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Extraction and Metabolic Profiling

of Phenolic Acids, Flavonoids, and

15 Carotenoids from Green Leafy

Vegetables: A State of the Art of the
Recent Techniques Used and Their
Application
Bonta Ramesh Kumar1*, Samsur Ali Dafadar2,
Saikat Sen3 and Srinivas Oruganti4

Introduction: Importance of Bioactive

Components in Vegetables
Vegetables are a broad category of plants high in phytochemicals and minerals. The
root, stem, leaf, immature flower bud, and fruit are the most appetizing components
of this food group. Leafy vegetables, also called “greens”, “vegetable greens”, or
“leafy greens”, contain a diverse range of bioactive chemicals. Although present
in small quantities, these metabolites serve a crucial function in plant secondary
metabolism (Pateiro et al., 2023). Leafy vegetables contain vitamins, proteins,
minerals, and antioxidants (Barba et al., 2014). Vegetable phytochemicals and
antioxidants are predicted to promote health by decreasing the formation of free
radicals, which are responsible for the early stages of various chronic disorders
(Kumar, 2017). Because of consumer awareness of health and nutraceutical benefits
of green leafy vegetables (GLVs), there is an expanding body of studies on some
of their active metabolites. Many of them are classed as bioactive compounds.
Bioactive compounds present in plants are divided into many categories based
on their molecular structure. They consist of dietary fiber, antioxidant vitamins,
polyphenols, terpene derivatives, sulfur compounds, alkaloids, phytoestrogens,
minerals, and polyunsaturated fatty acids (Barba et al., 2014). Figure 1 represents
the most common bioactive components found in GLVs.
Phenolic compounds are distributed throughout the plant as secondary
metabolites. They play significant roles in the plant’s defensive systems against
stress, infections, and UV radiation (Bang et al., 2024). They are an enormous

1-4
Center for Process Research and Innovation (CPRI), [Link]’s Institute of Life Sciences,
University of Hyderabad Campus, Gachibowli, Hyderabad 500046, Telangana, India.
* Corresponding author: [Link]@[Link]; rameshkumarb@[Link]

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Extraction and Metabolic Profiling of Phenolic Acids, Flavonoids, and Carotenoids ... 421

Phenolic
acids

Flavonoids
Alkaloids

Bioactive
compounds Terpenoids
Stilbenes

Phyto- Tannins
estrogens

Fig. 1 Most common bioactives found in Green Leafy Vegetables.

group of bioactive compounds (about 10,000), formed by the shikimic acid

pathway. They are distinguished by at least one aromatic ring connected to one or
more hydroxyl groups. Plant cell walls contain phenolic acids as a byproduct of
the monolignol pathway and lignin breakdown products. They can be classified as
hydroxy-cinnamic or hydroxy-benzoic acids based on their carbon structure. Green
leafy vegetables’ (GLVs’) aerial portions (leaf, root, stem, and fruit) accumulate
benzoic and cinnamic acid derivatives. While hydroxybenzoic acids are commonly
found as glycosides, products derived from hydroxycinnamic acid are simple
esters with a sugar moiety such as glucose. Hydroxycinnamic acids have a C6-
C3 configuration, which consists of coumaric acid, sinapic acid, caffeic acid, and
ferulic acid. Gallic acid, protocatechuic acid, syringic acid, and vanillic acid are the
primary components of hydroxy-benzoic acids, which consist of a C6-C1 structure.
Flavonoids’ structural backbone consists of C6-C3-C6. They are composed of 15
carbon atoms linked by a heterocyclic C-ring to aromatic rings A and B. They are
classified as flavonols, flavones, isoflavones, and anthocyanidins based on the
C-ring substitution pattern. They are found in plant cell vacuoles and undergo a
variety of chemical transformations, including hydroxylation, glycosylation, and
methylation, to yield their corresponding derivatives. The chemical structure of
flavonoids allows them to donate hydrogen (radical scavenging) and chelate metals
(Kumar, 2017). Some of the important phenolic acids, including hydroxy-benzoic
acids (Figure 2) and hydroxy-cinnamic acids (Figure 3), and flavonoids (Figure 4)
found in GLVs are represented below.

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422 Bioactive Compounds in Leafy Vegetables

Fig. 2 Hydroxy-benzoic acids.

Fig. 3 Hydroxy-cinnamic acids.

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Extraction and Metabolic Profiling of Phenolic Acids, Flavonoids, and Carotenoids ... 423

Fig. 4 Flavonoids.

Carotenoids are tetraterpene pigments that exist in yellow, orange, red, and
purple shades. Carotenoids can be found in algae, plants, animals, some archaeal
and fungal species, and photosynthetic bacteria. They are typically composed of a
polyene chain with an end group at either end and nine conjugated double bonds

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424 Bioactive Compounds in Leafy Vegetables

Fig. 5 Carotenoids.

(Maoka, 2023). They are classified into two types: xanthophylls and carotenes.
These are known for pigmentation and aroma (Zeb, 2017). Carotenes are made
up of hydrocarbon molecules. There are over 50 different types of carotenes in
nature, including lycopene, α-carotene, and β-carotene. However, xanthophylls
are carotenoids that contain oxygen atoms in the form of hydroxy, carbonyl,
aldehyde, carboxylic, epoxide, and furanoxide groups. As a result, xanthophyll
structure varies greatly. There are around 800 different types of xanthophylls in
nature, including lutein, zeaxanthin, lactucaxanthin, astaxanthin, fucoxanthin,
and β-cryptoxanthin (Maoka, 2023). The important carotenoids are represented
in Figure 5.
Plant-based pharmaceuticals, which have a yearly market value of more than
$100 billion, emerged as a result of research into plant-based therapies. Despite the
rise of synthetic drugs, herbal remedies are still popular due to their accessibility,
tolerance, and absence of adverse effects. Mohd Zaid et al. (2022) found that
44% of new drugs use plants as lead ingredients. Phytochemicals are useful
molecules found in a variety of industries such as agriculture, food and beverage,
cosmetics, pharmaceuticals, and nutraceuticals. They are used in the production
of pharmaceutical medications, nutritional supplements, functional foods, herbal
therapies, and cosmetic formulations to fulfill the diverse demands and preferences

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Extraction and Metabolic Profiling of Phenolic Acids, Flavonoids, and Carotenoids ... 425

of customers. The demand for a highly sensitive and selective analytical approach
to identify polyphenols is increasing. Despite multiple investigations, the extraction
and quantification of diverse polyphenols continues to be a challenge. The origin
of vegetal bioactive compounds for novel therapeutic formulations is a continuing
concern for the food and pharmaceutical industries, as identifying their identity and
dosage is required to determine health benefits (Mungwari et al., 2025).
This chapter gives current information on the analytical determination
of phenolic acids, flavonoids, and carotenoids from GLVs. It emphasizes
chromatographic and spectroscopic methodologies, as well as contemporary
extraction methods. The significance of the extraction and chromatographic
processes described in this chapter provides a thorough grasp of the study of GLVs
in terms of bioactive profile for medical and industrial applications. Due to the
numerous applications in this field, this chapter discusses a few key and relevant
published studies and demonstrates the state of the art in analytical methodologies.

Leafy Vegetables: Significance, Distribution, and

Nutritional Composition
Vegetables are one of the most important resources for preserving the body’s
alkaline reserve. The primary reason people value vegetables is because of
their high vitamin, carbohydrate, and mineral content. Ascorbic acid, folic acid,
carotenes, riboflavin, and minerals, including calcium, phosphorus, and iron are
all abundant in vegetables (Randhawa et al., 2015). A diverse profile of bioactive
compounds (BACs) is provided by leafy vegetables, sometimes referred to as
“greens”, “vegetable greens”, or “leafy greens” (Pateiro et al., 2023). GLVs have
been domesticated for hundreds or thousands of years due to the desirable traits
of their leaves, such as morphology, quantity, and flavor. Many widely cultivated
and consumed leafy crops, particularly from the Brassicaceae, Asteraceae, and
Amaranthaceae families, belong to this group. GLVs, especially phenolic acid,
which makes up 47.4% of vegetable polyphenols, are the primary source of
polyphenols in non-Mediterranean parts of Europe, accounting for 41.2% of total
intake (Masiala et al., 2024). A study by Pérez-Jiménez et al. (2010) stated that
among the 452 foods in the Phenolic Explorer database, the 100 foods highest in
polyphenols (average of the polyphenols determined by High Performance Liquid
Chromatography (HPLC)) contain 17 foods made from plant leaves, nine of which
are GLVs.
Many species of the Asteraceae family, which are widely cultivated
worldwide, such as lettuce (Lactuca sativa), endive (Cichorium endivia), and red
and green chicory (Cichorium intybus var. foliosum), are examples of GLVs that
are frequently eaten raw in salads. The glucosinolates of GLV leaves, which belong
to the Brassicaceae family, give them a unique peppery flavor and are especially
well-liked when consumed raw in salads. Numerous health benefits have been
demonstrated for these sulfur compounds, which are abundant in Brassicaceae
(Bell and Wagstaff, 2014). Vegetables from the Brassica genus (Brassicaceae

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426 Bioactive Compounds in Leafy Vegetables

family) are some of the most consumed vegetables in the world (Lin and Harnly,
2009). The Brassicaceae family includes several species, including watercress
(Nasturtium officinale) and cultivated rocket (Eruca sativa) (Masiala et al., 2024).
Amaranthaceae is a significant family of GLVs. A significant member of this
family, spinach (Spinacia oleracea), is one of the most popular leafy vegetables
consumed worldwide and is valued for its high nutritional content. Spinach leaves
have been consumed since ancient times after being domesticated in the Middle
East more than 2,000 years ago. China was the primary producer (92.59%) of the
33.12 million tons produced worldwide in 2022 (FAO, 2022).

