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Quantitative Determination of thymoquinone in Nigella Sativa and its nano
formulation using validated stability indicating HPTLC densiometric method
Mohamad Taleuzzaman1, Syed Sarim Imam2 and *Sadaf Jamal Gilani1
1Department of Pharmaceutical Chemistry, Glocal School of Pharmacy, Glocal University, Saharanpur -247121. (UP), India
2Department of Pharmaceutics, Glocal School of Pharmacy, Glocal University, Saharanpur -247121. (UP), India
INTRODUCTION
Nigella sativa Linn. (Ranunculaceae) very often known as
black seed or black cumin, is an herbaceous plant, mostly
found in the middle east, Central Europe, and Western
Asia. It is broadly used in indigenous system of medicine
for treatment of numerous disorders for over 2000 years
(Alam et al., 2013). Thymoquinone (TQ, Figure 1) is the
precious constituent of the volatile oil of Nigella Sativa
seeds, has shown vital medicinal properties, and has long
been used in traditional and folk medicines (Ragheb et al.,
2009). The main bioactive constituent of Nigella Sativa is
TQ and various biological activities have been reported in
different research (Akhtar et al., 2014; Sayeed et al., 2017).
TQ belongs to the family of monocyclic monoterpenes,
having the molecular formula (C10H12O2) and IUPAC
name 2-methyl-5-propane-2-yl-cyclohexane-2, 5-diene-
1,4-dione phenol. Most properties of the whole seeds or
their extracts are mainly attributed to the presence of qui-
none constituents in the volatile oil and the most
abundant component found is thymoquinone (about 27–
57%) (Ali et al., 2003). Other pharmacologically active con-
stituents, present were dithymoquinone,
thymohydroquinone and thymol in low cont ent
(Ghosheh et al., 1999).
The most preferred analytical tool for fingerprints
and quantification of marker compounds in herbal drugs
used is HPTLC. This technique has shown accuracy, sen-
sitivity, suitability for high-throughput screening,
reliability in the quantification of analytes from nano-
gram to microgram levels (Tayade et al., 2007; Kaur et al.,
2008). It has several advantages in comparison to high-
performance liquid chromatography (HPLC), it can ana-
lyze several samples, use the small quantity of mobile
phase, as well as it facilitates repeated detection (scan-
ning) of the chromatogram, analyze different kinds of
samples, which reduces the time, and cost of analysis
(Kaur et al., 2008) (Dixit et al., 2008). Although several
methods such as thin-layer chromatography (AbouBasha
et al., 1995) gas chromatography (Houghton et al., 1995).
HPLC (Ghosheh et al., 1999). HPTLC (Michelitsch et al.,
2003). and differential pulse polarography (Reelma et al.,
2011) have been used to quantify the TQ in black seed oil.
But till now not a single stress induced HPTLC method
was developed to quantify TQ.
The International Council for Harmonisation (ICH)
guideline entitled Stability Testing of New Drug Sub-
stances and products requires the stress testing to be
carried out to elucidate the inherent stability characteris-
tics of the active substance (Stolarczyk et al., 2010). There
are different stability parameters (oxidation, hydrolytic
and the photolytic) used for the stress stability studies. An
ideal stability- indicating method is one that quantifies
the standard drug alone and also resolves its degradation
products (Zammataro et al., 2010). The basic acceptance
criteria for evaluation of validation experiments based on
practical experience for planar chromatographical proce-
dures, which may be used at different levels either in
qualitative identity testing, assays, semi-quantitative
limit tests or quantitative determination of impurities
(Bakshi et al., 2011). Form this approach, the aim of the
present study to develop and optimize a simple and se-
lective HPTLC method for the quantification of TQ for
purity assessment in bulk as well as in in-house devel-
oped nanoformulation. The proposed method was
validated as per ICH guidelines (Ranganathan et al., 2002;
Sethi et al., 1996) and its updated international convention
(ICH, Q2A, 1994).
