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1096

Jaldip et al. World Journal of Pharmacy and Pharmaceutical Sciences

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

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

and validated for the determination of Resveratrol and Piperine in

pharmaceutical compositions. The chromatographic separation was

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

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

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

Resveratrol and Piperine respectively. The Retention times of

Resveratrol and Piperine were found 2.808 and 3.508minute

respectively. The method was found to be linear over the range of 10-

50 µg/ml and 1-5 µg/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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Article Received on 10 March 2014, Revised on 05 April 2014,

Accepted on 26 April 2014

*Correspondence for Author

Jaldip Jasoliya

Department of Pharmaceutical

Sciences, Saurashtra

University, Rajkot Gujarat,

India-360005


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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′-trihydroxystilbene—possesses 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 RP-

HPLC 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 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 1000µg/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 µg/ml of Resveratrol and 100 µg/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 100µg/ml and 10µg/ml of Resveratrol and Piperine respectively. After that

filter with whatman filter paper to remove unwanted particle. Then the sample solution was

filtered through 0.45µm cellulose acetate filter paper (0.45µm).


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Preparation of Mobile Phase

Methanol was sonicated for 10 min and filter through 0.2µm 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 0.2µm 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 10-

50µg/ml and 1-5 µg/ml respectively. 1, 2, 3,4, 5 ml of stock solution(100µg/ml) of

Resveratrol and 1, 2, 3, 4, 5 ml of stock solution (10µg/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 µg/ml for Resveratrol and 1-5 µg/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 µg/ml for Resveratrol and 3

µg/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 µg/ml for Resveratrol and 3

µg/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 (10:1µg/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 Concentration(µg/Ml) Area

Resveratrol Piperine Resveratrol Piperine

1 30 3 4892096 609945 2 30 3 4893126 612689 3 30 3 4892259 598402 4 30 3 4900135 608931 5 30 3 4889580 616734 6 30 3 489627 599876

Mean 4893912 607762.8

SD 3734.64 7220.84

% RSD 0.076

1.18


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Table 2: Intraday Precision of Resveratrol and Piperine by HPLC

Table 3: Interday Precision of Resveratrol and Piperine by HPLC

Table 4: Recovery studies of Resveratrol and Piperine

Level of

Recovery Mean of % Recovery

Resveratrol Piperine 80% 99.79 100.073

100% 99.54 99.79 120% 99.32 99.69

Table 5: Assay Results for Tablets Using the Proposed Method

Formulation Amount of Amount of Amount found drug taken (mg) drug found (mg)% (n=3) ±SD

Capsules Resveratrol Piperine Resveratrol Piperine Resveratrol Piperine 1 10 1 10.083±0.035 0.997±0.0561 100.83±0.3511 99.68± 1.1229

Table 6: System suitability parameters by HPLC

S.no System suitability parameters Criteria Results

RESVERATROL PIPERINE 1 Theoretical plates More than 2000 3380.7 4001 2 Resolution More than 2 4.2 3 Tailing factor ≤2 1.701 1.11

Conc. (µg/Ml) Conc. (µg/Ml)

Mean Area ± SD (N=3)% %RSD Mean Area

± SD (N=3)% %RSD

Resveratrol Piperine Resveratrol Resveratrol Piperine Piperine 10 1 1640890±18533 1.134 199106±3612 1.81 30 3 4895087±47423 0.970 597214±6543 1.09 50 5 8190879±73955 0.902 975527±4447 0.45

Conc. (µg/ml)

Conc. (µg/ml)

Mean Area ± SD (n=3)% %RSD Mean Area

± SD (n=3)% %RSD

Resveratrol Piperine Resveratrol Resveratrol Piperine Piperine 10 1 1637450±16537.6 1.09 201106±2072 1.03 30 3 4905087±54823.2 1.11 602267±6822.4 1.13 50 5 8157179±63955.0 0.78 941127±7127.1 0.75


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Table 7: Summary of validation parameter for the proposed method

Parameter RP-HPLC method

RESVERATROL PIPERINE Linearity Range 10-50 µg/ml 1-5 µg/ml

Linearity equation Y=31170X+3786 y=34446X+44477 Correlation co efficient 0.999 0.999 Repeatability (%RSD) 0.076 % 1.18 %

Intraday (%RSD) 0.90-1.13% 0.45-1.81% Interday (%RSD) 0.78-1.11% 0.75-0.1.03%

% Recovery 99.49-100.25% 99.52-100.53% Robustness 0.43-0.97% 0.40-1.09%

Specificity(%RSD) 0.70-0.984% 0.80-1.068% LOD 0.26 µg/ml 0.18µg/ml LOQ 0.78 µg/ml 0.55µg/ml

%Assay 99.48% 100.02%

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 min

-2.5

-2.0

-1.5

-1.0

-0.5

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5mAU

254nm,4nm (1.00)1/

2/

3/

4/

5/ 6/

7/

Fig 3: Chromatogram of Blank Mobile Phase

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 min

-25

0

25

50

75

100

125

150

175

200

225

250

275mAU

306nm,4nm (1.00)

1/RT2

.779

2/RT3

.710

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


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0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 min

-50

0

50

100

150

200

250

300

350

400

450

500

mAU306nm,4nm (1.00)

1/

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

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 min-7.5

-5.0

-2.5

0.0

2.5

5.0

7.5

10.0

12.5

15.0

17.5

20.0

22.5

25.0

27.5

30.0

32.5

35.0

37.5

mAU340nm4nm (1.00)

1/

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

3.50 3.75 4.00 min

0

10

20

30

40

50

60

mAU

331nm326nm321nm316nm311nm306nm301nm296nm291nm286nm281nm

Fig 7: Peak purity of Piperine


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2.50 2.75 3.00 min

0

100

200

300

400

mAU

331nm326nm321nm316nm311nm306nm301nm296nm291nm286nm281nm

Fig 8: Peak purity of Resveratrol

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 min0

100000

200000

300000

400000

500000

600000

700000

800000

900000

1000000

1100000

uV

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 µg/ml (r2 =0.999) & and 1-5µg/ml (r2 =0.999) respectively

(Fig.6). The LOD and LOQ were found to be 0.26 and 0.78 µg/ml and 0.18and 0.55µg/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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