Accumulation and Distribution of Phenolic Acids,

Flavonoids, and Carotenoids in GLVs
GLVs are an important source of phenolic compounds in vegetables. They are a
large source of bioactive polyphenols; hence, there is a growing interest in studying
their phenolic content and antioxidant properties. A study by Tresserra-Rimbau et
al. (2013) on the population of Mallorca revealed that lettuce leaves are the fourth
largest source of polyphenols among vegetables and account for 6% of flavonols
among all foods. Green and red chicory leaves (Chicorium intybus var. foliosum
L.) are widely consumed across the world. Their leaves are commonly used in
human cuisine due to their characteristic bitterness caused by the presence of
phenolic compounds and other useful nutrients. Red chicory contains the highest
proportion of hydroxycinnamic acid (183 mg/100 g FW). A variety of flavonol
glycosides, flavones, and other glycosylated anthocyanins, primarily found in
red chicory types, have also been reported (Masiala et al., 2024). Kaempferol
derivatives make up the most prevalent flavonoid category, with a range of 8.47–
26.0 g per kg dw (77–88% of total phenolics). The primary flavonoid among them
is kaempferol-3,4-diglucoside, which ranges from 8.07 to 23.68 g per kg dw.
The second flavonoid, isorhamnetin-3,4-diglucoside, has a range of 1.07–4.75 g
per kg dw (9–16.3% of total phenolics) (Pasini et al., 2012). In 2022, the globe
produced 72.60 million tons of cabbage, making it one of the most popular green
leafy vegetables in the world (FAO, 2022). It contains several subspecies of
Brassica oleracea and Brassica rapa that have been chosen for their edible leaves,
including kale (B. oleracea var. sabellica), Chinese cabbage or bok choy (B. rapa
var. chinensis), Chinese white cabbage or Pe-tsai (B. rapa var. pekinensis), and
head cabbage (Brassica oleracea var. capitata). Flavonoids, especially flavonols,
are abundant in the leaves of most Brassica species. Kaempferol and quercetin
glycosides, as well as their derivatives linked to hydroxycinnamic acids and sinapic
acid, are among the many flavonoid glycosides that have been found. Sinapic
acid (421.9 μg/g DM), caffeic acid (84.6 μg/g DM), p-coumaric acid (63.1 μg/g
DM), and ferulic acid (25.7 μg/g DM) were the primary phenolic acids detected
in green/white cabbage leaves cultivated outdoors in the spring (Park et al., 2014).
Flavonols like quercetin (601.3 μg/g DM) and kaempferol (448.7 μg per/ g DM)

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are also found in high concentrations in the same plant matrix. For instance, kale
accounts for 28% of flavonols in Brazil, making it the primary source (Carnauba
et al., 2021). Flavonols, especially derivatives of kaempferol and quercetin, are
mostly found in its leaves. Kaempferol-3-sinapoyl-diglucoside-7-diglucoside and
quercetin-3-sinapoyl-diglucoside-7-diglucoside constituents, with 159 mg of rutin
equivalent/100g of FW and 144 mg of rutin equivalent /100 g FW, respectively
(Olsen et al., 2009). Bok choy leaves, like those of other cabbage cultivars, are high
in flavonols, especially glycosylated Kaempferol. Petonidin-3-O-galactoside and
petonidin-3-O-glucoside make up the majority of the purple leaves of the cultivar
B. rapa var. chinensis, but there are also trace amounts of various glycosylated
anthocyanins, including derivatives of delphinidin, cyanidin, peonidin, and
malvidin (Zhang et al., 2014). Gymnanthemum amygdalinum, an Asteraceae
member whose leaves are commonly known as “bitter leaf”, is mainly consumed
in several sub-Saharan African countries, notably Cameroon. They are used in
the preparation of “Ndolé”, in Cameroon and Nigerians prepare bitter leaf soup
appellé “Onugbo”, as a popular dish. Their leaves are well-known for having a high
concentration of hydroxycinnamic acid, especially derivatives of caffeoylquinic
acid: 3,4-dicaffeoylquinic acid, 1,5-dicaffeoylquinic acid, 3,5-dicaffeoylquinic
acid, and 4,5-dicaffeoylquinic acid (Johnson et al., 2011). Ocimum gratissimum
leaves, often known as “false basil”, are consumed in some Sub-Saharan African
countries, particularly in the western region, as well as in India, Latin America, and
the Caribbean. The plant’s ethanolic leaf extract (EtOH 70%) contained the highest
quantities of p-coumaric acid, followed by caffeic acid, myricetin, quercetin, and
kaempferol (Ganguly et al., 2021). Vicenin-2, luteolin-7-O-glucoside, quercetin-
3-O-glucoside, and quercetin-3-O-rutinoside were the predominant flavonoids
in the leaves, corresponding to the principal flavonoid profile published by other
authors (Ola et al., 2009).
Carotenoids are secondary metabolites that play crucial roles in plants
and are nutritionally relevant. They have received a lot of interest due to their
potential antioxidant properties. They are commonly referred to as provitamin
A, because this vitamin is a result of carotenoid metabolism. Despite being
overshadowed by chlorophyll, leafy vegetables contain the most β-carotene, a
vitamin A-rich compound. There are trace quantities of zeaxanthin, α-carotene,
and antheraxanthin. Although roughly 600 carotenoids have been identified in
nature, about 50 have provitamin A activity, and only about 40 are found in the
average human diet (Kiokias et al., 2016). A study in Andhra Pradesh, India,
measured the levels of lutein and β-carotene in 10 green vegetable species. The
leafy vegetables used during the study were Allmania nodiflora, Alternanthera
sessilis, Amaranthus cruentus, Amaranthus tricolor, Amaranthus viridis, Celosia
argentea, Chenopodium album, Dallocardia muricata, Hibiscus cannabinus, and
Rumex vesicarius. Further, the reported consumption of the 10 selected GLV
species provides 1489 μg of β-carotene, contributing over 40% of the daily intake
recommended by WHO/FAO (Bélanger et al., 2010).

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Extraction Methods: Widely Used Methods with Recent

Advancements, Applications, and Limitations
Conventional Extraction Methods
The extraction method allows for the recovery of components from a solid mixture
or solution. It is essential in sample preparation, experimental research, and the
qualitative analysis of bioactive compounds. The initial step in the extraction
process is known as pre-extraction. Pre-extraction can be performed on both
fresh and dried materials (Zahran and Najafi, 2020). Fresh plant samples used in
medicinal studies tend to be more sensitive and decay faster than dried samples
(Kozłowska and Gruczyńska, 2018). For solid materials, pre-extraction may involve
several operations such as screening, milling, grinding, and homogenization. In
contrast, liquid samples can be filtered or centrifuged before extraction to ensure
successful analysis.
The solubility of bioactive substances is influenced by the degree of phenolic
polymerization and the type of solvent used, whether it is polar, nonpolar, or less
polar. To effectively extract these substances from complex plant matrices, it is
essential to use solvents with varying polarity. Generally, the complexity of the
plant material determines the duration of the extraction process; however, longer
extraction times may lead to the oxidation of phenolic compounds. Phenolic acids
and their derivatives are typically extracted using polar and organic solvents,
including acetone, methanol, ethanol, water, and ethyl acetate. Additionally,
alkaline hydrolysis can be employed to remove phenolic compounds that are
attached to cell walls. Furthermore, lipid fractions are typically present in extracts
that have been vacuum-concentrated. As a result, lipid extraction frequently uses
solvents such as n-hexane, acetone, petroleum ether, and diethyl ether. One easy
way to inhibit or prevent additional enzymatic activity in plant samples is to freeze
them with liquid nitrogen. Frozen samples can then be extracted directly or crushed
into a powder. This process helps isolate substances at the endocellular level.
Polyphenols are best extracted from leafy vegetables using an aqueous solution
of methanol, ethyl acetate, and acetone (Kumar, 2017). In dry samples, reducing
particle size enhances the surface contact between the samples and extraction
solvents. When samples are ground into smaller and more uniform particles, they
exhibit improved interaction with the extraction solvents (Oubannin et al., 2024).
The most widely used conventional extraction techniques include maceration,
Soxhlet extraction, and hot water bath methods (Giacometti et al., 2018). These
traditional techniques are often solvent-intensive and more suitable for small-scale
research.
Maceration involves soaking the plant material, whether ground or powdered,
in an airtight container filled with a solvent, known as the menstruum. The mixture
should be stirred frequently until the soluble components dissolve, and it can be
stored at room temperature for up to three days (Manousi et al., 2019). Afterward,
to remove contaminants, the mixture is filtered (Jovanović et al., 2017). Infusion
and decoction are similar to maceration, involving the immersion of soluble

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materials in either cold or boiling water until they dissolve (Manousi et al.,
2019). However, infusion typically requires a shorter steeping time. Percolation
is another extraction method that shares similarities with decoction; it involves
transferring powdered and dried material into hot water for a two-hour maceration
(Sultana et al., 2018). The Soxhlet extractor is the most common device used to
extract bioactive compounds from plants (Oubannin et al., 2024). This equipment
dissolves weakly soluble compounds from solid matrices through a continuous
extraction process. The solvent repeatedly passes through the product, dissolving
the desired compounds by heating, vaporizing, and condensing the solvent. This
cycle continues until the extraction is complete. Despite their widespread use,
conventional extraction methods have several drawbacks. They often require
multiple extraction cycles to achieve good yields due to their low extraction
efficiency. This inefficiency can lead to sustainability issues, resulting in longer
processing times, higher energy costs, and greater solvent consumption (Mungwari
et al., 2025).