ORIGINAL RESEARCH ARTICLE OPEN ACCESS
International Current
Pharmaceutical Journal
ABSTRACT
An accurate and stability indicating high-performance thin layer chromatographic method (HPTLC) was developed and validated for
the quantification of thymoquinone (TQ) as per the ICH guidelines. The analysis was carried out on the aluminum plate using n-
hexane: ethyl acetate: methanol (7:2:1 v/v/v) as the mobile phase and the densitometric determination was carried out by TLC scanner
(CAMAG) at 254 nm. The developed method was validated for different parameters like linearity, precision, recovery, robustness, and
stressed stability study. The developed analytical method was found to be linear in the concentration range of 75-500 ng band-1 with
regression value closer to unity (r2 = 0.997). The developed system was found to give compact spots for thymoquinone (Rf 0.77) with
the limit of detection and limit of quantification (18 and 54 ng band-1) respectively. Further, the study showed accuracy, precision and
repeatability were all within the required limits. The stress degradation study of TQ showed well separated degraded peak from the
pure thymoquinone. The mean recoveries measured at three concentrations were more than 95% with RSD 3%. The method has been
successfully applied in the analysis and routine quality control of herbal material and formulations containing TQ.
Key Words: Nigella Sativa, thymoquinone, HPTLC, validation, stress degradation, Nano formulation.
*Corresponding Author:
Dr. Sadaf Jamal Gilani, Associate Professor
Department of Pharmaceutical Chemistry
Glocal School of Pharmacy
Glocal University, Saharanpur 247121 (UP), India
E-mail: gilanisadaf@gmail.com
Contact No.: +91-9997939221
INTRODUCTION
54
MATERIAL AND METHODS
Materials
Pure Thymoquinone and Mature seed powder of Nigella
Sativa were purchased from Sigma Aldrich, Mumbai and
from Sunpure extract private Ltd. (Delhi) respectively. Pre-
coated silica gel 60 F254 HPTLC plates were purchased
from E. Merck, Germany. All the solvents used were of
chromatography grade and other chemicals used were of
analytical reagent (AR) grade. Water was purified by a
Milli-Q system (Millipore Corporation, Bedford, MA,
USA).
Methods
HPTLC instrumentation and condition.
The samples were spotted in the form of bands of width 6
mm with a Camagmicrolitre syringe on precoated silica gel
aluminum plate 60 F-254, (20 cm × 10 cm with 250 µm thick-
ness; E. Merck, Darmstadt, Germany, supplied by
Anchrom Technologists, Mumbai). Methanol was used to
wash the plates and activated at 60°C for 5 min prior to the
study. A constant sample application with the rate of 140
nL s−1 was employed and space between two bands was 6
mm maintained. The slit dimension was kept at 5 mm × 0.45
mm and 20 mm/s scanning speed was employed. The mon-
ochromatic bandwidth was set at 20 nm, each track was
scanned thrice and a baseline correction was used. The op-
timized mobile phase composition (20mL) consisted n-
hexane: ethyl acetate: methanol (7:2:1 v/v/v) was used for
the analysis. Linear ascending development was carried
out in 20 cm × 10 cm twin trough pre-saturated glass cham-
ber (Camag, Muttenz, Switzerland). The HPTLC plate
development was carried out for twice with the same mo-
bile phase to get high resolution. The optimized chamber
saturation time for mobile phase was 30 min at room tem-
perature (25 ± 2°C) at the relative humidity of 60 ± 5%.
Densitometric scanning was performed on Camag TLC
scanner III in the reflectance-absorbance mode at 254 nm
and operated by WinCATS® software (Version 1.2.0).
Preparation of standard and quality control (QC) samples
A standard stock solution of TQ (10 mg/mL) was prepared
in methanol and further dilution of the standard was done
in the concentration range of 0.1 to 1.0 mg/mL. The calibra-
tion of TQ standard solution (1-10 µL) was applied to an
HPTLC plate to furnish amounts in the range 75-500 ng
band-1. The data of peak area vs. drug concentration were
treated by linear least square regression analysis. QC sam-
ples as low, medium and high at the concentration level of
250, 350 and 400 ng band-1 were taken to carry out valida-
tion of the method. Three replications of each calibration
standard were performed (n = 3).
