WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES

Jaldip et al.

World Journal of Pharmacy and Pharmaceutical Sciences

Volume 3, Issue 5, 1096-1107.

Research Article

ISSN 2278 4357

METHOD DEVELOPMENT AND VALIDATION OF RP-HPLC

METHOD FOR SIMULTANEOUS ESTIMATION OF RESVERATROL
AND PIPERINE IN COMBINED CAPSULE DOSAGE FORM
Jaldip Jasoliya*, Aashka Jani
Department of Pharmaceutical Sciences, Saurashtra University, Rajkot Gujarat,
India-360005.
ABSTRACT

Article Received on
10 March 2014,

A simple, precise, cost effective RP-HPLC method has been developed

Revised on 05 April 2014,

and validated for the determination of Resveratrol and Piperine in

Accepted on 26 April 2014

pharmaceutical compositions. The chromatographic separation was

achieved on hibarR 250-4, C-18 columns (250mm 4.6mm,5um) using

*Correspondence for Author

a mobile phase consisting of Acetonitrile+water(95+5) : Methanol

(60:40v/v) with pH 4.0 adjusted with Acetic acid at a flow rate of

Jaldip Jasoliya
Department of Pharmaceutical

1ml/min. Detection wavelength was found 305 and 336 nm for

Sciences, Saurashtra

Resveratrol and Piperine respectively. The Retention times of

University, Rajkot Gujarat,

Resveratrol and Piperine were found 2.808 and 3.508minute

India-360005

respectively. The method was found to be linear over the range of 1050 g/ml and 1-5 g/mlwith correlation co-efficient (r2) 0.999 & 0.999

for Resveratrol and Piperine respectively. Percentage recoveries obtained for both the drugs
were 99.49-100.25% and 99.52-100.53% for Resveratrol and Piperine respectively. The
%RSD for precision and accuracy of the method was found to be less than 2%.The method
was validated according to the ICH guidelines with respect to specificity, linearity, accuracy,
precision and robustness. The method developed can be used for the routine analysis of
Resveratrol and Piperine from their combined dosage form.
Key Words: Resveratrol, Piperine, ICH guideline, simultaneous determination, RP-HPLC
method.
INTRODUCTION
The international conference on harmonization (ICH) guide-lines emphasizes that the purity
and assay of drug susceptible to change during storage, must be determined by using
validated

methods. In recent year there is interest in the possible role of nutrient in

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prevention of disease.In context of antioxidant, especially derived from natural sources such
as Indian medicinal plant and herbal drugs derived from them require special attention.
Antioxidant have many potential application especially in relation to human health, both in
term of prevention and treatment of disease.
Resveratrol is chemically 3,5,4-trihydroxystilbenepossesses antioxidant activities in
vitro. It is obtained from Roots of polygonum cuspidatum and Leaves of veratrum
grandiflorum. It dose-dependently inhibited the generation of peroxyl, hydroxyl, peroxides,
and lipid peroxidation products in cell free systems. Oxidative burst of whole human blood
stimulated with PMA, fMLP, OpZ, and A23187 was inhibited in a concentration-dependent
way, indicating suppression of both receptor and nonreceptor activated chemiluminescence
by resveratrol. Results from isolated human neutrophils revealed that resveratrol was active
extracellularly as well as intracellularly in inhibiting the generation of reactive oxygen
species. Resveratrol is useful as Anti-cancer, Anti-diabetic, Anti-inflammatory, Anti-viral,
Cardioprotective effect, Neuroprotective effect etc.., Molecular weight of Resveratrol is
228.4 gm/mol and formula is C14H12O3.
.

Piperine is chemically 1-[5-(1,3-Benzodioxol-5-yl)-1-oxo-2,4-pentadienyl] piperidine. It is

obtained from Fruits of piper longum and

piper nigrum. When Piperine is added with

Resveratrol, It increaseas its bioavailability and intracellular residency timefound that the
degree of exposure (i.e. AUC) to resveratrol was enhanced to 229% and the maximum serum
concentration (C(max) ) was increased to 1544% with the addition of piperine. it is official in
Indian pharmacopeia 2010. Molecular weight of Piperine is 285.34 gm/mol and formula is
C17H19NO3. Literature Review revels that there was no reported Stability Indicating RPHPLC method for Simultaneous Estimation of Resveratrol and Piperine in combined dosage
form

[9-13]

. So the present work is aimed for To develop an accurate, specific, repeatable

Stability Indicating RP_HPLC method for Simultaneous estimation of Resveratrol and

Piperine in bulk and Pharmaceutical Dosage form.

