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9.2 Chromatographic Techniques 171
a standardization protocol for Ayurvedic formulations,
ensuring accurate identification and evaluation of the formulation’s quality and potency. Therefore, it is imperative
to implement contemporary analytical techniques to
standardize and ensure the quality of Ayurvedic polyherbal formulations, thereby enhancing their effectiveness and safety for therapeutic use.
M. Karpakavalli conducted the extraction of piperine
using microwave-assisted extraction and HPLC techniques
for separation. The Indian Herbal Pharmacopoeia recommended the reflux method for extracting piperine for
30 minutes, with an additional 3 hours for andrographolide,
and utilized HPLC for quantitative determination. The
study also discussed the use of microwave-aided extraction
to extract limonoids from Azadirachta indica, saponins
from chickpea, taxanes from Taxus brevifolia, and camptothecins from Nothapodytes fetida. Microwave extraction is
advantageous as it facilitates rapid breakdown of plant
tissue and cells, reducing the likelihood of oxidation or
decomposition of important plant ingredients. This
approach significantly saves time compared to prolonged
heating methods, offering a valuable cost- and time-saving
solution for both industry and educational curriculums.
Herein, A Shimadzu RP-C18 liquid chromatographic system, which included a column oven preheated to 35 °C, a
degasser, quaternary pumps with low-pressure gradient, an
auto-injector, and a multi-wavelength UV array detector,
was used to perform chromatographic separation. The
mobile phase of methanol and water (65:35) with flow rate
1.5 mL per minute for piperine detection at a wavelength of
343 nm was employed. While for andrographolide, mobile
phase methanol and water (65:35) at 1 mL per minute at
223 nm was operated [34].
Warjeet S. Laitonjam conducted a study on the estimation of phytoconstituents from Heiña (Ficus pomifera
klall.). The alkaloid composition of khat (Catha edulis
Forsk) was determined using HPLC, while phenolic components in castor seeds were identified and characterized.
Additionally, HPLC was used to quantify or identify several
flavonoids, including kaempferol, rutin, and quercetin in
hop extract, with the free and glycoside forms of kaempferol and quercetin determined. A liquid chromatography–
electrospray ionization–tandem mass spectrometry
(LC–ESI–MS/MS) method was utilized to calculate Panax
ginseng’s neopanaxadiol amount. It was discovered that
the concentration of triterpenoids was 8.64 ppm in
2000 ppm of the extract when assuming that every component was eluted. This technique holds promise for isolating
and determining novel bioactive compounds through preliminary analysis. In this study, A UV-visible detectorassisted HPLC system was filled with some of the petroleum
ether extracts. Two milliliters of acetic acid mixed with the
acetonitrile and water (mobile phase) were used. A wavelength of 254 nm, an isocratic elution mode, was used for
detection. A specified amount of the sample was dissolved
in an acetonitrile:water mixture to create the sample solution, which was then filtered using a Millex-GV syringedriven filter (0.22 µ), as mentioned in Table 9.1 [35].
9.2.2 Gas Chromatography
GC is frequently utilized in the estimation of phytochemicals in extracts, as it is highly sensitive, selective, and reproducible. In GC, volatile and semi-volatile compounds are
separated based on their affinity for the stationary phase
and their vaporization characteristics [36]. After extraction
of phytochemicals from the plant material, GC analysis
involves injecting the sample into a heated injector, where
it vaporizes and enters the GC column. The interaction of
the vaporized substances and stationary phase, while traveling through the column, leads to separation according to
their molecular characteristics [37]. Detection is typically
performed employing a detector that produces signals corresponding to the abundance of each compound, such as
an MS or flame ionization detector (FID). By comparing
peak areas and retention times with known standard compounds, the identification and quantitation of phytochemicals in the sample can be done [38]. GC is particularly well
suited for analyzing compounds, such as terpenoids, fatty
acids, and volatile organic compounds, commonly found
in plant extracts [39].
Species of Punica, belonging to the Lythraceae family,
are esteemed medicinal herbs renowned for their diverse
therapeutic properties. Pomegranates, in particular, are
known for their antioxidant, antiviral, antiproliferative,
and anticancer attributes. Here, GC–MS analysis was carried out using ethanolic extracts produced from the peel
seeds of Punica protopunica (PP) and Punica granatum
(PG), two species of Punica. The examination showed that
there were 21 and 14 chemicals in the peel seeds of PG and
PP, respectively. Primary chemical components identified
in PG peel seeds included propanoic acid, methyl amine,
methoxypropionic acid, and benzenedicarboxylic acid.
Similarly, PP peel seeds exhibited comparable amounts of
propanoic acid, benzoic acid, and benzenedicarboxylic acid.