Extraction process
The weighed quantity of NS seed powder was packed in a
muslin cloth and kept in the beaker containing sufficient
quantity of methanol for 72 hrs. Thereafter the methanolic
extracts were filtered through Whatman paper no. 42 and
the resultant filtrates were concentrated under reduced
pressure using the rotary evaporator to yield a concen-
trated mass which was labeled as meth-NS and preserved
in airtight amber colored glass container at 4°C until use.
The fingerprinting of methanolic TQ extract was enact by
spotting 10 µL of suitably diluted sample solution on an
HPTLC plate.
Method validation
The developed method was validated as per ICH guide-
lines for linearity range, precision, accuracy as recovery,
robustness, limits of detection (LOD), limits of quantifica-
tion (LOQ) and stability study (Ali et al., 2016; Mittal et al.,
2015).
Precision
The inter-day precision study was done by repeating the
study on same day whereas in intraday assay the same
study was repeated for three different days. In both, the
study, precision of the developed method was evaluated by
performing replicate analyses (n = 6) of QC samples at low
(250 ng band-1), medium (350 ng band-1) and high levels
(400 ng band-1). The result of precision was expressed as the
percent recovery with the coefficient of variation of meas-
ured concentrations for each level.
Accuracy
The accuracy was determined by standard additions
method at three different levels, i.e., by multiple level re-
covery studies. The recovery studies were performed with
the addition of extra percentage level of 50, 100 and 150%
from the initial level (Ali et al., 2016). It was performed by
application of test sample (n=6) of known concentrations of
TQ that had been prepared from stock solutions. The per-
cent recovery was calculated using regression equation at
different levels in the sample.
Limit of detection (LOD) and quantification (LOQ)
The limit of detection and limit of quantification are ap-
plied for the determination of low levels of the compound
in sample matrices and is used particularly for the determi-
nation of impurities and degradation product in the
quantitative assay. To estimate the limits of detection (LOD)
and quantification (LOQ), blank methanol was applied
(n=6) and the standard deviation (σ) of the analytical re-
sponse was determined. The LOD and LOQ values were
calculated from the calibration curves as kσ/b where k = 3
for LOD and 10 for LOQ, σ is the standard deviation of the
intercept and b is the slope of the calibration curve.
Robustness
Robustness was determined by introducing small changes
in the different parameters such as mobile phase composi-
tion, mobile phase saturation time, and mobile phase
volume and their effects on the results were examined. The
study was performed in triplicate at 350 ng band−1 and the
effects of changes were examined on the results of peak ar-
eas and Rf value. The mobile phase composition n-hexane:
ethyl acetate: methanol in different proportions (7.5:1.8:0.7
and 6.8:2.1:1.1 v/v/v) and mobile phase volume (13, 17 and
15 ml) and duration of saturation (10, 20 and 30 min) were
investigated. Further, the plates were prewashed with
methanol and activated at 60 ± 5°C for 2, 5 and 7 minutes.
Figure 1: Chemical structure of Thymoquinone.
MATERIALS AND METHODS
55
Specificity
The specificity of the method was ascertained by analyzing
standard drug and sample. The spot for TQ in the sample
was confirmed by comparing the RF and spectra of the spot
with that of the standard. The peak purity of TQ was as-
sessed by comparing the spectra at three different levels,
i.e., peak start (S), peak apex (M) and peak end (E) positions
of the spot.
Analysis of TQ from Developed nano Formulation
The validated developed method was applied to the quan-
tification of TQ in-house developed TQ loaded
nanoformulation. The estimation of the contents of TQ in
nanoformulation, 5 mL of nanoformulation was extracted
with 10 mL methanol. To ensure complete extraction of the
drug, it was sonicated for 30 min and centrifuged at 3000
rpm for 10 min. The supernatant of each sample was suita-
bly diluted to give desired concentration (400 ng spot–1).
After that, it was applied on TLC plate followed by the de-
velopment. The result of the peak area obtained
corresponding to TQ was used for quantification in sam-
ples using regression equation. The results of the triplicate
analysis were expressed as an average amount of TQ in %
w/w. The possibilities of excipient interference in the anal-
ysis were studied.
