Asian Journal of Pharmaceutical Analysis.

8(2): April- June, 2018

ISSN 2231–5667 (Print) Available online at

2231–5675 (Online) www.anvpublication.org
DOI: 10.5958/2231-5675.2018.00020.0
www.asianpharmaonline.org
Vol. 08| Issue-02| Asian Journal of Pharmaceutical Analysis
April- June 2018
Home page www. ajpaonline.com

RESEARCH ARTICLE

Development and Validation of RP-HPLC Method for Simultaneous

Estimation of Piracetam and Vinpocetine
Payal Patil, Mukesh Patil, Dipak D Patil
H. R. Patel Institute of Pharmaceutical Education and Research, Near Karwand Naka, Shirpur, Dist. - Dhule,
Maharashtra 425405, India
*Corresponding Author E-mail: dipakpatil888@gmail.com

ABSTRACT:
Designed for the simultaneous estimation of Piracetam (PIRA) and Vinpocetine (VINP) combination a validated
Reversed Phase-High-Performance Liquid Chromatography (RP-HPLC) method was developed. The separation
and analysis were performed on a reversed phase C18 column (250 mm×4.6 mm) with particle size 5 μm as the
stationary phase. The mobile phase selected was consisting of potassium dihydrogen phosphate buffer (0.05M,
pH 6.0): methanol (50:50, v/v) with flow rate 1.0 ml/ min. For the detection of analytes, 225 nm was selected as
suitable wavelength. The retention times of 3.52 min and 7.41 min was found for PIRA and VINP respectively.
The proposed method exhibited good linearity over the concentration range of 80-480 μg/ml for PIRA and 2 - 12
μg/ml for VINP. The correlation coefficient (r2) value of 0.999 and 0.996 for PIRA and VINP were found
respectively. The ICH Q2 (R1) guidelines were followed for validation of this developed method. The %
recovery of piracetam and vinpocetine was found 101.38 ± 0.71 % and 102.04 ± 0.58 %. The % RSD for the
method was found to be less than 2 shows the technique is precise one. The developed method was precise,
accurate, robust, selective and rapid for simultaneous estimation of PIRA and VINP. Finally, the optimized
method was used for analysis of in-house bilayered tablet formulation. The percentage purity was found to be
103.63 ± 0.02 for VINP and 100.92 ± 0.67 for PIRA in a tablet.

KEYWORDS: Piracetam, Vinpocetine, Simultaneous estimation, RP-HPLC, Method validation.

Received on 12.12.2017 Accepted on 21.02.2018
© Asian Pharma Press All Right Reserved
Asian J. Pharm. Ana. 2018; 8(2):103-108.
DOI: 10.5958/2231-5675.2018.00020.0
INTRODUCTION:
Piracetam (2-oxo-1-pyrrolidine acetamide) is a
universally recognized as the ‘Smart’ or ‘nootropic’
drugs (Fig. 1a). It is a water-soluble cyclic derivative of
neurotransmitter GABA. 1-4 This drug is responsible to
improve memory and cognition, improve blood flow and
supply of oxygen to brain, delay brain aging, support
stroke recovery, and improve Down syndrome,
Alzheimer's, dementia, dyslexia and is also used for
schizophrenia treatment. It improves cognitive function
without leading to sedation or stimulation and also
protects the cerebral cortex against hypoxia.2 For the
analysis of piracetam in biological fluids various
methods were developed like thin layer densitometric

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determination , capillary electrophoresis and micellar HPLC Instrumentation