Fig1:Chemical Structure of Resveratrol

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Fig2:Chemical Structure of Piperine

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MATERIALS AND METHODS

Experimental Instruments and Apparatus
The chromatography was performed on Shimadzu instrument equipped with PDA detector
and LC Solution software, hibarR 250-4 C-18 columns (250mm 4.6mm, 5um) was used as
stationary phase. Shimadzu analytical balance and ultra sonicator were used during the
research work.
Reagents and materials
Standard samples of Resveratrol and Piperine were obtained from Sami labs Pvt. Ltd, (
Bangalore).Combination capsule formulation containing Resveratrol200 mg and Piperine 20
mg was procured from Metabolic maintainance. Triple distilled water, Acetonitrile,
Methanol, and 0.45 membrane filters (Millipore) used were of HPLC grade. Acetic Acid used
were AR grade.
Chromatographic Conditions
The mobile phase containingAcetonitrile+water(95+5ml) : Methanol (60:40v/v) with pH 4.0
adjusted with Acetic acidat a flow rate of 1ml/min..Detection wavelength was found305nm
and 336nmrespectively. The Retention times of Resveratrol and Piperine were found 2.805
and 3.505 minutes respectively.
Preparation of working standard
10mg of each of Resveratrol and Piperine were weighed separately and transferred in two
different 100 ml volumetric flasks. Both the drugs were dissolved in 10 ml of Methanol by
ultra sonication and then volume was made up to the mark with Methanol to obtain final
concentration 1000g/ml of each component. 1ml of stock solution was withdrawn
&transferred in 10 ml volumetric flask and volume was made up with methanol to achieve
100 g/ml of Resveratrol and 100 g/ml of Piperine.
Preparation of Sample Solution
Powder of tablet formulation equivalent to 10mg of Resveratrol and 1mg of Piperine were
transferred to a 100ml volumetric flask and volume was made up with methanol. The mixture
was mixed and sonicated for 10 min and made up to the mark with methanol, to get final
concentration of 100g/ml and 10g/ml of Resveratrol and Piperine respectively. After that
filter with whatman filter paper to remove unwanted particle. Then the sample solution was
filtered through 0.45m cellulose acetate filter paper (0.45m).

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Preparation of Mobile Phase

Methanol was sonicated for 10 min and filter through 0.2m syringe filter and kept in bottle
B. then 95 ml of Acetonitrile is mixed with 5 ml of Water, then pH was adjusted to 4 with
Acetic Acid filter through 0.2m syringe filter and kept in bottle A. and these components
were used as mobile phase in 60:40 v/v (A:B) respectively in gradient elution mode.
Calibration curve of standard solution
Calibration curve of Resveratrol and Piperine were plotted over concentration range of 1050g/ml and 1-5 g/ml respectively. 1, 2, 3,4, 5 ml of stock solution(100g/ml) of
Resveratrol and 1, 2, 3, 4, 5 ml of stock solution (10g/ml) of Piperine were transferred in
10ml volumetric flask and volume were made up with methanol up to 10ml. Each solution
was injected in to column to get chromatogram.
Validation of the method
Validation of the optimized RP-HPLC method was carried out with respect to the following
parameters.
Linearity
The linear response of was determined by analyzing six independent levels of the calibration
curve in the range of 10-50 g/ml for Resveratrol and 1-5 g/ml for Piperine. Result should
be expressed in terms of Correlation co-efficient.
Precision
A) Repeatability
Precision of method was determined by analyzing 6 different solution of same concentration
at same time and relative standard deviation is shown in table1 .
B) Intra-day precision
Variation of results within same day is called Intra-day precision. The Intra-day precision was
determined by analyzing 3 different solution of concentration 30 g/ml for Resveratrol and 3
g/ml for Piperine at variant times of same day on Relative standard deviation (%RSD) is
given in table 2.
C) Inter-day precision
Variation of results among days called Inter-day precision. The Inter-day precision was
determined analyzing 3 different solution of concentration 30 g/ml for Resveratrol and 3
g/ml for Piperineat different days (n=3) it is given in table 3.