Stability studies
The stress testing of the drug substance can help identify
the likely degradation products, the stability of the mole-
cule and also validate the stability and specificity of the
analytical procedures. The degradation studies were car-
ried out as per by subjecting the standard thymoquinone
sample to oxidation, wet heat, dry heat and photo-degra-
dation to evaluate the stability indicating properties of the
developed HPTLC method.
Hydrogen peroxide-induced degradation
To 25 ml of a methanolic stock solution of thymoquinone,
10 ml of 50 % w/v hydrogen peroxide were added. The so-
lution was heated in boiling water bath for 15 min to
remove completely the excess of hydrogen peroxide and
then refluxed for 2h at 70°C. The resultant solution (200 ng
band-1) was applied to TLC plate and the chromatograms
were run for the analysis as described above.
Dry heat and wet heat degradation
The standard drug was placed in the oven at 100°C for 7
days to study dry heat degradation, and the stock solution
was refluxed for 12h on boiling water bath for wet heat deg-
radation. The resultant solution (200 ng band-1) was
applied on TLC plate. Further, the HPTLC study was per-
formed and the chromatograms were run for the analysis.
Photochemical and UV degradation
The photochemical stability of the thymoquinone was also
studied by exposing the stock solution (250 g ml-1) to direct
sunlight for 3 days on a wooden plank and kept on the ter-
race. The thymoquinone solution was also exposed to UV
radiation for 15 days in UV stability chamber. One micro-
litre (200 ng band-1) from each sample was applied on TLC
plate and chromatograms were run as described above.
RESULTS AND DISCUSSION
Mobile phase optimization
There are different solvent systems were tried for the sepa-
ration of thymoquinone on the TLC plates. Initially, the
different solvent system used as the solvent system to get a
high-resolution peak, and get better peak resolution. The
optimized mobile phase system was found n-Hexane: ethyl
acetate: methanol in the ratio of (7:2:1 v/v/v). The Rf value
found with this mobile phase composition was 0.77 for all
the samples shown in Figure (2 & 3). The resolution be-
tween spots of standard and depredates appeared better,
when the TLC plates were pretreated with methanol, acti-
vated at 60°C for 30 min, and were saturated with conc.
ammonia vapors for 30 min in TLC chamber prior to appli-
cation. It was required to eliminate the edge effect and to
avoid unequal solvent evaporation losses from the devel-
oping plate that can lead to various types of random
behavior usually resulting in lack of reproducibility in Rf
values. So the attempt has been taken to develop and vali-
date a cost-effective simple and robust HPTLC technique
to quantify thymoquinone in the methanolic extract of Ni-
gella Sativa. The methanolic extract peak of thymoquinone
was well resolved at Rf value 0.77 depicted in Figure 3. The
developed method was found to be quite selective with
good baseline resolution of each compound.
Method Validation
Linearity
The drug concentration versus peak area was plotted be-
tween 75 - 500 ng band−1 (n = 3) to construct a standard
curve of TQ (Figure 4). The graph was found to be linear,
i.e., adherence to the system to Beer’s law. The polynomial
regression for the calibration plots showed the good linear
relationship with the coefficient of correlation r2 = 0.997 and
regression equation 16.58x - 224.2 over the concentration
range studied depicted in in Table 1. There is no significant
difference (ANOVA; P < 0.05) observed in the slopes of the
standard curves in this range. The LOD and LOQ of the de-
veloped method were (18 and 54 ng band-1) which showed
that the method was sensitive to detect and quantitative the
thymoquinone.
Precision
The intra-day and inter-day precision of TQ were ex-
pressed as % recovery and percentage relative standard
deviations (% RSD) at three different levels were shown in
Table 2. The percentage recovery for both was found to be
in the range of (98.5-99.7 %) indicates the method was ac-
curate by satisfying the acceptance criteria. The results for
intra-day and inter-day variation of TQ recovery were
studied at three different concentration levels 250, 350 and
400 ng spot–1. The % RSD value was found to be <2% in all
the cases. The low values of % RSD are indicated the high
precision of the method.
