Saudi Pharmaceutical Journal 28 (2020) 487–494

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Saudi Pharmaceutical Journal

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Original article

Central composite design expert-supported development and validation

of HPTLC method: Relevance in quantitative evaluation of protopine in
Fumaria indica
Ajaz Ahmad a, Mohd Amir b, Abdulrahman A. Alshadidi c, Muhammad Delwar Hussain d, Anzarul Haq e,
Mohsin Kazi c,⇑
a
Department of Clinical Pharmacy, College of Pharmacy, King Saud University, Riyadh 11451, Saudi Arabia
b
Department of Natural Product & Alternative Medicines, College of Clinical Pharmacy, Imam Abdulrahman Bin Faisal University, Dammam 1982, Saudi Arabia
c
Department of Pharmaceutics, College of Pharmacy, King Saud University, Riyadh 11451, Saudi Arabia
d
Department of Pharmaceutical and Biomedical Sciences, College of Pharmacy, California Health Science University, Clovis, CA 93612, USA
e
College of Pharmacy, Prince Sattam Bin Abdulaziz University, Alkharj, Saudi Arabia

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

Article history: The current study was executed for method development, validation and to estimate the concentration of
Received 29 October 2019 protopine in methanolic extract of Fumaria indica by high-performance thin-layer chromatography
Accepted 26 February 2020 (HPTLC). Isolation of bioactive compounds was carried out using the mobile phase, toluene:ethyl acet-
Available online 4 March 2020
ate:diethyl amine (8:2.5:0.5 v/v/v), and detected at wavelength 290 nm. This method was validated for
precision, specificity, linearity, limit of detection (LOD), limit of quantification (LOQ), etc. The calibration
Keywords: range was found to be 500–5000 ng/spot for the bioactive compound. Protopine was separated with an Rf
Protopine
value of 0.22 ± 0.03. The method was validated for linearity (r2
0.996 ± 0.082), accuracy 98.75–
Fumaria indica
HPTLC
102.12%), and RSD of precision (0.49–2.07) with a calibration curve range of 500.00–5000.00 ng/spot.
Central composite design The LOD and LOQ were found to be 83.92 ng/spot and 254.30 ng/spot., respectively. The Central
DPPH Composite design expert was applied for the validation of robustness. Three independent variables such
as the volume of toluene in solvent system, chamber saturation time and wavelength were investigated.
The results indicated that a slight change in these variables had no significant effect on the peak response.
This developed HPTLC method is simple, precise, robust, specific, rapid, and cost effective. It could be used
for quality control study and quantification of protopine in the plant extract and different herbal formu-
lations containing the plant species.
Ó 2020 The Author(s). Published by Elsevier B.V. on behalf of King Saud University. This is an open access
article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction lized as antidiabetic, antioxidant, hepatoprotective, etc. (Amir

et al., 2013). Fumaria indica belongs to the family Fumariaceae
Plant derived natural drugs are well-known from the start of and called as Haussk in India. It is an annual weed, sub-erect, pos-
human civilization for their quality, security, usefulness, cultural sibly just scandent and cultivated over the most parts of the plains
suitability and less adverse effects. Because of their natural source, as well as lower hills of India, Turkey, Iran, Baluchistan and Afgha-
it is assumed that they will show better compatibility with biolog- nistan. The whole plant is widely used in traditional and folkloric
ical systems. Since ancient time numerous herbal plants were uti- systems of medicine. This plant is used as anthelmintic, laxative,
diuretic, to purify blood, diaphoretic, and in liver obstruction in
traditional medicine (K
ırtikara and Basu, 1918; Chopra and
⇑ Corresponding author at: Department of Pharmaceutics, College of Pharmacy, Chopra, 1958). Pharmacological evaluation reported that F. indica
P.O. Box-2457, King Saud University, Riyadh 11451, Saudi Arabia.
possesses antidiarrheal (Gilani et al., 2005), anti-inflammatory
E-mail address: [email protected] (M. Kazi).
and anti-nociceptive (Rao et al., 2007), hepatoprotective (Rathi
Peer review under responsibility of King Saud University.
et al., 2008), anxiolytic and central nervous system depressant
(Singh and Kumar, 2010), and chemopreventive effect (Hussain
et al., 2012). Recently, it has been reported that F. indica is safe
Production and hosting by Elsevier and non-toxic through different toxicity analysis (Kumar et al.,