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Accuracy
The accuracy of the methods was determined by calculating recoveries of Resveratrol and
Piperine by the standard addition method.
LOD&LOQ
The limit of detection (LOD) and the limit of quantification (LOQ) of the Resveratrol and
Piperine were calculated using the standard deviation of responses (N) and slopes (S) of
respective calibration curves using signal-to-noise ratio.
LOD = 3.3 N/SLOQ = 10 N/S
Robustness
The evaluation of robustness should be considered during the development phase and
depends on the type of procedure under study. It should show the reliability of an analysis
with respect to deliberate variations in method parameters. Robustness of method is
determine by change in Saturation Time, Analytical Wavelength & mobile phase composition
there is no change in peak area & shape data show in table 4.
Assay of the tablet dosage form
The proposed validated method was successfully applied to determine Resveratrol and
Piperine in capsule dosage form. Three replicates of sample solution (10:1g/ml of each)
were injected. From the peak area of the Resveratrol and Piperine, amounts of drugs in
samples were computed. %Assay is given in to table 5.
RESULTS AND DISCUSSION
Table 1 :Data of Repeatability of Resveratrol and Piperine by HPLC(n=6)
Sr.No

1
2
3
4
5
6
Mean
SD
%
RSD

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Concentration(g/Ml)
Resveratrol

Piperine

30
30
30
30
30
30

3
3
3
3
3
3

Area
Resveratro
Piperine
l
4892096
609945
4893126
612689
4892259
598402
4900135
608931
4889580
616734
489627
599876
4893912
607762.8
3734.64
7220.84
0.076
1.18

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Table 2: Intraday Precision of Resveratrol and Piperine by HPLC

Conc. (g/Ml)
Resveratrol
10
30
50

Conc.
(g/Ml)
Piperine
1
3
5

Mean Area
SD (N=3)%
Resveratrol
164089018533
489508747423
819087973955

Mean Area
SD (N=3)%
Piperine
1991063612
5972146543
9755274447

%RSD
Resveratrol
1.134
0.970
0.902

%RSD
Piperine
1.81
1.09
0.45

Table 3: Interday Precision of Resveratrol and Piperine by HPLC

Conc.
(g/ml)

Conc.
(g/ml)

Mean Area
SD (n=3)%

%RSD

Mean Area
SD (n=3)%

%RSD

Resveratrol

Piperine

Resveratrol

Resveratrol

Piperine

Piperine

10
30
50

1
3
5

163745016537.6
490508754823.2
815717963955.0

1.09
1.11
0.78

2011062072
6022676822.4
9411277127.1

1.03
1.13
0.75

Table 4: Recovery studies of Resveratrol and Piperine

Level of
Recovery
80%
100%
120%

Mean of % Recovery
Resveratrol Piperine
99.79
100.073
99.54
99.79
99.32
99.69

Table 5: Assay Results for Tablets Using the Proposed Method

Formulation Amount of
drug taken (mg)

Amount of
drug found (mg)%

Amount found
(n=3) SD

Capsules Resveratrol Piperine Resveratrol Piperine

Resveratrol
Piperine
10.0830.035 0.9970.0561 100.830.3511 99.68 1.1229
1
10
1
Table 6: System suitability parameters by HPLC
S.no

System suitability parameters

Criteria

1
2
3

Theoretical plates
Resolution
Tailing factor

More than 2000

More than 2
2

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Results
RESVERATROL PIPERINE
3380.7

4001
4.2

1.701

1.11

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Table 7: Summary of validation parameter for the proposed method

Parameter
Linearity Range
Linearity equation
Correlation co efficient
Repeatability (%RSD)
Intraday (%RSD)
Interday (%RSD)
% Recovery
Robustness
Specificity(%RSD)
LOD
LOQ
%Assay

RP-HPLC method
RESVERATROL
PIPERINE
10-50 g/ml
1-5 g/ml
Y=31170X+3786 y=34446X+44477
0.999
0.999
0.076 %
1.18 %
0.90-1.13%
0.45-1.81%
0.78-1.11%
0.75-0.1.03%
99.49-100.25%
99.52-100.53%
0.43-0.97%
0.40-1.09%
0.70-0.984%
0.80-1.068%
0.26 g/ml
0.18g/ml
0.78 g/ml
0.55g/ml
99.48%
100.02%
2/

mAU
3.5 254nm,4nm (1.00)