Furthermore, in vitro evaluations of the hydroalcoholic
extract’s antioxidant properties were conducted, revealing
significant differences in phenolic and total flavonoid contents (TFC) between the two studied extracts. This research
was conducted by Gehan A. Elgaaly. Herein, GC analysis, a
Perkin Elmer was used for the procedure. The Perkin Elmer
Elite-5 capillary column with a dimension of 30 × 0.25 mm
and film thickness of 0.25 mm was used. It was made up of
Table 9.1 High-performance liquid chromatography analysis of phytoconstituents obtained from a variety of sources with details of chromatographic conditions.
Phytoconstituents
Quercetin,
Ocimum
sanctum, and
Tinospora
cordifolia
Ashwagandhadi
Lehyam
corresponding to
standard
Withaferin-A
Buchanania
lanzan Spreng
Piperine,
andrographolide
Heiba (Ficus
pomifera Wall.)
Chromatographic
conditions
Mobile phase: Methanol
and acetonitrile
(50:50 v/v), Flow rate: 1
mL per min, Detection
wavelength: 256 nm
Mobile phase:
Acetonitrile: Buffer
(35:65) (v/v), flow rate:
1.8 mL per min, column
temperature: 32 °C,
detection wavelength:
227 nm
Mobile phase: Methanol:
0.005 mM phosphate
buffer (70:30),
Mobile phase: methanol
and water (65:35), Flow
rate: 1.5 mL per min for
piperine and 1 mL per
min for andrographolide,
column temperature:
25 °C, detection: 343 nm
for piperine and 223 nm
for andrographolide
Mobile phase:
acetonitrile-water with
2 mL acetic acid, flow
rate: 1 mL per min,
detection: 254 nm
Sample Instrumentation
Sample matrix:
Flavonoid
(quercetin) and
stem extract of
Ocimum
HPLC System:
Column: C18
(250 mm ×
4.60 mm), UV
detector
sanctum,
Tinospora
cordifolia,
injection
volume: 20 μL
Injection
volume: 10 μL
HPLC system:
Agilent 1200,
column: C18
eclipse, XDB,
4.6 mm ×
150 mm, 5 μm
particle size, diode
array detector
(DAD)
Injection
volume: 20 µL
HPLC system:
Shimadzu
LC-10ATVP
Column: C18,
110 Å, 250 ×
4.6 mm
Injection
volume: 50 µL
HPLC system:
Shimadzu liquid
chromatographic
system (LC2010 A, HT),
column: C18
column (250 ×
4 mm, 5 µL)
Injection
volume: 25 μL
Column:
Whatman Partisil
10-ODS-3 reverse
phase C18 (250 ×
4.6 mm, 5 μ)
Retention time
(Std.) (min)
Retention time
(Test) (min)
Peak area
Concentration
(μg/mL)
2.596 2.452 142.55 5–25 Linearity:
Method
validation
parameters
References
[31]
R2 = 0.99
5.050 5.015 —0.17
−0.010625 mg
per mL
Linearity:
R2 >
0.9999
— — — — Linearity:
[32]
[33]
R2 = 0.999
Piperine: 6.150,
andrographolide:
2.458
Piperine:
5.792, andrographolide:
2.458
Piperine:
2914859,
andrographolide:
— — [34]
20567404
— — — — — [35]
9.2 Chromatographic Techniques 173
95% dimethyl polysiloxane. Helium was used as the carrier
gas, using injection volume and flow rate of 1μL and 0.5 mL
per min, respectively. The analysis was done using electron
impact ionization (70 eV) while data was evaluated using
total ion count (TIC) for the determination of the compound’s identity and quantity [36].
9.2.3 Thin-layer Chromatography and High-performance Thin-layer Chromatography
TLC and HPTLC are widely employed techniques in phytochemical estimation due to their simplicity, costeffectiveness, and versatility [40]. TLC comprises the
separation of compounds in a blend on the basis of their
degree of difference in migration rates on an adsorbent
material’s thin layer, typically cellulose or silica gel [41].
After applying the sample to the TLC plate, it is developed
in a solvent system, allowing the individual components
to migrate. Visualization of separated compounds is
achieved through techniques such as UV absorption,
chemical staining, or fluorescence. TLC is valuable for
qualitative analysis, identification of compounds, and
assessing the purity of natural extracts [42]. HPTLC, a
modern advancement of TLC, offers improved resolution,
sensitivity, and reproducibility. It utilizes specialized
instrumentation, such as automated sample application
and development chambers, and high-performance stationary phases with uniform particle size distribution
[43]. HPTLC provides enhanced separation efficiency and
allows for precise quantification of compounds through
densitometric analysis. It is particularly useful for analyzing complex mixtures and trace-level components in
plant extracts [44]. Both TLC and HPTLC are invaluable
tools in phytochemical analysis, including rapid screening for identity, and quantity of bioactive compounds present in plants, herbal extracts, and natural products [45].
