics carotenoids and their role in human health. Aimed at collating valuable information about recent analytical
Profiling methodologies for carotenoids, this review mainly focuses on studies released in the past five years (2009–13).
Ó 2014 Elsevier Ltd. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
2. Carotenoids in human diet . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
2.1. Carotenoid content of food . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
2.2. Bioaccessibility and bioavailability. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
2.3. Evaluation of intake of carotenoids in humans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52
3. Analytical methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
3.1. Extraction methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
3.1.1. Saponification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
3.1.2. Carotenoid extraction in human samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
3.1.3. Carotenoid extraction in food samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
3.1.4. Other extraction techniques. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
3.2. Determination methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
3.2.1. Separation methods: liquid chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
3.2.2. Detection and identification methods: mass spectrometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66
3.2.2.1. LC-MS interfacing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66
4. Modern approaches and foodomics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67
5. Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69

1. Introduction

Carotenoids are lipid-soluble pigments responsible for the color

q
This paper was originally submitted to the recently published Special Issue: of a wide variety of foods. They may be divided into two groups:
‘Modern Food Analysis and Foodomics’ (http://www.sciencedirect.com/science/
journal/01659936/52).
⇑ Corresponding author. Tel.: +34 982 285900x22484; Fax: +34 982 254592.  xantophylls, molecules containing oxygen, such as lutein and
E-mail address: patricia.regal@usc.es (P. Regal). zeaxanthin; and,

http://dx.doi.org/10.1016/j.trac.2013.12.011
0165-9936/Ó 2014 Elsevier Ltd. All rights reserved.
50 K.T. Amorim-Carrilho et al. / Trends in Analytical Chemistry 56 (2014) 49–73

 carotenes, non-oxygenated molecules such as a-carotene and following sections, we review in detail the current analytical
lycopene [1]. methodologies and extraction procedures for determination of
carotenoids. Table 1 shows some of the most important carotenoid
Some of them are pro-vitamin A carotenoids, subsequently compounds.
transformed into vitamin A, which can prevent serious eye dis-
eases, such as night blindness, susceptibility to infection, rough,
2. Carotenoids in human diet
scaly skin, and retarded tooth and bone development. There are
about 700 carotenoids in nature, but only about 50 have pro-vita-
2.1. Carotenoid content of food
min A activity. Of those 50 compounds, we found the three most
important precursors of vitamin A in humans to be a-carotene,
Although carotenoids are present in many common human
b-cryptoxanthin and b-carotene, which are the major pro-vitamin
foods, deeply pigmented fruits, juices and vegetables constitute
A components of most carotenoid-containing foods [2,3].
the major dietary sources with:
The consumption of carotenoids, provitamin A or not, has been
associated with a number of health benefits, such as cancer chemo-
 yellow-orange vegetables and fruits providing most of the b-
protection [4], prevention of heart and vascular disease [5], and
carotene and the a-carotene;
prevention of other chronic diseases (e.g., cataracts and age-related
 orange fruits providing a-cryptoxanthin;
macular degeneration) and degenerative diseases (e.g., Alzheimer’s
 dark green vegetables and egg yolk providing lutein and zea-
Disease) [6–8].
xanthin and tomatoes and tomato products lycopene [29].
Almost all carotenoids, to a greater or lesser degree, show
scavenging properties against excessive numbers of free radicals
As examples, good sources of b-carotene include collard, turnip,
that may be produced over the course of a cell’s life cycle [9]. This
spinach, lettuce, mangos, cantaloupe melons, peppers, pumpkin,
antioxidant capacity has been the most investigated and it has been
carrots and sweet potato. a-Carotene is found in a limited number
suggested as the main mechanism of action of the carotenoids.
of orange vegetables, such as carrots, sweet potato, pumpkin, and
However, other mechanisms have been reported and some of the
dark green vegetables, such as broccoli, green beans and spinach.
most exciting progress in understanding the actions of carotenoids
Food sources of a-carotene also tend to be good food sources of
have come out of recent investigations on the effect of carotenoids
b-carotene. Lycopene occurs primarily in tomatoes, as stated be-
on cellular processes [10] (e.g., gene modulation, cell cycle, cell-cell
fore, in watermelon and guava. b-Cryptoxanthin can be found in
communication, cytotoxicity and apoptosis) [11–17]. As an
mandarin, orange, papaya, corn, peas and egg yolks. Good sources
example, Teodoro et al. [18] treated human cell lines with lycopene,
of lutein and zeaxanthin are leafy greens, such as spinach, collard
showing its capacity to inhibit cell proliferation, to arrest cell cycles
and kale, and other foods, such as corn, persimmons, and broccoli.
in different phases and to increase apoptosis, mainly in breast,
Carotenoids can also be found in the animal kingdom (bird
colon and prostate lines after 96 h of treatment. The authors
plumage, fish, crustaceans, and insects), and astaxanthin and can-
indicated that the anti-proliferative effect of lycopene depended
taxanthin are found in salmon and crustaceans. However, animals,
on cellular type, time and dose.
(including humans) cannot synthesize carotenoids, so food is their
Some studies have shown that carotenoids may mediate their
only source of these compounds [4,30–32].
effects via gap-junction communication (GJC), which allows the
But, the level of a particular phytochemical, such as carotenoids,
direct transmission of ions, small hydrophilic metabolites, and
varies in different fruits and vegetables, according to the variety
messengers <1–2 kDa in size among neighboring cells. GJC plays
cultivated. In addition to dependence on cultivar variation, the con-
an important role in normal development and physiology, and
tent of phytochemical substances is influenced by numerous fac-
failure of GJC has been related to various human diseases and
tors, such as ripening time, genotype, cultivation techniques, and
pathologies [5].
climatic conditions that occur during the pre-harvest period [33,34].
Several studies have highlighted the ability of carotenoids to
Carotenoid content may vary even according to the part of the
modulate gene expression [13,18–24]. Genetic variations may be
plant. For example, the studies performed by Ranveer et al. [35]
involved in inter-individual variability in carotenoid metabolism
on tomato revealed that the peel has the highest amount of lyco-
and carotenoid status [25], and responsiveness to specific caroten-
pene, followed by industrial waste, whole tomato and pulp.
oid diets were associated with individuals with genetic variants of
Other than pre-harvest factors, various post-harvest steps,
the carotenoid-metabolizing enzyme b-carotene 15, 150 -monooxy-
including food-processing operations, also have a great influence
genase [26].
on the stability of phytochemicals in fruit and vegetables and in
Regarding the role of carotenoids in DNA integrity, Azqueta
their products. Conventional (thermal), modern or non-thermal
and Collins [27] reviewed the positive effects of vitamin A and
(e.g., high-pressure processing, pulsed electric field, ultrasound/
pro-vitamin A carotenoids (the carotenes and b-cryptoxanthin)
sonication, ozone and ultraviolet), domestic (e.g., washing, peeling,
and non-pro-vitamin A carotenoids (lycopene, lutein, astaxanthin
and cutting) and industrial (e.g., canning, and drying) processing
and zeaxanthin) on DNA damage. Santocono et al. [9] evaluated
may significantly degrade the level of phytochemicals in food
the ability of the zeaxanthin, lutein, and astaxanthin to protect
products [36]. Also, the results obtained by some authors suggest
SK-N-SH human neuroblastoma cells against DNA damage induced
that cooking methods may influence the carotenoid content [35].
by different RNOSs (reactive nitrogen oxide species) and found that
their ability to reduce DNA damage depends on the type of RNOS
donors and the carotenoid concentration used. The immune 2.2. Bioaccessibility and bioavailability
response was reviewed by Hughes [28] and, considering its impor-
tance, that needs to be updated. Many bioactive compounds (e.g., carotenoids) must be released
It is important to highlight that cell-line and animal findings from the food matrix and reach their site of action to exert their
cannot be directly extrapolated to humans and that the definitive biological effects, so bioaccessibility and bioavailability are critical
scientific evidence will come from human studies. Providing more features in assessing the role of these compounds in human health
details about the mechanisms of action of carotenoids and their [37,38]. Bioaccessibility represents the maximum amount of
function on human health was not the aim of this review which carotenoids released from the food that are available for absorption
can be easily found in the review articles cited above. In the in the enterocytes [39], while the fraction of the dose entering the
 K.T. Amorim-Carrilho et al. / Trends in Analytical Chemistry 56 (2014) 49–73 51

Table 1
Names, chemical formulas, structures and molecular weights (MW) of some of the main carotenoid compounds

Basic carotenoid structure

Compound R Y MW
(average)
Lycopene /w,w-Carotene C40H56 536.873

b-Carotene/b,b-Carotene/b,b-Carotene C40H56 536.873

Astaxanthin/(3S,30 S)-Astaxanthin/(3S,30 S)-3,3’-Dihydroxy-b,b-carotene-4,40 -dione C40H52O4 596.838

Fucoxanthin/(3S,5R,6S,30 S,50 R,6’R)-5,6-Epoxy-30 -ethanoyloxy-3,50 -dihydroxy-60 ,70 -didehydro- 658.906

5,6,7,8,50 ,60 -hexahydro-b,b-caroten-8-one C42H58O6

Zeaxanthin/(3R,30 R)-Zeaxanthin/(3R,30 R)-b,b-Carotene-3,30 -diol C40H56O2 568.871

