Artifacts in Nuclear Medicine with Identifying and resolving artifacts.
Simultaneous Estimation of Withaferin A and Z-Guggulsterone in.pptx
1. Simultaneous Estimation of
Withaferin A and Z-Guggulsterone in
Marketed Formulation by RP-HPLC
Name: Shruti Suresh Mandle
Class: M. Pharm 2nd Year Q.A.
Roll No: 13
Guided By: Dr. Vineeta Vivek Khanvilkar
5/5/2022
1
Journal Club Activity
Bharati Vidyapeeth’s College of Pharmacy, C.B.D. Belapur
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Introduction:-
Withaferin A is a steroidal lactone derived from Acnistus arborescens,Withania
somnifera and other members of family Solanaceae.
Withaferin A has wide range of pharmacological activities including
cardioprotective, anti-inflammatory, immunomodulatory, anti-angiogenesis, anti-
metastasis and anti-carcinogenic properties.
Withaferin A, is the chief constituent present in the plant Withania somnifera L.
commonly known as Ashwagandha.
The plant is reputed to have immunomodulatory activity and antidepressant
activity. It is also reported to possess various other activities such as
hypoglycemic, diuretic and anti-inflammatory activities.
Z-Guggulsterone is a phytosteroid found in the resin of the guggul plant.
Z-Guggulsterone is the chief constituent present in the plant Commiphora wightii
belonging to family Burseraceae, commonly known as Guggul.
The plant is traditionally used in the management of hypercholesterolemia and
obesity. Other activities reported in the plant are antiarthritic activity, anticancer,
antioxidant, etc
a) Withania somnifera
b) Commiphora wightii
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Figure 1: Structure of Withaferin A Figure 2: Structure of Z-Guggulsterone
Chemical Name: 4b,27-dihydroxy-1-oxo-5b,6b-epoxywitha-2, 24-dienolide
Molecular formula: C28H38O6
Molecular weight : 470.606 g·mol−1
Chemical Name: 4,17(20)-(trans)-pregnadiene3,16-dione
Molecular formula :C21H28O2
Molecular weight :312.4 g·mol−1
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Highlights:-
Standard Material:- Withaferin A and Z-guggulsterone (standard) was obtained from Sigma-Aldrich.
Sample Material:- A polyherbal formulation (tablets), Yogaraaj Guggul(500 mg), containing W. somnifera and
C. wightii extracts, manufactured by Ayurvedic Rasashala, was purchased from local market in Mumbai.
Chemicals used:- HPLC-grade methanol and acetonitrile were obtained from Merck, India. Water was double-
distilled.
Instrument:-HPLC analysis was performed with a Jasco (Hachioji, Tokyo, Japan) system consisting of an
intelligent pump (PU-1580, PU-2080), a high-pressure mixer (MX-2080-31), a manual sample injection valve
(Rheodyne 7725i) equipped with a 20-mL loop and a UV– visible detector (UV-1575).
Column :- 250 mm 4.6 mm i.d., 5-mm particle, Hibar LiChrocart Purospher Star RP-18 endcapped column
(Merck, Darmstadt, Germany)
Injection Volume :- 20 µL
Mobile Phase composition:-. A gradient system consisting of acetonitrile(A) and water(B) was used : 0 –7 min,
50% A in B; 7 –9 min, 50–80% A in B; 9 –20 min, 80% A in B
Flow rate :- 1 mL min-1
Wavelength used for scanning:- 235 nm
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Significance:-
HPLC method is simple, rapid and highly sensitive method.
Separation is fast and efficient (high resolution power) and hence saves time.
In HPLC, we have a wide variety of stationary phases.
Early recovery and easy visualization of separated component.
It has good reproducibility and repeatability using the same column.
We can continuously monitor the column effluent.
Both aqueous and non aqueous samples can be analyzed with little or no sample pre treatment.
A variety of solvents and column packing are available, providing a high degree of selectivity for specific
analyses and it provides a means for determination of multiple components in a single analysis.
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Preparation of standard stock solution
Standard solution of withaferin A and Z-guggulsterone were prepared by dissolving 10 mg of
each standard in 10 mL methanol (1000 μg/mL).
Further dilutions were prepared from the stock solutions, by diluting a required volume of stock
solution with methanol to get a final concentration range of 50–250 mg/mL for withaferin A and
10–60 mg/mL for Z-guggulsterone.
Preparation of sample solution
1
• Powder equivalent to 10 tablets was weighed and extracted with methanol using a
Soxhlet apparatus.
• The extract was concentrated and filtered through Whatman filter paper no. 41.
2
• The filtrate was transferred to a 25-mL volumetric flask. Volume was made upto the
mark with mobile phase methanol.
• From above stock solution, 1 mL was transferred to volumetric flask of 10 mL
capacity and then diluted to 10 mL with methanol.