This enables quality control, authentication, and standardization of botanicals of traditional medicine, pharmaceuticals, and dietary supplements [28].
Dr. Shweta P. Ghode’s study focused on detecting the
phytoconstituent curcumin in Curcuma longa rhizomes
using TLC and HPTLC. Numerous C. longa crude medications, extracts, and formulations have been found to
include curcumin, which is well known for its antioxidant,
anti-inflammatory, and anticancer qualities. The TLC and
HPTLC methods utilizing chloroform:ethanol:glacial acetic acid (90:5:1 v/v, mobile phase) successfully detected
curcumin, with an approximate Rf value of 0.37. The study
also explored strategies to improve curcumin’s bioavailability, noting challenges like inadequate absorption, quick
metabolism, and removal. Numerous methods have
been investigated to enhance curcumin’s bioavailability,
including inhibiting its metabolic pathways. It was
observed that curcumin concentrations in different species may vary due to factors like collection time, regional
variation, genetic diversity, growing conditions, and preservation methods. In this, a specific sample and standard
quantity was applied as bands on the TLC plate using
Linomat 5. The development of plate using a mobile phase
was done until it reached approximately 70 mm. After
development, the chromatographic plate was dried using a
hairdryer. The plate was then scanned at 425 nm with a
Camag TLC plate Scanner 3. The determination of curcumin content was done by comparison of the peak areas
of the standard and sample spots [46].
In another study, Rashmi et al. conducted a study on the
phytochemical standardization of seeds of Diploknema
butyracea using the HPTLC system to investigate phytochemical components and create a fingerprinting profile.
The HPTLC method was developed for extracting active
ingredients from seed extracts, assessing their qualitative
and quantitative distribution. Lipids, tannins, alkaloids,
phenols, flavonoids, steroids, and saponins were detected by
preliminary phytochemical screening. Curcumin, a phytoconstituent, was detected by TLC and measured by HPTLC
in several C. longa crude medication, extract, and formulation samples. In the TLC approach, the author coated glass
plates (20 cm × 20 cm) with silica gel (Qualigen fine chemicals) to a thickness of 0.5 mm. Samples of each extract, dis-
1
solved at a 5 mg mL
concentration in methanol, were
applied onto the coated plates. Chromatography was conducted using 100% chloroform, following the procedure outlined by Harborne. After chromatography, concentrated
sulfuric acid was sprayed on the plates as a reagent to help
see the spots, and then the plates were heated to 100 °C for
10 minutes. The color that the spots formed after reacting
with the spray reagent was then used to identify them. For
the HPTLC approach, chromatographic separation was conducted using a stationary phase of aluminum plates (10 cm
× 10 cm) precoated with silica gel 60F254 (Merck). The
application of constant 10 μL volume for different extracts
with separate solvent systems was chosen. Scanning was
performed at a wavelength of 366 nm and also in the visible
range. A saturation time of 25 minutes was allowed before
the chromatographic run. The sample was spotted on the
TLC plate in triplicate using an automatic TLC applicator
system, as depicted in Figure 9.3 [47]. This study aims to
define and quantify the plant’s basic phytochemical ingredients to establish its scientific foundations and compare its
bioactive principles with other species. In all, HPTLC fingerprinting profiling serves as an essential tool for standardizing herbal drugs and accurately identifiying medicinal
plants, opening new avenues for pharmacological activities
and clinical trials.
174 9 Modern Analytical Techniques for Quality Control and Chemical Identification of Phytochemicals
(a)
[AU]
(b)
600
[AU]
400
300
200
100
0.0
–0.20
800
600
500
400
300
200
100
0.0
–0.20
0.00
0.00
All trades @ 200
0.20
0.40
All trades @ 200 nm
0.40
0.60
0.60
0.80
[Rf]
[Rf]
1.00
1.00
0.0
0.0
10.0
10.0
20.0
30.0
20.0
40.0
30.0
50.0
40.0
60.0
50.0
70.0
60.0
80.0
70.0
[mm]
80.0
800
[AU]
600
500
400
300
200
100
0.0
100.0
[mm]
600.0
[AU]
400.0
300.0
200.0
100.0
0.0
100.0
Figure 9.3 Densitograms for kernels (a) chloroform extract and (b) methanol extract. Source: Reprinted with permission from Ref.
[47]. Copyright 2015.
