Journal of AOAC INTERNATIONAL, 104(4), 2021, 1167–1180

doi: 10.1093/jaoacint/qsab013
Advance Access Publication Date: 30 January 2021
Article

NATURAL PRODUCTS

Quantification of Scopoletin from the Roots of Argyreia

Speciosa (Linn. F) Sweet Using HPLC Through the

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Concept of Design of Experiment
Preksha Patel,1 Manan Raval, 2,
* Nidhi Patel,1 Samir Patel, 1
Niraj Vyas, 2

and Amit Patel3

1
Department of Pharmaceutical Chemistry and Analysis, Ramanbhai Patel College of Pharmacy, Charotar
University of Science and Technology, CHARUSAT Campus, Changa, 388421, Anand, Gujarat, India,
2
Department of Pharmacognosy, Ramanbhai Patel College of Pharmacy, Charotar University of Science and
Technology, CHARUSAT Campus, Changa, 388421, Anand, Gujarat, India, 3Department of Pharmaceutics and
Pharmaceutical Technology, Ramanbhai Patel College of Pharmacy, Charotar University of Science and
Technology, CHARUSAT Campus, Changa, 388421, Anand, Gujarat, India

*Corresponding author’s e-mail: [email protected]

Abstract
Background: The roots of Argyreia speciosa (Linn. F) Sweet (family: Convolvulaceae) are used in Ayurveda to treat male
reproductive and nervous system disorders.
Objective: Isolation of scopoletin from the roots of Argyreia speciosa, and development and validation of an analytical method
using HPLC for the quantification of scopoletin from the root powder of Argyreia speciosa.
Method: Scopoletin was isolated from chloroform fraction prepared from hydrolyzed methanolic extract and identified using
spectral studies. A reverse-phase HPLC-based analytical method was developed and optimized using the Design of Experiment
(DoE) approach to estimate scopoletin from the roots of Argyreia speciosa. Scopoletin was separated and quantified using HPLC
containing the C18 column and a PDA detector. The optimized mobile phase was methanol: water (pH3.2) [25: 75, %v/v].
Results: The Box–Behnken design was used to optimize chromatographic parameters and the extraction procedure. The
validation studies showed a linear relationship (r2¼0.998) in the range of 1–40 mg/mL. The LOD and LOQ were found to be
0.28 mg/mL and 0.84 mg/mL, respectively, and the recovery values were found to be between 91.94 and 97.86%. The developed
analytical method was found to be robust as well. The amount of scopoletin was estimated to be 0.024 6 0.0016%w/w from
dried root powder.
Conclusion: The recorded chromatogram and amount of scopoletin determined would serve as one of the standardization
parameters to access the quality of raw material containing Argyreia speciosa.
Highlights: Developed analytical method may be adopted as a part of the standardization procedure for Argyreia speciosa in
the quality control laboratory.

Argyreia speciosa (Linn. F) Sweet (family: Convolvulaceae) (A. spe- debilities, nervous system diseases, chronic ulcers, etc. (1).
ciosa) is indicated in Ayurveda and found all over India. It is tra- Studies have shown that methanol extract from different
ditionally used in the treatment of male reproductive function parts of A. speciosa has antibacterial, anti-cancer, anti-diarrheal,

Received: 29 September 2020; Revised: 15 November 2020; Accepted: 6 January 2021

C AOAC INTERNATIONAL 2021. All rights reserved. For permissions, please email: [email protected]
V

1167
 1168 | Patel et al.: Journal of AOAC INTERNATIONAL Vol. 104, No. 4, 2021

anti-inflammatory, and nootropic activity (2, 3). The methanol Experimental

extract of A. speciosa showed the possible aphrodisiac potential
Collection of Plant Material, Authentication, and
in rats (4) by up-regulating the synthesis of testosterone in
Leydig cells (5). The fraction of A. speciosa containing flavonoids
Processing
showed anti-stress activity (6); moreover, flavonoid glycosides The whole plant of A. speciosa, including roots, were collected
of A. speciosa exhibited antimicrobial activity against M. tuberculi from nearby tribal areas (Coordinates 22.5823659,
(7). Phytochemical analysis of seed powder has shown the pres- 72.7207995,19.25z). The plant material was identified by Dr. A.M.
ence of trace quantities of ergoline alkaloids (8). Furthermore, Patel Taxonomist, J. and J. Science College, Nadiad, Gujarat, and
N-methyl ergometrine was isolated from the alkaloidal fraction issued an authentication certificate (Voucher Specimen No.
prepared from A. speciosa (5). Scopoletin and other flavonoids 2011/NV/AS). The roots were separated, cleaned, and washed
were reported in the roots of A. speciosa (9, 10); in addition to with water to remove the adhering sand and other earthy
this, the plant was also reported to contain carbohydrates, tan-
particles. The roots were dried under shade for fifteen days, cut
nins, resin, sterol, and saponins (11).
into small pieces, and powdered using a laboratory grinder.
Scopoletin is a coumarin derivative which is classified as a
The coarse root powder was stored in a dry and dark place in