Table 1: Linear regression analysis data for the developed
HPTLC method of Thymoquinone.
Parameter
Values
RF
0.77
Linearity range (ng band-1)
75-500
Regression equation
16.58x - 224.2
Correlation coefficient (r2)
0.997
Slope ± SD
0.0068±0.0067
LOD
18ng band-1
LOQ
54 ng band-1
Recovery (%)
99.15 ± 1.44
Robustness
Robust
Specificity
Specific
RESULTS AND DISCUSSION
56
Figure 2: HPTLC chromatogram of standard thymoquinone (Rf 0.77).
Figure 3: HPTLC chromatogram of thymoquinone extract (Rf 0.77).
57
Figure 4: Comparative 3D scan of thymoquinone extract.
Figure 5: Degraded Chromatogram (A). H2O2 treated thymoquinone peak with degraded peak 1: degraded, Rf: 0.29, peak
2, Rf: 0.36, peak 3: degraded, Rf0.46, peak 4: degraded, Rf: 0.68, peak 5: degraded, Rf: 0.73); (B). dry heat and wet heat
treated thymoquinone with degraded peak (peak 1: degraded, Rf: 0.33, peak 2, Rf: 0.36, peak 3: degraded, Rf0.64, peak 4:
degraded, Rf: 0.85); (C). photochemical and uv rays treated thymoquinone with degraded peak (peak 1: Rf: 0.48, peak 2:
Rf: 0.64, peak 3: Rf- 0.67, peak 3: Rf- 0.69).
A
B
C
58
Table 2: Intra-day and Inter-day precision data of the Thymoquinone (N=6).
Nominal
concentration
Intra-day batch
Inter-day batch
Final conc.
Precision
% Recovery
SD
%CV
Final conc.
Precision
% Recovery
SD
%CV
250
247.25
0.90
98.9
0.81
0.28
247.75
0.71
99.1
0.58
0.23
350
348.25
0.37
99.5
0.67
0.19
344.75
0.20
98.5
0.29
0.08
400
398.80
0.48
99.7
0.81
0.20
397.80
0.31
99.4
0.41
0.10
SD- Standard Deviation, cv- Coefficient Variation
Table 3: Accuracy data of the Thymoquinone (n=6).
Conc. added to
analyte (%)
Theoretical (ng)
Added (ng)
Detected (ng)
Recovery (%)
SD
%CV
50
250
125
374.01
99.73
0.055
0.014
100
250
498.86
99.77
0.364
0.073
150
375
623.95
99.83
0.756
0.121
Accuracy (%) = [concentration found)/(nominal concentration)] × 100
Table 4: Robustness of the developed HPTLC method of Thymoquinone (n=6).
Mobile Phase
Conc.
ng/ml
n-Hexane: ethyl acetate : methanol
Results
Original
Used
Area
SD
%RSD
Rf
350
(7:2:1)
7.5:1.8:0.7
154.83
1.24
0.80
0.75
7:2:1
156.86
1.40
0.90
0.77
6.8:2.1:1.1
155.75
1.32
0.85
0.76
Mobile Phase Volume (15 ± 2 ml)
Conc.
ng/ml
Volume
(ml)
Result
Area
SD
%RSD
Rf
350
17
156.83
1.39
0.89
0.78
15
155.86
1.49
0.96
0.77
13
157.75
1.73
1.1
0.76
Duration of Saturation
Conc.
ng/ml
Time
(min)
Result
Area
SD
%RSD
Rf
350
10
157.83
1.89
1.2
0.77
20
158.86
1.41
0.89
0.79
30
155.75
1.49
0.96
0.76
Activated at 60 ± 5°C
Conc.
ng/ml
Time
(min)
Result
Area
SD
%RSD
Rf
350
2
155.83
1.21
0.78
0.78
5
156.86
1.28
0.82
0.77
7
154.75
1.47
0.95
0.76
59
Specificity
The specificity of the method was ascertained by analyzing
the pure TQ and methanolic extract. The spot for TQ in the
sample was confirmed by comparing the Rf values and
spectra of the spot with that of the standard. The peak pu-
rity of TQ was assessed by comparing the spectra at three
different levels, i.e., peak start (S), peak apex (M) and peak
end (E) positions of the spot, i.e., r2 (S, M) = 0.989 and r2 (M,
E) = 0.991. A good correlation (r2 = 0.997) was also obtained
between standard and sample spectra of TQ, therefore, the
method was considered specific.