https://doi.org/10.1016/j.jsps.2020.02.011
1319-0164/Ó 2020 The Author(s). Published by Elsevier B.V. on behalf of King Saud University.
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
488 A. Ahmad et al. / Saudi Pharmaceutical Journal 28 (2020) 487–494

2011). This plant showed significant antifungal and antiviral activ- 99.5% w/w) was purchased from Sigma Aldrich, United States. All
ity (Orhan et al., 2007). Phytochemical study indicated the pres- solvents used were of chromatography grade and all chemicals
ence of alkaloids, viz. copticine, fumaritine, fumaramine, used were of analytical reagent grade. Precoated silica gel 60 F254
fumariline, protopine, parfumine, cryptopine, paprafumicin, HPTLC plates were purchased from Merck, Darmstadt, Germany.
paprarine, papracinine, fuyuziphine, narlumidine, narceimine,
papraline, fumarophycine, narlumicine; steroids, viz. campesterol, 2.2. Extraction of plant material
b-sitosterol and stigmasterol; organic acids viz. fumaric acid and
caffeic acid (Pandey et al., 2008). The biological activity of Fumaria The plant of Fumaria indica was dried in sun light properly. The
spp. is generally associated with the presence of isoquinoline alka- dried plant was powdered using mixer grinder. These crude plant
loids, the most essential isoquinoline alkaloid found is protopine powder (50 g) was defatted separately with 300 mL of petroleum
(Fig. 1). This alkaloid has strong anti-hepatotoxic action (Rathi ether by the maceration method. The defatted powder was then
et al., 2008); inhibits histamine H1-receptors and platelet aggrega- extracted with methanol (300 mL) for 6 h by Soxhlet method. This
tion (Saeed et al., 1997); inhibits serotonin transporter and nora- extracts were concentrated by rotary evaporator (Buchi, R-215;
drenaline transporter, and has an antidepressant (Xu et al., 2006), Flawil, Switzerland). The concentrated extract was kept in air tight
antimicrobial, antiviral (Orhan et al., 2007) and anti-inflammatory glass container at 5–10 °C for further study. The % extraction yield
action (Saeed et al., 1997). It was also reported that protopine was calculated by applying the given formula below:
ingestion down regulated glutamine level in rat brain by activation
of glutamate dehydrogenase (Lee et al., 2005) and could assist the Weight of dried extract
%Extraction yield ¼  100
fortification against transient cerebral ischemic injury (Hu, 2005). Weight of drug sample
Response surface methodology (RSM) is a statistical tool, in
which several variables are tested simultaneously (Srinubabu
2.3. Preparation of standard stock solution
et al., 2007; Parajo et al., 1992). The multivariate approach has ben-
efits included decrease in the number of investigational runs,
Stock solution of the reference compound protopine was made
enhances statistical justification potentials and specifies whether
by dissolving 10 mg of accurately weighed protopine in 10 mL
parameters interact or not. Central Composite Design (CCD) is
HPLC grade methanol in a volumetric flask to get a stock solution
widely used statistical experimental design. Although ratability is
of 1 mg/mL concentration. This standard stock solution was filtered
an anticipated property of a CCD, in the case of difficulty to extend-
by 0.45 mm syringe filters and used for HPTLC analysis.
ing the star points beyond the experimental region defined by the
upper and lower limits of each factor (Lundstedt et al., 1998).
2.4. Preparation of samples
Several techniques have been used for the determination of pro-
topine in plant extracts, but until now no study has been reported
Dried methanolic extract (10 mg) of F. indica extracted by Soxh-
on HPTLC-densitometry method for quantitative determination of
let method was dissolve in 100 mL HPLC grade methanol. This
protopine in crude extracts from F. indica. HPTLC-densitometry is
sample solution was then filtered by 0.45 mm syringe filters and
a suitable method for screening of huge number of samples for
used for HPTLC study.
selection of high-producing plants as it is cheaper, faster and sim-
pler than HPLC. Also, the TLC-densitometry method has suitable
sensitivity and consistency (Satpathy et al., 2017). The aim of the 2.5. Instrumentation and chromatographic conditions
present study was to develop a HPTLC based detection and quan-
tification of protopine in the extracts of F. indica and to assess Chromatographic study was done, as mentioned in previous
the robustness of the chromatographic method for the quantitation methods (Lundstedt et al., 1998; Satpathy et al., 2017). Briefly,
of protopine, in the extracts of F. indica, using RSM, and regulate 20 cm  10 cm alumina HPTLC plates pre-coated with 200 mm lay-
the analytical constraints that present higher effect in the final ers of silica gel 60F254 (E. Merck, Darmstadt, Germany) were used
results of the analysis. for the HPTLC study. Samples were applied as bands 5 mm wide
and 10 mm apart by means of Camag Linomat V sample applicator
(Muttenz, Switzerland) set with a 100 mL syringe. The stable appli-
2. Material and methods cation rate was 150 nL/s. Linear climbing development with
toluene: ethyl acetate: diethyl amine (8: 2.5: 0.5 v/v/v) as solvent
2.1. Plant material and reagents system was done in a 20 cm  10 cm HPTLC chamber formerly sat-
urated with solvent system for 30 min at 60 ± 4% humidity and
The fresh plant of Fumaria indica was collected from the field 25 ± 2 °C temperature. The distance travelled by mobile phase
area of New Delhi, India in the month of December 2018 and was 80% in near about 10 min. The plates were dried at normal
was authenticated by taxonomist, College of Science, King Saud temperature and heated at 110 °C for 5 min to recognize compact
University, Riyadh, Saudi Arabia. Standard protopine (purity: bands. Densitometric study was done at 290 nm in reflectance
manner with a Camag TLC scanner III controlled through WinCATS
software (Version 1.2.0). The slit dimensions were 4 mm  0.45
mm and the scanning speed of 15 mm/s.

