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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

2. Contents
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 Introduction
 Highlights
 Significance
 Method
 Result and Discussion
 Conclusion
 References

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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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Figure 3: Jasco (Hachioji, Tokyo, Japan) HPLC system

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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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Method Validation
Linearity
Precision
Accuracy
Specificity
Limit of Detection (LOD)
Limit of Quantification (LOQ)

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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

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Image References:-
1. https://www.google.com/search?q=withania+somnifera
2. https://www.google.com/search?q=commiphora+wightii
3. https://www.google.com/search?q=structure+of+withaferin
4. https://www.google.com/search?q=structure+of+z-guggulsterone
5. https://www.google.com/search?q=Jasco+HPLC+system

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