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phenyl propanoid. It is often present in the form of glycoside
an airtight glass container at room temperature until
(7, 9, 11). The International Union of Pure and Applied
further processing.
Chemistry (IUPAC) name of scopoletin is 7-Hydroxy-6-methoxy-
2H-1-benzopyran-2-one (12). Scopoletin contains a benzo-
pyrone (chromane) ring structure. The presence of benzene ring Chemicals
fused with pyran ring and pyran ring substituted at C2 position Scopoletin was procured from Sigma Aldrich (Purity > 95%) and
with a keto group made coumarin fluorescent in UV light. was used without any further purification. All solvents used for
Scopoletin was reported to be estimated from Convolvulus spp. extraction and separation purposes were of AR grade and used
(13), a herbal drug product containing asiaticoside along with after distilling using a mercury bulb at the respective boiling
scopoletin (14), hydro alcoholic extract of oak wood and wine point. Methanol, water, and acetonitrile used for analytical pur-
(15), extract prepared from Aegle marmelos (16), and bamboo poses were of HPLC grade. All solvents and chemicals were pro-
leaves (17) using HPLC coupled with a UV detector. There have cured from Loba Chemie, Mumbai, India. All the extractions and
been reports for the estimation of scopoletin from plasma using separations were carried out under diffused light and the
HPLC (18, 19). Scopoletin was also estimated from tobacco sample and standard solutions were stored in amber-colored
smoke using HPLC (20) and from a mixture of polyphenols from volumetric flasks.
tobacco extract using HPLC coupled with an ESI–MS detector
(21). Scopoletin was estimated using one-dimensional (22) and
Instruments
two-dimensional TLC-based analytical methods (23). Scopoletin
was also estimated using a spectrofluorometric method from The HPTLC system containing Camag linomat V, Camag TLC
medicinal plant extracts (24) as well as using capillary zone Scanner IV, Camag UV cabinet with dual-wavelength lamp,
electrophoresis from the plant extract of Saussurea superba (25). Hamilton 100 mL syringe, and Camag Win CATS 1.4.7 Software
The literature review revealed that scopoletin was present in were employed to assess the purity of isolated scopoletin. The
the roots of A. speciosa (6) and the extracts prepared from the HPLC system equipped with a PDA detector (LC 2010-HT,
roots of A. speciosa containing scopoletin as well as scopoletin Shimadzu, Kyoto, Japan), system controller, column oven, and
alone showed biological potential (9, 10). It suggested that Rheodyne manual injector (20 mL) was used for analytical stud-
scopoletin might serve as a biological marker for the standardi- ies. LC solution software was used to control the HPLC, record,
zation of the raw material containing the roots of A. speciosa. and integrate the chromatogram. The chromatographic separa-
Recently, we reported an analytical method for the estimation tion was achieved using Phenomenex HypercloneTM
of scopoletin from the dried root powder of A. speciosa using (250  4.6 mm, 5 mm) C18 column. A pH meter (Mettler Toledo,
HPTLC. Being an open chrome system, it might have limited the Switzerland) was used to adjust the pH of the mobile phase. A
potential to be adopted for routine quality control process (26). rotary vacuum evaporator (Heidolph, Germany) was used for
The HPLC-based analytical method was reported for estimation evaporation of the solvent, and a Soxhlet extractor was used for
of scopoletin from hydro alcoholic extract prepared from the the extraction.
roots of A. speciosa. The extract was then screened for potential
neuroprotective activity (27). The method employed for estima-
Extraction of Plant Powder and Preparation of Fraction
tion of scopoletin from freeze-dried hydro alcoholic extract was
for Column Chromatography
applicable to estimate only the non-glycosidic form of scopole-
tin, which limits the adoptability of the method as part of the An accurately weighed quantity (500 g) of dried plant powder
standardization process of the plant material. It was, thus, was initially extracted using petroleum ether (60–80 C)
thought to isolate scopoletin from the roots of A. speciosa and (1200 mL) for 3 h. The powder was dried and finally extracted
develop an analytical method for estimation of total scopoletin with methanol (1200 mL) for 12 h using a soxhlet extractor at the
content from the plant material, which would be accurate, pre- boiling point of methanol. The extract was collected, filtered,
cise, selective, and sensitive to be adopted for routine quality and concentrated using a rotary vacuum evaporator at 40 C.
control purpose. Though Quality by Design has been an integral The concentrated extract was evaporated to dryness on a hot
part of optimizing different processes as well as optimizing the water bath and preserved in an airtight glass container in a des-
pharmaceutical formulation formula, it is not used routinely in iccator. The dried methanolic extract (35 g) was added to 250 mL
the field of analytical chemistry, an attempt was made here to of 1% v/v hydrochloric acid in a conical flask. The flask was
implement the Design of Experiment (DoE) in optimizing the tightly capped and placed in a shaking water bath for 60 min.
extraction process and optimizing the chromatographic param- The temperature of water in the bath was set at 70 C. The oscil-
eters of an analytical method (28, 29). lating movement of a flask in the shaker was set at 25 RPM. The
 Patel et al.: Journal of AOAC INTERNATIONAL Vol. 104, No. 4, 2021 | 1169

content was cooled at room temperature and partitioned with Estimation of Scopoletin from Methanolic Extract of
100 mL chloroform using a 500 mL capacity separating funnel. A. speciosa
This procedure was repeated five times. The content was
shaken vigorously for 10 min and kept aside for phase (a) Optimizing chromatographic conditions for the HPLC method
Few pre-experiments were carried out with the help of the sam-
separation. The chloroform layers were collected and mixed.
ple solution (chloroform fraction (accurately weighed about
The collected chloroform was passed through anhydrous so-
20 mg) diluted using methanol (10 mL)) to resolve the peak cor-
dium sulfate and evaporated to dryness using a rotary vacuum
responding to scopoletin using HPLC. The optimization of chro-
evaporator at 40 C (30).
matographic parameters was carried out by adopting the Box–
Behnken design (31). Percentage of methanol (% v/v) in mobile
Column Chromatographic Separation phase (A ¼ 20%, 25%, and 30%), pH of mobile phase (B ¼ 3.0, 3.2,
and 3.4), temperature (C ¼ 27, 30, and 33 C), and flow rate (FR)
Scopoletin was separated from chloroform enriched fraction
(D ¼ 1.0, 1.2, and 1.4 mL/min) were selected as independent vari-
using column chromatography. The silica gel (150 g) for column
ables to be optimized. The selection of the variable was based