2.6. Validation of HPTLC method

The developed HPTLC process was validated with respect to the

following Parameters. The validation process was done as per the
ICH guideline (ICH, 2005).

2.6.1. Linearity
Standard solution of protopine was ready in methanol to obtain
Fig. 1. Chemical structure of protopine, MW: 353.4 g/mol. a concentration of 1 mg/mL. Different quantities of standard
 A. Ahmad et al. / Saudi Pharmaceutical Journal 28 (2020) 487–494 489

solution were selected for application on the TLC plate to obtain an ker by linearity equation. The results of triplicate evaluation were
absolute dilution of 500–5000 ng/spot for protopine. All concentra- uttered as mean quantity of protopine in % w/w.
tion was applied six times on the TLC plate. This TLC plate was then
developed by the previously mentioned solvent system. Calibra-
2.7. Assessment of % free radical scavenging activity by 2,2-diphenyl-
tion curves were obtained by plotting the peak area verses differ-
1-picrylhydrazyl (DPPH)-UV method
ent concentrations of the protopine. Linear standard plot was
made by least-squares linear-regression study.
The free radical scavenging power of the plant sample was
observed using DPPH-UV method (Mujeeb et al., 2012). DPPH
2.6.2. Specificity
(0.004%) was freshly dissolved in methanol and used as the control.
The capability of an analytical procedure to clearly assess the
The DPPH solution (1 mL) was added to a mixture of 1 mL of plant
analyte in the presence of other components can be verified by
extract (10 mg/mL) and 3 mL of methanol. Quercetin (1 mg/mL)
evaluating specificity. The specificity of the HPTLC process was
was used as a standard and follow the same procedure as per plant
determined by analyzing standard drug and test samples. The spot
extract (Mujeeb et al., 2012). The solution mixtures were shaken
for protopine in the samples was confirmed by comparing the Rf
vigorously and kept at 20 °C in the dark light for 10 min. The
value and peak of the spot to that of a standard. The peak purity
decline in absorbance of the resulting solutions were measured
of protopine was determined by comparing the peak at three var-
at 517 nm after 10 min. The decrease of absorbance between the
ious regions of the spot that is peak start, peak apex and peak end.
control and the plant extract/standard was applied for calculating
the % free radical scavenging activity. Each result was examined
2.6.3. Precision
in triplicate.
According to the guidelines of ICH, precision should be analyzed
at two phase, inter-day and intra-day precision. Inter-day precision ðAC  AS Þ
observes to applying the analytical method in different days over a Free Radical Scavengingð%Þ ¼  100
AC
specific period of time by the same analyst with unchanged instru-
ment. Intra-day was determined by the applying of analytical where AC = Absorbance of control, AS = Absorbance of sample.
method within a day at different period of time by the same ana-
lyst with the same instrument.
3. Results and discussion
2.6.4. Accuracy
3.1. Extraction
To evaluate the consistency and appropriateness of the devel-
oped procedure, recovery studies were performed. This study
The quantity of plant extract that an extraction yields in a speci-
was performed by standard adding procedure. Known quantity of
fic solvent is frequently an approximate assess of the quantity of
reference marker protopine was added to pre-studied samples
particular compound that the plant material contains. The extrac-
and its recovery was matched with the theoretical value.
tion process was done by Soxhlet method by using methanol as
solvent. During the study, time of extraction, temperature of