Advanced Extraction Methods

Researchers are constantly seeking new and creative techniques to extract and
concentrate bioactive substances, driven by the growing demand for natural
products in cosmetics, nutraceuticals, and pharmaceuticals. To meet these needs,
advanced extraction techniques have become essential, offering solutions that
are both effective and environmentally friendly. Several sophisticated extraction
methods have been developed that effectively separate and concentrate these
important components from their sources, surpassing the capabilities of traditional
approaches. The value of modern techniques lies in their ability to increase yields
of bioactive components by reducing extraction time, enhancing efficiency, and
using fewer solvents. Additionally, these methods often lead to improved selectivity
and help preserve sensitive molecules, enabling the extraction of powerful, high-
quality bioactives. Importantly, they also minimize the use of hazardous solvents,
contributing to more environmentally sustainable practices (Bhadange et al.,
2024). Microwave-Assisted Extraction (MAE), Supercritical fluid extraction
(SFE), Ultrasound-Assisted Extraction (UAE), Solid-Phase Microextraction
(SPME), and Subcritical solvent extraction (SSE) are some of the most important
advanced extraction methods being recently used in natural products extraction
from plant matrices. Table 1 summarizes the principal mechanisms, advantages,
and limitations of these extraction techniques.
While contemporary techniques have largely replaced traditional extraction
methods in many areas, the latter are still used in small-scale, artisanal, or resource-
limited contexts. Traditional extraction methods offer several advantages, including
simplicity, cost-effectiveness, and low equipment requirements (Brewer et al.,
2014). However, they often lack the efficiency of modern methods and typically
produce smaller yields of the target compounds (Gori et al., 2021). Advanced
extraction techniques usually require specialized tools and technology, which can
be expensive and challenging for smaller businesses to access (Yu et al., 2024).

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Table 1 Advanced extraction techniques used for bio-actives with advantages and limitations.
Method of Principle Mechanism Advantages Limitations Main Target Class of References
Extraction Compounds
Microwave Analysts are separated 1. The solvent and sample are 1. The depth to which Phenolic acids, Bhadange et al.
Assisted from the sample matrix to be heated quickly, which microwave energy can Flavonoids, (2024)
Extraction into the solvent by heating speeds up the extraction penetrate the sample is Terpenoids, and
solvents in contact kinetics and may increase the limited. Essential oils.
with the sample using yields of bioactive chemicals. 2. Particularly in complex
microwave energy. 2. Depending on the polarity matrices, this may result
and dielectric characteristics in uneven heating and
Bioactive Compounds in Leafy Vegetables

of a molecule, microwave insufficient extraction of

scan targets it specifically. bioactive chemicals.
3. It makes laboratory and 3. The quality of the extracted
industrial applications bioactive compounds may be
convenient by doing away impacted when interfering
with the requirement for compounds are released
intricate setups and lengthy as a result of microwave
extraction periods. interaction with matrix
components.
Contd...

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 Table 1 Contd...
Method of Principle Mechanism Advantages Limitations Main Target Class of References
Extraction Compounds
Supercritical It is a procedure that 1. It offers a high level of 1. The limited solubility Phenolic acids, Bhadange et al.
Fluid uses supercritical fluids, selectivity. of supercritical CO2 for Flavonoids, (2024)
Extraction mainly CO2, as the 2. Because SFE is usually polar compounds reduced Tannins, Vitamins,
extraction solvent to performed at comparatively extraction efficiency for Essential oils, and
separate components moderate temperatures, there specific molecule types. Carotenoids.
from a is less chance that heat- 2. The effectiveness of the
matrix. sensitive chemicals would extraction process and the
degrade. quality of the final product.
May be impacted by this
method’s sensitivity to
moisture.
Contd...
Extraction and Metabolic Profiling of Phenolic Acids, Flavonoids, and Carotenoids ...
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Table 1 Contd...
Method of Principle Mechanism Advantages Limitations Main Target Class of References
Extraction Compounds
Ultrasound This technique uses 1. By improving mass transfer 1. Energy consumption may Phenolic acids, Bhadange et al.
Assisted ultrasonic waves to and dissolving cell walls be taken into account in Flavonoids, (2024)
Extraction improve the extraction of and embraces, ultrasound large-scale applications since Carotenoids,
bioactive components by increases the effectiveness ultrasonic extraction uses Saponins,
causing cavitation, which of bioactive ingredient energy to generate high- Proteins,
breaks down the cell extraction from a variety of frequency waves. Polysaccharides.
membrane and makes it materials. 2. Excessive ultrasonic
easier for the components conditions might cause
to be released into the sample degradation,
solvent medium. especially for compounds
Bioactive Compounds in Leafy Vegetables

that are heat-sensitive or

fragile.
Subcritical This technique uses 1. SSE usually employs water 1. Some types of compounds Phenolic acids, Bhadange et al.
Solvent hot water as a solvent as a solvent, obviating the with restricted solubility may Flavonoids, (2024)
Extraction to extract bioactive requirement for potentially not be effectively extracted Carotenoids,
compounds from environmentally hazardous by them. At the higher Polysaccharides.
medicinal plants under organic solvents. By varying temperatures employed in
atmospheric pressure or the temperature and pressure, SSE, certain substances may
at higher temperatures SSE’s selectivity can be be thermally sensitive and
(between 100°C and changed. prone to breakdown.
374°C) and pressures
(less than 5 MPa).
Contd...

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 Table 1 Contd...
Method of Principle Mechanism Advantages Limitations Main Target Class of References
Extraction Compounds
Solid-phase A solid phase fiber 1. The SPME method requires 1. The extraction efficiency Volatile and Semi- Bhadange et al.
Micro coated with a thin layer very little sample preparation. may be impacted by high volatile organic (2024)
Extraction of suitable sorbent 2. Analytes can be quickly molecular weight analyses of compounds.
material is this solvent- extracted and concentrated the SPME fiber.
free technique to extract from the sample matrix. 2. The extraction efficiency
and concentrate target 3. Because SPME focuses and selectivity of SPME
analytes from a variety of analytes onto a limited may be impacted by matrix
matrices. volume of the stationary interferences caused
phase, it can achieve high by complicated sample
sensitivity and enhance matrices.
detection limits.
Extraction and Metabolic Profiling of Phenolic Acids, Flavonoids, and Carotenoids ...
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434 Bioactive Compounds in Leafy Vegetables

Additionally, achieving selectivity for desired molecules can be difficult

due to the wider range of phytochemicals that may be extracted (Zhou et al.,
2024). Nonetheless, precise and focused extraction techniques can help minimize
extensive purification processes and waste production (Li et al., 2024). Figure 6
represents the steps involved in obtaining the bioactives from crude plant samples
via extraction, purification, and characterization.

Sample preparation

Pretreatment

Centrifugation Freeze-drying Homogenization

Dilution Filtration Grinding

Extraction

Liquid-Liquid SPE Solid-Liquid (SFE, UAE, MAE, PLE,

SWE, HSCCC, QuEChERS, EAE< HHP, PEF)

Cleanup

Instrumental Analysis

UV-VIS, IR, FID, XRD, NME HPLC, LC-MS, GC-MS, MALDI

Data Analysis

Identification (Comparison
with Standards, Databases

Fig. 6 Flowchart diagram of the steps involved in the extraction, purification, and
characterization of bioactive compounds from plant matrices.

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Role of Liquid Chromatography and Mass Spectrometry

Techniques in the Metabolic Profiling of Phenolic Acids and
Flavonoids from Green Leafy Vegetables
Liquid Chromatography-Mass Spectrometry (LC-MS) has become an essential
tool in natural product research because it can identify and accurately determine
the molecular weight of substances from small amounts of plant extracts, even
when concentrations are very low. In standard HPLC procedures for phenolic
compounds, reverse-phase (RP) octadecyl silica columns are typically employed.
During RP-HPLC analysis, polar compounds, such as phenolic acids, elute earlier
in the gradient than low-polarity molecules. Due to their significant structural
diversity, there is a possibility of overlap among individual members of various
classes. However, the general elution order is as follows: phenolic acids < cinnamic
acids < flavonoids. The mobile phase and structural complexity significantly
influence the elution time of compounds. Primary phenolic acids, such as ferulic
acid, sinapic acid, and p-coumaric acid, can be separated using RP-HPLC within
40–50 minutes, although some analyses may extend to 70–90 minutes. The number
of hydroxyl groups at the fourth position, followed by those at the third and second
positions, increases the polarity of cinnamic and other phenolic acids. During
HPLC analysis, compounds are separated in a single run despite their differing
polarities, which requires gradient elution with higher concentrations of an organic
modifier. Free phenolic acids, including caffeic acid, vanillic acid, syringic acid,
ferulic acid, chlorogenic acid, protocatechuic acid, p-hydroxybenzoic acid, and
p-coumaric acid, can be effectively separated using isocratic elution with a mixture
of water, methanol, and acetic acid (Kumar, 2017). The phenolic profile of some
of the most popular GLVs consumed in China was analyzed using the HPLC-MS
technique. This study included vegetables from several families, such as
Brassicaceae, Amaranthaceae, and Fabaceae (Zhang et al., 2019). To separate the
components, three distinct extraction techniques were employed, targeting free
phenolic acids, bound phenolic acids, and free esters of free phenolic acids. The
analysis utilized a reversed-phase C18 column maintained at 45°C, and a diode
array detector (DAD). The mobile phase consisted of acetonitrile and 0.1% aqueous
formic acid. The HPLC system was coupled online with a mass spectrometer using
an electrospray ionization (ESI) source in multiple reaction monitoring (MRM)
mode. Seventeen primary phenolic acids were identified in these leafy vegetables,
including gallic acid, 3,5-dihydroxybenzoic acid, chlorogenic acid,
p-hydroxybenzoic acid, gentisic acid, 2,3,4-trihydroxybenzoic acid, vanillic acid,
syringic acid, caffeic acid, protocatechuic acid, p-coumaric acid, sinapic acid,
ferulic acid, 3-hydroxycinnamic acid, isoferulic acid, 2-hydroxycinnamic acid, and
salicylic acid. Under optimal conditions, 15 of the identified phenolic compounds
eluted within 15 minutes. In natural plants, most phenolic acids exist as trans-
isomers. However, when exposed to ultraviolet (UV) light, they gradually convert
into cis-isomers. To separate cis-isomers from trans-isomers, an RP-stationary
phase, which includes an optically active molecule in the mobile phase, is typically

