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9
Modern Analytical Techniques for Quality Control and Chemical Identification of Phytochemicals
Rakesh E. Mutha1, Mohan Kalaskar 2, Zamir G. Khan
1
H. R. Patel Institute of Pharmaceutical Education and Research, Shirpur, India
2
R. C. Patel Institute of Pharmaceutical Education and Research, Shirpur, India
1
9.1 Introduction
In the current scenario, a growing recognition of the therapeutic potential and health benefits offered by plant-derived
phytochemicals and bioactive compounds is evident [1].
The interest in natural remedies and plant-based therapies
continues to surge, and ensuring the quality, safety, and efficacy of phytochemical-based products has become paramount [2]. Modern analytical techniques play a pivotal role
in meeting this demand, offering powerful tools for quality
control and chemical identification of phytochemicals [3].
From high-performance liquid chromatography (HPLC) to
mass spectrometry (MS) and nuclear magnetic resonance
(NMR) spectroscopy, these advanced methodologies enable
precise characterization, quantification, and validation of
phytochemical compositions, ensuring compliance with
regulatory standards and fostering confidence among consumers and healthcare professionals alike [4]. This introductory chapter sets the stage for exploring the diverse array
of modern analytical techniques employed in the quality
control and chemical identification of phytochemicals,
highlighting their significance in advancing research,
industry, and clinical applications in the realm of natural
products and herbal medicine.
Plant-derived phytochemicals are also known as phytonutrients or secondary metabolites [5]. These compounds
indirectly involved in the plant’s growth or reproduction,
play a fundamental role in the plant’s defence in connection with pathogens, ultraviolet (UV) radiation protection,
and attracting pollinators [6]. Moreover, phytochemicals
contribute to the color, flavor, and aroma of plants, making
them integral components of the human diet. They encompass a diverse array of chemical classes, including polyphenols (e.g. flavonoids and phenolic acids) [7], alkaloids (e.g.
caffeine and nicotine), terpenoids (e.g. carotenoids and
saponins) [8], and sulfur-containing compounds (e.g. glucosinolates) [9].
9.1.1 Background and Significance of Phytochemicals
Phytochemicals, the bioactive compounds derived from
plants, hold significant importance in human health disease management [10]. These compounds, found abundantly in various fruits, vegetables, whole grains, nuts,
seeds, and herbs, are responsible for the vibrant colors, flavors, and aromas of plant-based foods [11]. Their diverse
array of health-promoting properties, including antimicrobial, antioxidant, anti-inflammatory, and anticancer potential, underscores their significance in healthcare [12].
Phytochemicals play a crucial role in neutralizing harmful
free radicals [13], alleviating inflammation, inhibiting cancer cell growth [14], and protecting against cardiovascular
and neurodegenerative diseases [15]. Additionally, ongoing research continues to unveil new therapeutic applications of phytochemicals, highlighting their potential in
personalized medicine and disease management [16].
Incorporating a variety of phytochemical-rich foods into
168 9 Modern Analytical Techniques for Quality Control and Chemical Identification of Phytochemicals
the diet is essential for reaping the numerous health benefits offered by these bioactive compounds, ultimately promoting overall well-being and longevity [17].
9.1.2 Importance of Quality Control and Chemical Identification
Despite the significant health benefits attributed to phytochemicals, ensuring their quality control and accurate
identification is imperative. As these bioactive compounds
play a pivotal contribution in human health management
and disease prevention, it is essential to verify their composition and purity [18]. Quality control measures are necessary to guarantee that phytochemical-based products meet
regulatory standards and are safe for consumption.
Additionally, accurate identification of phytochemicals is
vital for conducting research, developing effective herbal
remedies, and formulating evidence-based dietary supplements [19]. Without robust quality control and precise
identification techniques, there is a risk of variability in
phytochemical content, adulteration, and contamination,
which can compromise the efficacy and safety of products
derived from plants [20]. Therefore, implementing stringent quality control protocols and employing advanced
analytical techniques are essential steps in connecting the
full exposure of phytochemicals in the promotion of wellbeing of human health. Overall, many researchers have
been working on the phytochemical analysis and quality
control of phytochemicals since 2002, and the details of a
number of publications per year published as per PubMed
data are highlighted in Figure 9.1.