Lutein/LuteinA/(3R,30 R,60 R)-b,e-Carotene-3,30 -diol C40H56O2 568.871

b-Cryptoxanthin/(3R)-b,b-Caroten-3-ol C40H56O 552.872

systemic circulation to participate in physiological functions (or
stored for later use) is called bioavailability [40]. Bioaccessibility
is part of bioavailability. Nowadays, there is no clear consensus
on the best approach to estimate carotenoid bioavailability. Con-
sidering that no animal model completely mimics human absorp-
tion and metabolism of carotenoids, and that human studies are
labor intensive and expensive [41], in-vitro studies are less expen-
sive and faster tools for bioavailability studies. Also, application of
in-vitro digestion and assimilation procedures is suitable to gain
information about factors that modulate different steps of caroten-
oid bioaccessibility and bioavailability [42].
In-vitro digestion models can be classified as static or dynamic.
The static models characterize chemical digestion but they do not
take into account the muscle contractions of the gastrointestinal
(GI) tract, which generate the mechanical forces and fluid motions
that promote not only the mechanical break-down of the food, but
also its chemical digestion, absorption and transport [43].
Accordingly, dynamic in-vitro models have the advantage of closely
mimicking in-vivo conditions. Despite this advantage, the most
widely-used model to evaluate bioaccessibility of carotenoids has
been static in-vitro digestion coupled with a Caco-2 cell absorption
[44]. This model is based on a human-colon adenocarcinoma-cell
line, which has many characteristics of the small intestinal epithe-
lium. This pioneer model, developed by Garrett et al. in 1999 [45],
was the first in-vitro digestion assay specifically applied to esti-
mate carotenoid bioaccessibility. Several researchers have applied
in vitro methods to simulate the digestion process in the GI tract
and to estimate the release of carotenoids from food after digestion
[46–48], in some cases using modifications of Garrett’s method
[49,50]. Thus, most models used nowadays in carotenoid-bioacces-
sibility studies are static and are not standardized. These models
present similar general steps but different methodological details.
Apart from the restricted digestion simulation of these static
in-vitro models, the use of different sub-clones, different time
points of post-confluence (2–21 days) and the way of introducing
carotenoids to the cells (e.g., various concentrations, digesta or
artificial micelles, and different culture conditions) constitute a
further obstacle to the comparison of results between static studies
and studies from different laboratories [51].
To overcome the limitations of static digestion procedures and
to get more realistic results, some studies have developed in-vitro
dynamic digestion models, most of which are computer controlled
and some of which have been applied to evaluate bioavailability of
carotenoids in food. For example, the application of the Human
Gastric Simulator (HGS) model permitted the observation of a
greater release of b-carotene from oil-fortified almond butter than
in the same study performed with static models [44]. HGS is a dy-
namic gastric digestion model where the effect of peristaltic move-
ments of stomach walls, pH, gastric emptying, contraction, and the
size of food particles in digesta and nutrient release and rheological
properties can be studied simultaneously [52].
TNO GI tract model (TIM-1) reproduces the stomach and the
three compartments of the human small intestine. The mixing of
the chime and its transit through compartments, as well as the
pH, enzymes and salts, are computer-monitored and continuously
controlled [53]. In particular, the TIM-1 system has shown its use-
fulness in studying the digestive stability of carotenoids from dif-
ferent food matrixes throughout the GI tract. Blanquet-Diot et al.
[54] assessed the digestive stability of the lycopene and other
carotenoids from different food matrixes using this dynamic
digestion model. Considering that the TIM-1 system simulates
the absorption of foods by dialysis, a method that allows only
water-soluble compounds to be absorbed, Deát et al. [55] devel-
oped a TIM-1 system coupled to the Caco-2 cell-culture model.
This newly-developed system permitted assessment of lycopene
and a-tocopherol bioavailability from a whole food containing
52 K.T. Amorim-Carrilho et al. / Trends in Analytical Chemistry 56 (2014) 49–73
red tomatoes and sunflower oil. This approach permits a more sen-
sible representation of the bioavailability of carotenoids from foods
and it is very useful for future research.
The TNO group also developed the TIM-2 model, a proximal co-
lon simulator. The importance of this method relies on bioactive
compounds, such as carotenoids, being able to experience notable
modifications as a result of the action of the intestinal microbiota.
The incorporation of colonic and large-intestine fermentation is
desirable, since it offers a better approximation to the in-vivo situ-
ation and allows the study of the effect and the interaction be-
tween these compounds and the intestinal microbiota [56].
However, few examples of including colon fermentation in in-vitro
models have been reported [57]. Besides its cost, the difficulty of
establishing a representative human-gut microbiota in vitro is also
a limitation of dynamic models. Moreover, the availability of the
system, the prolonged time involved, its laboriousness, the use of
large working volumes and long residence times are other limita-
tions [56]. Consequently, the increasing complexity of the dynamic
models makes their use hardly practical.
The literature strongly supports that bioaccessibility and bio-
availability of carotenoids depend on several factors including,
among others:
 the type of carotenoid ingested [54,58,59];
 the location within the plant/food matrix [60–63];
 the type and the amount of co-consumed lipids [64–68]; and,
 the type and the extent of food processing and preparation
[41,66,69–76].
The factors that can influence the bioavailability of carotenoids
are summarized in the mnemonic ‘‘SLAMENGHI’’ (i.e., Species of
carotenoids, Linkages at molecular levels, Amount of carotenoid,
Matrix, Effectors, Nutrient status, Genetics, Host-related factors
and Interactions among these variables) [74]. The food matrix
and linkages at molecular levels are assumed to play a major role
among these factors {e.g., b-carotene bioavailability is affected by
chromoplast structure in mature plant tissues, with globular chro-
moplasts providing the best bioavailability and crystalline types
providing the poorest bioavailability of b-carotene [77]}.
With regard to carotenoid species, the extent of incorporation
into micelles is considered inversely related to their hydrophobic-
ity. For example, lutein and other xanthophylls are known to be
better absorbable into micelles and thus to have higher bioaccessi-
bility than b-carotene or lycopene, which are more hydrophobic
[78]. With regard to the amount of carotenoids, Goltz et al. [79]
suggested that carotenoid absorption may be higher when daily
recommended vegetables are consumed in one meal, compared to
smaller doses over multiple meals. Similarly, Granado-Lorencio
et al. [80] observed that the higher the amount of lutein esters in
esterified fermented milk, the higher the amount of free lutein
released and micellized.
In recent studies, the in-vitro digestion models were used as
analytical tools to optimize the formulation of oil/water emulsions
containing carotenoids as functional ingredients [81]. The food
industry is using knowledge of the digestibility of lipids within
the human GI tract to design food-grade delivery systems to encap-
sulate, to protect, and to release bioactive compounds, such as
carotenoids. These applications of in-vitro models could lead to
the creation of specific functional foods designed to control human
satiety, by controlling the rate and the extent of lipid digestion in
different regions of the GI tract. Moreover, these models would
permit the development of functional foods designed to increase
the digestibility of lipids in individuals with impaired digestive
process and to deliver lipids in a specific location in the GI tract.
There has been continual interest in the bioavailability of carote-
noids in oil emulsion in recent years [81–87].
In conclusion, the diversity of factors affecting carotenoid bio-
availability cited before and the lack of consensus concerning the
in-vitro methodology result in comparability between the different
studies often being limited. For that reason, there is a clear need for
harmonization of the in-vitro methods. This task is currently being
carried out at the European level within the project ‘‘Improving
health properties of foods by sharing our knowledge on the diges-
tive process’’ (INFOGEST, 2011-2015) (FAO COST Action FA 1005)
(http://www.cost-infogest.eu/ABOUT-Infogest).
Furthermore, and in spite of their limitations, dynamic models
have a promising role in understanding carotenoid bioavailability.
In-vivo studies related to subject-specific factors, such as genetics,
nutritional status and host-related factors, of the bioavailability of
carotenoids have also been performed and discussed [88–90].
Moreover, considering that in-vivo studies cannot be replaced by
in-vitro studies, the validation of different in-vitro procedures un-
der a variety of conditions and additional comparison with human
absorption of carotenoids (in vivo) may provide important data on
the relation between the subject-specific factors and bioavailabil-
ity of carotenoids in foods. The validation of data from in-vitro
models using in-vivo studies is especially important due to the lim-
itations of the in-vitro methods.
2.3. Evaluation of intake of carotenoids in humans
Due to the strong evidence of their biological activities and their
importance in human nutrition, provitamin A carotenoids (a-caro-
tene, b-carotene, and b-cryptoxanthin) and non-provitamin A
carotenoids (lycopene, lutein, and zeaxanthin) have had their food
content compiled in several tables and databases. These nutritional
tools allow the evaluation of carotenoid ingestion, which is neces-
sary to detect carotenoid deficiency and to study the relationship
between carotenoid consumption and human health. The tables
and the databases are valuable in developed and developing coun-
tries, enabling the study of different diseases related to carotenoid
deficiency. The establishment of appropriate databases of caroten-
oid content may be considered more important in countries where
the existing knowledge from carotenoid analysis is absent or weak
[91]. In these circumstances, the absence of data makes the use of
non-specific databases or tables very common. As a result, estima-
tion of carotenoid intake may be inaccurate because the carotenoid
content of food varies widely due to many factors, including the
sensitivity and the specificity of different analytical methods, sea-
sonal variations and food-processing methods.
In addition to the six most studied carotenoids, mentioned at
the beginning of this section, there are other less studied carote-
noids, which are important to human health, such as phytoene or
phytofluene. These compounds should also be included in tables
and databases [92]. For example, Biehler et al. [93] measured the
presence of violaxanthin, neoxanthin and carotenoid-precursor
phytoene and phytofluene in frequently consumed fruits and veg-
etables, and their contribution to the total carotenoid intake in
Luxemburg. In 2013, Estévez-Santiago et al. [94] developed a soft-
ware application that enables evaluation of the dietary intake of
carotenoids with potential beneficial effects on health, such as
carotenes c-carotene, phytoene, phytoene and neurosporene, and
xantophylls b-cryptoxanthin, a-cryptoxanthin, neoxanthin, viola-
xanthin, cantaxanthin, astaxanthin, capsanthin, capsorubin, anter-
axanthin, lactucaxanthin and echinone.
The generation and the inclusion of new data on the less com-
mon carotenoids might provide valuable information for future
investigations about the functions of these compounds in human
health. In spite of the recognition of the beneficial role of carote-
noids in human health, they are not considered essential nutrients
and there are no dietary reference intake (DRI) values assigned to
them [29]. The adequate levels of these components have not been
 K.T. Amorim-Carrilho et al. / Trends in Analytical Chemistry 56 (2014) 49–73
established because the positive health effects may be due to other
constituents that are co-ingested with carotenoids [42]. However,
there are recommendations for the consumption of carotenoids
as supplements. For b-carotene, 15 mg/daily, with a safe dose
range of 15–60 mg/daily. Supplements of 6–20 mg/daily of lutein
and 2–5 mg/daily of zeaxanthin are recommended in age-related
macular degeneration, and 15 mg/weekly of lutein to improve
performance in those with cataracts. Doses of up to 60 mg/day of
lycopene may be required for specific conditions, such as
hypercholesterolemia [95].
3. Analytical methods
3.1. Extraction methods
A wide variety of food products contain carotenoids (see sub-
section 2.1 for details), as do different genotypes of an organism
or a product. Also, there may be differences between the different
parts of the same food or sample. As a consequence, there is no
generally accepted method or standard method for carotenoid
extraction in laboratories [96,97]. However, most extraction
methods follow a common path involving the release of desired
components from their matrices by disrupting tissue, followed
by removal of the unwanted components and a liquid–liquid or
liquid–solid extraction [98].
For carotenoids, the most common procedure to collect these
compounds for further analysis is the one-step application of an
organic solvent that would extract them from the matrix. By con-
trast, solid-phase extraction (SPE) is rarely reported in carotenoid
studies, and, when it is, the typical SPE sorbents include C30 and
C18, while diol and silica cartridges have only had good retention
for lutein, a more polar carotenoid [1,99–102]. Also, SPE can be
applied to enhance the recovery efficiency of organic solvent
extraction of carotenoids.
However, many different organic solvents have been used in the
analysis of carotenoids and selection of the appropriate one is not
always easy. Besides the difficulties explained at the beginning of
this section, the different polarities of the existing carotenoids
and the structure of the analytical matrix and its components also
play important roles when selecting a solvent for extraction.
Usually, non-polar solvents, such as hexane, are a good choice for
non-polar (carotenes) or esterified carotenoids, while polar sol-
vents, such as ethanol and acetone, are more appropriate for polar
carotenoids (xanthophylls). Also, the susceptibility of carotenoids
to oxidation has to be considered when developing a method for
carotenoid extraction. These molecules are relatively stable in the
matrix, but carotenoids in solution may be very sensitive to light,
heat, acid or oxygen exposure.
3.1.1. Saponification
The xanthophyll group is the most complex carotenoid group in
terms of number of compounds and variation in their structure.
These compounds can be found in their free form (like carotenes)
or in a more stable fatty-acid esterified form. The naturally com-
plex availability of xanthophylls is increased by the formation of
these carotenoid esters. That is why these compounds have fre-
quently been analyzed after saponification [103], which is an
extraction step aimed at removing chlorophylls and lipids to re-
lease carotenoids eventually in a clean preparation for analysis,
free from conjugated forms, fatty acids and lipids that make the
chromatography separation difficult. Saponification is also used
for hydrolyzing carotenoid-esterified forms. Depending on the nat-
ure of the carotenoid and the food type, saponification may result
in destruction or structural transformation [102,104].
53
Rasmussen et al. [105] have tested the possibility of forming
meso-zeaxanthin, a xanthophyll also found in the macula, and zea-
xanthin, from the saponification of the standard lutein. The sapon-
ification of pure lutein resulted in the formation of peaks with
spectrum and chiral normal-phase retention times that matched
those of meso-zeaxanthin and zeaxanthin.
Irakli et al. [102] observed high recoveries of b-carotene and lu-
tein after cereal saponification and lower recoveries (ranging from
46.7% for b-carotene to 74.5% for lutein) after extraction without
saponification. However, Sagratini et al. [100] found little recover-
ies for b-carotene after saponification in a very recent study on
table olives. Similarly, Divya et al. [104] compared the concentra-
tion of b-carotene before and after saponification and observed
the loss of 20–30% of b-carotene and 50% of other carotenes during
saponification in coriander. Conversely, Watanabe et al. [106]
found no differences in carotenoid contents between saponified
and unsaponified samples of cabbage.
With regard to the saponification solutions, García-de-Blas et al.
[107] reported the best results with 0.02 M KOH in methanol and
observed lower yields of free carotenoids when higher concentra-
tions were used. Chan et al. [108] related that the addition of
2.5% KOH as the saponifying agent, resulting in a nearly two-fold
increase in the extracted lutein content, compared to that obtained
using only deionized water. The increase in the KOH from 2.5% to
80% did not influence the lutein content, suggesting that a low per-
centage of KOH is sufficient to convert the esterified lutein forms to
their free forms.
Inbaraj et al., 2008 [109] reported a large amount of carotenoid
esters was still present after simultaneous extraction and saponifi-
cation of L. barbarum fruit for 12 h, concluding that this method
was not applicable to extract free carotenoids. For that reason,
extraction and saponification should be carried out separately.
The content of carotenoid esters decreased gradually with saponi-
fication time and no carotenoid esters were detected after 6 h and
8 h. A longer saponification time (8 h) resulted in a lower yield of
carotenoids than 6 h, probably because of carotenoid degradation
after prolonged saponification.
To summarize, recoveries of carotenoids depend on whether
saponification is performed or not, the time used in the saponifica-
tion procedure, and the concentration of the alkaline treatments.
Also, considering the lack of commercially-available carotenoid-
derivative standards, their formation by saponification may be
very useful for their analysis in food and human samples, and con-
sequently for the study on their benefits to human health.
3.1.2. Carotenoid extraction in human samples
The most common solvent employed for plasma extraction of
carotenoids is n-hexane [110,111], or a mixture of n-hexane and
other solvents {e.g., hexane/ether [112], hexane/ethanol/acetone/
toluene (HEAT) [113,114], hexane/dichloromethane [115] and hex-
ane/chloroform [116]}. The suitability of n-hexane for carotenoid
extraction was confirmed in a previous review article that high-
lighted nine studies between 1990 and 2002 in which n-hexane
was used as the main extraction solvent. For carotenoid extraction
in serum, n-hexane was also the most commonly applied solvent
[117–121]. For example, Hsu et al. [122] used a mixture of hexane
and ethyl acetate, while heptane was the solvent selected by Con-
nolly et al. and Matsubara et al. in their studies [123,124]. Tetrahy-
drofuran (THF) was used for carotenoid extraction by Ferreiro-Vera
et al. [99].
Most studies with human samples have used butylhydroxytol-
uene (BHT) to prevent carotenoids from oxidation and echinone
as internal standard. Also, ethanol was used to precipitate proteins
in serum and plasma in most of the studies, since these proteins
may cause clogging of the standards in the HPLC columns after a
few injections and interfere with chromatographic separation.
54 K.T. Amorim-Carrilho et al. / Trends in Analytical Chemistry 56 (2014) 49–73
A combination of protein precipitation by organic solvents, salts
or acids, followed by centrifugation, is the most widely applied
technique for protein removal from blood-derived materials
[125]. Most of the studies follow these extraction steps:
 deproteination;
 extraction and vortex;
 centrifugation and concentration; and,
 re-dissolution.
See Table 2 for a brief summary of the existing methods for
carotenoid analysis in human samples that have been reported
during the past five years.
3.1.3. Carotenoid extraction in food samples
The use of various solvents has been observed in the different
procedures developed to extract carotenoids from food samples:
acetone, ethanol, THF, hexane, toluene, diethyl ether, methanol,
ethyl acetate, dichloromethane, petroleum ether, chloroform and
butanol. Most studies have included hexane as extraction solvent,
followed by acetone, methanol and, finally, THF. Less frequently,
petroleum ether, diethyl ether, dichloromethane, ethyl acetate
and ethanol were used. A wide variety of solvent combinations
have been used by different researchers (e.g., acetone/dichloro-
methane, acetone/ethanol, acetone/hexane, acetone/petroleum
ether, hexane/diethyl ether, hexane/ethanol/acetone/toluene,
methanol/THF, and hexane/ethyl acetate). However, ethanol/hex-
ane (4:3) has been one of the most common combinations for food
samples, as shown in Table 3.
In a comparative study of carotenoid extraction from algae in
different solvents, Sarkar et al. [128] found that hexane, dichloro-
methane, diethyl ether and THF showed better extraction effi-
ciency after homogenization of the sample and that mixing these
solvents with ethanol, methanol or butanol decreased extractabil-
ity. However, when acetone was used in combination with metha-
nol or ethanol (7:3), the extractability remained the same as with
acetone only. As methanol and ethanol are considered green sol-
vents, Sarkar et al recommended the application of solvent systems
of acetone with these solvents instead of acetone only. In a very re-
cent study in tomato, Ranveer et al. [35] observed that the tri-mix-
ture of hexane, acetone and ethanol (50:25:25, v/v/v) achieved the
highest lycopene recovery among all the solvents tested individu-
ally. Similarly, Carrilho et al. [129] analyzed b-carotene, lutein and
fucoxanthin in Wakame (Undaria pinnatifida) and Kombu (Lami-
naria sp.) seaweed using a combination of methanol, hexane and
dichloromethane (50:25:25, v/v/v) and a second extraction with
acetone.
Recently, Rivera and Canela [96] studied the influence of sample
processing on the analysis of carotenoids in maize. They reported
that combinations of methanol with other less polar solvents were
more effective than ethanol, acetone and acetone/ethanol/hexane.
They also observed that samples that were hydrated before ace-
tone extraction showed greater extraction efficiency, which might
be attributable to water enhancing acetone penetration into the
endosperm. Kamffer et al. [34] developed a method for extraction
of carotenoids and chlorophylls in grape berries and stated that
these compounds were more stable in diethylether/hexane than
in acetone. Fewer degradation products of carotenoids and chloro-
phylls were found when a 30-min extraction with diethylether/
hexane was used, the optimal extraction time that increased the
extraction of more prevalent carotenoids without an increase in
degradation products.
Warkoyo and Saati [130] studied solvent effectiveness in the
extraction process from Eucheuma cottonii seaweed and found that
acetone tends to produce the highest content of carotenoid in the
extract for the green and brown seaweeds compared to ethanol,
petroleum benzene and hexane:petroleum benzene solvents.
Meanwhile, for red seaweed, acetone and ethanol produced the
same high pigment content.
Lucini et al. [131] studied the best extraction conditions in to-
mato and observed that ethyl acetate was the best extraction sol-
vent for b-carotene, while dichloromethane was the most efficient
for extracting the other carotenoids. Due to them being less toxic
than dichloromethane, ethyl acetate and cyclohexane/ethyl acetate
(1:1, v/v) were chosen as extraction solvents. Significant concen-
trations of lycopene were detected until the third extraction, while
b-carotene concentration was only detectable until the second
extraction.
Similarly, Strati et al. [132] found out that ethyl acetate/hexane
(55:45) combination gave the highest carotenoid yield in tomato-
waste extraction. In this case, the combination of hexane with eth-
anol or ethyl acetate improved the carotenoid yield compared with
that obtained by these solvents individually. The highest percent-
age of carotenoid separation for lycopene and b-carotene was ob-
tained by the apolar solvent hexane and hexane/ethanol extract,
respectively, while for lutein, a polar carotenoid, the maximum
percentage was found in ethanol extracts. The authors also high-
lighted that there was no synergistic effect from the hexane-ace-
tone solvents and a cumulative action of acetone and hexane,
since the mixture showed lower carotenoid yield than acetone
extraction and was almost in the middle compared with the sum
of the carotenoid yields after individually extracting with acetone
and hexane.
In general, as highlighted above, there is no failure-free rule to
select a solvent for carotenoid extraction. When preferring a single
solvent, hexane is frequently selected, especially for carotenes. In
turn, ethanol/hexane (4:3) is one of the most frequently applied
solvent combinations to extract carotenoids from food samples.
However, due to the great variability in content and composition
of carotenoids in food and their different polarities, continual opti-
mization for different matrices and analytes is required. The influ-
ence of extraction time and the number of extractions, the stability
of these compounds in the extract, the application or not of indi-
vidual solvents, and the synergistic effect of solvents on extraction
efficiency are recurrent topics in carotenoid analysis. Also, due to
the large volumes that are usually employed in these extractions,
the toxicity of the selected solvent should also be considered. Last,
BHT is by far the most commonly used antioxidant added to the
solvents to protect carotenoids. Other antioxidants like pyrogallol,
butyl-hydroxyanisol (BHA), ascorbic acid and butyl-hydroquinone
(TBHQ) are also used (see Tables 2 and 3). See Table 3 for a brief
summary of the existing modern methods for carotenoid detection
in food and vegetable samples.
3.1.4. Other extraction techniques
Although liquid-liquid and solid-liquid extraction are the most
frequently applied techniques in human and food samples, other
extraction methods have been reported for carotenoid analysis
{e.g., supercritical-fluid extraction (SFE) [184], pressurized liquid
extraction (PLE) [185], accelerated solvent extraction (ASE) [186],
pressurized hot-water extraction (PHWF) [187], ultrasound-as-
sisted extraction (UAE) [184,188,189] and microwave-assisted
extraction (MAE) [75,190–192] have been used as an environmen-
tally friendly extraction alternatives}. These options are beneficial
in the laboratory, because traditional extractions with organic sol-
vents are characterized by using high volumes of solvents and long
extraction times, and they often present low extraction yield of
bioactives, low selectivity and reproducibility.
However, the application of mechanical forces (e.g., sonication,
autoclave and bead-beaters) is also an interesting option to im-
prove carotenoid extraction. For example, greater cell disruption
and higher lutein content were observed when using a bead-beater
 K.T. Amorim-Carrilho et al. / Trends in Analytical Chemistry 56 (2014) 49–73 55