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436 Bioactive Compounds in Leafy Vegetables

used. The primary methods for detecting phenolic acids include fluorescence,
DAD, and UV-visible detectors. Gallic acid and syringic acid have absorption
maxima at 271 nm and 275 nm, respectively, while most benzoic acids have
maximum absorption between 246–262 nm. Cinnamic acids exhibit their highest
absorption in two wavelength ranges: 225–235 nm and 290–330 nm. For phenolic
compounds and their derivatives, the detection range of 280 nm is most commonly
used. The separation of flavonoids in RP-HPLC is based on hydrophobicity.
Variations in selectivity depend on the polar groups attached, the carbon loading,
and the characteristics of the stationary phase. The order in which various classes
of flavonoids elute is as follows: flavanols < flavanones < flavonols < flavones. A
higher number of methoxy groups on the flavonoid backbone increases the retention
duration of the compounds, while a greater number of hydroxyl groups decreases
it. Flavonoid analysis employs both binary and isocratic gradient elution techniques;
however, isocratic gradient elution has certain limitations. During RP-HPLC
analysis, acylated compounds tend to extend the elution duration, whereas
glycosylated flavonoids slightly reduce the retention time. Additionally, a higher
pH leads to the ionization of flavonoids, which shortens the retention period during
RP separation. To prevent crystallization, small amounts of acetic acid (2%–5%)
and trifluoroacetic acid (0.1%–1%) are added to the solvent mixture, enhancing
the resolution and reproducibility of each separation (Kumar, 2017). The accurate
molecular weights of the phenolic acids isolated via RP-HPLC can be determined
using mass spectrometry (MS). This technique allows for the collection of MS data
during the analysis, as well as the simultaneous monitoring of the retention time
of the isolated chemical peak. As a result, LC-MS is now a recognized technique
for phenolic acid identification and characterization (Kumar, 2017). Phenolic acids
and flavonoids were extracted from the adult stems of Basella alba and Basella
rubra using HPLC and electrospray ionization-mass spectrometry (ESI-MS)
methods. Water and acetonitrile with 0.1% trifluoroacetic acid (TFA) were used as
mobile phases to separate the flavonoids and phenolic acids on a reverse-phase
C18 column. The identified phytochemicals included 4-hydroxybenzaldehyde,
caffeine, ferulic acid, gallic acid, galloyl shikimic acid, p-coumaric acid, salicylic
acid, trans-cinnamic acid, and diosmetin. Furthermore, the ESI-MS technique was
employed for the provisional identification of these compounds. Notably,
diosmetin—an O-methylated flavone—was reported in these plant species for the
first time. The detected bioactives also demonstrated strong antioxidant, anti-
angiogenic, and anti-cancer properties (Kumar et al., 2018). In a related
investigation, the red Basella cultivar, B. rubra, was found to contain flavonoids
and phenolic acids. These compounds were extracted from the fruits of B. rubra
using a reverse-phase C18 column. The phenolic acids detected included gentisic
acid, chlorogenic acid, sinapic acid, ferulic acid, and coumaric acid at 280 nm.
Flavonoids such as myricetin, apigenin, kaempferol, luteolin, and quercetin were
observed at 320 nm. Additionally, betacyanins were identified in the flesh of B.
rubra fruits using HPLC-Quadrupole Time of Flight Mass Spectrometry (HPLC-
QTOF-MS). The study also documented significant antioxidant and anticancer

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Extraction and Metabolic Profiling of Phenolic Acids, Flavonoids, and Carotenoids ... 437

effects against human cervical cancer cell lines (Kumar et al., 2015). The detection
of flavonoids is influenced by their chemical characteristics and the sensitivity of
the analytes. To identify certain substances, researchers use the UV absorption
spectra and retention times of reference standards. Even when two compounds are
closely related and elute simultaneously, they can be differentiated using multiple
wavelength detection methods that rely on variations in their absorption spectra.
Acetonitrile and methanol, with low UV cut-off λmax values at 190 and 205 nm,
do not interfere with the detection of UV-Vis absorption bands between 240–285
nm and 300–560 nm for the A and B rings of flavonoid aglycones. The wavelength
range of these bands can vary slightly among flavones, depending on the substitution
patterns of hydroxy and methoxy groups, as well as the types of glycosides
(C-glycosides or O-glycosides). Metabolite profiling of phenolic acids,
anthocyanidins, and flavonols has been conducted on Brassica oleracea var.
capitata (Park et al., 2014). The study utilized LC-MS and HPLC methods to
identify and quantify key compounds, including flavonols (quercetin and
kaempferol), anthocyanidins (cyanidin and peonidin), and phenolic acids (caffeic,
p-coumaric, ferulic, and sinapic acid). Phenolic compounds were separated using
an HPLC-Photodiode Array (PDA) detector, featuring a C18 column along with a
guard column. The mobile phase consisted of methanol and 0.1% formic acid in
water. Measurements for anthocyanidins, phenolic acids, and flavonols were taken
at wavelengths of 530 nm, 330 nm, and 360 nm, respectively. For phenolic acids
and flavonols, ESI-MS was performed in negative-ion mode, while for
anthocyanidins, it was operated in positive-ion mode. Notably, the primary phenolic
acids identified in Brassica vegetables were derivatives of sinapic acid. Through
this combined technique, the study successfully identified and quantified phenolic
acids, flavonoids, and anthocyanins across nine different Brassica species. The
best ionization sources for the analysis of flavonoids are Atmospheric Pressure
Chemical Ionization (APCI) and ESI. These ionization methods facilitate the
coupling of HPLC with MS. Flavonoids in plants are predominantly found in their
glycosidic forms, which can be categorized into two main types: O-glycosides and
C-glycosides. Typically, the mass spectra of flavonoid glycosides exhibit similar
fragmentation pathways, regardless of the ionization method used. However, in
many cases, the desired fragment ions may not be generated due to contaminants
in the sample or the compounds being analyzed. In such situations, Collision-
Induced Dissociation (CID) MS/MS techniques can be beneficial as they help
produce the appropriate fragment ions. Identifying the structure of acylated
flavonoid glycosides can be challenging because these compounds are often
acylated with either aliphatic (e.g., acetic acid) or aromatic acids, which can attach
directly to the glycosidic or aglycone portions. Although increasing the cone
voltage may sometimes lead to their fragmentation, ESI spectra of glycosidic
compounds typically show an aglycone ion, a pseudo-molecular ion such as
([M+H]), and ions associated with the solvent (Kumar, 2017). The phenolic,
vitamin, and antioxidant profiles of several Amaranthus lines were examined by
Sarker and Oba (2021). Phenolic compounds were identified using LC-ESI-MS

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438 Bioactive Compounds in Leafy Vegetables

and HPLC techniques. The HPLC system consisted of an ODS-II column and a
UV-Vis detector. The mobile phase was composed of 6% acetic acid in water as
Solvent A and acetonitrile as Solvent B. The MS system was integrated with the
HPLC and UV-Vis detector, with an online ESI ionization source. The detector
was calibrated to continuously monitor flavonoids, cinnamic acids, and benzoic
acids at wavelengths of 360 nm, 370 nm, 280 nm, and 254 nm. From the leaves,
25 phenolic acids and flavonoids were identified, including protocatechuic acid,
salicylic acid, gentisic acid, gallic acid, vanillic acid, p-hydroxybenzoic acid,
chlorogenic acid, ellagic acid, syringic acid, ferulic acid, coumaric acid, trans-
cinnamic acid, caffeic acid, sinapic acid, quercetin, kaempferol, myricetin,
apigenin, isoquercetin, rutin, hyperoside, and catechin. It was concluded that the
hyphenated techniques employed in this study provided a comprehensive analysis
of the flavonoids and phenolic acids from four Amaranthus genotypes. When
analyzing flavonoids using ESI in positive mode, sodium and potassium adducts
are commonly observed. These adducts form when the samples are kept in a glass
solution and are particularly noted in the analysis of flavanol-3-O-glycosides and
isoflavones (Kumar, 2017). Additionally, a combined loss of a methyl group and
water, represented as [M+H-33]+, is also detected. In the case of C-glycosylated
flavonoids, complete removal of the sugar moiety is not observed. Instead,
fragments or portions of the sugar ring are eliminated. The intensity of the
protonated molecule ions [M+H]+ seen in the mass spectrum depends on the
number of sugar moieties attached to the aglycone. Specifically, the strength of
[M+H]+ ions decreases as the number of sugar rings increases. The bond that
connects sugars to the flavonoid ring is a strong, acid-resistant C-C bond. The
results of these analyses may be improved by conducting collision experiments
using appropriately high and low energy levels. Moreover, 6-C-glycosyl flavonoids
tend to lose more water molecules compared to 8-C-glycosyl flavonoids for
[M+H]+ and [M-H]– ion fragmentation patterns at high collision energy (Kumar,
2017). The phenolic profiles of red and green oak-leaf lettuce cultivars were
thoroughly characterized using Ultra-High Performance Liquid Chromatography
(UHPLC) in combination with ESI, Quadrupole-Time of Flight Mass Spectrometry
(QToF/MS), and DAD (Viacava et al., 2017). For this investigation, an ultra-
performance liquid chromatography Ethylene Bridged Hybrid (UPLC-BEH) C18
VanGuard™ pre-column (1.7 μm) and a reverse-phase UPLC-BEH C18 column
(100 mm × 2.1 mm; particle size 1.7 μm) were employed. The mobile phases
consisted of acetic acid in water (Solvent A) and methanol (Solvent B), both at
0.1% (v/v). Several classes of compounds were identified using the UV-Vis detector
at different wavelengths: hydroxybenzoic acids at 254 nm, hydroxycinnamic acids
and coumarins at 320 nm, flavanones at 280 nm, flavonols and flavones at 370 nm,
and anthocyanins at 500 nm. A High Definition Mass Spectrometry (HDMS)
system with an ESI source, operating in both positive and negative modes, was
used for all MS data acquisitions. For the first time, 48 compounds were identified
in lettuce with a reasonable degree of certainty. The identified phenolic compounds
included caffeoyl quinic acids, p-coumaroyl quinic acids, caffeoyltartaric acids,
p-coumaroyl tartaric acid, caffeoylmalic acid, dicaffeoylquinic acids, caffeoylquinic