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chromatography (60–120 #) was poured in the glass column af-
on observations noted during pre-experiments. Area, retention
ter preparing a slurry in chloroform. The column was allowed to
time (RT), tailing factor (TF), and resolution of the peak corre-
settle overnight. The hydrolyzed fraction (7 g) was dissolved in
sponding to scopoletin were selected as dependent variables.
methanol, mixed intimately with a minimum amount of silica, R
Design ExpertV software was used to create a randomly distrib-
and dried. The fraction mixed with silica was then loaded onto
uted series of experiments. Two-way ANOVA was performed
a column. The column was eluted initially with chloroform,
using Design Expert to evolve the level of significance. The com-
followed by a gradual increase in the amount of methanol
bined effect of selected factors on responses was determined
(0.5–7% v/v). The fractions (20 mL) were collected in test tubes.
and graphically represented as a perturbation graph as well as
All the fractions were subjected to TLC studies using optimized
an overlay graph. The fitness of the model was ensured using
chromatographic conditions. Few consecutive fractions, con-
statistical analysis, and the results are summarized and tabu-
taining 5% methanol, showed a single blue-colored spot in long-
lated. The validation of the developed statistical model was car-
wave UV light. These fractions were collected, concentrated,
ried out by comparing the values of responses obtained from
and mixed. The purity of the isolated constituent was assessed
the overlay graph with the values obtained after performing the
using TLC. The chromatographic parameters were optimized to
experiments under similar conditions.
separate the spot of scopoletin from nearby spots in the sample
track. The stationary phase was pre-coated TLC sheets
R (b) Optimization of extraction conditions
ALUGRAMV Xtra SIL G/UV254, and the mobile phase was petro-
The Box–Behnken design was also applied to evolve optimized
leum ether: ethyl acetate: formic acid (7:6:0.3, v/v/v). These opti-
extraction conditions. The methanolic extract was hydrolyzed
mized chromatographic conditions were used to assess the
through heating the extract with aqueous hydrochloric acid.
purity of isolated constituent.
The selected independent variables, thus, were heating time
(A ¼ 10, 30, and 50 min), heating temperature (B ¼ 50, 70, and
90 C), and concentration of hydrochloric acid (C ¼ 3%, 5%, and
Spectroscopic Identification of Scopoletin
7% v/v) (31). The dependent variable was the percentage
(a) IR spectroscopy amount of scopoletin in hydrolyzed fraction. The data was
IR spectrum was recorded within the range of 400 cm1 to inserted in Design Expert and yielded 17 runs. The experiments
4000 cm1 using an IR spectrophotometer (Nicolet 6700, Thermo were performed by adopting the altered value of variables, and
Scientific, Waltham, MA). A small amount of compound was the sample solution was prepared each time by following the
ground with dried potassium bromide (IR grade), and the trans- procedure mentioned under the heading preparation of the
lucent disk was prepared using a hydraulic press. The IR spec- sample solution. This solution was injected in HPLC, and the
trum of scopoletin was recorded, and major IR stretches were amount of scopoletin was determined. Each experiment was
identified, inferred, and compared with the stretches observed performed thrice to get the mean value of the response. The
in the reported IR spectrum of scopoletin (30). results were analyzed to determine the statistical significance
of the model as well as to get a perturbation chart, which was
used to analyze the effect of variables on the amount of scopo-
(b) 1H NMR spectroscopy
letin. The contour plot and overlay plot were created to opti-
NMR spectroscopy was performed using 400 MHz FT-NMR
mize the experimental conditions as well to get design space.
Avance III (Bruker, Switzerland) instrument equipped with
The design was validated by performing a few experiments by
Topspin 2.1 Software. The sample solution was prepared in deu-
selecting the set of variables from the design space and the
terated methanol. Recorded 1H NMR was compared with the
amount of scopoletin was determined each time.
reported 1H NMR spectrum of scopoletin (30).

(c) Preparation of standard solutions

(c) MASS spectrometry Scopoletin (100 mg) was accurately weighed and dissolved in
The mass spectrometry was performed using Shimadzu 5 mL methanol using sonication. The final volume was made up
LC-MS-MS-2020 with APCI & ESI Probe, Shimadzu equipped to 10 mL using methanol (10,000 mg/mL). This preparation would
with LC/MS solution software. The isolated constituent (1 mg) serve as a reference standard solution. The reference standard
was dissolved in methanol (10 mL) and subjected to mass solution was further diluted using methanol to get the working-
spectrometry. standard solution (1000 mg/mL).
 1170 | Patel et al.: Journal of AOAC INTERNATIONAL Vol. 104, No. 4, 2021

(d) Preparation of serial dilutions (b) Linearity

Various aliquots were taken from working standard solution Different replicates were prepared from a standard solution in a
and diluted to prepare a series of dilutions containing scopole- range of 1–40 mg/mL. The peak of scopoletin covered by a peak
tin concentration in the range of 1–40 mg/mL. corresponding to each concentration was noted. The calibration
curve was established by plotting the peak area versus concen-
tration graph, and the regression equation was calculated by
(e) Preparation of sample solution
adopting the least square method and the linearity was estab-
Accurately weighed quantity of A. speciosa root powder (100 g)
lished by determining the regression coefficient. The experi-
was extracted in the soxhlet apparatus. The powder was defat-
ments were repeated six times. The average value of correlation
ted by extracting it with 500 mL petroleum ether (60–80 C) for
coefficient (r2), y-intercept, and slope were determined along
3 h. The resulting marc was dried and further extracted using
with standard deviation.
500 mL methanol up to 8 h. This methanolic extract was filtered
and concentrated using a rotary vacuum evaporator at 40 C.
(c) LOD and LOQ
The concentrated extract was evaporated to dryness using a
LOD and LOQ for scopoletin were determined initially from the
water bath and preserved in a vacuum desiccator. Accurately