Table 2
Analytical methods for detecting carotenoid compounds in human samples

Sample Analyte Analytical method Ref.

Extraction protocol Detection system
Plasma Lutein 4 mL of centrifuged blood + 5 ml of saline and LC-PDA [112]
Zeaxanthin ethanol (1:2 ratios). Extraction twice with 5 mL of C30 column (3 lm, 4,6  150 mm)
b-criptoxanthin hexane and ether (1:1 ratio). Vortexing for 1 min. Mobile phase: MeOH (containing 0.1%
a-carotene Centrifuging at 2000g for 10 min at 4 degrees. ammonium acetate) and MTBE
b-carotene Collecting upper layer. Pooled extractions Gradient elution
Lycopene concentrated to dryness under nitrogen,
Phytoene reconstituted in 100 lL of ethyl acetate
Phytofluene
36 different carotenoid isomers
Serum 30 carotenoids 1mL of blood into a 10 mL brown vial + 1 mL of LC-PDA-APCI (+)/MS [122]
0.01% Vit. C in ethanol + 1 mL ethyl acetate and C30 column
3 mL hexane. Vortex (30 s), shaking (10 min at Mobile phase: (A) MeOH/ACN/water (84:14:4, v/
200 rpm), centrifuging (20 min at 3000 rpm and v/v) and (B) DCM
4°C). Gradient elution
To the residue, 3 mL of hexane was added and the
procedure repeated three times
Serum 9 carotenoids 200 lL of serum + 400 lL of THF. Vortex mixed for LC-PDA [99]
1 min. Settled for 10 min. Centrifuged for 6 min at C18 (100 mm  3.9 mm, 4 lm) column
85 g. Supernatant transferred to an auto sampler Mobile phase: (A) ACN-water (90:10) and (B) THF
vial. Gradient elution
Solid phase extraction (SPE) for clean- up pre-
concentration
Serum Lutein 0.1 mL of water, 0.2 mL of ethanol (including 10 ng/ SFC/MS/MS [124]
a-carotene mL echinone as an internal standard) and 1 mL of ODS (250 mm  4.6 mm, 5 lm) column
b-carotene heptane with 5 lL/mL dibutylhydroxytoluene Mobile phase: Carbon dioxide (99.99% grade).
b-cryptoxanthin added to 0.1 mL of serum sample Modifier: MeOH with 0.1% (w/v) ammonium
b-cryptoxanthin epoxidized formate
products
Serum Lutein 500 lL serum + 500 lL internal standard (8-apo-b- LC-UV-Vis [119]
Zeaxanthin carotenal) + 500 lL ethanol pippeted into 5 mL C30 (4.6 mm  25 mm, 5 lm) column, guard C30
Lycopene disposable tubes. Vortex mixed for 1 min. column
b-carotene Mixture + 700 lL hexane (three times). Upper layer Mobile phase: (A) MeOH: ACN (40:60, v/v) (B)
collected and evaporated under N2 at 4°C. MeOH: ACN (25:75, v/v) and (C) MTBE
Residue dissolved in solvent A (MeOH: ACN, 40:60, Gradient elution
v/v)
Serum a-carotene Serum centrifuged at 3000xg at 4°C for 10 min. LC-PDA [117]
b-carotene 200 mL + 200 lL ethyl alcohol containing 0.5 lmol/ ODS 2 C18 (3 mm  250 mm, 3 lm) column
cis-b-carotene L echinone and 4 lmol/L tocol with 30 mg/L BHT in connected with a ODS 2 guard column
an amber vial. Vortex mixed for 2 min + 800 lL Mobile phase: ACN: methylene chloride: methyl
hexane; mixed for 10 min at 1500 rpm by alcohol (7:2:1,v/v/v)
mechanical vortex; centrifuged for 10 min at Isocratic elution
1500 rpm. Supernatants transferred into amber
vials. Hexane extraction repeated.
Supernatants combined and evaporated under N2.
Residue reconstituted in 100 lL mobile phase
containing 30 mg/L BHT without ammonium
acetate. Vortex 2 min; Ultrasonic bath 2 min and
20 lL injected into HPLC column
Serum a-carotene 200 lL + 200 lL ethanol-butylated hydroxytoluene LC-PDA [118]
b-carotene containing 50 lL internal standard cocktail. C18 ODS 4.6 mm column
Lycopene Vortexed (60 s), extracted (1000 lL n-hexane), Mobile phase: MeOH 60%; ACN, 20%; and DCM,
Lutein dried (N2, 10 min). 20%
Reconstituted in 200 lL ethanol-butylated Isocratic elution
hydroxytoluene solution
Plasma Lutein 0.5 mL + 0.5 mL ethanol (0.1% BHT) + 2,5 mL of LC-PDA [113]
Zeaxanthin HEAT (hexane/ethanol/acetone/toluene, 10:6:7:7, C30 (150 mm  4.96 mm, 5 lm) column
b-carotene v/v/v/v) Mobile phase: (A) MeOH/MTBE/H2O/2% aqueous
Lycopene ammonium acetate solution, 88:5:5:2, v/v/v/v)
Red blood cells Lutein 2,5 mL + 2,5 water + 5 ml pyrogallol (0–400 mmol/L LC-PDA-APCI(+) /MS [115]
Zeaxanthin in ethanol) + 1 mL aqueous potassium hydroxide C30 (250 mm  4,6 mm I.D, 5 lm) column
a-carotene (0–7 mmol/L) and 40 lL of echinone (1 lmol/L in Mobile phase: (A) MeOH/MTBE/water (83:15:2, v/
b-carotene ethanol); sonicated for 5 min; vortexed for 2 min, v/v) containing 3,9 mmol/L ammonium acetate
Lycopene incubated at various temp. (20–70) for different (B) MeOH/MTBE/water (8:90:2, v/v/v) containing
b-cryptoxanthin time periods (0–24 h). 2,6 mmol/L ammonium acetate
Mixed; extracted with 1.25 mL of 0.1 mol/L sodium
dodecyl sulfate aqueous solution and 15 ml of
hexane/dichloromethane (5:1, v/v) containing
1.2 mmol/L BHT; sonicated, vortexed, centrifuged at
1000g for 10 min. Supernatants collected,

(continued on next page)

56 K.T. Amorim-Carrilho et al. / Trends in Analytical Chemistry 56 (2014) 49–73

Table 2 (continued)

Sample Analyte Analytical method Ref.

Extraction protocol Detection system
extraction repeated.
Supernatants evaporated under N2; reconstituted in
3 mL of hexane/acetone (2:1, v/v) and eluted with
7 mL of hexane/acetone (2:1, v/v); eluent
evaporated and residue dissolved in 100 lL of
MeOH/MTBE (2:3, v/v)
Plasma Lutein and Zeaxanthin 100 lL plasma + 1100 lL 20% mixture of n-hexane LC-PDA [116]
and chloroform + 100 Si60 (250 mm  4 mm, 5 lm) column
lL water + 200 lL ethanol. Centrifugation. Mobile phase: n-hexane and acetone (19% by vol)
800 lL supernatant dried under N2 and
reconstituted in 200 lL of n-hexane and acetone;
19% by vol)
Plasma Lutein/Zeaxanthin 200 lL plasma extracted twice with 1 mL of hexane LC-PDA [111]
Lycopene containing 0.01% BHT 8 0.167 lL/mL of echinone. C18 column
a-carotene Dried under N2; reconstituted in 100 lL of mixture Mobile phase: (A) 0.0125% ammonium acetate in
b-carotene CH3Cl:MeOH:CH3CN (30:35:35) MeOH, (B) 100% CHCl3 and (C) CH3CN with 0.1%
b-cryptoxanthin triethylamine
Gradient elution
Serum Lycopene 200 lL + 5 mL of hexane + 1 mL of ethanol. Hexane LC-PAD [121]
a-carotene layer evaporated under N2. Residue dissolved in C18 ODS-2 (100  4.6 mm, 3 lm) column.
b-carotene 200 lL of the mobile phase (ACN-MeOH-chloroform Mobile phase: ACN/MeOH/chloroform 47:47:6, v/
47:47, v/v/v) v/v
Isocratic elution
Serum Lutein Serum samples + H2O + ethanol containing b-apo- LC-PAD [120]
Zeaxanthin 80 carotenal and extracted into hexane. RP C18
lycopene Organic layer was removed, evaporated to dryness Mobile phase: MeOH/THF/H2O(94:5:1)
a-carotene and resolved in chloroform/ethanol (1:19)
b-carotene
b-cryptoxanthin
Serum Lutein 400 lL + 300 lL ethanol containing 0.25 g/L BHT LC-PDA [123]
Zeaxanthin 200 lL a-tocopherol acetate (0.25 g/L) + 500 lL RP (250 mm  4.6, 5 lm) column connected with
heptane an in-line guard-column
Vortexed for 2 min; centrifuged at 2000 rpm for Mobile phase: 97% MeOH, 3% THF
5 min Isocratic elution
Second heptane extraction; combined heptane
layers evaporated under N2 and reconstituted in
200 lL MeOH containing 0.25 g/L BHT
Plasma Lutein 100 lL plasma diluted with 100 lL of H2O + 200 lL Reversed phase HPLC [110]
Zeaxanthin of ethanol. Vortex mixed for 30 s, extracted twice C30 (250  4.6 mm, 5 lm) column in line with a
Lycopene with 1 ml n-hexane stabilized with 0.05% BHT; C18 pre-column
b-cryptoxanthin vortex mixed for 10 min and centrifuged for 10 min Mobile phase: (A) MeOH/water (90:10, v/v, with
b-carotene at 1500g 0.4 g/l ammonium acetate in H2O) (B) MeOH/
a-carotene, Supernatants pooled, evaporated under N2 and MTBE/water (8:90:2 v/v/v, with 0.1g/l ammonium
reconstituted in 200 lL isopropanol acetate in H2O).
Plasma Apo-lycopenals 2 ml + HEAT (hexane/ethanol/acetone/toluene, UPLC-PDA-APCI(-)/MS [114]
10:6:7:7, v/v/v/v) + equal volume of ethanol C30 (150 mm  4.96 mm, 5 lm) column
containing 0.1% butylated hydroxytoluene + 0.5 ml Mobile phase: (A) MeOH/0.1% aqueous formic
of a saturated NaCl solution + 10 ml of HEAT. acid solution (80:20) (B) MTBE /MeOH/0.1% aq.
Vortex, centrifuged for 5 min at 300xg. Upper non- formic acid solution (78:20:2)
polar layer removed. Remaining aqueous plasma
extracted two more times.
Non-polar layer was combined and dried under
argon.
Serum Lutein Serum: 500 ll spiked with 375 lL of echinone LC-APCI (+)/MS [126]
(1.25 lM) + 4 ml chloroform/MeOH (2:1, v/ RP C30 (150 mm  4.6 mm, 3 lm)
v) + 0.5 ml saline solution (0.85%). Vortexed, Mobile phase: (A) MeOH/tert-butyl methyl ether/
centrifuged for 10 min at 4°C and 3000 rpm. water (83:15:2, v/v/v) and (B) MeOH/MTBE/water
Chloroform layer removed. 5 ml of hexane added. (8:90:2, v/v/v)
Vortexed, centrifuged. Gradient elution
Extract combined and dried in a water bath at 40°C
and dissolved in 300 lL
Plasma Astaxanthin 0.5 ml + 0.5 ml of water + 1 ml of ethanol + 3 ml HPLC-APCI(+)/MS [127]
hexane with 4 lmol/L of an echinone ethanol C30 (4.6  250 mm, 5 lm) carotenoid column
solution (50 lL). Vortexed for 30 s, centrifuged at Mobile phase: (A) MeOH/MTBE/water (8:90:2)
1000 g for 10 min at 4°C. Upper hexane collected. containing 2.6 mmol/L of ammonium acetate
Extraction repeated. Combined hexane layers dried
under N2 and re-dissolved in 100 lL of MeOH/MTBE
(2:3, v/v)

ACN, Acetonitrile; BHT, Butylhydroxytoluene; DCM, Dichloromethane; MTBE, Methyl tert-butyl ether; THF, Tetrahydrofuran; SFC, Supercritical fluid chromatography
 K.T. Amorim-Carrilho et al. / Trends in Analytical Chemistry 56 (2014) 49–73 57

Table 3
Analytical methods for detecting carotenoid compounds in food samples

Sample Analyte Analytical method Ref.