2.6.5. Robustness by design expert
extraction and plant sample: solvent ratio remained constant.
A Central Composite statistical screening design (Parajo et al.,
The % extractive yield of F. indica was found to be 9.41 ± 0.096%
1992), was applied to optimize the compositional parameters
w/w.
and to estimate quadratic effects of the toluene volume in solvent
system (mL), chamber saturation time (min) and wavelength (nm).
Seventeen experiments with three center points were performed 3.2. Optimization of method
by selection of three factors, toluene volume in solvent system
(A), chamber saturation time (B), wavelength (C) and peak area TLC or HPTLC is mainly used as an economical technique for iso-
was chosen as the response. The supposed value of these three fac- lation, separation, qualitative detection, or semi-quantitative
tors A, B and C was 8 mL, 30 min and 290 nm; respectively. The visual study of samples. Accordingly, TLC is generally described
statistics generated were examined via Design-Expert (Version as a pilot technique for HPLC (Rozylo and Janicka, 1996). However,
11.0.3.0, Stat-Ease Inc., Minneapolis, MN, USA) statistical software. now a day the TLC and HPTLC method can be applied to solve
The significance of the factors was calculated by using Fisher’s sta- numerous qualitative and quantitative analytical problems in an
tistical test for Analysis of Variance (ANOVA) model that were extensive range of fields, like environmental analysis, biotechnol-
expected. These components were then used to calculate an F- ogy, medical science, food as well as nutrition analysis, toxicology,
ratio that estimates the usefulness of the model. If the F-ratio pos- biochemistry, chemistry and pharmaceutical science (Weins and
sibility is low, the model is considered a better statistical robust for Hauck, 1996) utilization of TLC/HPTLC has extended significantly
that data. Every experiment was carried out in randomized order because of the improvement of forced flow (FF) and gradient TLC
to reduce the bias effects of uncontrolled factors. techniques, advance stationary phase and selection of solvent sys-
tem, as well as development of new methods for quantification
2.6.6. Limit of detection and limit of quantification (Poole and Poole, 1994). Composition of mobile phase and ratio
The limit of detection (LOD) and the limit of quantification of solvents were studied as variables to optimize the chromato-
(LOQ) were estimated by the blank determination process. In order graphic circumstances. The chromatograms were recorded as well
to determine the LOD and LOQ, blank methanol was spotted six as the Rf value and resolution were calculated. The chromato-
times and the area calculated. The signal-to noise level was esti- graphic conditions for HPTLC like detection of wavelength and sol-
mated. LOD was considered as 3:1 and LOQ as 10:1. vent system composition were optimized to give perfect, exact and
reproducible results for the analysis of protopine. A scanning
2.6.7. Estimation of protopine in plant sample wavelength of 290 nm for protopine was obtained from UV spec-
The amount of protopine in F. indica was examined via devel- trum. An excellent resolution was achieved by using an optimum
oped and validated procedure by calibration curve. The plant solvent system consisted of toluene:ethyl acetate:diethyl amine
extract solution was applied in triplicates on the TLC plate and area (8.0:2.5:0.5 v/v/v). Protopine was adequately resolved with Rf value
of each triplicate samples were used for study of quantity of mar- at 0.22 ± 0.03 (Fig. 2).
490 A. Ahmad et al. / Saudi Pharmaceutical Journal 28 (2020) 487–494