Yogesh V. Ushir conducted a study utilizing the HPTLC
fingerprint profiling for the quantitation of different phytochemicals in animal species, specifically targeting
quercetin, catechin, and stigmasterol. Densitometric scanning at various wavelengths was employed to identify and
quantify each phytoconstituent. The study aimed to standardize these species using the fingerprints, particularly
focusing on Anisomeles species, for which no published
reports on HPTLC methods for quantifying phytoconstituents were available. The research established the densitometric HPTLC technique as reliable for the phytochemical
estimation of herbal medications, enabling the measurement of phytoconstituents from different extracts of
Anisomeles species. A comparison between Anisomeles
indica and Anisomeles malabarica was conducted based
on the identified phytoconstituent amounts. The suggested HPTLC method for quantitative assessment was
found to be quick, easy, and accurate, facilitating the
determination of crude medication quality and aiding in
the separation and isolation of constituents for further
chromatographic analysis in future research. From the
HPTLC fingerprints, the following peaks were observed:
quercetin at Rf 0.60 (AUC = 3756.9 for 20 µL), β-sitosterol
at Rf 0.39 (AUC = 3503.5 for 10 µL), catechin at Rf 0.20
(AUC = 1710.2 for 20 µL), and ovatodiolide at Rf 0.69
(AUC = 9686.4 for 20 µL) [48].
9.3 Spectroscopic Techniques 175
In another study, Jayita Saha and Taniya Mitra investigated the universal remedy Saraca asoca (Roxb.) Wilde,
commonly known as Ashok, in various traditional medicinal practices like Ayurveda, homeopathy, and Unani. They
developed a sensitive and reliable technique, HPTLC, to
determine the gallic acid content, a pharmacologically significant active ingredient, in S. asoca (Roxb.) Wilde’s dried
flowers and leaves. This method enabled qualitative assay
and detection of standard gallic acid. The study confirmed
that the gallic acid was present in Saraca asoca leaves and
flowers utilizing an HPTLC experiment with methanolic
extracts. Recognizing the availability of leaves year-round
compared to flowers, the concentration of gallic acid in
leaves becomes crucial for the effective utilization of this
ancient plant. Silica gel 60 F254 precoated chromatographic plates were used, with the mobile phase added to a
pre-saturated glass tank utilizing a CAMAG twin trough
chamber. The mobile phase consisted of toluene, ethyl acetate, formic acid, and methanol in a ratio of 6:6:1.6:0.4
(v/v/v/v), applied to a height of 86.2 mm in an ambient
environment. The developed spots were seen at 254 and
280 nm using CAMAG UV cabinet after they had dried followed by scanning by WINCATS software-enabled
CAMAG TLC scanner 3. The leaf and flower’s methanolic
extract containing gallic acid was verified by UV spectra
superimposition of samples and standards having same
retention factor. HPTLC plates measuring 10.0 × 10.0 cm
were used for this analysis. The applications of standard
gallic acid and extracts were done using TLC applicator
automated with CAMAG Linomat 5 having a bandwidth of
−1
8 mm with a delivery speed of 150 nL s
assisted by nitrogen flow [49]. The comparative data of all chromatographic
HPTLCs is summarized in Table 9.2.
9.3 Spectroscopic Techniques
UV-Vis, FTIR, and NMR Spectroscopy are instrumental
techniques in phytochemical estimation and quality control
[50]. UV-Vis spectroscopy is widely used to analyze the
presence and concentration of phytochemicals based on
their absorption of ultraviolet and visible light [51]. It provides qualitative and quantitative information about compounds like phenolics, flavonoids, carotenoids, alkaloids,
tannins, antioxidants, etc. FTIR spectroscopy is mainly
employed for the identification of functional groups of phytochemicals by computing the IR absorption [52]. It allows
for the characterization and qualitative analysis of various
chemical bonds and groups, aiding for the identification of
specific compounds and assessing their purity. NMR spectroscopy offers thorough evidence regarding the structure
and composition of phytochemicals by analyzing the magnetic properties of atomic nuclei in a molecule [53].
It is particularly useful for elucidating complex structures, determining stereochemistry, and identifying
unknown compounds, thereby ensuring the quality and
authenticity of herbal products. Together, these spectroscopic techniques play crucial roles in the determination of
quality and quantity of phytochemicals, facilitating identification, characterization, and quality control in medicinal
plants and herbal products.
9.3.1 Ultraviolet-visible Spectroscopy
Masarrat Mukadam conducted a study on the primary ingredients found in various plant parts, including chlorophyll,
proteins, sugars, and amino acids, with terpenoids and alkaloids as secondary components. These phytochemicals
exhibited direct therapeutic actions and could potentially
serve as raw materials for creating more sophisticated semisynthetic chemicals. UV-visible spectroscopy was utilized to
facilitate the identification of certain chemical components
in pure or biological samples through qualitative analysis.