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line equation, by applying the formula given below:
weighed quantity of methanolic extract (1000 mg) was taken in
a 20 mL aqueous solution of hydrochloric acid (5% V/V). The LOD ¼ 3:3 r=S
mixture was heated at 70 C for 30 min using a rotary shaking
water bath. The shaking speed for the flask was set at 25 RPM.
LOQ ¼ 10 r=S
The content was cooled at room temperature after hydrolysis.
The mixture was subjected to partition using a separating fun-
where r ¼ Standard deviation of the y-intercept and S ¼ slope
nel with 20 mL chloroform. The funnel containing the mixture
of the calibration curve (m).
was shaken vigorously for 10 min. It was then kept aside for
The LOD and LOQ values were calculated from the equation
phase separation. The same procedure was repeated three
and further ensured by recording chromatogram and determin-
times. Each time, the chloroform layer was collected and
ing S/N value in the chromatogram. S/N values for establishing
washed with the water. Collected chloroform was passed
LOD and LOQ were selected 3:1 and 10:1, respectively.
through anhydrous sodium sulfate and evaporated to dryness
using a water bath. An accurately weighed quantity (100 mg) of (d) Precision
chloroform fraction was dissolved in methanol (5 mL) using a The precision of the developed analytical method was deter-
bath sonicator, and the final volume was made using methanol mined by measuring inter-day and intra-day variation in the
(10 mL). The mixture was filtered using a syringe filter (0.45 mm, area of a peak corresponding to scopoletin, for selected three
PTFE). The filtered solution was injected in HPLC, and the concentrations (5.1, 10.2, and 20.4 mg/mL). Intra-day variation
amount of scopoletin was determined. was determined by recording peak area of scopoletin, six times
in a day, while the same was determined for six days, once a
day to decide inter-day precision. The data set obtained for each
System Suitability
concentration was subjected to statistical treatment to deter-
The area of peak, number of plates, resolution, and tailing factor mine RSD, %.
of the peak corresponding to scopoletin were selected as system
suitability parameters. These values were determined from the (e) Accuracy
chromatogram of the sample solution. The experiments were Accuracy studies were performed by the standard addition
repeated six times, and the mean value for each parameter method. A known amount of scopoletin was spiked in the pre-
along with standard deviation is tabulated. analyzed sample solution. The spiked and un-spiked sample
solutions were analyzed for the content of scopoletin. The
experiments were carried out at three different levels of spiking.
Validation of the Analytical Method
Each experiment was repeated thrice. The mean value of
The developed HPLC method was validated using ICH Q2 (R1) percentage recovery was determined.
Guideline (32). The results of the studies are summarized and
tabulated. (f) Robustness
Robustness of the developed analytical method was evaluated
(a) Selectivity by assessing the effect of small changes in critical chromato-
The selectivity of the analytical method was ensured by mea- graphic parameters such as FR, mobile phase composition, tem-
perature, and pH on peak area and retention time. Each
suring the peak purity of the analyte using the PDA detector.
experiment was performed thrice, and the data sets were sub-
The chromatogram of the sample solution was recorded using
jected to F-test. The null hypothesis considered as “the mean
the optimized chromatographic parameters. The peak corre-
value for the individual data set was statistically not
sponding to scopoletin was analyzed for peak purity parame-
different from each other.”
ters. Peak purity was evaluated using software by comparing
the threshold value calculated from the noise component. The
purity index, for the peak of scopoletin, greater than or equal to Estimation of Scopoletin from Roots of A. speciosa
0.95, was considered as an acceptance criterion. The peak Scopoletin was estimated from chloroform fraction prepared by
purity index was determined after the auto integration of the adopting optimized extraction conditions from hydrolyzed
scopoletin peak with a total peak purity method. The reference methanolic extract prepared from dried root powder of A. spe-
wavelength range was 380 nm to 220 nm. ciosa by adopting external standardization method. The sample
 Patel et al.: Journal of AOAC INTERNATIONAL Vol. 104, No. 4, 2021 | 1171

solution was injected in HPLC and chromatogram was recorded showed a single spot. Figure 2 suggested the absence of constit-
by following optimized chromatographic conditions. The uents other than the isolated constituent. These fractions were
chromatogram recorded after injecting the standard solution of finally mixed and evaporated to dryness which yielded 27 mg of
scopoletin (40 mg/mL) and the sample solution (10 mg/mL chlo- the isolated constituent.
roform fraction) is presented for comparison. Peak correspond-
ing to scopoletin was integrated and the area covered by the Spectroscopic Studies
peak was recorded. The amount of scopoletin was calculated by
inserting the peak area in a line equation to get the concentra- Wavenumber corresponding to various stretches from recorded
tion of scopoletin in the injected sample solution, which will IR spectra of the isolated compound, as shown in Figure 3, and
further yield the amount of scopoletin present in dried plant those were from reported IR spectra of scopoletin are summa-
material. Five independent experiments were performed and rized in Table 1. The stretch corresponding to the hydroxyl
the mean value for the percentage of scopoletin present on a group in recorded spectra was not sharp as well as appeared at
dried weight basis of plant material was reported. wavenumber lesser than reported for a hydroxyl group. It sug-
gests that the hydroxyl groups might be hydrogen-bonded in
the constituent. Stretch appeared at 3025 cm1 as well, as over-