Fig. 2. (A) Densitogram of standard Protopine (1000 ng; Rf = 0.22 ± 0.03); (B): sample extract (Rf = 0.22 ± 0.06).

3.3. Method validation 3.3.2. Specificity

Assessment of analyte in the occurrence of interference to
3.3.1. Linearity ascertain peak purity in sample chromatogram is a very complex
The linearity of an analytical procedure is its facility to draw task. Specificity is a determination of the degree of interference
analysis results that are directly, or by means of precise mathemat- from other active ingredients, degradation materials, impurities
ical alteration, proportional to the concentration of standard in sam- and additives. Specificity in a process ensures that a peak response
ples within a set range. The linear regression data found for the is because of a single component only. Specificity is commonly cal-
standard plot (n = 6) explained an outstanding linear relationship culated and documented in a separation by the resolution and tail-
over different concentration level 500–5000 ng/spot for protopine ing factor. The specificity of the method was determined by
(Fig. 3) with respect to area of chromatogram. The plot shows the analyzing the standard drug and extract. The spot of protopine in
y-intercept values were low and standard deviations of slope were the plant sample was proved by comparing the Rf values and peak
also quite low. The r2 value was found to be 0.996 ± 0.082. of the spot with that of the standard.
 A. Ahmad et al. / Saudi Pharmaceutical Journal 28 (2020) 487–494 491

Fig. 3. Calibration plot with respect to peak area by HPTLC at different concentration levels of standard protopine.