3.0

2.5

2.0

3/

1.5

7/

5/

0.5

6/

1/

4/

1.0

0.0

-0.5

-1.0

-1.5

-2.0

-2.5

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

min

4.5

min

Fig 3: Chromatogram of Blank Mobile Phase

mAU
306nm,4nm (1.00)

1/RT2.779

275

250

225

200

175

150

125

100

75

2/RT3.710

50

25

-25

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

Fig 4 : Chromatogram of RESVERATROL&PIPERINE Standard Solution(10:1ppm)

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1/

mAU
306nm,4nm (1.00)
500

450

400

350

300

250

200

150

100

50

-50

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

min

Fig 5: Chromatogram of RESVERATROL Standard solution (10 PPM)

1/

mAU
340nm4nm (1.00)
37.5
35.0
32.5
30.0
27.5
25.0
22.5
20.0
17.5
15.0
12.5
10.0
7.5
5.0
2.5
0.0
-2.5
-5.0
-7.5
0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

mi n

Fig 6: Chromatogram of PIPERINE Standard solution (1 PPM)

mAU
281nm
60 286nm
291nm
296nm
50 301nm
306nm
311nm
40 316nm
321nm
326nm
30 331nm
20
10
0
3.50

3.75

4.00

min

Fig 7: Peak purity of Piperine

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mAU
281nm
286nm
291nm
400 296nm
301nm
306nm
311nm
300 316nm
321nm
326nm
331nm
200

100

0
2.50

2.75

3.00

min

Fig 8: Peak purity of Resveratrol

uV
1100000
1000000
900000
800000
700000
600000
500000
400000
300000
200000
100000
0
0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

min

Fig 9: Chromatogram of Linearity of RESVERATROL and PIPERINE Solution

Fig 10: Calibration graph for the Resveratrol (Peak Area Vs Conc.)

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Fig 11 : Calibration graph for the Piperine (peak area VsConc.)

DISCUSSION
RP-HPLC method was optimized with a view to develop a simple, accurate method for
estimation of drug in pharmaceutical formulation and in bulk drug. UV scanning at 200-450
nm for both Resveratrol and Piperine show that 305 and 335nm is the suitable wavelength for
detection of drugs.(Fig.3) The mobile phase of Acetonitrile+Water (95+5v/v) ph:4 adjustedby
Acetic acid: Methanol(60:40)at a flow rate of 1ml/min was selected because it gave highest
resolution, minimum tailing and Rt values of 2.808 and 3.505 min for RESVERATROLand
PIPERINE respectively (Fig.5). Resveratrol and Piperine both showed linearity in the
concentration range of 10-50 g/ml (r2 =0.999) & and 1-5g/ml (r2 =0.999) respectively
(Fig.6). The LOD and LOQ were found to be 0.26 and 0.78 g/ml and 0.18and 0.55g/ml,
respectively for Resveratrol and Piperine. Repeatability of measurement of peak area was
determined by six replicate and six time measurement of working standard of Resveratrol and
Piperine and %RSD was found to be 1.61 and 1.13% respectively (Table.1). Intraday & Inter
day Precision was found below 2%. (Table 2&3). Recovery studies of the drugs were carried
out for the accuracy parameter. These studies were carried out at three levels (80%, 100%,
and 120%) by standard addition method. Recovery was found to be 99.49-100.25%% and
99.52-100.53% for Resveratrol and Piperine respectively (Table 4). To confirm the
specificity, The standard solution with varying amount of Lactose was added. There is no
interference of peak Piperine and Resveratrol in presence of Lactose (Excipient) was noticed,
indicating the specificity of the method. The assay values for Resveratrol and Piperine are
presented in (Table 5). The result of dosage form analysis by developed method was
compatible with the labelled amount of each component of tablet. There was no interference

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of excipient for analysis of dosage form.

CONCLUSION
The proposed HPLC method is one of the simple, rapid, reproducible, accurate
andeconomical method for determination of Resveratrol and Piperine in pharmaceutical
formulations simultaneously. This method can reduce the time for routinequality control
analysis of Resveratrol and Piperine in their dosageform.
ACKNOWLEDGEMENTS
The authors would like to thank, Sami Labs Pvt. Ltd. Bangalore, India for providing a gift
sample of standard Resveratrol and Piperine. The authors would like to thank to Department
of Pharmaceutical Scinces, Saurashtra University,Rajkot, India for providing necessary
facilities to carry out the work.
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