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Results and Discussion tones observed between 2000–1600 cm1 confirmed the pres-
Scopoletin is a benzo-pyrone derivative (lactones of o-hydroxy ence of an aromatic ring in the structure. Stretch observed at
cinnamic acid). All plant coumarin, including scopoletin, has 2945 cm1 (Sp3 C-H stretch) suggested the presence of the
hydroxyl substitution at position 6, as shown in Figure 1. methyl group. Carbonyl bond in C ¼ C-C ¼ O was reported to be
Scopoletin is often present in the form of glycoside, where the less enone in nature and so weaker, which yielded a stretch at
aglycone part is soluble in chloroform, ethyl acetate, and meth- 1704 cm1. A profound stretch observed at 1219 cm1 might be
anol but insoluble in water (30). Coumarin glycosides undergo attributed to asymmetric C(alk)-O-C(Ary), while a stretch at
acid hydrolysis to yield aglycone, thus, the methanolic extract 1018 cm1 was attributed to symmetric C(alk)-O-C(Ary) (33). This
was fractionated by hydrolyzing the extract to convert the gly- confirmed presence of aryl alkyl ether in structure. Comparative
cosides to scopoletin (an aglycone) from a possible glycosidic IR spectral data of isolated constituent and scopoletin, thus, in-
structure. The hydrolyzed methanolic extract was then parti- dicated that the isolated constituent might possess the back-
tioned with chloroform to separate scopoletin from an aqueous bone structure of coumarin.
acidic portion and the chloroform layer was washed with dis- 1
H- NMR spectra of the isolated constituent, as shown in
tilled water to remove any remaining traces of acid. As a non- Figure 4, indicated hydrogen might present in six different elec-
glycosidic compound, scopoletin was separated using a polar tronic environments. Summary of the chemical shift along with
stationary phase composed of silica gel using chloroform with
the integration of the peak for 1H- NMR spectrum of the isolated
an increasing proportion of methanol as eluent while the
constituent is shown in Table 2 along with those reported for
amount of methanol in the mobile phase was increased only
the 1H- NMR spectrum of scopoletin for comparison purposes. A
when elute was devoid of any constituent. All the fractions
singlet at peak d ¼ 3.81 ppm was attributed to the methoxy
were collected and subjected to TLC analysis, moreover the frac-
group attached to C5. Two doublets were observed at d ¼ 6.12 ppm
tion number 98–123 containing 5% v/v methanol in chloroform
and d ¼ 7.7 ppm. These signals, typical of hydrogen attached to
vinyl carbon, were assigned to hydrogen attached to C2 and C3.
Hydrogen on C3, being attached to the carbon of aromatic ring,
was de-shielded as compared to hydrogen on C2 and showed a
downfield signal. The inference derived from the spectral inter-
pretation (34) and the comparison of recorded NMR with reported
NMR spectra (30) suggested that the isolated compound might be
scopoletin.
Mass spectra of the isolated constituent were recorded
using positive ion mode at 45 eV. The recorded mass spectra,
Figure 1. Chemical structure of scopoletin. as shown in Figure 5, showed a molecular ion peak (Mþ) at 192
(m/z ¼ 192) indicated the molecular ion peak for scopoletin
(C10H8O4). The peak at 177 (m/z ¼ 177) was attributed to
Mþ-(-CH3). A peak was seen at 149 (m/z ¼ 149) and might be
attributed to the removal of methoxy and a hydroxyl group
Mþ-(-OCH3 - OH). Benzopyran (C9H4O) showed a peak at 126
(m/z ¼ 126) (30). The position of molecular ion peak, fragmenta-
tion pattern, as well as comparative studies of IR and NMR spec-
tra, ensured that the isolated constituent was scopoletin.

Optimization of Chromatographic Parameters

An analytical method was developed to standardize the dried
root powder of A. speciosa with reference to scopoletin content,
a principal constituent of A. speciosa, as a marker for standardi-
Figure 2. TLC-based purity assessment of scopoletin, isolated using column
zation. As the fraction contained many constituents apart from
chromatography. The isolated scopoletin was detected in fluorescence mode
(long-wave UV) in UV cabinet and scopoletin appeared as a single spot in the scopoletin, it was challenging to develop the RP-HPLC method,
track. which could resolve the peak of scopoletin from nearby peaks.
 1172 | Patel et al.: Journal of AOAC INTERNATIONAL Vol. 104, No. 4, 2021

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Figure 3. IR spectrum recorded for isolated scopoletin.

Table 1. Summary of different stretches of IR spectra and inference

Peaks Functional group Peaks Functional group

Sr. No. Reported spectra [30] Recorded spectra

-1 a -1 a
1 3480 cm OH 3336.80 cm OH
2 – – 3025,3010cm-1 a Cyclic vinyl C-H
3 – – 2945, 2850 cm-1 a Methyl C-H
4 1715 cm-1 a C¼O 1704 cm-1 a C¼O
5 1610 cm-1 a C¼C 1608.05 cm-1 a C¼C
6 1540 cm-1 a , 1403 cm-1 a Aromatic 1511.36, 1432.54 cm-1 b Aromatic
7 – – 1219 cm-1 b C(alk) – O–C(ary) (assy)
8 – – 1018 cm-1 b C(alk) – O–C(ary) (sym)

a
s ¼ small
b
m ¼ medium

The methanolic extract was subjected to acid hydrolysis to get as the basis of the selection of independent variables for DoE,
sample solution containing total scopoletin. which might constitute 34 full factorial design having 81 experi-
The HPLC method was developed using a trial-and-error ap- mental runs. As Box–Behnken design yielded a fewer number of
proach to yield well resolved, sharp, and Gaussian peak corre- experiments, it was selected to evolve design space, which
sponding to scopoletin, from a solution prepared from yielded 29 runs, as shown in Table 3, along with the value of the
hydrolyzed fraction. Initial trials suggested that the methanol– selected dependent variable for each run. As shown in Table 4,
water mixture yielded better peak shape as compared to the when two-way ANOVA was applied to determine the level of
acetonitrile–water mixture, furthermore the temperature of the significance for the model, yielded p-value less than 0.05 sug-
column oven affected the peak shape. The wavelength was se- gested that the model was significant statistically and, the pre-
lected based on the UV spectrum of the peak and the mobile dicted R2 value for most of the factors was in reasonable
phase having a pH of more than four could not resolve the peak agreement of adjusted R2 value. Signal to noise ratio was repre-
of scopoletin from nearby peaks. These observations remained sented by determining Adeq precision and the value of Adeq
 Patel et al.: Journal of AOAC INTERNATIONAL Vol. 104, No. 4, 2021 | 1173

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Figure 4. 1H-NMR spectrum recorded at 400 MHz for isolated scopoletin.