electrokinetic chromatography methods.5,6 In Indian The RP-HPLC system of Agilent Technologies 1200
Pharmacopoeia7 and British Pharmacopoeia 2003 a series consisted of gradient pump LC-20AC, Rheodyne
liquid chromatography method is mentioned for injector 7725I with 20 μl loop, UV-VIS detector SPD-
estimation piracetam as bulk drug.8 The piracetam 20A. The C18 Princeton SPHE-100 C-18 100 A
impurities using TLC and FT-IR spectroscopy was also (dimension: 250 mm×4.6 mm) with 5 μm particle size
determined. 9, 10 was selected as the stationary phase. The selected mobile
phase was composed of 0.05M potassium dihydrogen
Vinpocetine (VINP) is chemically related to and derived phosphate buffer (pH 6.0): methanol (50:50, v/v) with
from vincamine, an alkaloid found in the plant source 1.0 ml/ min of flow rate. The analytes detection was
Vinca minor L., (family: periwinkle). It is chemically, done at wavelength 225 nm.
14-ethoxycarbonyl-(3α, 16α-ethyl-14, 15-eburnamenine)
which is used as a vasodilating agent for the Other equipment used was Digital pH
management of stroke, parkinson disease and meter; Ultrasonicator (Enertech Electronics Pvt. Ltd),
Alzheimer’s disease (Fig. 1b).2,11 Vinpocetine can be Balance (Shimadzu AUX-120).
estimated in bulk drug form, or in amalgamation with
other drugs and in biological fluid using various Methods:
techniques such as HPLC method, spectrophotometric Preparation of in-house Piracetam and Vinpocetine
method and GC-MS (gas chromatographic-mass Bilayer tablet:
spectrometric) method.12-15 The bilayer tablet of Piracetam and Vinpocetine was
prepared using the wet granulation method as reported 2
On the basis of the literature, it was realized that a new by R. T. Jadhav et al. The bilayer tablet contains 400 mg
analytical method for piracetam and vinpocetine of Piracetam in one layer and 5 mg Vinpocetine in
simultaneous estimation was required to be developed as another layer. The excipients used for bilayer tablet
no method was reported for investigation of this includes maize starch, P.V.P K-30, sodium methyl
combination till date. Therefore authors planned to paraben, sodium propyl paraben, talc, sodium starch
develop a validated RP-HPLC method for simultaneous glycolate, magnesium stearate, aerosil, lactose,
estimation of piracetam and vinpocetine in bulk drug as microcrystalline cellulose. The prepared bilayer tablets
well as in-house developed bilayered tablet formulation. were used as a pharmaceutical formulation for further
analysis.

Preparation of Potassium dihydrogen phosphate

buffer:
Accurately weighed (1.701 gm) of potassium dihydrogen
phosphate was dissolved in doubled distilled water to
produce 250.0 ml potassium dihydrogen phosphate
buffer solution (0.05M). The pH was attuned to pH 6.0
with orthophosphoric acid.

Preparation of stock and standard solution

Figure 1. Chemical structures of (a) PIRA (b) VINP.
To prepare 1000 µg/mL PIRA and VINP stock solution,
100 mg of both drugs were accurately weighed and
dissolved separately in 100 ml methanol in volumetric
flasks. The standard solutions range of 80-480 µg/mL for
PIRA and in 1-6 µg/mL for VINP was prepared from
stock solution by the serial dilution.

EXPERIMENTAL: System suitability parameter

Chemicals and Reagents
Piracetam and Vinpocetine both acquired as generous
gift sample from M. J. Biopharma Pvt. Ltd, India.
Potassium dihydrogen phosphate and O-Phosphoric acid
of AR Grade, HPLC Grade Methanol, HPLC Grade
Acetonitrile all these chemicals were bought from Merck
Ltd., Mumbai, India.
In the HPLC method development checking system
suitability is a key parameter as it helps to authenticate
the efficiency of system for performing analysis. Various
factors like a resolution, capacity factor and number of
theoretical plates for PIRA and VINP were assessed as
presented in Table No. 1.