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Extraction and Metabolic Profiling of Phenolic Acids, Flavonoids, and Carotenoids ... 439

acid glycosides, p-coumaroylcaffeoylquinic acids, di-caffeoyltartaric acid,

hydroxycinnamic acid derivatives, hydroxybenzoic acid derivatives,
hydroxyphenylacetic acid derivatives, quercetin derivatives, kaempferol
derivatives, luteolin derivatives, apigenin derivatives, eriodictyol-O-glucuronide,
and cyanidin derivatives. The UHPLC-DAD-ESI-QToF/MS method proved to be
a valuable and robust technique for effectively characterizing phenolic compounds.
Tables 2 and 3 provide recent studies on profiling and characterizing phenolic acids
and flavonoids from GLVs using HPLC and MS methods.
When used alongside ESI-MS, HPLC columns with a small diameter offer
increased sensitivity because they elute the concentrated analyte in a smaller
volume. This design also minimizes the introduction of impurities and buffers
into the MS, which helps maintain the ionization source’s sensitivity for a longer
time. Guard columns are vital for enhancing the longevity and performance of
analytical columns, as they protect these columns by removing strongly binding
components like chlorophyll and other endogenous compounds commonly found
during the analysis of crude plant samples. Advanced HPLC techniques, such as
high-temperature liquid chromatography (HTLC), ultra-high-performance liquid
chromatography (UHPLC), and two-dimensional liquid chromatography (LC
× LC), utilize new types of columns to detect flavonoids and phenolic acids in
plant samples. Recent advancements in instrumentation have made LC × LC and
hydrophilic interaction liquid chromatography (HILIC) popular for analyzing plant
samples. HILIC may be particularly effective when combined with MS due to its
higher compatibility with the mobile phase used for separation.
In traditional Chinese medicine, polar and semi-polar fractions have been
successfully detected using a combination of 2-D LC × HILIC and 2-D LC ×
RP-LC. The online coupling of HPLC and MS using ESI allows for the high-
resolution characterization of various polar substances. Recent innovations, such
as modifications to sprayer techniques (including pneumatically aided electrospray,
ultrasonic nebulizer electrospray, and nanoelectrospray), have further optimized
the application of ESI techniques (Kumar, 2017).

Role of Liquid Chromatography and Mass Spectrometry

Techniques in the Metabolic Profiling of Carotenoids from
Green Leafy Vegetables
The extraction of carotenoids from the sample matrix is essential for accurate
analysis. Because carotenoids are inherently unstable, it is crucial to minimize
degradation, oxidation, and isomerization during this process to ensure reliable
results. Their high degree of conjugation makes carotenoids susceptible to damage
from heat, light, oxygen, and metal ions. For example, in an aerobic environment,
β-carotene rapidly breaks down when exposed to visible and UV light. Therefore,
samples should be handled under low light or red light, and solvents should be
evaporated using gases like helium, argon, or nitrogen. Additionally, it is important
to store samples in a dark place at temperatures below –20°C (Kopec et al., 2012).
Most carotenoids absorb light in the 400–500 nm range due to their conjugated

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Table 2 HPLC profiling of phenolic acids and flavonoids.
Plant Name Parts Extraction Method Stationary Phase Mobile Phase Flow Rate (mL) Phenolic Compounds References
Used Solvent and Wavelength Identified
(nm)
Amaranthus sps. Leaves 75% UPLC- BEH C18 Water and 0.25 mL/min, Phenolic acids: Bang et al.
(A. methanol PDA Column (2.1mm Acetonitrile 260 nm. Gallic acid,3,4- (2024)
hypochondriacus, ×100 mm, 1.7 with 0.1% dihydroxybenzoic acid,
A. cruentus, μm) acid. 4-hydroxybenzoic acid,
A. caudatus, 2,4-dihydroxybenzic acid,
A. tricolor, A. vanillic acid, syringic acid,
dubius, A. blitum, benzoic acid, caffeic acid,
A. crispus, A. p-coumaric acid, ferulic acid,
hybridus, and A. sinapic acid, and cinnamic
Bioactive Compounds in Leafy Vegetables

viridis) acid.
Flavonoids:
Rutin, quercetin-3-β-D-
glucoside, kaempferol-3-O-
β-rutinoside, quercetin, and
kaempferol.
Basella alba and Stem Aqueous HPLC RP-C18 Water and 1.0 mL/min, Phenolic acids: Kumar et al.
Basella rubra methanol column (250 x Acetonitrile 254 nm, Gallic acid, p-Coumaric acid, (2018)
4.6 mm) with 0.1% 280 nm, Salicylic acid, Caffeic acid,
TFA. and 330 nm. Ferulic acid, Trans-cinnamic
acid, Galloyl shikimic acid.
Flavonoids: Diosmetin.
Contd...

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 Table 2 Contd...
Plant Name Parts Extraction Method Stationary Phase Mobile Phase Flow Rate (mL) Phenolic Compounds References
Used Solvent and Wavelength Identified
(nm)
Tinosporia Stem and 80% UHPLC C18 column Formic acid 0.2 mL/min Phenolic acids: Mueed et al.
cordifolia, Leaves methanol (150 x 2.0mm) in water Vanillic acid, Ferulic acid, (2023)
Mentha arvensis. (0.1%) and Chlorogenic acid, p-coumaric
acetonitrile in acid, gallic acid, rosmarinic
water (0.1%) acid, sinapic acid, 3-feruloyl
quinic acid, and neo-
chlorogenic acid.
Flavonoids:
Luteolin-7-O-
neohesperidoside, Quercetin-
3,7-O-diglucoside, Luteolin-
5-O-glucoside, Genkwanin-
5-O-glucoside, Hesperidin,
Diosmin, Didymin, Rutin,
Quercetin dihydrate,
Kaempferol, and Quercetin.
Contd...
Extraction and Metabolic Profiling of Phenolic Acids, Flavonoids, and Carotenoids ...
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Table 2 Contd...
Plant Name Parts Extraction Method Stationary Phase Mobile Phase Flow Rate (mL) Phenolic Compounds References
Used Solvent and Wavelength Identified
(nm)
Amaranthus Leaves 80% HPLC STR-ODS-II 6% v/v acetic 1.0 mL/min, Phenolic acids: Sarker &
(VA14, VA16, methanol (150 x 4.6 mm) acid and 254 nm, 280nm, Protocatechuic acid, salicylic Oba (2021)
VA11, VA6) with 1% Acetonitrile 260 nm, acid, gentisic acid, gallic
acetic acid. and 380 nm. acid, vanillic acid,p-hydroxy-
benzoic acid, chlorogenic
acid, ellagic acid, syringic
acid, ferulic acid, coumaric
acid, trans-cinnamic acid,p-
coumaric acid, caffeic acid,
Bioactive Compounds in Leafy Vegetables

and sinapic acid.

Flavonoids:
Kaempferol, quercetin, iso-
isoquercetin, rutin, naringenin,
myricetin, and hyperoside.
Indian spinach Fruits Water HPLC C18 column Water with 1.2 Phenolic acids: Kumar et al.
(Basella rubra) (250mm × 4.6 acetic acid mL/min, 280 Chlorogenic acid, (2015)
mm) (pH 2.6), nm, and Sinapic acid, Ferulic acid, and
and 80% 320 nm. Coumaric acid.
acetonitrile Flavonoids:
Apigenin, Kaempferol,
Luteolin, Myricetin, and
Quercetin.
Contd...

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 Table 2 Contd...
Plant Name Parts Extraction Method Stationary Phase Mobile Phase Flow Rate (mL) Phenolic Compounds References
Used Solvent and Wavelength Identified
(nm)
Green and Red Whole Methanol- UHPLC- UPLC BEHC18 0.1% (v/v) 0.5 mL/min, 254 Phenolic acids: Viacava et
Oak leaf lettuce plant water- DAD Column (100mm acetic acid nm, Caffeoyl quinic acids, al. (2017)
(Lactuca sativa) acetic acid × 2.1 mm) in water and 280 nm, 320 p-coumaroyl quinic
(30:65:5, 0.1% (v/v) nm, 370 nm acids, caffeoyltartaric
v/v/v) acetic acid in and 500 nm. acids,caffeoyltartaric acids,
methanol. p-Coumaroyl tartaric
acid, caffeoylmalic acid,
dicaffeoylquinic acids and
caffeoylquinic acid glycosides,
p-Coumaroylcaffeoylquinic
acids, di-caffeoyltartaric
acid, hydroxycinnamic acid
derivatives, hydroxybenzoic
acid derivatives,
hydroxyphenylacetic
derivatives.
Flavonoids:
Quercetin derivatives,
Kaempferol derivatives,
Luteolinderivatives, Apigenin
derivatives, Eriodictyo-O-
Extraction and Metabolic Profiling of Phenolic Acids, Flavonoids, and Carotenoids ...

glucuronide, and Cyanidin

derivatives
443

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 Table 3 MS profiling of phenolic acids and flavonoids.
444
Plant Name Part Ionization Phenolic Retention Observed Observed Scan Range Reference
Used Method Compounds Identified Time ( Minutes) [M+H]+ [M-H]- (m/z)
Basella alba and Stems ESI-MS Phenolic acids 2.51 - 121.1 50–1000 Kumar et al.
Basella rubra 4-hydroxybenzaldehyde (2018)
Caffeic acid 2.55 - 179.2
Ferulic acid 10.00 195.97 -
Gallic acid 3.43 - 169.10
Galloylshikimic acid 11.03 - 325.1
p-Coumaric acid 3.85 - 163.0
Salicylic acid 4.36 - 137.02
Trans-cinnamic acid 9.25 149.3 -
Bioactive Compounds in Leafy Vegetables

Flavanoids 10.86 - 301.4

Diosmetin
Amaranthus (VA14; Leaves ESI-MS Phenolic acids Sarker & Oba
VA16; VA11; VA6). 2,4-dihydroxybenzoic acid 4.28 - 154.11 0–1000 (2021)
3,4-dihydroxybenzoic acid 2.33 - 154.13
2,5-dihydroxybenzoic acid 3.82 - 154.13
2-hydroxybenzoic acid 48.23 - 137.21
4-hydroxybenzoic acid 31.34 - 137.02
3,4,5-trihydroxybenzoic acid 9.24 - 169.12
4-Hydroxy-3-methoxybenzoic 30.13 - 167.17
acid
Contd...