Extraction protocol Detection system
Leafy vegetables Lutein 0.6 g heated at 85°C/5 min in ethanol with BHT LC-PDA [133]
a-carotene (0.1% w/v); saponification with 400lL KOH (80% in C30 (4.6  250mm, 3 lm) column
Zeaxanthin water); vortex mixed 20 sec; water bath (85°C) Mobile phase: (A) MeOH:water (92:8, v/v) with
b-criptoxanthin 10 min; placed in ice + 3 mL deionized water. b- 10 mmol/L ammonium acetate and (B) MTBE
All-trans-carotene 13- Apo-80 carotenyl decanoate added post-
cis-carotene saponification.
9-cis-b-carotene Extract four times with hexane. Dried under argon,
reconstituted in 1 mL (50:50) MeOH: DCM
Glabrous canary seeds Lutein Re-extraction until colorless with 25 mL of acetone/ LC-PDA [134]
Zeaxanthin ethanol (1:1, v/v) with 200 mg/L BHT. Centrifuged YMC Carotenoid column
b-carotene at 1500 for 15 min at 4–5. Mobile phase: (A) MTBE (B) 1% water in MeOH
Supernatants brought to 100 mL with the
extraction solvent
Tomatoes Lycopene Extraction twice until colorless with 10 mL THF LC- UV/VIS [135]
Lutein (0.01% BHT). Centrifuged at 3000 rpm for 10 min. RP 18 (5 lm, 125  4.6 mm) column
a-tocopherol Evaporated to dryness under N2. Mobile phase: MeOH:ACN: DCM (50:48:2, v/v/v)
b-carotene Residue dissolved in 3 mL of Chloroform and Isocratic elution
diluted with mobile phase (MeOH:ACN:DCM,
50:48:2)
Chinese herb 25 carotenoids Extracted with 30 mL hexane/ethanol/acetone/ LC-PDA-APCI(+)-MS [136]
Taraxacum toluene (10:6:7:7, v/v/v/v). Shaken for one hour. C30 (250 mm  4.6 mm, 5 lm) column
formosanum 2 mL of 40% methanolic potassium hydroxide for Mobile phase: (A) MeOH/ACN/water (79:14:7, v/v/
saponification for 16 hours under N2. 15 mL v) and (B) methylene chloride
hexane-shaken for 10 min. 15 mL 10% sodium
sulfate solution-shaken for 1 min.
Supernatant + 15 mL of hexane shaken for 10 min
(repeated 4 times until colorless).
Supernatants evaporated to dryness, dissolved in
5mL of MeOH:methylene chloride (1:1, v/v)
Orange juice 34 carotenoids Extracted with MeOH/acetone/hexane (25:25:50, v/ LC-PDA [137]
v/v) containing 0.1% of BHT. Centrifuged 2500 g for C30 column (5 lm, 250 mm  4.6 mm)
10 min. Upper phase treated with 10% ethanolic Mobile phase: MeOH and tert-butyl- methyl ether
potassium hydroxide for 1 hour and washed 4 (TBME) both containing 0.1%BHT and 0.5%
times with water (saponification). trietylamine) and water.
Carotenoid extracts taken to dryness and Gradient elution
reconstituted in 1 mL of acetone:MeOH (1:2, v/v,
with 0.1% BHT)
13 different cultivars of All-trans carotenoids 0.5 g dried tomato powder + 5 mL of acetone/ UPLC-PDA [138]
tomato Cis-isomers ethanol (1:1, v/v). Shaking (150 rpm for 1 h), C18 (100 mm  2.1 mm, 1.7 lm) connected with a
centrifuging. Re-extraction until the residue was C18 guard column
colorless. Mobile phase: (A) MeOH/MTBE/water (90:5:5, v/v/
Supernatants brought to 10 mL with the extraction v) and (B) MeOH/MTBE/water (90:5:5, v/v/v)
solvent.
Cereal grains Lutein 200 mg of ground samples + 2 mL of water- LC-PDA [139]
Zeaxanthin saturated butanol. Vortex for 30 s, shaking for S-3 (3 lm, 4.6  100 mm) carotenoid column
b-cryptoxanthin 15 min at speed 40 using a horizontal rotary shaker. Mobile phase: (A) MeOH/MTBE/Milli Q water
13-cis lutein Left to stand for 60 min at 25°C. Homogenized and (81:15:4,v/v/v) and (B) MTBE/MeOH (90:19, v/v)
9-cis lutein shaking for another 15 min. Stand for another Gradient elution
90 -cis lutein 60 min. 1.8 mL of extract transferred into 2 mL
brown micro-centrifuge tubes and centrifuged at
4000 and 20°C.
Supernatant filtered, stored overnight at 20°C
before analysis
Melon Lutein Carotenoids were extracted using the method LC-PDA [140]
a-carotene described by Bushway and Wilson (1982) C18 (200 mm  4.6 mm, 5 lm) column with a C18
b-carotene guard column
Phytoene Mobile phase: (A) ACN/dicloromethane/ethyl
Phytofluene acetate 40:25:35, v/v/v and (B) ACN
Gradient elution
Teas Lutein 200 mg + 5 mL of 1:1 n-hexane: diethyl LC-PDA [141]
b-carotene isomers ether + 25 lL of APO (170 mg/L in 100% ethanol), C18 (250 mm  4.6 mm, 5 lm) with a C18 guard
Chlorophylls vortex mixed for 1 min. Stand for 20 min at 20°C, column
Chlorophylls vortex mixing every 5 min. Mobile phase: (A) 90% ACN with 10% ultra-pure
degradations products Centrifuged at 4000 rpm, 20°C, 10 min. Re- water and (B) ethyl acetate
extraction twice with 5 mL 1:1 n-hexane: diethyl Gradient elution
ether (in the second and third extractions, no
additional APO).
Pooled supernatants evaporated to dryness (water
bath set at 40°C). Residue reconstituted in 1 mL of
1:1 n-hexane:acetone

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58 K.T. Amorim-Carrilho et al. / Trends in Analytical Chemistry 56 (2014) 49–73

Table 3 (continued)

Sample Analyte Analytical method Ref.

Extraction protocol Detection system

Tomatoes after 10 carotenoids 0.2 g lyophilized tomato fruits + 5 mL of ethanol: LC-UV-Vis-API (+)/MS [142]
moderate-intensity hexane (4:3, v/v), sonicated for 5 min, centrifuged C30 (250 mm  4.6 mm, 5 lm). Post-column for
pulsed electric field (4000 rpm at 4°C) for 15 min. Re-extraction. delivery of 100 lL/min of a LiCl solution 500 ppm
Supernatants combined and evaporated under N2 Mobile phase: (A) water, MeOH, (C) MTBE
flow. Gradient elution
Residue reconstituted with up to 1 mL of MTBE,
filtered into a vial for HPLC
20 tomato cultivars All-trans-lutein 0.5 g dried tomato powder + 10 mL ethanol:hexane UPLC-PDA [143]
and breeding lines Lycopene (4:3, v/v), shacken at 150 rpm for 1 h, centrifuged, C18 (1.7lm  100, 2.1 mm) column with a C18
b-carotene supernatant separated, re-extraction twice with guard column
b-carotene cis-isomers 10 mL hexane. Combined supernatant washed with Mobile phase: (A) MeOH-MTBE-water (90:5:5, v/v/
50 mL distilled water and 50 mL 10% aq. NaCl v) and (B) MeOH-MTBE-water (90:5:5, v/v/v)
solution separately. Supernatant collected and Gradient elution
evaporated to dryness under N2.
Re-dissolved in 1 mL MeOH-MTBE-water (90:5:5,
v/v/v)
Carrot juice, raw and a-carotene Samples ground for 1 min with 10 mL of ethanol/ LC-PDA [59]
cooked spinach b-carotene hexane (4:3, v/v) + 100 lL of internal standard C30 (4.6 mm  250 mm, 5lm) column
violaxanthin13-cis- (echinone 5 mg/L in acetone) and 10 mL of NaCl Mobile phase: (A) MeOH: MTBE:milliQ water,
carotene 10% (p/v). Vortex mixed; centrifuged during 10 min 28,5:67,5:4 (v/v/v)
b-carotene at 875 g. Upper phase washed twice with 10 mL of Gradient elution
9-cis-b-carotene distilled water. Settled for 10 min, dried under N2
Residue dissolved in 1 mL of acetone
Enriched fruits juice Retinol DLLMEC (dispersive liquid-liquid micro extraction) LC-UV-APCI (+)/MS [144]
Retinyl acetate 0.1–2 mL + 10 mL water + 2 mL MeOH containing RP C8 (15 cm  46 mm, 5 lm) column
Retinyl palmitate 150 lL of carbon tetrachloride. Manually shaken for Mobile phase: mixture of MeOH/water
b-carotene several seconds. Centrifugation for 2 min at Gradient elution
3000 rpm.
Sediment phase evaporated under N2 and
reconstituted with 50 lL of MeOH
Tamarillo fruit 36 carotenoids 3 g of puree or 5 g of nectar + 80 mL MgCO3, 15 mL LC-PDA-ESI (+)/MS [145]
(Solanum betaceum ethanol/hexane, 4.3 v/v with 0.1% BHT C30 (250  4.6 mm, 5 lL) column
Cav.) Stirred 5 min, filtered (porosity n.2), and washed Mobile phase: (A) water/20 mm ammonium acetate
with 15 mL ethanol/hexane, 4:3 v/v, 15 mL ethanol, (B) MeOH/20 mm ammonium acetate and (C) MTBE
15 mL hexane. Organic phase washed with 40 mL Gradient elution
10% sodium chloride and 2  40 mL distilled.
Hexane phase dried under anhydrous sodium
sulfate, filtered and evaporated at 40°C in a rotary
evaporator.
Residue dissolved in 500 lL DCM and 500 lL MTBE/
MeOH (80:20, v/v), saponification with 10% KOH
Arazá fruits 15 carotenoids 0.25 g lyophilized + 5 mL MeOH, homogenized at LC-PDA-APCI ()/MS [146]
5000 rpm for 2 min, centrifuged at 300 g for 10 min. C30 S-3 column (2 mm  150 mm, 3 lm)
MeOH extract separated. 5 mL hexane/acetone Mobile phase: (A) MeOH/water/2% aqueous
(1:1) + pellet, homogenized, centrifuged; hexane/ ammonium acetate, 80:18:2 (B) MeOH/MTBE/2%
acetone extract + MeOH extract. Pulp-hexane/ aqueous ammonium, 20:78:2
acetone twice; for peel, once more. 1 mL sodium Gradient elution
chloride + 5 mL water + pooled hexane/acetone/
MeOH extract. Upper hexane removed, dried over
sodium sulfate.
A fraction was dried under N2, reconstituted in
MeOH/MTBE (MTBE) (1:1, v/v)
Ketchup and gazpachos Lutein 0.5 mL ethanol/hexane (4:3, v/v) sonicated for LC-PDA-ESI (Li+)/MS/MS [147]
a-carotene 5 min. Centrifuged at 2140  g for 15 min at 4°C. C30 (250 mm  4.6 mm, 5 lm) column, connected
b-carotene trans- Extraction repeated; Supernatants combined and with a guard column for the delivery of 100 lL min
lycopene 5-cis- evaporated under N2 of lithium chloride
lycopene Reconstitution with MTBE up to 1 mL Mobile phase: (A) water, (B) MeOH and (C) MTBE
9-cis-lycopene Gradient elution
13-cis-lycopene
Red-ripe tomatoes Lycopene 10 g + 200 mg magnesium carbonate, 35 mL UPLC-MS/MS [148]
a-carotene ethanol/hexane (4:3, v/v), rotation for 15 min RP C18 (100  2.1 mm, 1.9 lm) column
b-carotene (42 rpm), filtration. Obtained residue re-extracted Mobile phase: isocratic (A) ACN, MeOH and
a-tocopherol with 35 mL ethanol/hexane (4:3, v/v), rotated for chloroform (50:35:15, v/v) containing 0.01% BHT
15 min, filtration, washed with 30 mL pure hexane. and 0.05% TEA; and gradient elution with mobile
Pooled filtrates + 150 mL distilled water, 100 mL of phase (B) ACN, MeOH and isopropanol
10% (w/v) sodium chloride solution.
Supernatants collected, lower phase extracted again
with 20 mL hexane.
Supernatants combined, 100 lL diluted with ACN/
MeOH (50.50, v/v) + 0.01% BHT until a final volume
of 1 mL
 K.T. Amorim-Carrilho et al. / Trends in Analytical Chemistry 56 (2014) 49–73 59