Table 2. Comparative summary of 1H -NMR spectra with inference

Values derived from reported 1H- NMR spectra of scopoletin [30] Values derived from reported 1H- NMR spectra

Sr. No. d, ppm Integration and assignment d, ppm Integration and assignment

1 3.96 3H,a C5-O- CH3 3.81 3H,a C5-O-CH3

2 6.28 1H,b C2H 6.12 1H,b C2H
3 6.86 1H,a C7H 6.68 1H, a C7H
4 6.92 1H,a C4H 7.02 1H,a C4H

a
s ¼ singlet
b
d ¼ doublet

precision was more than four in all the cases suggested that the the column oven. The perturbation graph, as shown in
model could be used to navigate design space. Predicted Figure 6A suggested that amongst the selected factors, FR (D)
Residual Error Sum of Squares Statistics (PRESS) is the measure had a notable impact on the area of the peak. It was also ob-
of the discrepancy between the data and an estimated model, served that except pH, all selected variables had a significant ef-
thus, PRESS is used to cross-validate the regression analysis. fect on RT. The perturbation chart shown in Figure 6B suggested
The optimal PRESS value for the dependent variables suggested that the amount of methanol in the mobile phase had maxi-
the appropriate selection of the model. The analysis of the mum impact on RT among all the selected factors. Initial obser-
responses suggested that the model was significant (P < 0.005) vations suggested that the temperature of the column oven
for the responses (31). The detailed statistical analysis sug- affected the shape and TF of the peak. The perturbation graph,
gested that the area corresponding to the scopoletin peak was as shown in Figure 6C, suggested that methanol proportion in
not affected significantly by incorporating alteration in the the mobile phase, the temperature of the column oven, and FR
amount of methanol in the mobile phase, pH, or temperature of affected the TF significantly. Plant extract and fractions form a
 1174 | Patel et al.: Journal of AOAC INTERNATIONAL Vol. 104, No. 4, 2021

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Figure 5. Mass spectrum recorded using isolated scopoletin.

Table 3. Studies performed to optimize chromatographic parametersa


Sr. No. % v/v methanol pH Temp, C Flow rate, mL/min Area RT , min Tailing factor Resolution

1 25 3.2 30 1.2 261925.7 14.92 1.24 3.87

2 20 3.2 30 1 335937 27.3 1.13 2.06
3 30 3.0 30 1.2 285804 9.17 1.061 3.13
4 25 3.2 33 1.4 250163.3 11.17 1.216 3.805
5 30 3.2 30 1.4 243812.3 7.958 1.131 3.907
6 20 3.2 30 1.4 292445 18.996 1.105 2.205
7 20 3.2 27 1.2 285252 25.39 1.349 1.931
8 20 3.2 33 1.2 292520 21.326 1.157 1.915
9 25 3.4 30 1.4 244670 12.026 1.247 4.867
10 25 3.0 33 1.2 280537 13.163 1.135 3.087
11 20 3.4 30 1.2 284404 23.177 1.225 2.116
12 25 3.0 27 1.2 290591 15.002 1.179 3.341
13 25 3.2 30 1.2 266707 14.035 1.281 3.018
14 25 3.4 33 1.2 272667 13.233 1.159 2.865
15 25 3.2 27 1.4 243341 12.93 1.351 4.859
16 25 3.0 30 1.4 250383 11.998 1.265 2.785
17 25 3.2 30 1.2 262844 14.037 1.301 3.28
18 25 3.2 30 1.2 260166 14.061 1.408 2.99
19 30 3.2 33 1.2 294296 8.706 1.06 3.019
20 25 3.2 27 1 357791 18.043 1.126 4.723
21 30 3.2 30 1 341526 11.013 1.043 5.239
22 20 3.0 30 1.2 502596 23.12 1.16 2.036
23 30 3.2 27 1.2 279454 9.832 1.11 3.508
24 25 3.2 30 1.2 260386 14.05 1.281 3.400
25 25 3.4 27 1.2 290777 15.169 1.146 5.204
26 30 3.4 30 1.2 289637 9.223 1.075 3.329
27 25 3.0 30 1 364962 16.691 1.134 3.222
28 25 3.4 30 1 349338 16.98 1.105 4.258
29 25 3.2 33 1 382247 15.633 1.049 4.304

(n ¼ 3) using sample solution.

Table 4. Selection of experimental model to optimize chromatographic parameters

Sr. No Dependent variable P-value for model R2 Adjusted R2 Adeq. precision PRESS Suggested model

1 Area 0.0023 0.4873 0.4018 8.168 65.03 Linear

2 RT <0.0001 0.9978 0.9956 78.971 6.45 Quadratic
3 TF 0.011 0.7835 0.5670 6.811 0.27 Quadratic
4 Resolution 0.0008 0.8595 0.7189 9.440 19.97 Quadratic
 Patel et al.: Journal of AOAC INTERNATIONAL Vol. 104, No. 4, 2021 | 1175

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Figure 6. Perturbation plots showing the relative effect of selected independent variable on dependent variables while optimizing the chromatographic parameters.