This study concluded that UV-Vis spectroscopy was effective
in both quantitative analysis of phytochemicals and the process of identifying certain chemical components in both
pure and combined samples. It was found to be particularly
useful in identifying bioactive chemicals in plants, including
phenolic compounds, flavonoids, alkaloids, carotenoids,
tannins, and antioxidants. The absorption spectrum facilitated the identification of structural components within
molecules through the presence of multiple absorption
bands. Phytochemical analysis involved the extraction,
screening, and identification of medicinally active compounds in plants [54].
Using a wide range of techniques of analysis, like GC/
MS, FTIR, UV-Vis spectroscopy, the study carefully examined the bioactive components found in the extract of
Mentha spicata. Employing UV-Vis spectroscopy, the
researchers conducted scans of the plant extract across a
spectrum ranging from 300 to 800 nm, revealing distinctive
peaks indicative of various chemical constituents. These
peaks, characterized by varying absorption levels, provided
insights into the complex composition of the extract.
Subsequently, FTIR examination revealed the existence of
a wide variety of substances, such as aromatic chemicals,
carbonyl, alkanes, alkenes, phenols, and alcohols, further
enriching the understanding of its chemical profile.
In the UV-Vis spectra as depicted in Figure 9.4, the occurrence of unsaturated groups with heteroatoms like S, O,
and N was evident due to the emergence of one or more
peaks between 200 and 400 nm range. Two peaks could be
seen in the M. spicata extract spectra at wavelengths of 353
and 407 nm. This verified that the M. spicata extract
included organic chromophores. However, the inherent
challenges of attributing the absorption peaks to specific
Table 9.2 HPTLC: a modern analytical tool for the estimation of phytoconstituents and chromatographic conditions.
Phytoconstituents
Chromatographic
conditions
Curcumin Mobile phase:
chloroform: ethanol:
glacial acetic acid
(90 : 5 : 1), Detection
wavelength: 425 nm
Diploknema
butyracea (Roxb.)
H.J. Lam. (Family
Sapotaceae)
Mobile phase: 3 MP
were used here Ethyl
acetate: Pet ether
(15 : 85), chloroform:
methanol: water
(13 : 7 : 2) and
chloroform: methanol:
water (16 : 6 : 1),
detection wavelength:
366 nm
Quercetin,
Β-sitosterol,
catechin and
ovatodiolide in
Anisomeles
Species
Moblie phase: 4 MP
were used Toluene:
ethyl acetate:
formic acid (5 : 4 : 1),
chloroform:
methanol10 (8 : 0.6),
toluene: ethyl acetate:
methanol (4 : 3 : 3),
toluene: ethyl acetate:
formic acid11 (7 : 3 : 1),
detection wavelength:
366 nm
Saraca asoca
(Roxb.) Wilde
Mobile phase: toluene:
ethyl acetate: formic
acid: methyl alcohol
(6 : 6 : 1.6 : 0.4 v/v/v/v),
detection wavelength:
280 nm
Sample information
Sample matrix:
Raw sample, extract
and formulation,
Sample
preparation:
Macerated in
methanol
Sample matrix:
powdered seeds,
Sample
preparation:
macerated with a
particular solvent
Sample matrix:
dried powder,
sample preparation:
extracted separately
with ethanol and
acetone using
Soxhlet apparatus
for 6 h
Sample matrix:
powder form,
sample preparation:
dissolved in
methanol then
sonicated and
finally air-dried.
Instrumentation
details
Plate: Precoated
silica gel 60F254
TLC aluminum
sheets, Scanner:
CAMAG TLC
plate scanner,
Plate: 10 cm ×
10 cm aluminum
plate precoated
silica gel 60F254,
scanner:
densitometer,
software:
WinCATS
Plate: 10 cm ×
10 cm aluminum
plates pre-coated
silica gel 60F254,
scanner:
densitometer,
software:
WinCATS
Plate: precoated
silica gel 60F254,
scanner:
densitometer,
software:
WinCATS
Rf value Peak area Concentration
0.37 Raw sample: 13326.3,
— Rf value [46]
Extract: 166 92.0,
Formulation: 14 167.4
0.35 — Samples of
every extract
with a
methanol
concentration
of 5 mg mL
Quercetin:
0.60,
β-sitosterol:
0.39,
catechin:
Quercetin: 3756.9,
β-sitosterol: 3503.5,
catechin: 1710 and
ovatodiolide: 9686.4
1 µg µL
-1
0.20 and
ovatodiolide
0.69
Standard:
— 50 mg mL
-1
0.44,
methanolic:
0.43
Method validation
parameters
Rf values and
finger print
profile data
-1
Rf values and
finger print
profile data
Rf value [49]
References
[47]
[48]
9.3 Spectroscopic Techniques 177
1. 6
1. 4
1. 2
1. 0
0.8
0.6
Absorbance
0.4
0.2
0.0
–0.2
300 400 500
Figure 9.4 UV visible spectrum of Mentha spicata extract (in
methanol). Source: Reprinted with permission from Ref. [55],
Copyright 2016.