Mature grapes Regio isomers of lutein 50 g + 25 lL BHA (12,66 mg/mL in EtOH). LC-PDA-MS (ESI+) [149]
cis-isomers of lutein Homogenized 5 min with 3 g of magnesium C30 Reversed phase column (3  250 mm, 5 lm)
cis-isomers of b- carbonate basic. Spiked with 200 lL of internal connected with a C30 guard-column
carotene standard (100lg of b-apo-8‘-carotenal). Diluted Mobile phases: (A) 0.05% TEA in H2O, (B) 0.05% TEA
5,6-epoxy xantophylls with 40 mL of water HPLC grade; Extracted with in MeOH and (C) 0.05% TEA in TBME
5,8-epoxy xantophylls 40 mL of hexane/diethyl ether (1:1, v/v). Pooled Gradient elution
diasteroisomers extract evaporated to dryness using a rotary
evaporator.
Residue dissolved in 2 mL MTBE/hexane (1:1, v/v)
Edible microgreens b-carotene Lutein, 0.05 g lyophilized samples + 7.5 mL of 1% BHT in LC-PDA [150]
Zeaxanthin ethanol and 500 lL of internal standard (86.82 lm C18 (150 mm  4.6 mm, 5 lm) column
Violaxanthin trans-b-apo-carotenal). Ultrasonic homogenization Mobile phase: ACN/ethanol (1:1, v/v)
15 s; Dry bath at 70°C for 15 min + 180 lL of 80%
KOH; mixed and flushed with N2. Dry bath at 70°C
30 min; vials cooled on ice. 3.0 mL of deionized
water and 3.0 mL of hexane/toluene solution (10:8,
v/v). Vortex 1 min; Centrifuged at 1000g for 5 min.
Top organic layers placed in a N2 evaporator at
30°C, flushed with N2. Bottom layer extracted 5
times with 3.0 mL of hexane/toluene solution (10:8,
v/v).
All supernatants evaporated, reconstituted in
500 lL of ACN/ethanol (1:1, v/v)
Bulgarian berries Lutein Zeaxanthin 2 g + 0.2 magnesium carbonate + 15 mL MeOH/ LC-PDA [151]
Lycopene tetrahydrofuran (1:1, v/v) containing 0.1% BHT. RP C18 (150  4.6 mm, 5lm) column connected
b-carotene Vortex mixed for 3 min at 2500 rpm; centrifuged with a C18 guard column
b-cryptoxanthin for 3 min at 3000 rpm. Supernatant collected; pellet Mobile phase, containing 0.1% BHT and 0.05%, TEA:
a-carotene re-extracted until colorless; reduced under N2; (A) ACN/MeOH (95:5, v/v) (B) ACN/MeOH/ethyl
dissolved in ACN/MeOH/ethyl acetate (60:20:20, v/ acetate (60: 20:20, v/v/v)
v/v). Saponification
Orange juice Lutein THF with BHT until colorless + APO. THF LC-PDA [152]
Zeaxanthin extracts + diethyl ether and salt water. Organic RP C18 (250  4.6 mm, 5 lm) column
b-cryptoxanthin layer combined, dried over anhydrous sodium Mobile phase: (A) MeOH/water (75:25, v/v) (B)
a-cryptoxanthin b- sulfate. Ethereal solution reduced to 30 mL + KOH ACN/DCM/MeOH (70:5:25, v/v/v)
carotene 16 h. Added sodium chloride solution and diethyl
a-carotene ether. Washed. Evaporated.
Redissolved in 2 mL of DCM.
Semolina Tocols 2 g samples + 5 mL ethanolic pyrogallol (60 g/l); LC-UV-Vis [153]
Egg Carotenoids stored at 20°C until analysis. Under N2, 2 mL of (250  4.6 mm, 5 lm) column
Samples during egg- Retinols 95% ethanol, 2 mL or 1 mL (egg samples) of sodium Mobile phase: (n-hexane/isopropyl alcohol (5%)
pasta production chloride (10 g/L) and 2 mL or 3 mL (egg samples) of Gradient elution
steps KOH (600 g/L); water bath at 70°C 45 min or 30 min
(egg samples). Cooled in ice bath + 15 mL of NaCl
(10 g/L). Suspension extraction twice with 15 mL of
n-hexane/ethyl acetate (9:1, v/v)
Organic layers collected, evaporated; residue
dissolved in isopropyl alcohol (10%) in n-hexane
Carrot b-carotene 10 g extracted with n-hexane/ethanol 96% (1:1, v/v) LC-PDA [154]
Carotenes until colorless; kept at 20°C and analyzed within RP-18, 250 mm column
Xantophylls 72 h Mobile phase: (A): 1% water in MeOH (B) MeOH
and(C) 10% n-hexane with ACN
Sauces based on b-carotene Amaranth leaves (150 mg), Palm nuts pulp LC-PDA [155]
amaranth leaves Lutein (200 mg), and cooking water (10 mL) homogenized C30 (4.6 mm  250 mm, 5 lm) column
and palm nuts Violaxanthin 4  with 10 mL ethanol-hexane 4:3 (v/v). Filtered Mobile phase: (A) MeOH and Milli Q-water, 60:40,
9-cis-b-carotene through a glass funnel, transferred to a glass tube, (v/v) and (B) MeOH/MTBE:Milli Q-water, 28,5:
13-cis-b-carotene washed with 10 mL of 10% NaCl, 10 mL distilled 67,5:4, v/v/v)
water; 10 min partitioned. Organic extract dried
with N2; reconstituted in 2 mL of acetone.
Crude and thermal-treated Red Palm Oil, diluted
with acetone, centrifuged (9°C, 900g, 15 min)
Rose hips (Rosa spp.) 14 carotenoids 1 g of lyophilized rose hips extracted in 20 mL of a LC-PDA [156]
solution containing ethanol (99,7%), n-hexane and RP (250  4.6 mm, 4lm) column equipped with a
BHT (75:25:0.01). The extraction was done in an C18 guard column
orbital shaker in darkness at 4°C for 20 h; Mobile phase: (A) 80% ACN, 15% MeOH and 5%
centrifuged at 10000 g for 10 min DCM, (v/v) (B) 30% ACN, 20% MeOH and 50% DCM,
(v/v)

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60 K.T. Amorim-Carrilho et al. / Trends in Analytical Chemistry 56 (2014) 49–73

Table 3 (continued)

Sample Analyte Analytical method Ref.

Extraction protocol Detection system

Table olives b-carotene 2,5 g homogenized for 1 min with 10 mL hexane; LC-PDA-ESI()/MS [100]
centrifuged at 4000 rpm for 5 min. Supernatant RP-18 (100  3 mm, 2 lm, 3 lm) column
organic solution purified on a Strata SI-1 Silica Mobile phase: (A) ACN/MeOH (7/3) (B) i-propanol
cartridge (6 mL, 1 g). Cartridge conditioned with
3  3 mL of n-hexane; n-hexane was loaded onto
the cartridge at a flow rate lower than 0.5 mL/min.
Elution performed using a 6  4 mL of n-hexane/
ethyl acetate (9/1) at a flow rate lower than 0.5 mL/
min. Eluted. Totally evaporated under N2. Residue
dissolved in 10 mL of i-propanol/tetrahydrofuran
(7/3). Saponification: n-hexane/ethyl acetate
extract evaporated under vacuum at 40°C and the
residue dissolved in 10 mL of i-propanol/
tetrahydrofuran (7/3)
Papaya b-carotene 0.5 g homogenized in 10 mL of hexane:DCM (1:1, v/ LC-PDA-APCI(+)/MS [157]
b-cryptoxanthin v);centrifuged at 9000g for 10 min at 5°C (3 times). RP C30 (150 mmx 4.6 mm, 3 lm) column
Lycopene 10 mL of methanolic KOH 40% (1:1, v/v) for 1 h at Mobile phase: (A) MeOH (B) MTBE
50°C in a stirring bath at 100 rpm. After
saponification, 10 mL of 10% sodium sulfate;
extracts left in the dark for 1 hour.
Extract evaporated in a rotary evaporator at 30°C;
reconstituted in 2 mL acetone
Fish and seafood Lutein 0.5–1 g samples lyophilized + 5 mL MeOH; LC-PDA [105]
Zeaxanthin homogenized and left to stand overnight in a C30 (150 mm  4.6 mm, 3 lm) column
Crypoxanthin refrigerator at 4°C; centrifuged at 800g for 10 min. Mobile phase: (A) MeOH/MTBE/water 895:3:2, v/v/
5- lycopene MeOH layer separated. (3 times) 5 mL of THF added v, with 1.5% ammonium acetate in water (B) MeOH/
9-lycopene to the residue; vortexed for 30 s and centrifuged fat MTBE:water (8:90:2, v/v/v, with 1.0% ammonium
13-lycopene 800g for 5 min THF layers transferred to 25 mL flask acetate in water)
15-cis-lycopene containing MeOH.
Trans-Lycopene 10 mL was dried under N2 at 40°C and reconstituted
a-carotene in 500 lL of ethanol; vortexed for 30 and injected
All-trans-carotene
b-carotene
9-cis-b-carotene
13-cis-b-carotene
Different citrus species Zeaxanthin Lutein 300–500 g (fresh weight) extracted three times LC-PDA [158]
b-cryptoxanthin with MeOH and twice with diethyl ether. Extracts C18 (250  4.6 mm, 5 lm) column
b-citraurin combined, evaporated and saponified in ether with Mobile phase: (A) 12% (v/v) H2O in MeOH (B) MeOH
b-carotene 30% KOH/MeOH (C) 30% (v/v) DCM in MeOH
a-carotene
Violaxanthin
(9z)-violaxanthin
Mutatoxanthin
Nopal (O. ficus-indica b-carotene 7 g of fresh Nopal or 14 g of marmalade was LC-PDA [159]
var. Copena F-1) and Lutein macerated with 45 mL of cold acetone (4°C) and ODS2 (25 cm  4.6 mm, 5 lm) column
nopal marmalade a-cryptoxanthin vacuum filtered. Residue re-extracted twice more Mobile phase: ACN/MeOH/tetrahydrofuran
with cold acetone. A small portion of the combined (58:35:7)
filtrates mixed with 50 mL of petroleum ether and
200 mL of bi distilled water added slowly.
(Repeated until all the extracts were transferred
into the petroleum ether.) Ether phase washed four
times with water to remove all the acetone.
Residual water in the ether eliminated with
anhydrous sodium sulfate.
Extract concentrated in N2 and reconstituted in
5 mL of acetone
Virgin olive oil Chlorophyll a An oil-bonded cartridge conditioned by the LC-PDA [101]
Chlorophyll b consecutive passing of 6 mL of MeOH and 6 mL of ODS 2 (250 mm  4.6 mm, 5 lm) column
Pheophytins a hexane. Mobile phase: (A) MeOH/water (8:2 v/v) containing
Pheophytins b 1.0 g + 0.001 g virgin olive oil + 4 mL of hexane. 0.025%ammonium acetate and 0.05% triethylamine
Lutein Solvent collected in a volumetric flask. Column (B) MeOH/acetone (1:1, v/v)
Anteraxanthinv washed with 5 mL of hexane; hexane phase
Violaxanthin retained the b-carotene fraction.
Neoxanthin Column eluted with 3 mL of acetone; solvent
b-cryptoxanthin evaporated in a rotary evaporator until dryness and
reconstituted in 0.3 mL of acetone
Saffron Trans and cis-isomers Raw saffron obtained by the extraction of 20 mg of LC-PDA-ESI (+)/MS [160]
of crocins dried stigmata with 5 mL of MeOH. The mixture SB-C18 (4.6  150 mm, 3.5 lm) column connected
was sonicated by 15 min and kept at 60°C for with a SB-C18 guard column
another 15 min Mobile phase: (A) 0.15% formic acid in water (B)
0.15% formic acid in MeOH
 K.T. Amorim-Carrilho et al. / Trends in Analytical Chemistry 56 (2014) 49–73 61

Amazonian fruits 60 different Pulps extracted with acetone, transferred to LC-PDA-APCI (+)/MS [161]
carotenoids petroleum ether (30-70°C)/diethyl ether and C30 (250  4.6 mm, 3 lm) column
saponified overnight at room temperature with 10% Mobile phase: MeOH with 0.1% Triethylamine/
Methanolic KOH. Prior to ether transference, the MTBE
carotenoid extracts were kept in the freezer Gradient elution
(18°C) for 2 h, followed by filtration and washing
with cold acetone
Extruded and non- Total carotenoids and (3X) 10 g + 30 mL MeOH-tetrahydrofuran (Me-THF, LC-PDA [162]
extruded sweet b-carotene 1:1, v/v) containing 0.1% BHT for 30 min at 45 rpm; 250  4.6 mm, 5 lm internal diameter column (no
potato flours centrifuged at 3000g, 12°C, 5 min. Extract separated more details given)
with petroleum ether (PE, 40 mL) and 300 mL Mobile phase: MeOH/THF/water (67:27:6)
MilliQ water for 15 min
The upper PE dried with 5g of anhydrous Na2SO4;
sample filtered, made up to 50 mL, concentrated
under N2 and redissolved in 1 mL of MeOH-THF
Fruit juice milk Cis-Violaxanthin 30 mg + 50 mL magnesium hydroxide LC-PDA [163]
beverage Anteraxanthin carbonate + 25 mg BHT and 35 mL ethanol:hexane RP C18 (250 mm  4.6 mm, 5 lm) column
cis-Anteraxanthin (4:3 v/v) under N2; agitation 45 min; filtration; Mobile phase: (A) MeOH/ammonium acetate 0.1 M
Lutein Residue washed with 35 mL of ethanol-hexane (4:3 (B) MilliQ water (C) MTBE and (D) MeOH
Zeaxanthin v/v), filtered twice with 12.5 mL of ethanol and once
a-cryptoxanthin with 12.5 mL of hexane (until colorless). All liquid
b-cryptoxanthin filtrates washed twice with 50 mL of sodium
b-carotene chloride solution (10%), three times with 50 mL
a-carotene water. Organic phase evaporated at 40°C; residue
under N2 + 10 mL of diethyl ether, 10 mL of
methanolic KOH 0.5 M with 0.1% BHT + 20 mL of
diethyl ether. Solution washed by the same
procedure before + 10 mL ethanol.
Solution evaporated at 45°C; residue dissolved with
4 mL of diethyl ether, evaporated under N2 and
reconstituted with 1 mL of MeOH:TBME (70:30, v/v)
20 watermelon Lycopene 3 g of slurry homogenized with 30 mL acetone. LC-PDA [164]
genotypes Lutein Homogenates washed with acetone on filter paper OSD2 (250 mm  4.6 mm, 5 lm) column connected
Violaxanthin until colorless. Acetone extracts + hexane with a guard column
Neurosporene (50 mL) + 300 mL water. Mobile phase: (A) ethylacetate (B) ACN/water (9:1,
Zeacarotene Hexane extracts (0.5 mL or 3 mL depending on the v/v) both containing 0.1% triethylamine
Phytofluene concentration) evaporated with N2 and
Phytoene reconstituted with 0.5 mL acetone
Soybean Lutein 100 mg soybean + 3 mL EtOH, 0.1% TBHQ; vortex LC-PDA [165]
10 s; water bath 85C  5 min. Add 0.10 mL 10 mol/ Phenyl (150 mm  3.9 mm, 5 lm) column
mL KOH; vortex 10 s; water bath 85°C  10 min, connected with a guard column
vortex 10 s. Add 3 mL 1 mol/L NaCl. Invert gently Ternary isocratic solvent system: ACN, MeOH and
5. Add 3 mL hexane, vortex 10 s; centrifuged at water (48:22.5:29.5, v/v/v). Isocratic conditions for
1000xg  5 min at 4°C. Repeat hexane extraction 40 min, followed by a 4 min linear gradient to 100%
2; Combined supernatants washed with 5 mL 5% MeOH for 7 min, subsequent 4 min linear gradient
Na2CO3 (w/v); centrifuged at 1000  g, x5 min at to initial conditions
4°C.
Wash supernatants with 5 mL ultrapure water;
evaporated under N2 and dissolved in 250 mL IPA
(isopropyl alcohol)
25 vegetables Lutein 10g of raw or cooked + 20 mL cold acetone LC-PDA [166]
Zeaxanthin 10 mL of internal standard b-apo-80 carotenal, 6 C30 (4.6 mm  250 mm, 5 lm) carotenoid column
b-carotene Trans/cis (E/ mg/200 mL hexane; homogenized for 60s; Mobile phase: (A) MeOH/MTBE (85:15, v/v), (B)
Z)-b-carotene centrifuged at 1500  g for 5 min. Precipitate MeOH/MTBE (6:94, v/v)
reconstituted in 20 mL acetone and homogenized
60s; centrifuged. Acetone extraction repeated until
colorless. Supernatants combined and dried under
vacuum at a temperature below 30°C.
Residue dissolved in acetone by ultrasonic agitation
to a final volume of 20 mL. (Ambersep 900 OH,
0.1 g) + acetone extract (20 mL)
Avocado paste 12 carotenoids (3X) 2 g + b-apo-80 carotenal + 10 mL acetone; LP-PDA [167]
ultrasonic bath 2 min; filtered under vacuum. C30 reverse phase (4,5 mm  250 mm, 5 lm)
Acetone extracts concentrated in a centrifugal column
evaporator at 37°C for 1 h. Concentrated acetone Mobile phase: (A) MeOH/MTBE/water (81:15:4, v/
extracts + 2 mL of 9 M KOH in ethanol/water (50%) v) (B) MTBE/MeOH/water (90:6:4, v/v)
for 10 h in dark vials with N2. Saponified
extracts + 10 mL bi distilled water; three
consecutive extractions with hexane (12 mL) in a
separator funnel. Hexane fraction washed 10 times
with 10 mL bi distilled water until neutrality.
0.5 mL of 30% (p/v) NaCl added if turbidity.
Hexane fraction concentrated at 37°C for 1 hour;
dried with N2 and re-suspended with 1 mL
isopropyl alcohol

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62 K.T. Amorim-Carrilho et al. / Trends in Analytical Chemistry 56 (2014) 49–73

Table 3 (continued)

Sample Analyte Analytical method Ref.