complex matrix while considering for chemical analysis. The to peak corresponding to scopoletin. The statistical analysis
sample solution showed a positive reaction for flavonoids and revealed that all selected factors, except the FR, affected the res-
phenolics, which might be attributed to the presence of phenyl olution of the peak, which is seen in the perturbation chart
propanoids. The fraction also contained alkaloids and steroids, shown in Figure 6D. The contour plot, as shown in Figure 7A,
including other terpenoids (35). Resolving scopoletin in the shows the effect of the proportion of methanol in the mobile
presence of other secondary metabolites was a challenging task phase and pH on the resolution of the peak corresponding to
and the most demanding feature of the proposed analytical scopoletin, when temperature and FR was kept constant. The
method. The range of the independent variables was selected to plot suggested that obtaining the value of resolution of the peak
have minimal effect on peak shape and resolution. The studies corresponding to scopoletin more than 2.0, the proportion of
showed that all selected independent variables had an impact methanol in the mobile phase would be more than 22% v/v, and
on the resolving ability of the analytical method with reference the pH of the mobile phase should be more than 3.0. The results
 1176 | Patel et al.: Journal of AOAC INTERNATIONAL Vol. 104, No. 4, 2021

obtained were subjected to graphical optimization. The value of using a laboratory bath sonicator. The FR was set at 1.2 mL/min,
independent variables was inserted and with the help of the with an injection volume of 20 mL. The chromatogram was
Design Expert an overlay graph was constructed, as shown in recorded at 345 nm, and the temperature of column oven was
Figure 7B. The overlay graph shows the area of possible re- set at 30˚C.
sponse values in the factor space. The level of significance was Statistical analysis of the results of the experiments, per-
selected as 0.05. The golden yellow region in Figure 7B shows formed to optimize the experimental variables involved in the
that the range of all intervals meets the specified criteria of sta- sample solution preparation, suggested that the model selected
tistical significance. Three random points were flagged from the for optimizing the variable was statistically significant
yellow area of the overlay graph, which yielded the value of in- (P ¼ 0.004). The variables had a statistically significant effect on
dependent variables as well as the determined value of depen- the selected response. The difference between adjusted R2 and
dent variables (P< 0.05). The chromatogram was recorded using predicted R2 was 0.4, with a good precision value of 8.89. PRESS
the same variables and the average value of responses were was determined to be 0.0001. The results of the statistical analy-
obtained. The deviation in each response was measured be- sis suggested that the adopted model was adequate, and design
tween the determined value and practically achieved value, as space could be navigated from the model. The perturbation

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shown in Table 5. The percentage difference was found within graph suggested that heating time had the maximum effect,
the range of ten percentage points, suggesting the validity of while heating temperature and concentration of acid had an al-
the model in optimizing and selecting the chromatographic most similar effect on the amount of scopoletin. Though the
conditions. Graphical responses and actual responses were crit- melting point of scopoletin was reported to be 204–206 C (36),
ically analyzed to finalize the chromatographic conditions as heating in the presence of hydrochloric acid might break glyco-
mentioned below. sidic linkage and liberated free scopoletin. This resulted in vari-
The optimized mobile phase, thus, consisted of two solvents, ation in the results corresponding to each run, as shown in
Solvent A comprised of water (pH was adjusted to 3.2 with the Table 6. The effect of factors is graphically depicted through
help of formic acid) and Solvent B, which was methanol. The ra- contour graph, as shown in Figure 8A. The contour graph
tio of solvent A and Solvent B was 75: 25, %v/v. The solvents suggested that, when the concentration of hydrochloric acid
were premixed, filtered using a 0.45 mm membrane filter, and was fixed at 5% v/v, the maximum amount of scopoletin was
kept aside for 30 min. The solvent mixture was then degassed determined in the experiment where the content was heated

Figure 7. Counter view and overlay plot for optimization of chromatographic parameters.

Table 5. Assessing the fitness of the suggested model in optimizing chromatographic conditions

Value of independent variable Difference between predicted and experimentally obtained value for dependent variables, %

Sr. No. Methanol, % v/v pH Area RT TF Resolution

1 25 3.3 8.17 8.84 8.59 6.78

2 28 3.1 9.61 8.96 6.92 9.39
3 26 3.1 5.62 9.54 2.38 8.36


(n ¼ 3) temperature ¼30 C, flow rate ¼ 1.2 mL/min, using sample solution (2.0 mg/mL)
 Patel et al.: Journal of AOAC INTERNATIONAL Vol. 104, No. 4, 2021 | 1177

Table 6. Results of the studies performed to optimize sample preparation parametersa

Sr. No. Time, min Temperature,  C % Hydrochloric acid, % v/v % Scopoletin

1 50 70 3 0.024
2 50 90 5 0.018
3 10 70 3 0.012
4 30 70 5 0.023
5 50 50 5 0.02
6 30 50 3 0.018
7 30 70 5 0.024
8 30 70 5 0.023
9 10 50 5 0.017
10 30 90 3 0.02
11 30 70 5 0.019
12 50 70 7 0.018

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13 30 70 5 0.023
14 10 90 5 0.011
15 10 70 7 0.012
16 30 90 7 0.011
17 30 50 7 0.018

a
(n ¼ 3)

Figure 8. Counter view and overlay plot for optimizing the variable for preparing the sample solution from the plant material.

for 30 min at 60 C. The overlay graph, as shown in Figure 8B was The chromatogram which was recorded using the optimized
generated from the data set, considering the significance level chromatographic conditions for standard scopoletin and the
0.05, suggested heating the content for more than 25 min at a sample solution are shown in Figure 9A and B, respectively.
temperature of more than 60 C, would yield the maximum These figures show the Gaussian and well-resolved peak corre-
amount of scopoletin. The model was validated by performing sponding to scopoletin.
experiments by selecting the variables from the design space,
as shown in Table 7. The results ensured the validation of the
Analytical Method Validation
model for the purpose. The experimental conditions, including
heating of 0.1 g methanolic extract in 20 mL, 5% v/v hydrochloric The results of the system suitability parameters, as shown in
acid for 30 min at 70˚C was selected as optimized conditions, Table 8, would be of importance while implementing the
based on graphical determination and practical method at different laboratories. The selectivity of the analyti-
experimentations. cal method is the ability to measure an analyte accurately in the
 1178 | Patel et al.: Journal of AOAC INTERNATIONAL Vol. 104, No. 4, 2021

Table 7. Assessing fitness of the suggested model for optimizing sample solution preparation process

Value of independent variable

Predicted value of % Actual value %

Sr. No. Heating time, min Heating temperature, C scopoletin, % w/w scopoletin, %w/w % Difference

1 30 70 0.022 0.024 9.090

2 40 70 0.023 0.021 8.695
3 30 65 0.022 0.021 4.545

(n ¼ 3) (concentration of hydrochloric acid ¼ 5% v/v)

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Figure 9. HPLC chromatogram of scopoletin: (A) chromatogram of standard scopoletin (40 mg/mL) and (B) chromatogram of sample solution (10 mg/mL chloroform frac-
tion). The peak corresponding to scopoletin was resolved using C18 column, mobile phase comprised of water (pH 3.2): methanol (75: 25, % V/V), flow rate 1.2 mL/min,
temperature of column oven was 30 C and chromatogram was recorded at 345 nm using a PDA detector.