353
407
665
504
Wavelength (nm)
609
535
600 700 800 900
system elements hampered the application of UV-visible
spectrophotometry in the investigation of complex media.
To allow accurate extract characterization and ingredient
identification, further analytical techniques like GC/MS
have to be added to the UV-Vis results.
Furthermore, the GC/MS study of the methanolic plant
fraction allowed for the identification of a remarkable 42
phytochemical components, each distinguished by distinct
peaks. These findings underscored the richness and complexity of the bioactive compounds present in M. spicata,
offering valuable insights into its potential therapeutic
properties. The detailed chemical analysis revealed a plethora of bioactive compounds, including volatile oils found
in M. spicata, with carvone being the main component.
Other volatile oils include ciscarveol, 1,8 cineol, carvyl acetate, limonene, cis-dihydrocarvone, and cis-sabinene
hydrate. Such exhaustive figuring of M. spicata’s chemical
composition lends credence to its historical usage in traditional medicine and supports its continued exploration for
potential therapeutic applications in modern healthcare
practices [55].
9.3.2 Fourier Transform Infrared Spectroscopy
FTIR plays a crucial role in phytochemical estimation by
providing detailed information about the functional groups
present in plant extracts. It enables rapid and accurate
identification of various phytochemical compounds based
on their characteristic absorption spectra, aiding in the
determination of the quality and quantity of phytoconstituents [56]. Additionally, FTIR facilitates the detection of
specific chemical bonds and structural features, enhancing
understanding of the chemical composition of a plant
extract’s potential bioactivity [57].
In one of the studies, an FTIR analytical tool was used to
analyze the compounds included in leaf extracts from
Grewia tilifolia (Vahl). Eight different solvents were used to
create the leaf extracts. Standard methods for FTIR analysis was employed for phytochemical analysis. The FTIR
spectroscopy testing revealed varied typical peak values
associated with different functional groups of relevant
extract’s constituent parts. The existence of numerous
functional groups like carboxylic acid, phenol, alcohol,
alkane, and aldehyde, with few others suggested the occurrence of important components in the plant material. A
similar study established an FTIR profiling of an important
medicinal herb, G. tilifolia [58]. Herein, as per Figure 9.5,
the FTIR investigation shows the existence of polyphenolic
and flavonoid compounds because of O—H stretching, terpenes because of the C—H group, and alkaloids because of
N—H stretching. The test plant was found to include the
following functional groups: ethers, carboxylic acids and
anhydrides, alcohols, phenols, aromatics, alkenes, amides,
amines, aldehydes, and organic halogen compounds.
These findings were established by the FTIR analysis,
which proposed the existence of O—H, C—H, N—H, C=C,
C—Cl, nitrates, and silicates stretching. The various medicinal properties of G. tilifolia could be attributed by the exist-
ence of characteristic groups, like carboxylic acid,
anhydride, alcohol, phenol, amine, amide, ester, ether, etc.,
as reported.
9.3.3 Nuclear Magnetic Resonance
NMR Spectroscopy is instrumental in phytochemical estimation by offering unparalleled insights into the molecular structure and composition of plant compounds [59].
Through NMR analysis, researchers can identify and characterize various phytochemicals, including alkaloids, flavonoids, and terpenoids, based on their unique chemical
shifts and coupling patterns. This technique enables precise quantification and elucidation of complex mixtures,
contributing to our understanding of bioactive constituents and potential therapeutic applications of plant
extracts [60].
NMR spectroscopy was reported to have proven effective
and valuable for researching herbal health items. This
technique, along with the procedures applied, can be effective for R and D of botanicals at all phases, from plant to
finished goods [61]. Plants not only benefited the global
ecosystem and environment, but also supplied essential
food such as daily consumables of our everyday lives, as
well as renewable assets for energy, raw materials, fodder,
and more [62].
178 9 Modern Analytical Techniques for Quality Control and Chemical Identification of Phytochemicals
100
80
6040200
Transmittance [%]
1730.12
1620.99
1449.15
1390.61
1239.59
1074.19
898.44
836.82
2923.98
3394.60
2855.73
4000 3500 3000 2500
2000 1500 1000 500
722.62
Wavenumber cm-1
Figure 9.5 FTIR spectrum of Grewia tilifolia leaf (methanolic extract). Source: Reprinted with permission from Ref. [58], Copyright 2019.