Extraction protocol Detection system

Dried peppers Violaxanthin 1 g of ground pepper + 20 mL cold acetone (5°C); LC-PDA [168]
b-cryptoxanthin vacuum filtered. Filtrate transferred to a separation ODS (25 cm  4.6 mm, 5 lm) column
b-carotene funnel together with petroleum ether and water. Isocratic, mobile phase: ACN/MeOH/
The upper ether layer washed several times with tetrahydrofuran (THF) (58:35:7)
water; added anhydrous sodium sulfate. Ether
phase adjusted to 10 mL with petroleum ether;
concentrated with N2 and re-suspended in 1 mL
acetone
Sweet potato b-carotene 2 g + 5 mL acetone + acetone/petroleum ether LC-UV-Vis [169]
(20:80); filtration. Procedure repeated until C18 RP-18 (4.6 mm  200 mm, 5 lm) column
colorless. Filtrate + anhydrous sodium sulfate. Mobile phase: ACN/MeOH/ethylacetate/
Concentrated in a rotary vacuum evaporator max. triethylamine (79:10:20:0.05)
35°C until 2 mL remained.
Dried under N2 and re-suspended in 5 mL
petroleum ether
Frozen carrot and Lutein 0.1 g of lyophilized samples + THF containing 0.01% LC-PDA [170]
spinach b-Carotene BHT ODS3 (250  4.6 mm  5 lm) column
a-Carotene Dried under N2 and re-suspended in DCM Mobile phase: (A) ACN/ n-hexane/MeOH and DCM
Phytofluene (2:1:1:1, v/v/v/v) (B) ACN
Phytoene
Tomato Lutein 100 mg + 3 mL ethyl acetate containing BHT Total individual carotenoid: LC-PDA [171]
Lycopene (100 mg/L); vortex mixing for 1 min; centrifuged C18 (3.9  150 mm, 5 lm) column
b-Carotene for 5 min Mobile phase: MeOH/ACN and THF (50:45:5, v/v/v)
Lycopene Upper hexane removed. Extraction with 2 mL of Lycopene and b-Carotene isomers: LC-PDA
b-Carotene isomers ethyl acetate (3 or 4) until colorless. Ethyl acetate C18 column
fractions combined, evaporated in a vacuum Mobile phase: MeOH/ACN/2-propanol (54:44:2, v/
evaporator. v/v)
Residue re-suspended in 25 lL of diethyl ether and
75 lL of MeOH/ACN/THF (50:45:5, v/v/v)
Sweet potato b-Carotene and 5 g extracted thrice with 50 mL of cold acetone. LC-PDA [172]
isomers Extracts pooled and transferred to a separation C30 (250  4.6 mm, 5 lm) carotenoid column
funnel and 40 mL PE added. Washed thrice with Isocratic elution with a mobile phase of MeOH/
200 mL of ultra-pure water. Upper PE-phase MTBE (80:20)
collected, dried with anhydrous sodium sulfate.
Sample filtered and concentrated to dryness in
vacuum (30°C) and under N2.
Re-suspended in 2 mL of acetone
Einkorn, emmer and Lutein 3 g + 15 mL of ethanol/acetone/hexane (1:1:2). LC-PDA [173]
spring wheat Zeaxanthin Overnight extraction Ultrasonic bath for 10 min. C30 (3.0  150 mm, 5 lm) Carotenoid column
varieties b-Carotene Samples filtered. Filter cake washed until colorless Mobile phase: MeOH/water/TBME
with 3 mL of extraction mixture. Filtrates
transferred into 25 mL flasks and made up with
extraction mixture.
Aliquots (20 mL) evaporated on a rotary evaporator
and the residue dissolved in 2-3 mL ethanol/
acetone (6:4) with 0.2% BHT
Whole wheat flour Lutein 0.10 g + 3 mL of 0.1% TBHQ in ethanol; vortex for 10 LC-PDA [174]
s and heated at 85°C for 5 min + 0.19 mL of KOH (3.9 mm  150 mm, 3.5 lm) column
(10M) and remained in a heated water for 10 min. Mobile phase: MeOH/ACN/water (22.5:48:29.5)
Tubes removed and placed on ice + 3 mL of 1 M
NaCl + 3 mL of hexane; vortex and centrifuged.
Hexane extraction repeated twice
Combined hexane supernatants washed with
sodium carbonate + pure water. Evaporated under
N2 and re-suspended in 0.25 mL of isopropyl
alcohol
Cereals Lutein 0.1 g (spiked with 0.01 to 1 lL/mL for b-Carotene, LC-PDA [102]
b-Carotene 0.01 to 4 lL/ mL for lutein) + 2 mL of ethanol + 0.1 g C18 (250 mm  4.6 mm, 5 lm) column
for ascorbic acid. Vortexed for 1 min, water bath for Mobile phase: MeOH/ACN/isopropanol
5 min + 100 lL (KOH 80% in water), vortexed for Gradient elution
20s, in water bath for 15 min and mixed every
5 min. Supernatant removed, residue re-extracted
with 2 mL of ethanol by sonication for 1 min,
centrifuged. Extraction repeated one more time
with 1 mL ethanol. Combined extract applied to SPE
cartridge (OASIS HBL) conditioned with 3 mL MeOH
and 3 mL of water. Extracts 8 + 2 mL water + 2 mL
DCM. Evaporation under N2 at 30°C
Reconstituted with 200 lL of methanolic solution of
a-tocopherol acetate (IS, 50 lm/mL)
 K.T. Amorim-Carrilho et al. / Trends in Analytical Chemistry 56 (2014) 49–73 63

Lycium barbarum 10 carotenoids 10 g powdered fruit sample + 50 mL of 10% of LC-MS [175]

(traditional Chinese hexane/ethanol/acetone/toluene (10:6:7:7, v/v/v/ YMC C30 column
herb) v) + 5 mL of 40% methanolic solution under N2 in Mobile phase: (A) MeOH/ACN/water (81:14:5, v/v/
the dark for 6 h v) (B) methylene chloride
Extract evaporated to dryness and dissolved in
20 mL of methylene chloride
Mango All-trans-violaxanthin 6 g + 0.2 g calcium carbonate + 15 mL MeOH. HPLC-PDA-APCI(+)/MS [176]
9-cis-violaxanthin Homogenates filtered by adding MeOH until C30 RP (4.6 mm  150 mm, 3 lm) column
All-trans-b-Carotene colorless. methanolic extract + 50 mL of hexane- Mobile phase: (A) water (B) MeOH (C) MTBE
acetone (1:1, v/v) containing 0.1% BHT. Vigorous
stirring + 40 mL of 10% sodium sulfate. Upper layer
separated, washed with water and evaporated in a
rotary evaporator at 35°C. Residue dissolved in
diethyl ether (30 mL) + 0.2 mL of 40% KOH in MeOH
(16 h in the dark). Extract washed with water,
evaporated and dissolved in 2-propanol (2 mL)
Crocus sativus Lutein diesters 2 g + 30 mL cold acetone, stirred for 3 h. Extract UPLC PDA-APCI(+)/MS [177]
filtered and residue re-extracted with 30 mL cold n- UPLC C18 (150 mm  2.1 mm, 1.8 lm) column
hexane for one hour. Filtrates centrifuged at 2200 g Mobile phase: (A) CH3OH/H2O (80:20,v/v) (B) ethyl
for 10 min at 4°C. Supernatants concentrated in a acetate
rotary evaporated. Residue dissolved in 20 mL
acetone. 1 mL acetone extract evaporated under
argon, dissolved in 50 lL of CH2Cl2 and completed
to 500 lL with CH3OH
Saponification:
2 mL acetone extract evaporated under argon,
dissolved in 2 mL n-hexane with 2 mL of CH3OH/
KOH (20% v/w), stirred, centrifuged at 2200  g for
5 min at 4°C. Upper hexane layer washed with
CH3OH and water. Lower methanolic phase washed
with diethyl ether, same volume water and washed
again with water
Extracts evaporated and dissolved in 50 lL of
CH2Cl2 and completed with CH3OH
Capsicum 52 carotenoids 10 g + 1 g sodium bicarbonate. Extract with acetone HPLC-PDA-APCI(+/)/MS [178]
until colorless. Combined acetone extracts C30 (250  4.6 mm, 5 lm) column
evaporated under vacuum at 35°C until 50 mL. Mobile phase: (A) MeOH/MTBE/water (82:16:2, v/
Concentrated into a separator funnel with 100 mL v/v) (B) MeOH/MTBE/water (10:88:2, v/v/v)
of diethyl ether, shacken and left to settle. NaCl
solution (10%) + several times with Na2SO4 solution
(2%). Ether phase evaporated to dryness at 30°C
Dry residue dissolved in MeOH/MTBE (1:1, v/v)
Apricots and pumpkins 25 carotenoids 1.5 g powdered plant material and purées + 50 mL HPLC-PDA-MS [179]
acetone/hexane (1:1, v/v) containing BHT (50 mg/ C30 RP (150  3 mm, 3 lm) column
100 mL) and butylated hydroxyanisole (50 mg/ Mobile phase: (A) MeOH/MTBE (MTBE)/water
100 mL). Extract filtered and residue washed with (81:15:4, v/v/v) (B) MeOH/MTBE/water (4:92:4, v/
75 mL of ethyl acetate. Combined extract v/v)
evaporated to dryness in vacuum and residue
dissolved in 2-propanol (2 or 5 mL) For jam: 3 g
lyophilized + 50 mL MeOH containing 0.1%
pyrogallol. Extract filtered and solvent concentrated
to 15–20 mL. Residue transferred to a separating
funnel + 25 mL of hexane (twice). Pooled organic
phases washed with 50 mL of water; dried with
sodium sulfate and evaporated in vacuum
Residue dissolved in 2-propanol and made up to
2 mL
Spinach and collard Lutein Spinach and collard greens: 500 mg of purée LC-APCI(+)/MS [126]
greens vegetables + 10 mL MeOH for 1 h in a shaking RP C30 (150 mm  4.6 mm, 3 lm)
incubator at 120 rpm. Homogenized for 30 s in an Mobile phase: (A) MeOH/TBME/water (83:15:2, v/
ice bath and washed with MeOH. Centrifuged at v/v) and (B) MeOH/TBME/water (8:90:2,v/v/v)
3000 rpm for 5 min. MeOH layer collected.
Extraction repeated four times with THF; vortexed,
centrifuged. THF layers combined with MeOH
layers. 1 mL dried under N2 and re-suspended in
1 mL of ethanol
Mandarin Free and Carotenoid 5 g oil + 0.1% (w/w) BHT + 5 mL of methanolic HPLC-PDA and HPLC-APCI(+)/MS [180]
esters potassium hydroxide (2M) Mixture left under For free carotenoids: Supelcosil LC-SI
stirring for 16 h. Extraction with three aliquots of (300 mm  1.0 mm, 5 lm) column
5 mL diethyl ether. Organic extracts combined, Mobile phase: (A) n-hexane and (B) ethyl alcohol
washed with water until neutral pH Second dimension: Chromolith RP-18
Solvent evaporated under vacuum at 30°C to 4 mL (100 mm  4.6 mm) monolithic column
⁄
For carotenoid esters: oil directly injected Mobile phase (A): 2-propanol (B) 20% (v/v) water in
ACN
Carotenoid esters: Discovery Cyano

(continued on next page)

64 K.T. Amorim-Carrilho et al. / Trends in Analytical Chemistry 56 (2014) 49–73

Table 3 (continued)

Sample Analyte Analytical method Ref.

Extraction protocol Detection system
(250 mm  1.0 mm, 5 lm)
Mobile phase (A) n-hexane, (B) n-hexane/butyl
acetate/acetone 80:15:5 (v/v/v)
Second dimension: same column and mobile as free
carotenoid
Water spinach b-carotene Extraction with a mixture of MeOH, acetone and TLC and HPLC-PDA [181]
Lutein petroleum ether (1:1:1, v/v/v) until colorless. Purified by TLC silica gel (activated for 2 hours at
Violaxanthin Saponified by 30% (w/v) KOH in MeOH for 16 h. 110C) with n-hexane: ethylacetate:acetone:MeOH
Extract washed until all KOH removed (27:4:2:2, v/v/v/v)
Concentrated in a rotary evaporator (30°C). Re- YMC C30 column (150 mm  4.6 mm, 3 lm)
suspended in acetone Mobile phase: (A) MeOH/water (9:1,v/v), (B) MTBE
Cabbage Lutein 5 g + 100 mL DCM:MeOH solution (2:1, v/v). LC-PDA-APCI (+)/MS [106]
b-carotene Centrifuged at 17000 g. Extraction repeated twice 5C18 ODS (4.6  250 mm)
Phytoene with 100 mL of DCM. Filtrated. Added 100 mL of Mobile phase: ACN/ethanol (8:2,v/v)
Prolycopene hexane and 100 mL of 5% NaCl. Supernatant
evaporated to dryness. Precipitated dissolved in
50 mL of diethyl ether + 5 mL of 60% (w/v) KOH.
Equal volume (50 mL) of hexane: diethyl ether
solution (1:1, v/v) and 5% NaCl added to saponified
solution
Organic solvent collected and treated with 5% NaCl,
evaporated to dryness and dissolved in diethyl
ether
Coriander (Coriandrim b-carotene Extraction with cold-acetone until colorless + 0.01% LC-PDA [104]
sativum L) BHT. Extract containing excess water + petroleum C18 RP (259  4.6 mm) column
ether centrifuged at 4°C. PE extract + KOH (10% w/ Mobile phase: ACN:MeOH:ethylacetate (80:10:10,
v). Extracted washed, concentrated at 40°C in v/v/v)
vacuum using rotary evaporator
Re-dissolved in 1 mL of mobile phase
Tomato paste All-trans 25 (+0.05) mg extracted three times using 4.5 mL of HPLC-PDA [182]
Lycopene MeOH-CHCl3 (2.5:2.0, v/v) and 2.5 mL of Tris buffer YMC C30 column with a gradient of MeOH and
b-carotene (pH 7.5) TBME
Lutein CHCl3 fractions pooled, dried under N2 gas and
taken up in ethyl acetate
Food complements Free lutein Extraction with ethyl acetate + 0.1% BHT in HPTLC (high-performance thin-layer [183]
Lutein esters ultrasonic bath for 30 min chromatography) and densitometry
Lycopene Centrifuged, supernatant filtered Thin layer chromatographic analysis performed on
b-carotene 20 cm  10 cm C18 RP HPTLC glass plates with
layer thickness 0.20 mm
TLC-MS: C18 RP HPTLC plate using MeOH-ethyl
acetate 3:1 (v/v)
Red chili peppers Free carotenoids and 200 g of homogenate extracted three times with NP-LC  RP-UHPLC with PDA and IT-TOF (APCI [208]
(Capsicum annuum carotenoid esters (>30) 300 mL of MeOH/ethyl acetate/petroleum ether ionization) MS
L) (1:1:1, v/v/v) Micro-bore cyano column (250 mm  1.0 mm,
Combined extracts dissolved in 4 mL of MeOH/tert- 5 lm) for 1D
butyl methyl ether (1:1, v/v) + filtration 0.45 lm C18 column (30 mm  4.6 mm, 2.7 lm) packed
acrodisc nylon membrane with fused-core particles for 2D
Mobile phase: 1D (A) n-hexane (B) n-hexane/butyl-
acetate/acetone (80:15:5, v/v/v); 2D (A) water/ACN
(10:90, v/v) (B) isopropanol
Yellow and red pepper 20 carotenoids 15–20 g homogenized sample + acetone, HPLC-PDA-MS [258]
(Capsicum annum L.) transferred to petroleum ether. Saponification 10% C18 (4.6  150 mm, 3 l) column.
KOH overnight (dark, ambient temperature) Mobile phase, gradient elution: ACN 0.05%TEA/
Extract washed free of acetone, anhydrous sodium MeOH/ethyl acetate
sulfate to remove water. Concentration and
evaporation until dryness with N2. Redissolved in
acetone