3.3.3. Precision 3.3.5. Robustness by design expert

The precision of an analytical procedure is the level of agree- The robustness of an analytical method is a measure of its abil-
ment among particular test results achieved when the process is ity to stay unaffected by introduction of small discrepancies in
useful to numerous sampling of a homogenous sample. It is an method constraints and provides assign of its consistency
evaluation of the reproducibility of the complete analytical method through-out normal procedure. For measuring robustness of the
under standard working conditions. The precision of sample appli- method, a number of chromatographic parameters, such as, flow
cation and observation of chromatogram area were showed in con- rate, detection wavelength, injection volume, compositions in
ditions of %RSD and results are illustrated in Table 1 that exposed mobile phases, or column temperature are varied within a convinc-
inter-day and intra-day difference of protopine at three various ing range and quantitative influence of the variables is examined or
dilution points of 2000, 3000 and 4000 ng/spot. determined. The robustness of current developed method was
examined/tested by using the CCD. The multivariate approach
using design of experiments was used in robustness testing, in
3.3.4. Accuracy order to study the concurrent dissimilarity of the factors on the
Accuracy articulated the closeness of understanding between responses of peak areas of protopine. RSM is an effective statistical
the values which is established as an ordinary genuine value or and mathematical procedure to examine and optimize composite
an established reference value and the observed obtained. This test processes (Bezerra et al., 2008). RSM provides a large amount of
permits the evaluation of probable bias. The selected method of information with less number of experiments for observing the
HPTLC was used for the assortment of accurate mobile phase for interface of the free variables on the response. CCD is broadly
estimation of protopine in selected herbal drug after injecting via employed because of its extraordinary competence with respect
50%, 100% and 150% of added references standard, which showed to the conventional practices that required large number of scores
best recovery of 98.75–102.12%. The results of % of drug recovered for sample analysis or optimization (Srinivas et al., 2008; Wang
and % RSD are shown in Table 2.

Table 1
Inter-day and intra-day precision of the HPTLC method (n = 6) for protopine (mean ± SD).

Amount (ng/spot) Inter-day precision Intra-day precision

Mean peak area ± SD %RSD Mean peak area ± SD %RSD
Protopine
2000 2476.08 ± 47.61 1.923175 2461.35 ± 51.07 2.07
3000 3474.35 ± 17.14 0.49335 3459.60 ± 26.23 0.75
4000 4876.58 ± 70.17 1.438938 4887.93 ± 52.84 1.08

RSD: Regressed Standard Deviation.

Table 2
Accuracy of the HPTLC method (n = 6) for Protopine (mean ± SD).

% Of standard spiked to the sample Theoretical content (ng) Amount of drug recovered ng ± SD % of drug recovered % RSD
Protopine
0 2500 2539.35 ± 19.24 101.57 0.75
50 3750 3746.03 ± 24.18 99.89 0.64
100 5000 5106.14 ± 98.66 102.12 1.93
150 6250 6172.22 ± 65.99 98.75 1.06

RSD: Regressed Standard Deviation.

492 A. Ahmad et al. / Saudi Pharmaceutical Journal 28 (2020) 487–494

et al., 2006). A CCD is a two factorial design involves 2 k factorial the response within the calculated range and to check the analyt-
runs with ±a beside each variable bloc, and at least one center ical validity of the model (Gunst et al., 1996). The varieties exam-
point (Srinubabu et al., 2007). Center point replications (more than ined were small deviations from the method settings and the
two) are mostly carried out to allow the estimation of pure error of parallel responses in the retention time considered were observed.
A three-factorial, Central Composite statistical experimental
Table 3 design was carried out by 17 experiments test run. The indepen-
Chromatographic factors for central composite response surface methodology. dent variables and the responses for all 17 optimized test experi-
Run Factor 1 Factor 2 Factor 3 mental runs are illustrated in Table 3. It was studied that the
A:Toluene volume B:Chamber saturation time C:Wavelength best-fitted model was the quadratic model and the relative values
(mL) (minute) (nm) of SD and % CV for the various projected models. Only statistically
1 8 26.63 290 significant (p < 0.05) coefficients are incorporated in the equations.
2 6 32 280 A positive value represents an effect that favors the optimization,
3 8 30 273.18 although a negative value indicates an opposite connection
4 8 33.36 290
5 4.63 30 290
between the factor and the response (Gunst et al., 1996). It is clear
6 8 30 306.81 from the equation that the factor toluene volume of mobile phase
7 10 28 280 (A) as well as wavelength (C) has a positive effect and chamber sat-
8 8 30 290 uration time (B) has a negative effect on the response (Y). The red
9 8 30 290
color intensity represents the concentration of protopine (Fig. 4). It
10 10 32 300
11 10 32 280 also confirms that the association between responses and factors is
12 11.36 30 290 not constantly linear. Used at different levels in study or when
13 6 32 300 more than one factor is changed simultaneously, a factor can pro-
14 6 28 300 duce different degrees of response. For an experimental design
15 8 30 290
16 6 28 280
with three variable factors, the appropriate model fitting to the
17 10 28 300 data was the quadratic model. The polynomial equations for the
response factors are given below:

Fig. 4. Response-surface 3D graphs showing the effect of (A) Chamber saturation time (min) versus toluene volume (ml) and wavelength remains constant (B) wavelength
(nm) versus toluene volume (ml) percentage and Chamber saturation time (min) remains constant (C) wavelength (nm) versus Chamber saturation time (min) and toluene
volume (ml) remains constant.
 A. Ahmad et al. / Saudi Pharmaceutical Journal 28 (2020) 487–494 493

Peak Area (Y) = +3408.64 + 33.34*A + 54.20*B + 76.36*C  22. useful for a rapid screening of antioxidant potential of compounds
04*A*B + 0.5200*A*C  48.61*B*C  58.94*A2  98.89*B2  61.13*C2. or radical scavenging activity of phytoconstituents.
Where A is the toluene volume of mobile phase (mL), B is the
chamber saturation time (min) and C is the wavelength (nm). 4. Conclusion
The data was examined by analysis of variance (ANOVA). The
reliability of the fitted model was assessed by the determination A validated HPTLC analytical method has been developed for
co-efficient (R2 = 0.9889), adjusted (R2a = 0.9799) and the analysis of protopine in F. indica. The projected method is accu-
(CV = 1.36%). A very high adjusted determination coefficient rate, simple, precise, less time consuming, cost effective and has
(R2a = 0.969) values confirmed that the model was very significant the capability to isolate the active compound from its other con-
(Parajo et al., 1992). In the present research study, values of R2a stituents. It also has good linearity range and limits of determina-
were found to be contiguous to the R2. High values of tion. Application of factorial design using CCD for validation of
R2 = 0.9889 presented an outstanding relationship between the robustness testing showed that a slight change in tested parame-
tested and projected responses (Lundstedt et al., 1998). The lower ters. However, a special consideration is prerequisite for firm mon-
CV value indicated that the abnormalities between tested and pre- itoring of the aforesaid tested factors during chromatographic
dicted values are low. Adequate precision was found to be 16.89 testing. Therefore, the developed method is proved to be rapid,
and greater than 4 is desirable in the design, confirms this design simple, and selective and could be used for routine analysis of pro-
is significant for determination robustness in chromatography topine in F. indica in bulk as well as in pharmaceutical formulations
(Beg et al., 2003). containing protopine.

3.3.6. Limit of detection and limit of quantification Acknowledgements

The limit of detection (LOD) is explained as the lowest amount
of a reference standard in a sample that can be detected, but not The author would like to extend their sincere appreciation to
essentially quantify. It is a limit test that indicates whether a refer- the Deanship of Scientific Research at King Saud University for its
ence standard is high or lowers a specific value. The limit of quan- funding through research group no- (RG#1435-017).
tification (LOQ) is explained as the lowest amount of a reference
standard in a sample that can be quantified with suitable accuracy Declaration of Competing Interest
and precision below the confirmed operational circumstances of
the process. In order to analyze the LOD and LOQ, blank methanol The authors have confirmed that there is no conflict of interest.
was spotted six times. The signal-to-noise level was examined.
LOD was measured as 3:1 and LOQ as 10:1. The LOD with S/N ratio
of 3:1 was obtained 83.92 ng/spot and LOQ with S/N ratio of 10:1
was obtained 254.30 ng/spot. References

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The amount of protopine in the extract of Fumaria indica was supported development and validation of HPTLC method: an application in
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content in the plant of F. indica was found to be 3.69 ± 0.46% w/ surface methodology (RSM) as a tool for optimization in analytical chemistry.
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