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 Table 3 Contd...
Plant Name Part Ionization Phenolic Retention Observed Observed Scan Range Reference
Used Method Compounds Identified Time ( Minutes) [M+H]+ [M-H]- (m/z)
3,5-Dimethoxy-4-
Hydroxybenzoic acid 34.57 - 197.12
3,4-dihydroxycinnamate 32.12 - 179.07
3-(3,4-Dihydroxycinnamoyl) 31.13 - 353.14
Quinic acid
4-Hydroxy-cinnamic acid 42.21 - 163.05
3-Methoxy-4-hydroxy
Cinnamic acid 47.88 - 193.16
3-Hydroxycinnamic acid 49.59 - 163.24
4-Hydroxy-3,5-dimethoxy 49.03 - 223.16
Cinnamic acid
3-Phenylacrylicacid 67.26 - 147.12
Flavanoids 26.71 - 271.05
Naringenin
Quercetin-3-O-glucoside 54.33 - 463.27
Quercetin-3-O-galactoside 53.29 - 463.44
Quercetin-3-O-rutinoside 53.14 - 609.28
Myricetin-3-O-rutinoside 4.58 - 626.31
Extraction and Metabolic Profiling of Phenolic Acids, Flavonoids, and Carotenoids ...

Kaempferol-3-O-rutinoside 17.77 - 593.43

Contd...
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Table 3 Contd...
Plant Name Part Ionization Phenolic Retention Observed Observed Scan Range Reference
Used Method Compounds Identified Time ( Minutes) [M+H]+ [M-H]- (m/z)
4,5,7-Trihydroxyflavone, 15.55 - 270.24
5,7- Dihydroxy-2-(4-
hydroxyphenyl)-4-
benzopyrone
2-(3,4-dihydroxy phenyl)-
3,5,7-trihydroxychromene- 7.54 - 301.12
4-one
Bioactive Compounds in Leafy Vegetables

Green radish; Purple Aerial ESI-MS Phenolic acids Zhang et al.

cabbage; Celery; parts Gallic acid 2.10 - 169.0 (2019)
Chinese cabbage,
Baby cabbage;
Amaranth, White
radish, Asparagus
lettuce; Radish,
Spinach,
Cauliflower,
Broccoli, Phaseolus
Vulgaris.
Contd...

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 Table 3 Contd...
Plant Name Part Ionization Phenolic Retention Observed Observed Scan Range Reference
Used Method Compounds Identified Time ( Minutes) [M+H]+ [M-H]- (m/z)
3,5-Dihydroxybenzoic acid 3.48 - 153.1
Chlorogenic acid 4.33 - 353.0
p-Hydroxybenzoic acid 4.47 - 137.0
Gentisic acid 4.54 - 152.9
2,3,4-Trihydroxybenzoic acid 3.56 - 141.0
Vanillic acid 5.07 - 167.0
Caffeic acid 5.21 - 179.0
Syringic acid 5.37 - 197.0
Protocatechuic acid 5.42 - 153.2
p-Coumaric acid 7.56 - 163.0
Sinapic acid 8.63 - 223.0
Ferulic acid 8.57 - 193.0
3-Hydroxycinnamic acid 9.00 - 163.1
Isoferulic acid 8.98 - 193.1
2-Hydroxycinnamic acid 10.26 - 163.1
Salicylic acid 10.43 - 137.1
Extraction and Metabolic Profiling of Phenolic Acids, Flavonoids, and Carotenoids ...
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448 Bioactive Compounds in Leafy Vegetables

double bonds; however, some precursors, such as phytoene and phytofluene,

primarily absorb in the UV range. Different carotenoid species can be distinguished
and identified through spectral analysis, but it is important to note that the UV/
Vis spectra for common carotenoids only reveal the features of the carotenoid’s
chromophore.
A distinctive characteristic of carotenoids is that, unlike many compounds
that have a single absorption band, most carotenoids exhibit three roughly separate
absorption peaks. The wavelength of maximum absorption and the fine structural
details can vary significantly between different carotenoids. The most common
method for separating carotenoids is RP-HPLC. However, several normal-phase
methods have been developed for separating specific combinations of xanthophylls
or carotenoid ketones, such as astaxanthin, which have a low affinity for C18 or
C30 columns designed specifically for carotenoids. While C30 columns generally
provide better separation, especially for closely related molecules like lutein
and zeaxanthin, C18 columns can sometimes suffice for distinguishing different
carotenoids. Carotenoids have also been analyzed using UHPLC, primarily in
conjunction with other fat-soluble vitamins. However, it is uncommon to find
UHPLC techniques specifically developed for quantifying a diverse range of
carotenoids (Kopec et al., 2012). The carotenoid profile of many leafy vegetables,
including Basella rubra L., Peucedanum sowa Roxb., Moringa oleifera Lam.,
Trigonella foenum-graecum L., Spinacia oleracea L., Sesbania grandiflora (L.)
Poir., and Raphanus sativus L., was assessed. Open column chromatography (OCC)
on a neutral alumina column was employed to purify the recovered carotenoids,
allowing for their identification based on distinctive UV-visible absorption spectra.
The separated carotenoids were analyzed using RP-HPLC on a C18 column with
UV-visible photodiode array (PDA) detection under isocratic conditions. The
mobile phase consisted of a 60:20:20 v/v/v mixture of acetonitrile, methanol, and
dichloromethane, with 0.1% ammonium acetate. Both methods identified lutein,
α-carotene, violaxanthin, neoxanthin, and zeaxanthin as the main carotenoids
present (Lakshminarayana et al., 2005). RP-LC is a popular technique that achieves
great resolution by utilizing C18 and C30 columns with non-polar stationary phases
(Xu et al., 2023). While C30 phases, including YMC C30 and Prontosil C30, provide
greater separation of structurally identical molecules and geometric isomers, C18
phases, like Spherisorb ODS-2 and BEHC18, are appropriate for hydrophobic
carotenoids (Delpino-Rius et al., 2014; Pop et al., 2014; Gebregziabher et al., 2021;
Londoño-Giraldo et al., 2021; Feng et al., 2022; Grujić et al., 2022). The polarity
and solubility of carotenoids influence the selection of mobile phases in HPLC.
Commonly used solvents include methanol (MeOH), acetonitrile (ACN), methyl
tert-butyl ether (MTBE), tetrahydrofuran (THF), acetone, and water. Ammonium
acetate (NH4OAc) is often used as a buffer modifier, while ethyl acetate (EtoAc) is
employed in some procedures to improve solubility (Lourenço-Lopes et al., 2022).
HPLC was utilized to examine the presence of β-carotene in the leaves of spinach,
cabbage, carrot, brinjal, tomato, and bitter gourd (Zahra et al., 2022). The HPLC
system operated in isocratic flow mode and utilized a UV-Vis detector along with

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Extraction and Metabolic Profiling of Phenolic Acids, Flavonoids, and Carotenoids ... 449

a reversed-phase C18 column. The separation was achieved using a mobile phase
composed of acetonitrile, dichloromethane, and methanol in a ratio of 70:20:10.
This study demonstrated that HPLC is a reliable method for analyzing carotenoids.
Most C30 techniques utilize a gradient to achieve optimal separation of various
carotenoid species and their isomers. These methods often involve a mixture of
MTBE, methanol, and a small quantity of water. Some C30 techniques can also
simultaneously separate tocopherols and chlorophylls, along with carotenoids
and their isomers. Others may use a combination of THF, acetonitrile, methanol,
isopropyl alcohol, ethyl acetate, and dichloromethane. Generally, a temperature of
23°C ± 1°C is sufficient for separating various carotenoid species, while 30°C has
been identified as the optimal temperature for isomer separation. It is important to
note that cis isomers typically exhibit reduced fine structure and absorb maximally
at wavelengths that are about 2–6 nm lower than those of the all-trans carotenoid
(Kopec et al., 2012). Another study identified carotenoids from the leaves of
Brassica oleracea L. var. botrytis, which were cultivated in four different regions
of Pakistan’s Malakand division: Buner, Swat, Malakand, and Lower Dir. The
researchers used an RP-HPLC with a DAD system to separate and identify the
carotenoids at 450 nm. The mobile phase consisted of a combination of solvents:
Solvent A was a mixture of methanol and deionized water in a 92:8 ratio, v/v,
containing 10 mM ammonium acetate; Solvent B included 0.1 mM ammonium
acetate and deionized water; and Solvent C was pure MTBE. The study identified
several compounds, including phytoene, all-E-neoxanthin, 9-Z-neoxanthin, all-
E-lutein, 9-Z-lutein, 9ʹ-Z-lutein, α-carotene, 9ʹ-Z-neoxanthin, γ-carotene, and all-
E-β-carotene. The major carotenoids found across all varieties were β-carotene,
lutein, fucoxanthin, δ-carotene, neoxanthin, γ-carotene, and phytoene (Zeb et al.,
2022).
Carotenoids, which are compounds with parent ions typically ranging from
536 to 600 Daltons, can be accurately identified and characterized using HPLC-
MS and HPLC-MS/MS. Most applications commonly utilize ESI and APCI as
ionization sources in mass spectrometry. APCI is particularly effective for ionizing
lipophilic substances and is often used for nonpolar carotenoids such as β-carotene,
α-carotene, and lycopene (Pintea et al., 2020). This method operates at higher
temperatures, which reduces fragmentation and enhances the ionization of volatile
compounds; this often results in a dominant [M+H]+ molecular ion. In contrast,
oxygenated carotenoids, known as xanthophylls, such as lutein, zeaxanthin, and
violaxanthin, are better suited for ESI. This technique provides gentler ionization,
minimizing fragmentation and yielding intact molecular ions in both positive
([M+H]+) and negative ([M-H]−) modes (Kurek et al., 2025). The majority of
M+ and [M+H]+ ions are found in hydroxy carotenoids, including zeaxanthin
and β-cryptoxanthin. The sodium adduct ions [M+Na]+ and [M+H]+ are mostly
provided by keto-carotenoids like astaxanthin. The protonated molecule [M+H]+
and the alkaline metal adductions [M+Na]+ and [M+K]+ are the main products of
Violaxanthin’s ESI-MS. Both carotenes and xanthophylls exhibit the protonated
molecule [M+H]+ as a molecular mass ion in APCI-MS. Xanthophylls exhibit the