1
4.75
5.00
6.37
4.45
3.31
3.26
3.19
3.14
3.09
3.05
2.94
2.89
3.34
–0.0
0.00
8500
8000
7500
7000
6500
6000
5500
5000
4500
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3500
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0
–500
6.5 6.0 5.5 5.0 4.5 4.0 3.5
3.0 2.5 2.0
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0.5 0.0
f1 (ppm)
Figure 9.6 1H NMR spectrum of Garcinia gummi-gutta. Source: Reprinted with permission from Ref. [63]. Copyright Nature, Scientific
Reports 2018.
Additionally, chemical components in complicated
combinations, including plant extracts, medications, and
herbal medicines, were analyzed using NMR methods.
Since NMR spectroscopy is very reliable, strong, and quantitative by nature, it does not require the chromatographic
separation of many components beforehand. The method
had been used to find, recognize, and quantify adulterants
in dietary supplements intended to help with weight
reduction. For the purpose of authenticating herbal medicines, combining spectroscopic techniques with DNA barcoding improved the resolution of species identification
and combination analysis. In one investigation, Garcinia
fruits and dietary supplements were authenticated using
1H NMR spectroscopy and DNA barcoding. 1H NMR was
deemed to be beneficial for the determination of quality
and quantity of phytoconstituents. Nonetheless, compared to other chromatographic techniques, NMR has not
been used as extensively in official food supplement testing and pharmaceutical control laboratories. This is mostly
because of the expensive apparatus and the perception of
NMR’s complexity. Nevertheless, much instrumental
advancement in NMR has been made since the last decade
so that it can be applied for routine purposes. As per Figure
9.6, the 1H NMR spectrum of G. gummi-gutta water
extract clearly displayed distinctive signals suggesting the
presence of (-)-hydroxycitric acid [63].
9.4 Mass Spectrometry 179
9.4 Mass Spectrometry
The MS’s contribution in phytochemical estimation is evident by its role in the identification, quantification, and
structural revealing of complex compounds present in
plant extracts [64]. This technique provides high sensitivity
and specificity, allowing for the detection of trace-level
compounds and differentiation of isomeric species, thus
enhancing our understanding of the chemical composition
and bioactivity of natural products [65].
9.4.1 Structural Elucidation of Phytochemicals by Mass Spectrometry
Faten Hameed Thamer and her colleagues studied
Citrullus colocynthis (L.), which is well-known for a variety of pharmacological qualities linked to its secondary
metabolites or phytochemicals. The study’s authors used
dichloromethane to extract the C. colocynthis seeds, and
then they used GC-MS to screen the oil extract that was
left behind to determine which phytoconstituents were
active. The unknown phytoconstituents were identified
using their GC-MS spectra comparison with spectra kept
in WILEY library of GC-MS and the National Institute of
Standards and Technology Mass Spectral database
(NIST-MS database). Twenty-four out of fifty-five different
compounds were found to be bioactive when studied using
GC-MS chromatogram. The major categories of the compounds found in oil were carotenes, phenols, esters, and
steroids. As per the available literature, some of these
compounds were found to have pharmacological activities. The major component revealed in the C. colocynthis
seed oil extract was isooctylphthalate (58%), which exhibited a potential antimicrobial effect [66]. Herein, the composition of C. colocynthis seed oil extract was analyzed
using GC-MS. In this study, the dichloromethane seeds oil
extract of C. colocynthis was utilized for GC-MS analysis.
The GC-MS analysis revealed approximately 55 phytoconstituents of pharmacological importance, which were
characterized by their molecular formulas, molecular
weights, compositions (%), and retention times determined from their peak areas as depicted in Figure 9.7.
9.4.2 Quantitative Analysis and Quality Control Measures
The MS has materialized as a critical tool of phytochemical
investigation owing to its remarkable sensitivity, specificity, and accuracy [64]. Two fundamental aspects concerning quantitative analysis and quality control measures
employing MS for phytochemicals are elaborated in the
following text.
9.4.2.1 Quantitative Analysis for Phytochemicals
Quantitative analysis utilizing MS entails determining the
concentration or amount of a specific phytochemical within
a sample. This is accomplished by comparing the intensity
of the mass spectral signal of the target compound with that
of a known standard [67]. The principal methods for quantitative analysis using MS are the following.