Accuracy
The recoveries of the drugs were determined by standard
addition method. The proposed method was used for ex-
traction and subsequent estimation of TQ after spiking
with 50, 100 and 150% of the additional drug in the extract,
and the afforded recovery was obtained 99.49 to 100.37%
(Table 3). The result suggests that the method can be con-
sidered accurate, as the %RSD of all the determinants were
to be <2% which indicated that the method was accurate
and also there was no interference of the excipients present
in tablets.
Robustness
Table 4 describes the robustness of the proposed method
and their SD and % RSD was calculated for the change in
mobile phase composition, mobile phase volume, and du-
ration of saturation and activation of prewashed-TLC
plates at concentration levels of 350 ng band-1 (in triplicate).
The result of the study showed that the small deliberate
change in the chromatographic condition gives no signifi-
cant effect on the area and Rf value. The low values of %
RSD (less than 3) indicated the robustness of the developed
method.
Estimation of TQ in nanoformulation
A single spot at Rf = 0.77± 0.03 was observed in the chroma-
togram of the TQ isolated from the extract along with other
constituents. The presence of single spot confirms that
there was no interference from the excipients found in the
developed nanoformulation. The drug content was found
to be 99.15 ± 1.44 with % RSD of 0.0877 for nanoformulation
for three replicate. The low value of % RSD indicated the
suitability of this method for routine analysis of TQ in
pharmaceutical dosage forms.
Stability studies
The results of the forced degradation study of thymoqui-
none using are summarized in Table 5. Hydrogen
peroxide-induced degradation. The sample degraded with
50 % w/v hydrogen peroxide showed additional peaks at
Rf value of 0.29, 0.36, 0.46, 0.68 and 0.73 (Figure 5A). The
spots of degraded products were well resolved from the
parent compound spot.
Dry heat and wet heat degradation
The samples degraded under dry heat and wet heat condi-
tions showed additional peaks at Rf values of 0.33, 0.36, 0.64
and 0.85, respectively (Figure 5B). The spots of degraded
products were well resolved from the parent compound
spot.
Photochemical and UV degradation
The photodegraded sample showed one additional peak at
Rf value of 0.48, 0.64, and 0.67 (Figure 5C) when thymoqui-
none solution was left in daylight for 3 days. The
thymoquinone was degraded when exposed to UV irradi-
ation for 15 days and showed additional peaks at Rf value
of 0.69. The spot of UV degraded product was well resolved
from the standard.
CONCLUSION
HPTLC is a simple, rapid and accurate method for analyz-
ing plant material. This method can be used for
phytochemical profiling of plants and quantification of
compounds present in plants, with increasing demand for
herbal products as medicines and cosmetics there is an ur-
gent need for standardization of plant products. The
presented study clearly gave evidennce of the bioactive
thymoquinone in methanolic extracts of Nigella Sativa. The
developed method is simple, precise, specific, sensitive,
and accurate. Further, this method can be effectively used
for routine quality control of herbal materials as well as for-
mulations containing this compound.
CONFLICT OF INTEREST
None.
ACKNOWLEDGEMENTS
Thanks to Mr. Manish (Sunpure Extract Pvt. Ltd. Delhi) for
providing the herbal sample for the analysis study.
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developed method.
Degradation
condition
No. of degradation
products
Rf value
Peroxide-induced
degradation
3
0.38, 0.40,0.45
Dry heat-wet heat
(100°C)
4
0.50, 0.55,
0.64, 0.69
Photochemical
degradation
1
0.74
UV degradation
1
0.79
CONCLUSION
CONFLICT OF INTEREST
ACKNOWLEDGEMENT
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