ACN, Acetonitrile; APO, Trans-b-apo-80 -carotenal; BHT, Butylhydroxytoluene; DCM, Dichloromethane; HPTLC, High-performance thin-layer chromatography; MeOH,
Methanol; MTBE, Methyl tert-butyl ether; NP, Normal phase; RP, Reversed phase; TLC, Thin-layer chromatography; UPLC, Ultra-high-performance liquid chromatography.

treatment [108]. Sun et al. [193] compared four sample-prepara-
tion methods for the evaluation of lutein content in the chicken li-
ver. Higher lutein concentration was found after UAE. The results
suggested that most lutein content in the chicken liver is present
within the liver cell and cannot be released by using only solvent
extraction. In addition, UAE may cause greater cell disruption
and stronger agitation. This might lead to a more efficient extrac-
tion by distributing more uniformly the solvent and avoiding
chemical-degradation reactions caused by alkali conditions in
saponification methods.
In 2011, Aktas and Yildiz [194] compared the effects of electro-
plasmolysis (EP) and heating in water-bath treatments on caroten-
oid extraction from spinach and tomatoes. The highest increase in
extraction yield for b-carotene and lycopene from tomato was ob-
tained by electrical-field treatment at 80 V/cm for 4 s. These same
conditions significantly decreased the b-carotene extraction yield
 K.T. Amorim-Carrilho et al. / Trends in Analytical Chemistry 56 (2014) 49–73
from spinach, for which the best extraction was achieved by a
water-bath process at 80°C. Conversely, all water-bath treatments
decreased the extraction yield of lycopene in tomato. Ranveer et al.
[35] optimized an extraction process of lycopene from tomato-pro-
cessing-industry waste by comparing the effectiveness of the en-
zyme treatments on lycopene extraction, which was higher at
1.5% and 2% for cellulose and pectinase, respectively. The authors
also fixed 4 h as an optimal incubation time when using cellulase
and pectinase enzymes, and stated that the finer the particle size
the higher the recovery of lycopene.
Very recently, Jeddi et al. [195] evaluated the extraction of
carotenoid pigments from shrimp wastes using four different
extraction techniques: solvent, acid, alkaline and enzyme. The re-
sults showed that alkaline extraction gave significantly higher
amounts of pigments than the other methods.
More information about innovation on carotenoid extraction
was gathered in an article from 2010 by Riggi [196], showing the
patents relating to the extraction of carotenoids presented in the
past decade.
3.2. Determination methods
The basic chemical structure of the carotenoids, based on
eight isoprenoid units with a conjugated double-bond system,
makes isomeric forms very common. Besides, their double bonds
in the carbon chain make carotenoids susceptible to some reac-
tions, such as oxidation and isomerization (cis–trans), especially
due to light, heat, acids, and oxygen [197]. Cyclization, hydroge-
nation, dehydrogenation or additions of lateral groups, among
others, are some modifications that lead to the existence of the
extremely complex variety of compounds with a common struc-
ture. Moreover, carotenoids can be found in nature both in their
free form and also in a more stable, esterified form with fatty
acids [198]. The high variability in their chemical structure and
their poor stability greatly contribute to the difficulty of caroten-
oid analysis. The lack of commercially-available standards, the
usually low concentrations found in biological samples, such as
human serum and tissue, and the presence of potentially-
interfering compounds [199] add difficulty to the development
of analytical methods to identify and to measure carotenoids in
real samples [200].
Carotenoid identity can be confirmed by liquid chromatography
coupled to mass spectrometry (LC-MS), comparing the mass spec-
tra with published fragment-ion abundances, or mass spectral li-
braries. In the case of unknown peaks, the coupling of on-line
PDA or UV-Vis detector and LC-MS/MS can provide valuable data
for their identification.
However, carotenoid quantification is usually based on a linear
relationship between the weight of standard injected and the
resulting peak area. The peak area is not very sensitive to chro-
matographic conditions, especially compared to peak height.
Carotenoid chromatograms are usually very complex, due to the
high variety of isomers and structurally related compounds and
metabolites. There are no standards available for all the carote-
noids that are susceptible to being analyzed or measured, and, in
many cases, only one isomeric form is commercially available.
Even though quality assurance (QA) issues are beyond the scope
of this review, some aspects may be commented upon. Carotenoid
concentration in natural samples cannot really be known, therefore
quality control (QC) measurements should demonstrate that the
method is capable of producing accurate results and uncertainties
are within expected limits. The main performance parameters that
should be assessed in an analytical method are accuracy, precision,
specificity, limits of detection and quantification, linearity of the
method and scope. When desiring to validate a method for mea-
suring carotenoids (or other compounds), some guides can be
65
found in the literature [201] and some validated methods for
carotenoids [202] can be followed as examples.
3.2.1. Separation methods: liquid chromatography
The monomeric octyl (C8) and octadecyl (C18) stationary phases
are the most widely and efficiently used packings for reversed-
phase chromatography. However, they have poor resolution for
the geometric cis-trans isomers. The C18 columns provide good
separation for a wide range of analytes, especially for those with
relatively short chains and low molecular weights. However, the
C30 columns with longer alkyl chains give better shape and selec-
tivity for long-chain analytes, so C30 stationary phases have shown
significant and often superior overall enhancement in molecular
shape separation and selectivity than the conventional C8 and
C18. C30 columns are a particularly good choice for better separa-
tion and high resolution of geometric isomers of the less polar
carotenes, mainly lycopene and b-carotene. However, when ap-
plied to the general analysis of carotenoids, including xantophylls
and carotenes from foods, polymeric C30 packings give profiles
similar to those obtained with conventional C18 columns, but with
longer chromatographic times [203]. The limitations on application
of C30 columns are therefore their efficiency to separate only geo-
metrical isomers of carotenoids with a limited range of polarity
and their long analytical times (generally 60–100 min, as opposed
to 10–25 min for C18 columns) [204]. Basically, high-performance
LC (HPLC) using reversed-phase C8, C18 or C30 bonded-phase col-
umns is the preferred approach for the separation of carotenoids
in sample extracts isolated from biological matrices.
Single-column (one-dimensional) chromatography has been
used for many years as a standard separation tool for analyzing
compounds in a broad variety of fields including food analysis.
However, it does not always provide the resolution and the separa-
tion power required to obtain the best results in terms of identifica-
tion of analytes in food samples, especially in very complex cases.
Multidimensional chromatography has emerged as an interesting
alternative to analyze complex matrices, since it allows the combi-
nation of two or more independent or nearly-independent separa-
tion steps, increasing significantly the separation power over the
corresponding one-dimensional techniques [205]. The record of
the detector at the outlet from the second-dimension column is
transformed into a 2D-chromatogram, which is represented as a
contour plot showing the separation time in the second dimension
versus the separation time in the first dimension. In the bi-dimen-
sional contour plot, each component is represented as an ellipse-
shaped peak, defined by doubled-axis retention-time coordinates.
The formation of a chemical-class pattern on the second-dimension
space plane offers great potential for the identification of ‘‘un-
known’’ peaks. The first application of comprehensive LC  LC
was used to elucidate the carotenoid pattern in saponified essential
oil of red orange, using a silica micro-HPLC normal-phase column as
first dimension, and a reversed-phase monolithic C18 column in the
second dimension [206]. Dugo et al. [180] also analyzed free (i.e.,
not esterified) carotenoids and carotenoid esters using a microbore
cyano column in the normal phase and a monolithic C18 column in
the reversed phase, with photodiode array (PDA) detection and an
APCI-MS detector. The study allowed identification of 40 different
carotenoids, without the need for any pre-treatment. In 2009, Dugo
et al. [207] used again two-dimensional LC to analyze epoxy-carot-
enoid esters in intact juices. More recently, Cacciola et al. [208] ap-
plied an UHPLC system with a micro-bore-cyano column for the
first-dimension separation, interfaced to a second-dimension C18
column packed with fused-core particles, to analyze the carotenoid
composition of red chili peppers, with PDA and MS detection. The
superior performance of the fused-core particles may be due to
the very narrow particle-size distribution of the superficially-por-
ous particles (SPPs) and perhaps their high particle density, which
66 K.T. Amorim-Carrilho et al. / Trends in Analytical Chemistry 56 (2014) 49–73
result in very homogeneous, efficiently packed beds. Also, expen-
sive, very high-pressure instruments and a special technique for
very high-pressure operation are unnecessary [209].
With regard to the solvent systems that have been suggested as
mobile phases for carotenoid analysis, most are modifications of
acetonitrile (ACN) and/or methanol that are primarily used. Polar-
ity, viscosity, volatility and toxicity are features that should be con-
sidered when selecting a mobile phase for any analyte. ACN is
frequently used due to its low viscosity, low absorbance under
UV light (low noise in UV detection), lower column pressure and,
in general, good peak shape. However, methanol has shown higher
recovery of carotenoids than ACN [210], and it is also more avail-
able, less expensive, and less toxic.
Usually, a small percentage of a less polar solvent is added as a
modifier of the primary solvent to obtain the desired retention, to
increase solubility of the analytes and to improve resolution. The
most frequently used are dichloromethane, THF, MTBE, ethyl ace-
tate, hexane, acetone, other chlorinated solvents (e.g., chloroform)
and water. Other solvents, such as methylene chloride, methyl
alcohol, 1-propanol, 2-propanol, ethanol and butyl acetate, are also
used (see Tables 2 and 3). The addition of ammonium acetate buf-
fer or triethylamine (TEA) to ACN-based solvents enhances carot-
enoid recovery, and THF is the most beneficial modifier for
methanol-based systems [91].
3.2.2. Detection and identification methods: mass spectrometry
A range of LC-based techniques have been used to analyze
carotenoids, most of them coupled to a PDA or UV-Vis detector
(see Tables 2 and 3 for references). Although LC separation coupled
to UV-Vis instruments has been the most common analytical
method for determining carotenoids qualitatively and quantita-
tively, the spectra of many carotenoids are very similar, so many
researchers have complemented the identification of carotenoids
using others detectors, such as MS. With UV and PDA systems, it
is impossible to provide molecular structure information for iden-
tification, especially for unknown carotenoids in complex sample
matrices. The MS instruments are used to overcome spectral inter-
ferences in UV-Vis and, therefore, to achieve high sensitivity in
complex mixtures [122] and to obtain molecular structure
information on the basis of the molecular mass and fragmentation
pattern under tandem MS (MS/MS and MS/MS/MS).
Although the fragmentation pattern depends on the ionization
technique and composition of the mobile phase used, characteris-
tic carotenoid fragments have been observed with various ioniza-
tion techniques. This approach can be used to distinguish
carotenoids with the same molecular mass but different fragmen-
tation patterns, such as structural and geometrical isomers
[136,138,149,160,166,176,211]. The study of geometrical isomers
of carotenoids is becoming to be of great importance since their
properties (vitamin A activity, susceptibility to oxidation, and bio-
availability) differ from those of their (all-E)-counterparts [212].
HPLC/MS-MS has also been used to distinguish between
structurally-related molecules and their epoxidized forms
[109,124,177], products of carotenoid oxidation, that are potential
oxidative stress markers and difficult to profile due to their small
amounts and the difficulty in separating them from hydroxy
carotenoids.
3.2.2.1. LC-MS interfacing. Several interfacing strategies have been
reported in the literature when using LC-MS as the detection sys-
tem. For carotenoids, the reported ionization methods include
electron impact (EI), fast atom bombardment (FAB), matrix-as-
sisted laser desorption/ionization (MALDI), electrospray ionization
(ESI), atmospheric pressure chemical ionization (APCI), atmo-
spheric pressure photoionization (APPI) and atmospheric solids
analysis probe (ASAP) [200]. Raman spectroscopy, ASAP and
MALDI-TOF-MS can be used for the direct analysis of samples,
without the need for sample preparation or chromatographic
separation [213], so MALDI-ToF-MS has been used to analyze
plant-derived metabolites and microbial metabolites, as it obtains
a profile of multiple carotenoid species from crude extracts
[214–216]. FAB, also known as liquid secondary ion mass spec-
trometry (LSIMS), is similar to MALDI but ‘‘softer’’, and it is often
used for compounds with high polarity and low volatility, which
are thermally and energetically labile. Mostly, analytes stay intact
during ionization and fragmentation reactions are rare [217]. FAB
proved to be most suitable for the analysis of samples containing
pre-formed ions in solution (i.e., protonated/deprotonated or
sodium adducts) [218]. Very few authors have analyzed carote-
noids (including esterified carotenoids) using FAB-MS [219–221].
The use of FAB ionization minimized degradation or rearrange-
ment of the carotenoid structures due to the inherent thermal
instability generally ascribed to these compounds.
Atmospheric pressure ionization (API) interfaces are the most
frequently used in carotenoid analysis, mainly due to their simplic-
ity and the direct on-line coupling of separation techniques to the -
mass spectrometer. API interfaces have replaced FAB ionization for
most MS applications [199].
ASAP is a relatively new technique with only a limited number
of applications reported to date. In 2005, McEwen et al. described
the qualitative detection of a range of chemicals, including carote-
noids in spinach leaves, using ASAP [222].
Among the ionization techniques compatible with LC, the most
successful for carotenoid studies have been ESI and APCI. One clas-
sic example of the use of APCI in plant studies has been the analysis
of lipid-soluble carotenoids. APCI generally has a greater ability to
ionize non-polar compounds and, if a study was particularly tar-
geted to oil/soluble pigments such as carotenoids, APCI would be
the most appropriate ionization method.
Alternatively, ESI is more suited to the ionization of more polar
compounds ([223]. Although carotenes and xanthophylls form
molecular ions or protonated molecules during positive-ion elec-
trospray, the hydrocarbon carotenes do not ionize when using neg-
ative-ion electrospray. However, APCI forms abundant positively-
or negatively-charged molecular ions or protonated and deproto-
nated molecules of both carotenes and xanthophylls [199]. For that
reason, this kind of ionization is frequently reported in the analysis
of carotenoids, and several authors have used LC-PDA-APCI-MS
methods to analyze and to identify a wide range of carotenoid com-
pounds in different matrices [109,136,143,176,177]. Recently, APPI
was introduced for the ionization of molecules with little or no
polarity, insufficiently ionized by either APCI or ESI sources [224].
Considering that unknown matrix components in the mobile
phase and sample may enhance or reduce the MS-ionization effi-
ciency, several studies have been performed to get a better under-
standing of the factors influencing the ionization process of
carotenoids [225]. For ESI, good results have been obtained using
additional chemicals that facilitate the ionization process, such as
ferrocene-based derivatives [226]. Other examples are:
 ammonium acetate, which has been used to increase the abun-
dance of deprotonated xanthophyll molecules using ESI in neg-