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Table 1 System Suitability Parameters and its average weight was calculated. All the tablets
Parameters PIRA VINP were triturated and an accurately weighed tablet powder
Retention time (tR) (min) 3.52 ± 0.02 7.41 ± 0.01
Peak Area 247127±1585 53467 ± 29.24
equivalent to about 40 mg of PIRA and 0.5 mg of VINP
Theoretical plate (N) 130771 ± 206 110689 ± 273 was shaken with 10 mL diluents and sonicated them for
Capacity factor (k’) 0.4 1.96 30 min. The volume was adjusted up to the mark using
Asymmetry 1.05 ± 0.015 1.09 ± 0.034 the diluents. The resulting solution was then filtered
Resolution 24.67 through the Whatman's filter paper; discarded first 5-6
Separation factor 4.9
mL of filtrate and then 0.4 mL of filtrate was diluted to
10.0 mL with diluents to obtain a concentration of 400
μg/mL of PIRA and 5 μg/mL of VINP respectively.
Linearity and Range
Initially, 100 μg/mL working standard solutions was Standard solution (4000 μg/mL of PIRA and 50
prepared. From this working standard, solution mixtures μg/mL of VINP)
in the range of 80–480 and 2–12 μg/ml for PIRA and Accurately weighed quantity of 0.5 mg VINP, 40 mg of
VINP respectively were acquired by diluting with PIRA was dissolved in 100 mL mobile phase in a
mobile phase. The samples were analysed thrice under volumetric flask.
optimized chromatographic conditions. The calibration
curves were plotted as the concentration of drug versus Working standard solution mixture (400 μg/mL of
the corresponding peak areas. The slope and Y intercept PIRA and 5 μg/mL of VINP)
value of the calibration curve were calculated. 16 From above standard solution (400 μg/mL of PIRA, 5
μg/mL of VINP), 1.0 mL was pipetted and diluted with
Application of the developed RP-HPLC method for mobile phase up to 10 mL.
evaluation of bilayered tablet
Analysis of PIRA and VINP from bilayered tablets Working solution
For quantification of PIRA and VINP in a bilayered Three sets of 10.0 mL volumetric flask were taken. To
tablet (label claim: 400 mg PIRA and 5 mg VINP per each set, the sample solution (0.8 mL) was pipetted. The
600 mg of the tablet) 20 tablets were taken and first set of 80 % of 3 volumetric flasks, to which 0.64
triturated. An amount of triturated powder corresponding mL of standard solution and working standard mixture
to 400 mg PIRA and 5 mg VINP was added to was added and the volume was adjusted to the mark. The
volumetric flask and dilute up to 50 mL with mobile second set 100 % of 3 volumetric flasks, to which 0.8
phase. The solution in volumetric flask was subjected to mL of standard solution and working standard mixture
sonication for 15 min to confirm thorough drug was added and the volume was attuned up to the mark.
extraction from tablet powder and then the volume was The third set of 120 % of 3 volumetric flasks, to which
made up to 100 mL with mobile phase. The resultant 0.96 mL of standard solution and Working standard
solution (4000 μg/mL of PIRA and 50 μg/mL of VINP) solution mixture was added and the volume was attuned
was passed through 0.45 µm filter paper. To up to the mark.
obtain sample solution containing 400 μg/mL of PIRA
and 5 μg/mL of VINP, 1 mL of above prepared solution Procedure
mixture was pipetted in a volumetric flask and diluted A fixed quantity 20.0 µl was injected as per optimized
upto 10 mL with mobile phase. This assay procedure chromatographic conditions for HPLC analysis and the
was repeated for five times. peak area was noted for each analysis for further
calculations.
Method validation
Validation of this proposed method was carried as per Precision
ICH Q2 (R1) guidelines considering different parameters Both the intraday and interday variation study was
like system suitability, linearity, range, precision, performed thrice. The results obtained were expressed in
accuracy, LOD, LOQ, Ruggedness and terms of % RSD (% relative standard deviation).
Robustness.15, 17, 18
LOD and LOQ
Accuracy The quantitation limit is a parameter of a quantitative
For PIRA and VINP, recovery study was carried out at assay for low levels of compounds in sample matrices
80 %, 100 % and 120 % of label claim using the and is used particularly for the determination of
standard addition method. 19 impurities and/or degradation products. The limit of
detection (LOD) and limit of quantitation (LOQ) were
Sample Solution determined using following formulae. 20, 21
Twenty tablets had been taken (each tablet containing
400 mg of PIRA and 5 mg of VINP) weighed separately

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230 wavelength regions and the concentration of VINP

is less as compared to PIRA. So, the wavelength of
detection λmax 225 nm was selected to have a better
simultaneous quantification of PIRA and VINP. The
optimized chromatographic conditions were capable of
resolving peak within short run time. The retention time
of piracetam and vinpocetine was found to be 3.52 min
and 7.41 min respectively (Fig. 2). A specific,
Specificity Study
reproducible and precise RP-HPLC method was
The specificity study of PIRA and VINP was performed
developed and optimized for simultaneous estimation
to check interference due to excipients like Maize starch,
of PIRA and VINP.
P.V.P K-30, sodium methyl paraben, sodium propyl
paraben, talc, sodium starch glycolate, magnesium
stearate, aerosil, lactose, microcrystalline cellulose, etc.
A precisely weighed amount of about 400 mg of PIRA
and 5 mg of VINP from a blend of both drugs and
excipients was dissolved in 100 ml methanol and further
subjected to sonication. The resulting solution was then
passed through Whatman filter paper for filtration.
Nearly 1.0 mL of above prepared solution was diluted
with diluents to three volumetric flasks with 10 mL
capacity. The solution was prepared was 400 ppm for
PIRA and 5 ppm for VINP. The samples were analyzed
by optimized HPLC method. The peak purity values of
each peak calculated to observe interference due to
commonly used excipients of tablet formulations. 22, 23 Figure 2. HPLC graph of PIRA AND VINP