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450 Bioactive Compounds in Leafy Vegetables

same fragment ions, including [M+H-H2O]+ and [M+H-AcOH]+. Furthermore,

the structural properties of the hydroxylated end group in carotenoids are reflected
in the intensities of these dehydrated fragment ions. Despite having the identical
chemical formula (C40H56O2), lutein (β,ε-carotene- 3,3′-diol), lactucaxanthin (ε,ε-
carotene-3,3′-diol), and zeaxanthin (β,β-carotene-3,3′-diol) displayed significantly
different ion peak intensities at m/z 569 [M+H]+, m/z 551 [M+H-H2O]+, and m/z
533 [M+H-2H2O]+. Zeaxanthin displayed a dehydrated ion at m/z 551 [M+H-
H2O]+ and a base peak at m/z 569 [M+H]+. On the other hand, zeaxanthin with
a 3-hydroxy-β- end group scarcely showed a fragment ion at m/z 533 [M+H-
2H2O]+, but both lutein and lactucaxanthin with a 3-hydroxy-ε-end group displayed
m/z 551 [M+H-H2O]+ as the most prevalent ion (Kaczor and Baranska, 2016).
Carotenoids containing hydroxyl or epoxy groups benefit significantly from ESI,
which enhances detection sensitivity in complex food matrices. Although both
ESI and APCI methods provide valuable information about the composition of
carotenoid pigments, ESI is more effective for analyzing polar carotenoids, while
APCI is better suited for nonpolar species (Wu et al., 2024). In contrast, triple
quadrupole (QqQ) and ion trap (IT) mass spectrometers offer more informative
data, enabling the execution of tandem mass spectrometry (MS/MS) and multi
stage mass spectrometry (MSn) experiments. Specific MRM studies allow for
both qualitative data collection and individual quantification of carotenoids present
at very low levels. Tandem setups, such as Quadrupole-Ion Trap (Q-IT) and
Quadrupole-Time of Flight (Q/ToF) mass spectrometry (IT-TOF-MS), are also
employed. Furthermore, some LC-MS systems utilize Q/Orbitrap and ion mobility
(IM)/MS for carotenoid investigations. Various LC-MS configurations exist, each
offering unique features that enable data collection at different levels of linearity,
sensitivity, mass resolution, and accuracy (Giuffrida et al., 2020).
A total of seven carotenoids were simultaneously identified in 12 Brassicaceae
plants, including broccoli, Brussels sprouts, cabbage, cauliflower, Chinese
cabbage, pak choi, choy sum, kailan, daikon radish, red radish, rocket salad, and
watercress. LC-APCI-MS/MS was used, and the carotenoids were separated on a
YMC Carotenoid C30 column connected to a triple quadrupole mass spectrometer
in positive mode with an APCI source. The mobile phases used were methanol:
water (9:1) as solvent A and isopropanol: methanol (7:3) as solvent B. The target
analytes were identified using optimized transitions in MRM mode. The carotenoids
identified included violaxanthin, neoxanthin, antheraxanthin, lutein, zeaxanthin,
β-cryptoxanthin, and β-carotene. The authors reported that the LC-MS approach was
user-friendly and demonstrated to be a quick and efficient method for simultaneously
determining carotenoids, tocopherols, and phylloquinone in Brassica species (Lee
et al., 2020). In a related study, the HPLC-APCI-MS/MS technique identified the
presence of eight carotenoids from 26 types of GLVs that are primarily consumed
in Southeast Asia (Lee et al., 2022). A YMC Carotenoid C30 column, along with
a guard column, was used as the stationary phase. The mobile phase consisted of
90% methanol (solvent A) and a mixture of isopropyl alcohol (IPA), and methanol
in a 70:30 (v/v) ratio (solvent B). To confirm the precursor and product ions, full

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Extraction and Metabolic Profiling of Phenolic Acids, Flavonoids, and Carotenoids ... 451

scan and product ion scans were conducted using separate standard solutions. The
analytes were quantified in MRM mode with optimized transitions. The identified
carotenoids included violaxanthin, neoxanthin, antheraxanthin, lutein, zeaxanthin,
β-cryptoxanthin, α-carotene, and β-carotene. Among the 26 GLVs studied, lutein
and β-carotene were the predominant carotenoids, followed by violaxanthin and
neoxanthin. This hyphenated approach successfully and accurately differentiated
between the two isomers, α-carotene and β-carotene. A recent study utilized
HPLC and LC-MS methods to analyze the carotenoid profiles in the leaves of
four different accessions of Amaranthus gangeticus (L3, L5, L7, and L9) (Sarker
and Oba 2021). The HPLC process involved an STR ODS-II column and a UV-
Vis detector. The mobile phase used for separating the carotenoids consisted of
acetonitrile, methanol, and dichloromethane in a ratio of 60:20:20 (v/v/v), with
0.1% ammonium acetate, under isocratic flow conditions. For the LC-MS analysis,
a TOF mass spectrometer was employed, which was connected online to an HPLC
system equipped with an ESI source operating in negative ion mode and measuring
in the m/z range of 0–1000. The carotenoids identified in this study included
neoxanthin, violaxanthin, lutein, zeaxanthin, and β-carotene. The hyphenated
techniques used in this investigation proved to be robust and reliable for the
accurate identification of carotenoids. A study on 18 conventional GLVs compared
the profiles of carotenoids and tocopherols using a single ion monitoring LC-MS
method to identify potential GLVs for healthy diet formulations (Eun-Young et
al., 2023). The GLVs used for the study were Amaranthus lividus, Angelica gigas,
Glehnia littoralis, Heracleum moellendorffii, Peucedanum japonicum, Pimpinella
brachycarpa, Aralia continentalis, Kalopanax septemlobus, Artemisia princeps,
Cirsium setidens, Ligularia fischeri, Petasites japonicas, Rudbeckia laciniata,
Solidago virgaurea var. asiatica, Taraxacum mongolicum, Adenophora triphylla
var. japonica, Allium victorialis var. platyphyllum, and Toona sinensis. A single
ion monitoring (SIM) method combined with LC-MS was used to analyze the
carotenoids. The study utilized a Q-TOF mass spectrometer with APCI in positive
ion mode as the ionization source. For the LC separation, a YMC C30 carotenoid
column was used. The mobile phases consisted of methanol and water in a ratio
of 95:5 with 5 mM ammonium formate, and a mixture of methyl tertiary butyl
ether, methanol, and water in a ratio of 90:7:3 with 5 mM ammonium formate.
The carotenoids detected included β-carotene, zeaxanthin, lutein, luteoxanthin,
neoxanthin, and violaxanthin. Among the conventional GLVs analyzed, all-E lutein
was found to be the most abundant carotenoid, comprising between 44.4–52.1% of
the total. It was followed by all-E violaxanthin and all-E β-carotene. These findings
demonstrate the reliability and validity of the LC-MS/SIM approach utilized in
this study.
Research clearly shows that LC-DAD-MS and MS/MS systems are effective
tools for analyzing natural carotenoids. The development of these systems is
progressing steadily. As the LC-DAD-MS and MS/MS technologies advance, we
can expect to gain more insights into the distribution and roles of small natural
carotenoids. Table 4 presents studies on the separation and identification of
carotenoids from GLVs using HPLC and LC-MS techniques.

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Table 4 Profiling of Carotenoids.

Plant Name Parts Extraction Compounds Method Retention Stationary Phase Mobile Phase Wavelength MS Observed Reference
Used Solvent Identified Used Time (nm) / Flowrate Condition V-alue
(Min) (mL/min)
Brassica oleracea Leaves Ethanol and Violaxanthin LC- AP CI- 6.88 YMC Methanol: 0.3 mL/min Triple 601.4 Lee et al.
var. italica; B. hexane: Ethyl MS/ MS Carotenoid C30 water (9:1) and quadrupole MS (2020)
Neoxanthin 7.23 601.4
oleracea var. acetate (1:1) Column, (150 IPA:methanol (7:3) with positive
gemmifera; B. and ethanol Antheraxanthin 10.39 mm× 2.1 mm) APCI source and 585.5
oleracea var. containing gas temperature
capitata; B. BHT Lutein 13.58 –200°C. 569.4
Bioactive Compounds in Leafy Vegetables

oleracea var. Zeaxanthin 15.73 Vaporizer

botrytis; B. temperature 569.4
oleracea var. –350°C, drying
alboglabra; B. rapa β-Cryptoxanthin 19.19 gas flow rate of
var. pekinensis; 2.33×10−4 553.5
B. rapa var. m3/s, nebulizer
β-Carotene 21.44
chinensis; B. rapa pressure of 1.38
537.4
var. parachinensis; ×105 Pa and
Raphanus sativus; capillary voltage
Nasturtium of 4500 V.
officinale

Contd...