9.1.3 Overview of Modern Analytical Techniques
Within the realm of phytochemical analysis, modern analytical techniques stand as indispensable tools for ensuring
the quality control and accurate identification of bioactive
compounds derived from plants [21]. HPLC, in conjunction with a variety of detectors comprising MS and ultraviolet-visible spectroscopy (UV-Vis), enables precise
quantification and characterization of phytochemicals
within complex mixtures. Gas chromatography (GC) complements HPLC for volatile compounds, while thin-layer
chromatography (TLC) offers rapid qualitative screening.
Spectroscopic techniques, including UV-Vis, Fourier transform infrared spectroscopy (FTIR), and NMR spectroscopy
[22], provide structural elucidation insights. MS enhances
identification capabilities with high-resolution mass spectra and fragmentation patterns. Hyphenated methods, like
gas chromatography-mass spectroscopy (GC-MS) and liquid chromatography-mass spectroscopy (LC-MS) [23],
integrate chromatography with mass spectrometry for
comprehensive analysis. Chemometric tools aid in data
100
86
79
80
60
48
44
42
40
No. of publications
20
777
5
2
0
Figure 9.1 Yearly number of publications on the topic of phytochemical analysis and quality control of phytochemicals (found in
PubMed database while searching the keyword: “phytochemical analysis and quality control” accessed 11 May 2024).
1
2004 2006 2008 2010 2012
2002
8
32
21
13
41
36
2014 2016 2018 2020 2022 2024
Year
45
32
31
79
75
48
42
9.2 Chromatographic Techniques 169
interpretation, facilitating quality control assessments, and
metabolomics [24] have enabled comprehensive profiling
of phytochemicals in biological samples, unveiling their
intricate interactions within the human body. Together,
these analytical methodologies empower researchers and
industry professionals to uphold the integrity and authenticity of phytochemical-based products, ensuring their efficacy and safety for various applications in healthcare,
nutraceuticals, and herbal medicine.
9.2 Chromatographic Techniques
Chromatographic techniques, including HPLC, GC, TLC,
and high-performance thin-layer chromatography
(HPTLC), play a pivotal role in phytochemical identification and quality control [25]. HPLC, a widely used technique, separates phytochemicals on the basis of their
chemical nature and retention times, enabling the quantification of individual compounds with high precision [26].
GC is particularly appropriate for volatile phytoconstituents
that provide complementary information to HPLC analysis
[27]. TLC serves as a rapid and cost-effective system with
respect to qualitative screening of phytochemicals [28],
while HPTLC offers better resolution with sensitivity in
comparison with traditional TLC [29]. Together, these chromatographic techniques empower researchers and industry
professionals to evaluate the consistency, purity, and content of products based on phytochemicals, ensuring their
safety and efficacy for various applications in pharmaceuticals, nutraceuticals, and cosmetics.
For example, Prabhakar et al. implemented various analytical techniques collectively to investigate the phytochemical composition of Carica papaya leaf extract (CPLE).
UV-visible spectral analysis was used to detect phytochemical compounds present in the extract. Additionally, FTIR
was applied for defining functional groups existing in
CPLE, facilitating separation of active components on the
basis of peak ratios. The application of these analytical
techniques enabled the researchers to gain treasured
understandings with respect to chemical composition and
presence of functional groups present in CPLE. Also, the
major polyphenols, including kaempferol, quercetin,
deoxy kaempferol, and deoxyquercetin, along with other
compounds, such as coumarin, cysteine sulphoxide, carpaine, folic acid, and L-glutamic acid, were identified
through UV–Vis, FTIR, TLC, and LC-MS techniques. These
compounds showed significant inhibition of α-amylase
and α-glucosidase, indicating potential as anti-diabetic
agents. The potential of improvement in pancreatic β-cell
function, regulation of carbohydrate metabolism, enhancement of insulin secretion, and reduction in oxidative stress
suggests their suitability as functional foods for diabetes
management [30].
9.2.1 High-performance Liquid Chromatography
Herein, Praveen Garg conducted a study on Ocimum sanctum (OS) and Tinospora cordifolia (TC), identifying these
plants as rich sources of flavonoids, particularly quercetin,
known for its antioxidant properties. Both plants were
selected for their traditional medicinal uses and potential
health benefits. The study involved quantitatively determining the flavonoid composition, specifically quercetin,
in the leaf and stem of TC and OS using HPLC analysis.