3
• The resulting solution was then analyzed by the proposed method.
• Each sample was injected and analyzed in triplicate.
Method of preparation:-
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Method Development:-
HPLC Instrumentation:- Jasco (Hachioji, Tokyo, Japan) system consisting of an intelligent pump
(PU-1580, PU-2080), a high-pressure mixer (MX-2080-31), a manual sample injection valve
(Rheodyne 7725i) equipped with a 20-mL loop and a UV– visible detector (UV-1575)
HPLC Column :- 250 mmx 4.6 mm i.d., 5-μm particle, Hibar LiChrocart Purospher Star RP-18
endcapped column
Mobile Phase :- Solvent A(Acetonitrile); Solvent B (Methanol)
0-7 min 50% Solvent A in B
7-9 min 50-80% Solvent Ain B
9-20 min 80% Solvent Ain B
Flow rate :- 1mL/min
Injection volume :- 20 µL
Detection wavelength :- 235nm
Retention time :- 5.32min (withaferin A) & 17.82 (Z-guggulsterone).
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Figure 4: HPLC chromatogram of withaferin A and Z-guggulsterone in standard mixture.
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Result and Discussion:-
1. Linearity:-
Different concentrations of the standard withaferin A and Z-guggulsterone solution was injected in triplicate and
linearity was evaluated.
Linearity of withaferin A and Z-guggulsterone was found over the concentration range of 50-250 µg/mL and 10-
60 µg/mL respectively.
The linear regression equation for two drugs : y = 14,987x -2 14,592 (r2 = 0.997) for withaferin A and y =
90,301x- 2 23,108 (r2 = 0.991) for Z-guggulsterone.
Summarized validation parameters of the developed method are given in Table II.
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2. Precision:-
The intra and inter-day studies were performed by injecting three different aliquots of the standard solution in
triplicate in a day and on three different days, respectively; the results were expressed as % RSD.
Intra- and inter-day precision in estimation of withaferin A showed RSD values 0.45% and 0.54% respectively.
Intra- and inter-day precision in estimation of Z-guggulsterone showed RSD values 2.12% and 1.26%
respectively.
RSD in the precision study for the withaferin A and Z-guggulsterone assay was < 2.0%, which confirmed that the
method was highly precise.
The results of precision are depicted in Table III.
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3.Accuracy:-
Standard drug solutions were added at three different concentration levels to a pre-analyzed sample solution.
The recovery rate was calculated using expected values obtained by adding known amounts of standards,
and actual values were obtained by HPLC analysis.
From the recovery study presented in Table I, it was clear that the method was very accurate for quantitative
estimation of withaferin A and Z-guggulsterone in a tablet dosage form, because all the statistical results
were within the acceptance range (i.e., RSD% < 3.0).
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4. Specificity:-
Specificity of the method was thus assessed by comparing the chromatograms obtained from
standard drugs (Figure 4) with the chromatograms obtained from tablet solution (Figure 5).
Because the retention time of standard drugs and the retention time of two drugs in sample
solutions were the same, the method was found to be specific.
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5. LOD & LOQ :-
LOD and LOQ were calculated based on the standard deviation (SD) of the response and the slope (S) of the
calibration curve according to the formulae: (LOD = 3.3 [SD/S] and LOQ = 10 [SD/S])
LOD and LOQ were derived from the linearity curve of withaferin A and Z-guggulsterone.
LOD and LOQ were found to be 1.76 μg/mL and 3.59 μg/mL for withaferin A and 0.38 μg/mL and 0.69 μg/mL
for Z-guggulsterone respectively (Table III). This indicated that the proposed method exhibits a good sensitivity
for the quantification of the above compound.
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Quantification of withaferin A and Z-guggulsteron from the polyherbal formulation
The developed and validated HPLC method was successfully used for quantification of withaferin A and
Z-guggulsteron from the polyherbal formulation .
The amounts of withaferin A and Z-guggulsterone in polyherbal formulation were found to be 0.740 and
0.106%, respectively (Figure 5).
Figure 5: HPLC chromatogram of Withaferin A and Z-guggulsterone in the formulation
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Conclusion:-
A new isocratic RP-HPLC method has been developed for simultaneous quantification of withaferin A
and Z-guggulsterone.
For the simultaneous measurement of withaferin A and Z-guggulsterone, a new isocratic RP-HPLC
method has been devised.
The key characteristics of this method are its low cost, environmental friendliness, faster analysis speed,
and satisfactory precision and accuracy.
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References:-
Poonam Agrawal, Rashmi Vegda, Kirti Laddha; Simultaneous Estimation of Withaferin A and Z-Guggulsterone
in Marketed Formulation by RP-HPLC; Journal of Chromatographic Science, (2015); 53: 940-944.
https://doi.org/10.1093/chromsci/bmu153