Robustness study System suitability

It is a measure of ability of developed method to stay The parameters for a drug combination were evaluated
unaffected by small, but deliberate variation in method and outcomes are stated in Table No. 1. From the results
parameter and provide an assurance of its consistency obtained it was observed that good peak area (above
during usage. To determine the robustness of a method 10000) with a symmetric peak (asymmetry value more
the experimental condition was deliberately changed. than 1) was obtained. The resolution obtained using this
The results for intraday and interday precision for PIRA method was also good. Therefore the developed
and VINP were compared for estimation of ruggedness technique was found appropriate for simultaneous
of the method. To perform this study evaluation was estimation of both drugs with reproducible retention
carried out on two different HPLC systems by two time.
different analysts in same laboratory conditions. Along
with this for proving the robustness of the method study Linearity and Range
was carried out under diverse situations including Least squares linear regression was used to establish
alterations of analyst, flow rate and detector wavelength,
linearity of the standard curve. From results obtained it
etc. 24, 25 was observed that the established standard curve was
linear over 80–480 μg/mL concentration range for PIRA
RESULT AND DISCUSSION: (n=5) and 2 – 12 μg/mL concentration range for VINP
The primary purpose of this research was to develop an (n=5).
validated and optimised RP-HPLC technique for
simultaneous estimation of piracetam and vinpocetine in Application of the established RP-HPLC method for
bulk and pharmaceutical formulation. tablet formulation assessment
Analysis of PIRA and VINP from bilayered tablets
Method optimization The RP-HPLC chromatogram of tablet sample analysis
The chromatographic conditions were optimized as as depicted in Fig. No. 2. No sign of interference was
stationary phase (C18 Princeton SPHE-100 C-18 100 A observed from the commonly used tablet excipients that
(250 mm × 4.6 mm x 5 μm particle size). The mobile were added to the formulations. The result for % drug
phase comprising of potassium dihydrogen phosphate content of tablet was found to be 100.92 ± 0.68 for PIRA
buffer (0.05 M, pH 6.0): methanol (50:50, v/v) with flow and 103.63 ± 0.02 and VINP.
rate 1.0 ml/min, detection wavelength 225 nm at ambient
temperature. The VINP has a higher absorbance at 200-

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 Asian Journal of Pharmaceutical Analysis. 8(2): April- June, 2018

Method validation quantifying PIRA and VINP in tablet formulation even

Accuracy in presence of its excipients.
The analytical method recovery is the propinquity of the
test outcomes to the true value. It has been estimated by Robustness study
applying recovery studies to the analytical procedure It was confirmed that the developed method was robust
where low, medium and high concentrations were with respect to measured variations made in flow rate,
analyzed. wavelength, and analyst. The % RSD on the peak area
was less than 1 suggests the method is a robust one.
The percent drug recovered for PIRA was 101.38 ± 0.42,
101.93 ± 0.13 and 102.81 ± 0.89 at 80, 100 and 120 CONCLUSION:
percent. The mean percent recovery for PIRA was It was established that the RP-HPLC method suggested
102.04 ± 0.72 with mean % RSD value 0.70. The percent for quantitative analysis of PIRA and VINP combination
drug recovered for VINP was 100.57 ± 0.30, 101.25 ± is simple, inexpensive and reproducible. It has short
0.22 and 102.32 ± 0.08 at 80, 100 and 120 percent. The retention time (less than 10 min) that allows the analysis
mean percent recovery for VINP was 101.38 ± 0.88 with of many samples in less time. The method was precise,
mean % RSD value 0.87. The measurement was repeated accurate, linear, robust and sensitive. From the results of
thrice for samples at each level. The value of percent % recovery and relative standard deviation it was
recovered was within limit and % RSD value less than 1 reflected that that the developed technique is highly
show the developed method is an accurate one. precise and accurate. Furthermore, the technique could
easily be applicable to wide range of concentration,
Precision besides being less time consuming. The recommended
It articulates the degree of scattering between successive RP-HPLC method is applicable for analysis of
quantifications acquired from numerous sampling of the formulation like a tablet.
similar homogeneous sample under the recommended
conditions. The results of interday precision for PIRA ACKNOWLEDGEMENTS:
achieved were 100.34 ± 0.2535 with 0.0025 %RSD and The authors are thankful to M. J. Biopharma Pvt. Ltd,
intraday precision results obtained were 100.98 ± 1.0227 India for providing a gift sample of standard PIRA and
with 1.0128 % RSD. On the hand, For VINP the interday VINP. The authors also thankful to Principal, H. R. Patel
precision results obtained were 101.2 ± 1.333 with 1.317 Institute of Pharmaceutical Education and Research,
% RSD and intraday precision results obtained were Shirpur for providing necessary facilities to carry out the
100.7 ± 0.847 with 0.841 % RSD. work.

Limit of Detection (LOD) and Limit of Quantification REFERENCES:

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