The assessment of quercetin in this study was carried out
utilizing chromatography under specific conditions:
employing an analytical column (RP-C18) and methanol
and acetonitrile (in a 50:50 v/v ratio) as a mobile phase,
with rate of elution of 1 mL per minute. Extracts were
injected into the HPLC system with a volume of 20 μL, and
generation of chromatograms was done using a UV detector at 256 nm. Each sample component’s peak value and
retention time were compared to standards for identification. Quantification of each sample was achieved using a
calibration curve derived from peak area assessments. The
results indicated that flavonoids were the major chemical
components in both plants, with variations observed
between the two species. Although both plants contained
quercetin, OS exhibited a higher concentration of flavonoids compared to TC. The analytical methods employed
in the study were appropriate in the accurate identification
and quantification of quercetin in the samples obtained
from both plant species [31].
Similarly, Ajay Kumar Meena conducted a study on
Ashwagandhadi lehyam, an important ingredient used in
Ayurvedic preparations containing Withania somnifera L.,
or ashwagandha, holds a significant place in Ayurvedic medicine and is valued for its tonic, hypnotic, sedative, and diuretic properties. It is considered one of the most precious
herbs in various systems of traditional Indian medicine.
Withania somnifera contains physiologically active compounds such as Withanolides and Withaferin-A, which are
known for their diverse therapeutic effects. Withaferin-A,
in particular, is a key bioactive component with reported
anti-inflammatory, anticonvulsive, anticancer, and antitumor activities. Due to the complex and naturally variable
composition of polyherbal formulations like Ashwagandhadi
lehyam, establishing quality control measures presents challenges. Two distinct mobile phase systems were employed
for running the sample and reference solutions. Various
ratios of acetonitrile, methanol, phosphate buffer, and water
were tested. It was determined that the 35:65 v/v ratio
170 9 Modern Analytical Techniques for Quality Control and Chemical Identification of Phytochemicals
5.650
mAU
250
200
150
100
50
0.708
0
1. 011
1 2
3
4
5 6
5.842
(a)
7
8
mi
mAU
400
350
300
250
200
150
100
50
1.002
0.718
1.111
1.605
1.470
1.284
2.029
1.793
0
2 4 6 8
2.367
2.547
2.790
3.147
3.868
3.499
4.322
5.015
4.122
5.861
6.123
6.448
7.053
7.541
8.280
(b)
9.388
mi
Figure 9.2 HPLC chromatograms of (a) Withaferin-A standard and (b) formulation of Ashwagandhadi lehyam. Source: Reprinted with
permission from Ref [32]. Copyright 2021, Elsevier.
of acetonitrile to buffer yielded a well-defined, symmetric
peak with excellent resolution, observed at retention times
of the standard compound taking 5.050 minutes, whereas
the formulation took 5.015 minutes. Here, the formulation
extract is eluted isocratically and a distinctive HPLC chromatogram was produced, showing a smooth, clean baseline
with good resolution and an identifiable marker peak.
Figure 9.2 depicts the Ashwagandhadi lehyam HPLC
chromatogram at a wavelength of 227 nm and a retention
period of 5.015 minutes, corresponding to the standard
Withaferin-A. Standardizing and developing reliable quality
protocols for Ayurvedic polyherbal formulations are essential tasks, especially considering the importance of ensuring
consistency and efficacy in traditional herbal remedies [32].
Jyoti Srivastava investigated the estimation and evaluation of phytoconstituents in plant extracts using HPLC.
Examples of these phytoconstituents include flavonoids,
carotenoids, tea polyphenols, vitamins, curcuminoids,
tannins, coumarins, chlorophyllin, porphyrins, and alkyl-
resorcinol, derived from plants and food ingredients.
These natural chemical compounds have been studied for
their antimutagenic properties. Additionally, Srivastava
explored phenols, which are compounds known to
strengthen the human immune system. These phenolic
chemicals induce apoptosis in cancerous or damaged
cells, highlighting the potential of herbal medications in
maintaining health by preventing or slowing the spread of
cancer. The quantity of total phenol was estimated by
HPLC method using gallic acid as standard drug. The relationship between diet and health is complex, as different
foods interact with the body through various pathways,
each serving a distinct purpose in promoting health. The
extract from Buchanania Lanzan bark maintained and
enhanced the concentration of flavonoids. In the future,
herbal items will be utilized to treat cancer because they
are safe and do not impair human health [33]. Verification
of bioactive markers through HPTLC fingerprint profiling
and quantification using HPLC was crucial in developing