ative-ion mode;
 acetic acid, which has been applied to increase the abundance of
protonated xantophylls using ESI in positive ion mode; and,
 halogen-containing eluents, which have been used to increase
the molecular ions of xantophylls and carotenes using ESI in
positive ion mode.
Xantophylls can also be detected when sodium acetate is added
to the mobile phase by forming adducts between its oxygen and
the sodium acetate [211]. Ionization of both carotenes and xanto-
 K.T. Amorim-Carrilho et al. / Trends in Analytical Chemistry 56 (2014) 49–73
phylls was achieved in 1998 by Rentel et al. using AgClO4 solution
as a post-column additive [227]. Similarly, Rivera et al. tested the
suitability of different ionization techniques (ESI, APCI, and APPI)
to ionize carotenoids under MS [228]. The results indicated that
APCI is a more powerful choice than ESI or APPI. Also, four dopants
– acetone, toluene, anisole and chlorobenzene – were evaluated
with APPI to enhance the carotenoid signal, and the signal strength
improved in almost all cases when a dopant was used, but caro-
tenes showed higher signals than xantophylls.
Formic acid and acetic acid were added to the eluent by Lech
et al. [160] in order to improve ionization efficiency of trans- and
cis- isomers of crocins in saffron analysis by ESI. Adducts with so-
dium salts of these acids and hydrochloric acid were also observed
in the spectra. Recently, Vallverdú-Queralt et al. [147] added post-
column lithium chloride to promote the positive ionization of
carotenoids in ketchup and gazpachos. A high limit of detection
was observed without the addition of lithium chloride, even when
other additives such as formic acid or acetic acid were used.
Mertz et al. [145] have analyzed by ESI a wide range of carote-
noids using the mobile phase water/20 nM ammonium acetate,
methanol/20 nM ammonium acetate and MTBE. Crupi et al. [149]
have also analyzed a wide variety of carotenoids using a small pro-
portion of TEA in mobile phases of water, methanol and tert-butyl
methyl ether.
4. Modern approaches and foodomics
The development and the application of new analytical method-
ologies and techniques in food analysis have grown notably in the
past decade [229]. Moreover, there is growing interest in the
health-related properties of some foods, functional ingredients
and nutraceuticals [230]. With regard to carotenoids, specific ana-
lytical techniques are required to address different issues, such as
food composition, food processing, modifications during storage,
and bioavailability and bioactivity of these components. In the past
decade, food science and nutrition studies have moved from classi-
cal approaches to modern and advanced methodologies. In this
context, foodomics has emerged as an innovative and promising
discipline that would include different omics possibilities (epige-
nomics and genomics, transcriptomics, proteomics, and metabolo-
mics) [231,232]. These new possibilities would join together
various analytical techniques and/or omics platforms, and bioin-
formatics tools, to deal with different issues. Due to the huge
amount of data usually obtained from these omics studies, bioin-
formatics has become a crucial tool in foodomics. In the omics
field, MS-based techniques are essential tools, especially for pro-
teomics and metabolomics [233].
Epigenomics studies aim to define the location and the nature of
the genomic sequences (genetic material known as epigenome)
that are epigenetically modified. Epigenetic alterations have been
identified as promising new targets for disease prevention and
nutriepigenetics has emerged as a new field. The dietary compo-
nents may influence the epigenome and thus help in the prevention
of related health problems [234]. The link between food and health
is well documented but the way that diet and genetics can influence
the delicate health–disease balance is still under study.
Carotenoids have been associated with a wide number of health
benefits. Based on extensive epidemiological observations, carot-
enoid-rich fruits and vegetables are thought to provide health ben-
efits by decreasing the risk of various diseases, particularly certain
cancers and eye diseases [10]. Recently, Karppi et al. [235] showed
that high serum concentrations of carotenoids may be protective
against early atherosclerosis by protecting the organism from oxi-
dation. In 2010, Bakker et al. [236] performed a nutrigenomics
study in urine, plasma and adipose tissue of overweight men. Using
67
large-scale profiling of genes, proteins and metabolites in different
matrices, the study proved that selected dietary products (includ-
ing carotenoids) affected inflammatory processes, oxidative stress
and metabolism in humans.
Nutrigenomics/nutrigenetics is defined as the application of the
genomics, transcriptomics, proteomics and metabolomics studies
to human responses to nutrition, especially the relationship be-
tween nutrition and health, focusing on the prevention of disease
through nutrition [237]. Nutrigenomics represents a new challenge
in order to account for inter-individual variations in disease sus-
ceptibility. Transcriptomics comprises the determination of thou-
sands of genes at the mRNA level. Current DNA-microarray
technology has provided a mechanism to circumvent the limita-
tions of single transcriptomics analyses by allowing researchers
to analyze the relative concentrations of thousands of mRNA tran-
scripts simultaneously. In the context of nutrition and micronutri-
ent research, transcriptomics methods have already been widely
applied, though primarily in studies using cell lines and animal
models [238]. With these approaches, genes regulated at the
mRNA level by dietary components have been identified.
Also, omics technologies are useful for food-engineering devel-
opment and evaluation of genetically-modified (GM) foods. Re-
cently, Farré et al. [239] performed a targeted mRNA and
metabolomics analysis of the carotenoid pathway of transgenic
maize plants expressing various carotenogenic gene combinations
and exhibiting distinct metabolic phenotypes. These studies sug-
gested genetic points that may allow the maize endosperm caroten-
oid pathway to be engineered for future bio-fortification strategies
that aim to increase particular carotenoids in maize endosperm.
The data obtained by Pan et al. [240] comparing transcriptome
with proteome showed a poor correlation, suggesting the necessity
to integrate both transcriptomics and proteomics approaches in or-
der to get a comprehensive molecular characterization of food. Pro-
teome analysis aims to characterize complete sets of proteins in a
sample using the classic two-dimensional gene electrophoresis,
LC-MS applications and antibody arrays. At present, MS represents
the most-powerful tool in proteomics because it requires no prior
knowledge of the proteins to be identified. The main mass analyzers
used in proteomics are TOF, quadrupole (Q), FT ion cyclotron reso-
nance (FT-ICR), and ion trap (IT), which are usually combined in one
mass spectrometer [233]. Also, capillary electromigration methods
have provided important contributions to proteomics analysis of
foods [241].
Recently, Li et al. [242] reported a proteomics study of caroten-
oid accumulation and stability during post-harvest storage of pota-
to tubers, using a two-dimensional separation strategy and an LTQ-
Orbitrap mass spectrometer. A proteomics approach has also been
applied to study the protein and carotenoids characteristics of eggs
produced under different housing and environmental conditions,
proving that cage-free eggs had a significant higher carotenoid
content [243]. Proteomics has also been useful to study the micro-
biological production of carotenoids in the food industry, providing
an overview of potentially important carotenogenesis-related pro-
teins by means of peptide-mass fingerprints generated by electro-
phoresis and MALDI-TOF-MS experiments [244]. Also, a recent
study of the proteome of various carotenoid-rich crops offered
new insights into the general metabolism and regulation of chro-
moplasts, plastids that massively accumulate carotenoids [245],
analyzing simultaneously abundant proteins and enzymes.
For transgenic foods, proteomics may be applied to analyze
complex protein samples from GM vegetables, enabling the study
of equivalences between different GM varieties in short times
[246]. However, it has also been applied to understand the mech-
anism of action of certain bioactive components of food that reduce
the risk of some diseases. Uppala et al. [247] performed a proteo-
mics study that demonstrated the inhibitory effect of lycopene
68 K.T. Amorim-Carrilho et al. / Trends in Analytical Chemistry 56 (2014) 49–73
on the growth of human breast-cancer cells, using electrophoresis
and MALDI-TOF/TOF. Some authors have reported the association
between human plasma proteome and plasma a-tocopherol and
ascorbic acid concentrations [248], suggesting novel physiological
effects of these vitamins in humans.
Metabolomics is ideally positioned to be used in many areas of
food science and nutrition research, including food-component
analysis, food quality/authenticity/safety assessment, food trace-
ability, food-consumption monitoring and physiological monitor-
ing in interventional food studies [249,250]. Metabolomics may
also enable understanding of not only the biochemical basis of
plant physiology but also maturation, ripening and storage, as al-
ready reviewed by Oms-Oliu et al. [250].
There are three possible metabolomics approaches: targeted
analysis, profiling and fingerprinting. Metabolomics fingerprinting
compares patterns of metabolites that change in response to spe-
cific situations. Metabolic profiling focuses on the analysis of a
group of previously selected metabolites of interest. The third ap-
proach would be targeted analysis of known compounds (e.g.,
carotenoids) and evaluation of their profile in different situations
and/or groups of samples.
Two analytical platforms are currently used for metabolomics
analyses: MS- and NMR-based systems. These techniques are
either stand alone or combined with separation techniques (typi-
cally, LC-NMR, GC-MS, LC-MS, and CE-MS) [233]. While extraction
for targeted analysis relies on previous knowledge, freeze-drying
and/or lyophilization and concentration steps are common choices
for untargeted metabolomics analysis, and the use and the testing
of several options are recommended [251]. Considering the impor-
tance of the carotenoids for the food industry and for the human
health, growing numbers of metabolomics studies of carotenoids
have been performed.
The multifaceted approach by Capanoglu et al. [182] revealed
that each processing step during tomato-paste production induces
specific alterations in the antioxidant metabolic profile. This work
comprised targeted analysis of specific antioxidants and carotenoids
through HPLC-PDA but also a broader overview using LC-QTOF-MS
for non-targeted metabolomics (see Table 3 for method details).
Similarly, Wehrens et al. [252] used targeted HPLC-PDA analysis
of the carotenoid profiling of carotenoids, combined with multivar-
iate curve resolution, and proved that TEA addition is beneficial in
carotenoid stability. In 2010, Rubio Díaz et al. [253] developed a
protocol for profiling tomato carotenoids involving ATR-IR (atten-
uated total reflectance in conjunction with infrared spectroscopy)
combined with soft independent modeling of class analogy
(SIMCA), a classification technique based on Principal-Components
Analysis (PCA). ATR-IR was able to identify differences in the
chemical structure of carotenoids and enabled SIMCA to generate
PC models that differentiate each genotype group from the other
based on their predominant carotenoid profile. The scores plot (a
projection of the original data onto the PC axes) allowed visualiza-
tion of clustering among samples.
Djuric et al. [254] have extracted plasma samples (200 lL)
twice with 2 mL of hexane; combined hexane extraction, dried,
reconstituted in 200 lL of ethanol/hexane (7:3) and conducted a
PCA by HPLC coupled both to visible and electrochemical detection
to examine patterns of changes in plasma carotenoid levels in
women following a Greek-Mediterranean diet. Lutein, a-carotene
and b-carotene, all high in vegetables, were clustered with each
other and termed vegetable pattern. Lycopene and saturated fat
were linked positively, indicating that this carotenoid was proba-
bly consumed in mixed dishes, such as pizzas, that have both
cheese and tomato sauces. Zeaxanthin and b-cryptoxanthin, high
in fruits, clustered together. These clusters of food could be useful
in understanding the types of dietary changes subjects make when
asked to follow a Mediterranean diet.
Meléndez-Martínez et al. [137], carried out a PCA to ascertain
whether it was possible to discern between juices differing in color
by considering some of the main carotenoids occurring in them.
Carotenoids were analyzed by HPLC-PDA and extracted with meth-
anol/acetone/hexane (25:25:50, v/v/v), containing BHT at 0.1% and
centrifuged at 2500 g for 10 min. The upper phase was recovered,
treated with 10% of ethanolic KOH for 1 h and then washed 4 times
with water to remove the alkali, dried and reconstituted in 1 mL of
acetone:methanol (1:2, v/v, containing 0.1% BHT). A clear separa-
tion was noticed between the ultra-frozen orange juice from con-
centrate samples, whereas the location of the squeezed orange
juice was somehow intermediate between those two groups of
samples, although some of the squeezed orange-juice samples ap-
peared within or very close to the group of the orange juice from
concentrate. The results indicated that it is possible to differentiate
between some kinds of commercial orange juices by considering
only some of the carotenoids occurring in them.
Szydlowska-Czerniak et al. [255] revealed three distinct clusters
by PCA in palm oil at various stages, which can be explained by the
fact that these oil clusters presented similar antioxidant capacities
and total phenolic and carotenoid content.
Also, van Ruth et al. [256] evaluated the authentication of or-
ganic and conventional eggs by carotenoid profiling. Samples were
extracted with ethanol and hexane, consecutively, vortexed and
centrifuged (2700 g, 5 min, 20°C) and extracted two more times
with 500 lL and 1000 lL of hexane. Combined hexane were evap-
orated and reconstituted in 500 lL of methanol to be analyzed in
HPLC-PDA analysis. Results from PCA scores showed that there is
a slight tendency for farms from the same the region to cluster
and that organic samples clustered apart from other samples in a
small region of the three dimensional plot.
Very recently, Park et al. [257] studied the phytochemical diver-
sity of cauliflowers through GC-TOF-MS profiling and identification
of various metabolites, including carotenoids. With their results,
they proved that metabolism and phenotypic variations may be
studied using metabolite profiling combined with chemometrics.
5. Conclusions
The health benefits of the carotenoids have encouraged re-
search on the potential effects of the individual carotenoids on hu-
man health by different ways. Also, bioavailability is a critical
feature in the assessment of bioactive compounds in human
health, and the bioavailability assessment of different forms of
carotenoids must be evaluated. The awareness about the role of
other less-known carotenoids, with the contribution of metabolo-
mics analysis of food, in turn, has encouraged the analysis of these
compounds in foods and the study of their contribution to the total
carotenoid intake. Analyzing carotenoids is not an easy task be-
cause of several factors. There are many naturally-occurring
carotenoids (enormous variety), food-carotenoid composition is
very complex and it varies qualitatively and quantitatively, with
some carotenoids at trace levels. Consequently, the analytical pro-
cedure, and the separation, in particular, must be adequate for the
type of sample. Also, carotenoids are susceptible to some reactions
of oxidation and isomerization under certain conditions (light,
heat, acids, oxygen). Thus, sample treatment must be thoughtfully
developed, in order to avoid degradation and transformation of the
initial carotenoid content. All these facts require new, environ-
ment-friendly methods of extraction and sensitive, selective ana-
lytical techniques for the determination of carotenoids, in both
human and food samples.
This review had a detailed description of recently reported
methodology (2009–13) for carotenoid analysis, and a brief sum-
mary of the best options for extraction and analytical determina-
 K.T. Amorim-Carrilho et al. / Trends in Analytical Chemistry 56 (2014) 49–73 69

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