100
90
80
70
60
50
40
30
Relative Abundance
20
10
0
Figure 9.7 GC-MS chromatogram of C. colocynthis (L.) dichloromethanolic seeds oil extract. Source: Reprinted with permission from
Ref. [66], Copyright Elsevier, 2023
5.33
10.44
5
0
10 15 20 25
19.27
23.99
Time (min)
32.42
30 35 40 45 50
46.26
50.17
55
180 9 Modern Analytical Techniques for Quality Control and Chemical Identification of Phytochemicals
9.4.2.1.1 External Calibration
This method entails constructing a calibration curve using
known concentrations of the target compound. Subsequently,
the concentration of the unknown sample is determined by
comparing its mass spectral signal to the calibration
curve [68].
9.4.2.1.2 Internal Standardization
This method involves adding a known quantity of an internal standard to the sample. The internal standard, exhibiting similar behavior to the target compound during
analysis, serves to rectify any fluctuations in the analytical
process [69].
9.4.2.1.3 Isotope Dilution Analysis
This method encompasses adding a known quantity of an
isotopically labeled version of the target compound to the
sample. The isotopically labeled compound functions as an
internal standard, enabling the precise determination of
the target compound’s concentration [70].
9.4.2.2 Quality Control Measures for Phytochemicals
Quality control measures are paramount for guaranteeing
the accuracy and precision with reliability of MS-based phytochemical investigation. These measures encompass various
aspects aimed at safeguarding the integrity of the analytical
process and the validity of the results obtained [20].
Instrument calibration stands as a fundamental practice
in MS analysis, involving the regular calibration of the
instrument to ascertain its proper functioning and accuracy of mass spectral signals. Through meticulous calibration, deviations or errors in the instrument’s performance
can be identified and rectified, ensuring the consistency
and reliability of the analytical data [71]. Sample preparation plays a pivotal role in MS analysis by standardizing
procedures to ensure uniformity in sample handling and
analysis. By adhering to standardized protocols, variations
in sample preparation can be minimized, thereby enhancing the reproducibility and reliability of the analytical
results [72]. Method validation serves as a critical step in
MS-based analysis, involving the rigorous assessment of
the analytical method’s accuracy, precision, and sensitivity.
This comprehensive validation process encompasses the
analysis of known standards to ascertain the method’s performance characteristics, including its limits of detection
and quantitation. Through method validation, the robustness and reliability of the analytical method can be confirmed, instilling confidence in the accuracy of the obtained
results [73].
Blind samples, comprising unidentified samples analyzed alongside test samples, serve as essential tools for
monitoring the performance of the analytical method and
identifying potential biases or errors. By incorporating
blind samples into the analysis, any discrepancies or deviations in the analytical process can be promptly detected
and addressed, thereby ensuring the integrity and reliability of the analytical data [74].
Data analysis, facilitated by specialized software, is crucial for processing mass spectral data and calculating the
concentrations of target phytochemicals. Regular validation and calibration of the data analysis software are essen-
analytical outcomes, thereby enhancing the overall quality
and integrity of the analysis [75].
9.5 Hyphenated Techniques
Hyphenated techniques, including LC-MS, GC-MS, and
liquid chromatography-nuclear magnetic resonance-mass
spectroscopy (LC-NMR-MS) synergize chromatographic
separation with sensitive detection methods like MS and
NMR, providing precise identity and quantitative determination of phytochemicals in complex phyto matrices [76].
Such methodologies enhance analytical resolution, offering insights into chemical structures and properties crucial
for understanding plant bioactivity and pharmacological
potential.
9.5.1 LC-MS and GC-MS Applications in Phytochemical Analysis
The Fabaceae family of plants includes the legume Vigna
unguiculata (L.) Walp., or cowpea, which is well known for
its nutritional and medicinal properties when consumed.
While cowpea leaves and pods received relatively little scientific focus, cowpea seeds are nutrient-rich source and
valued for their therapeutic capabilities. This study emphasized the identification of phytochemicals found in leaves
as well as the evaluation of their antioxidant and antibacterial potential. The methanol extract of cowpea leaves was
subjected to LC-MS, which shown the occurrence of
α-hederin, which is a newly suspected inhibitor of SARSCOV-2 (Severe acute respiratory syndrome coronavirus 2)
along with mycotoxin Zearlenone. The scientists noted that
the methanolic leaf extract has strong anti-Candida albi-
cans and anti-Streptococcus pyogenes qualities [77].
LC-MS offers exceptional advantages in phytochemical
analysis, combining the high-resolution efficiency of liquid
chromatography with sensitivity and structural elucidation
potential of mass spectrometry. Such technique enables the
identification of a wide range of phytochemicals in complex plant matrices with sensitive detection with specificity, making it indispensable for comprehensive profiling