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 Table 4 Contd...
Plant Name Parts Extraction Compounds Method Retention Stationary Phase Mobile Phase Wavelength MS Observed Reference
Used Solvent Identified Used Time (nm) / Flowrate Condition V-alue
(Min) (mL/min)
Chayote leaves; Leaves Ethanol Violaxanthin 9.73 YMC 90% methanol 0.3 mL/min Triple 601.45 Lee et al.
Celery; Garland and containing LC- AP CI- CarotenoidC30 and IPA: methanol quadrupole MS (2022)
chrysanthemum; Aerial BHT MS/ MS column (150 mm (70:30) with positive
Green amaranth; Parts. Neoxanthin 10.26 × 2.1 mm) with APCI source and 601.50
Iceberg lettuce; a guard column gas temperature
Kale; Leeks; Pea Antheraxanthin 15.01 –200°C. 585.45
shoots; Spinach; Nebulising gas
Sword leaf lettuce; flow –2.5L/
Sweet potato Lutein 18.67 min, Interface 551.40
leaves; Wolfberry temperature
leaves; Cassava –350°C,
Zeaxanthin 19.50 569.45
leaves; Kangkong; desolvation
Melinjau; Pegaga; temperature
Sweet leaf bush; β-Cryptoxanthin 21.36 –150°C and 553.45
Turmeric leaves; drying gas flow
Ulam raja; Agathi rate - 3L/min.
α-Carotene 22.50 537.45
leaves; Ceylon
spinach; Fenugreek
leaves; Gongura β-Carotene 23.69 537.45
leaves; Moringa
leaves; Ponnaganni

Contd...
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453

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 454
Table 4 Contd...
Plant Name Parts Extraction Compounds Method Retention Stationary Phase Mobile Phase Wavelength MS Observed Reference
Used Solvent Identified Used Time (nm) / Flowrate Condition V-alue
(Min) (mL/min)
Amaranthus lividus; Foliage Acetone: β-carotene ~ 32.5 YMC Methanol: water 0.5 mL/min. Quadrupole Eun-
Angelica gigas; ethanol: CarotenoidC30 (95:5) containing TOF-MS with 537.44 Young
Glehnia littoralis; cyclohexane column (150 5mM of ammonium APCI positive Ko et al.
Heracleum (1:1:2) mm formate and mode. Drying (2023)
Zeaxanthin ~ 21.5 569.43
moellendorffii; containing × 4.6 mm) methyltertiarybut-yl; gas flow - 10L/
Peucedanum 0.1% BHT LC- AP CI- ether:methanol: min, Nebulizing
japonicum; Lutein QT OF- ~ 20.0 water (90:7:3) gas flow - 3 L/ 551.42
Pimpinella MS/ SI M containing 5mM min, Interface
brachycarpa; ammonium formate temperature
Aralia Luteoxanthin ~ 17.0 –400°C, DL 601.42
continentalis; temperature
Kalopanax Neoxanthin ~ 15.2 –300°C, and 601.42
Bioactive Compounds in Leafy Vegetables

septemlobus; the Heat block

Artemisia princeps; temperature
Cirsium setidens; Violaxanthin ~ 14.8 –300°C. 601.42
Ligularia fischeri;
Petasites japonicas;
Rudbeckia
laciniata;
Solidago virgaurea
var. asiatica;
Taraxacum
mongolicum;
Adenophora
triphylla var.
japonica; Allium
victorialis var.
platyphyllum;
Toona sinensis

Contd...

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 Table 4 Contd...
Plant Name Parts Extraction Compounds Method Retention Stationary Phase Mobile Phase Wavelength MS Observed Reference
Used Solvent Identified Used Time (nm) / Flowrate Condition V-alue
(Min) (mL/min)
Amaranthus Leaves Ice cold Neoxanthin HPLC 2.52 CTO-10 Acetonitrile/ 450 nm, TOF-MS 438.54 Sarker
gangeticus acetone and LC- ESI AC STR ODS-II methanol/ 1 mL/min. with UV-Vis- & Oba
(L3,L5,L7 and L9) - MS column, dichloromethane Visdetector and (2021)
Violaxanthin 2.63 446.43
(150×4.6 (60:20:20, v/v/v) ESI source. Scan
mm) containing 0.1% range:
Lutein 3.84 ammonium acetate 0–1000m/z. 445.22
Needle voltage:
–2000V
Zeaxanthin 4.28 452.63

β-carotene 20.16 449.52

Brasicca capitates; Leaves Acetone β-carotene HP LC ~10.0 RP-C18 Acetonitrile 452 nm, NA NA Zahra et
Spinacia oleracea column dichloro- dichloro- 1 mL/min. al. (2022)
methane, and
methanol
(70:20:10)

Contd...
Extraction and Metabolic Profiling of Phenolic Acids, Flavonoids, and Carotenoids ...
455

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 Table 4 Contd... 456
Plant Name Parts Extraction Compounds Method Retention Stationary Phase Mobile Phase Wavelength MS Observed Reference
Used Solvent Identified Used Time (nm) / Flowrate Condition V-alue
(Min) (mL/min)
Brassica Leaves Ice-cold All trans HP LC- 5.2 RP-C18 Methanol: deionized 450 nm, NA NA Zeb et al.
compestris; acetone and anhydrolutein-III DA D Column water (92:8) with 1 mL/min. (2017)
Brassica rapa; ethanol (4.6 × 100 10mM ammonium
β-carotene-5,6- 8.5
Brassica juncea; mm) acetate, deionized
epoxide
Malva neglecta; water containing
Spinacia oleracea All trans 10.0 0.1mM
Neoxanthin Ammonium acetate
and MTBE.
All trans 10.7
violaxanthin
13-ciszeaxanthin 12.7

9-cis zeaxanthin 13.0

Bioactive Compounds in Leafy Vegetables

Lutein 15.3

9-cis-Lutein 17.6

9’-cis-Lutein 18.2

Apo-caroten-8-al 23.4

Β-cryptoxanthin 29.4
ester
Alltrans-β- 33.0
carotene
13-cis-β- 33.4
carotene

Contd...

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 Table 4 Contd...
Plant Name Parts Extraction Compounds Method Retention Stationary Phase Mobile Phase Wavelength MS Observed Reference
Used Solvent Identified Used Time (nm) / Flowrate Condition V-alue
(Min) (mL/min)
Brassica oleracea Leaves Ice-cold Phytoene HP LC- - C18 Methanol:deionized 450 nm, NA NA Zeb et al.
L. var. botrytis acetone and All-E-neoxanthin DA D column (4.6 water (92:8) with 1.0 mL/min. (2022)
ethanol 9-Z-neoxanthin × 100 10mM ammonium
All-E-lutein mm) acetate, ammonium
9-Z-lutein 9ʹ-Z- acetate (0.1mM)
lutein αcarotene with- ionized water
9ʹ-Z-neoxanthin and MTBE.
γ carotene
All-E-βcarotene
Extraction and Metabolic Profiling of Phenolic Acids, Flavonoids, and Carotenoids ...
457

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458 Bioactive Compounds in Leafy Vegetables

Conclusion and Future Perspectives

Natural product research is gaining popularity around the world. Medicinal plants
have attracted significant attention due to their rich sources of bioactive components,
such as phenolic acids, flavonoids, carotenoids, vitamins, and minerals (Mueed
et al., 2023). To understand the biochemical and pharmacological activities of
medicinal plants, functional foods, and other nutraceuticals, researchers need to
identify detailed chemical profiles. This information is crucial for evaluating their
therapeutic and preventive potential. GLVs are a vital part of the human diet, and
have become increasingly popular because they are a rich source of phytochemicals.
These compounds serve as important antioxidants and help prevent chronic diseases.
To maximize the health benefits of these phytocompounds from GLVs, they must be
extracted with high purity and thoroughly characterized. A comprehensive review
of various extraction methods—including solid-phase microextraction, subcritical
solvent extraction, MAE,, UAE, and SSE—provides valuable insights into their
applications and advantages. Together, these advanced methodologies enhance
the field of extraction techniques by offering specialized solutions for a wide
range of bioactive compounds and natural matrices. Incorporating these methods
into research and industrial processes improves efficiency, reduces environmental
impact, and enhances selectivity (Bhadange et al., 2024). Numerous studies
have investigated the phenolic profiles of various plant species using RP-HPLC.
However, recent advancements in MS have significantly enhanced the analysis of
complex biological samples. MS techniques require less sample dilution due to
reduced noise, improved detection sensitivity, and greater specificity. As a result,
several limitations of LC-UV—such as high detection and quantification limits, as
well as interference from complex samples—have been addressed (Chiriac et al.,
2021). LC-MS has become an essential tool in natural product analysis, particularly
for comprehensive profiling of secondary metabolites due to its unique advantages,
including effective chemical identification, high resolution, and sensitivity at a
relatively low cost. The rapid development of LC-MS technology can be attributed
to its ability to characterize molecules based on C-ring cleavages and to provide
precise data. It is important to note that HPLC parameters and ionization techniques
can influence fragmentation patterns. When investigating conjugated derivatives
of phenolic compounds, especially flavonoids, careful attention is necessary, as
even minor errors can lead to inaccurate identification of the target molecules
(Kumar, 2017). One of the significant advancements in carotenoid analysis is
the design and development of novel packing materials for chromatographic
columns. Unlike fully porous particles, core-shell technology utilizes superficially
porous particles to enhance separation efficiency by reducing band broadening and
increasing resolution. Additionally, C30 stationary phases have been improved to
enhance isomer separation, which is crucial for accurately distinguishing between
carotenoids with similar structures. By combining sophisticated chromatographic
techniques with high-resolution mass spectrometry (MS), methods such as UHPLC-

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Extraction and Metabolic Profiling of Phenolic Acids, Flavonoids, and Carotenoids ... 459

QTOF-MS, supercritical fluid chromatography-mass spectrometry (SFC-MS), and

bi dimensional chromatography coupled with MS facilitate greater structural
characterization and quantification. These approaches allow for detailed profiling
of carotenoids and the discovery of new isomeric and derivative forms. The
classification of closely related carotenoid compounds based on collision cross-
section values has been improved by integrating ion mobility spectrometry (IMS)
with MS (Giuffrida et al., 2020). Several research studies have employed LC-
MS techniques to investigate these bioactive compounds from GLVs. However,
most of the papers cited in this chapter represent only modest attempts at this
research. There is a pressing need for more studies on the distribution patterns
of phytocompounds in GLVs using these methodologies to better understand the
structural-functional relationship and their roles in the food and pharmaceutical
industries. Future advancements in the industry will undoubtedly rely on a
sophisticated understanding of the relationship between extraction parameters
and compound properties. This approach will lead to sustainable and optimized
systems for extracting bioactive compounds from natural sources. Researchers and
practitioners are encouraged to adopt these cutting-edge hyphenated techniques,
as they represent the future of extracting, identifying, and characterizing natural
products.

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