Table 8. System suitability parametersa presence of interference (32). The peak purity for the peak corre-
sponding to Scopoletin was measured using a PDA detector be-
Sr. No. Parameters Mean area 6 SD %, RSD tween 380 nm and 220 nm at all points of a peak. The peak
purity index was 0.995 when determined using software for
1 Number of plates 4896.4 6 60.71 1.24
peak corresponding to scopoletin after injecting sample solu-
2 Peak area 279582.5 6 986.50 0.35
3 Resolution 3.78 6 0.0175 0.45
tion indicated the spectral purity of the peak.
4 Tailing factor 1.09 6 0.0051 0.47 Summary of the results of validation studies, as shown in
Table 9, suggested that linearity was established from the value
a
(n ¼ 6) using sample solution (2.20 mg/mL) of the regression coefficient of a calibration curve. The calibra-
tion curve was established by plotting the graph of peak area vs.
 Patel et al.: Journal of AOAC INTERNATIONAL Vol. 104, No. 4, 2021 | 1179

Table 9. Summary of validation parameters

Sr. No. Parameters Results

1 Selectivity through peak purity analysis Peak purity more than 0.950
2 Linearity (1.02–40.8 mg/mL) Slope (m) 47924.33 6 141.41
y-intercept 7886.48 6 116.30
Regression coefficient (r2) 0.998 6 0.0008
Intraday precision (%, RSD) 0.26–0.54
Interday precision (%, RSD) 0.40–1.50
4 Sensitivity LOD 0.28 mg/mL
LOQ 0.84 mg/mL
5 Accuracy through Standard Addition Method (% recovery) 91.94–97.86%
6 Robustness (F-Test) Fexp < Fcrit, null hypothesis accepted Robust

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concentration of scopoletin. The concentration range selected root powder of A. speciosa contained 0.024 6 0.0016% w/w
for establishing the linearity was 1–40 mg/mL. The value of the scopoletin on a dried weight basis.
regression coefficient approaching one and the low value of
y-intercept confirmed the linearity of response (31) with
Conclusion
reference to a selected concentration range of scopoletin. The
values of LOD and LOQ were determined to be 0.28 mg/mL and A phenyl propanoid constituent was isolated from the roots of
0.84 mg/mL, respectively, as calculated mathematically from the plant A. speciosa and identified as scopoletin using spectral
equation. The results were then ensured by measuring the S/N studies. An analytical method was developed and validated to
ratio for the peak of scopoletin after injecting the solution of estimate scopoletin from dried root powder of A. speciosa. The
the same concentration. The value of LOD and LOQ suggested validation studies showed that the developed RP-HPLC method
that the developed method was sensitive enough to estimate was sensitive, selective, accurate, precise, robust, and yielded
the amount of scopoletin present in the sample solution. A low linear response with reference to the selected concentration
value of RSD, % value for intra-day precision and inter-day range. The amount of scopoletin was found to be
precision indicated the developed analytical method could es- 0.024 6 0.0016% w/w, which could be considered as one of the
tablish an agreement between the area of a peak correspond- standardization parameters for root powder of A. speciosa. The
ing to scopoletin and selected concentrations when subjected developed method may be utilized as a part of the standardiza-
to repeated estimation using optimized conditions. Recovery tion procedure to be adopted for the raw material of A. speciosa
studies were performed by adopting the standard addition in a quality control laboratory, in addition to this the developed
method. The average value for the recovery was found in the analytical method may serve as the basis for the development
range of 91.94–97.86%. It indicates that the developed method of new analytical method for estimation of scopoletin from
is capable of measuring the accurate amount of scopoletin in other plant species belong to the family Convolvulaceae.
the sample solution. The robustness of an analytical procedure
is a measure of its capacity to remain unaffected by deliberate Acknowledgments
changes in method parameters and indicates its reliability
The authors are thankful to Ramanbhai Patel College of
during normal usage (32). The robustness of the developed an-
Pharmacy, Charotar University of Science and Technology, for
alytical method was performed by incorporating the alteration
providing an instrumentation facility to carry out studies. We
in selected chromatographic parameters, one factor at a time
acknowledge the support of Dr. Archita Patel, Asst. Prof.,
(OFAT). The parameters were selected based on the relative
KBIPER, Gandhinagar in designing the experiments and data
impact of a variable observed while developing the analytical
analysis using Design Expert. The necessary funding was re-
method. The studies suggested that when the chosen
ceived from the PG contingency amount received from
parameters were altered within limits specified, e.g., FR
Ramanbhai Patel College of Pharmacy, CHARUSAT and
(6 0.2 mL/min), the proportion of methanol in the mobile
CHARUSAT Seed Research Grant sanctioned to Dr. Manan Raval
phase (6 3.0% v/v), temperature (6 2 C), and pH (6 0.2), the
[No. CSRG/RPCP/MAR/12].
mean value for area, and RT were not different significantly as
determined using one-way ANOVA. The results of statistical
analysis showed Fexp < Fcrit, led to accept the null hypothesis Conflict of interest
(31). It proved that the analytical method remained unaffected There is no conflict of interest to declare.
by incorporating the alteration in the selected chromato-
graphic parameters. The summary of validation studies is
shown in Table 9.
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