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 determination , capillary electrophoresis and micellar electrokinetic chromatography methods.5,6 In Indian Pharmacopoeia7 and British Pharmacopoeia 2003 a liquid chromatography method is mentioned for estimation piracetam as bulk drug.8 The piracetam impurities using TLC and FT-IR spectroscopy was also determined. 9, 10
Vinpocetine (VINP) is chemically related to and derived from vincamine, an alkaloid found in the plant source Vinca minor L., (family: periwinkle). It is chemically, 14-ethoxycarbonyl-(3α, 16α-ethyl-14, 15-eburnamenine) which is used as a vasodilating agent for the management of stroke, parkinson disease and Alzheimer’s disease (Fig. 1b).2,11 Vinpocetine can be estimated in bulk drug form, or in amalgamation with other drugs and in biological fluid using various techniques such as HPLC method, spectrophotometric method and GC-MS (gas chromatographic-mass spectrometric) method.12-15
On the basis of the literature, it was realized that a new analytical method for piracetam and vinpocetine simultaneous estimation was required to be developed as no method was reported for investigation of this combination till date. Therefore authors planned to develop a validated RP-HPLC method for simultaneous estimation of piracetam and vinpocetine in bulk drug as well as in-house developed bilayered tablet formulation.
Figure 1. Chemical structures of (a) PIRA (b) VINP.
EXPERIMENTAL:
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.
HPLC Instrumentation
The RP-HPLC system of Agilent Technologies 1200 series consisted of gradient pump LC-20AC, Rheodyne injector 7725I with 20 μl loop, UV-VIS detector SPD-20A. The C18 Princeton SPHE-100 C-18 100 A (dimension: 250 mm×4.6 mm) with 5 μm particle size was selected as the stationary phase. The selected mobile phase was composed of 0.05M potassium dihydrogen phosphate buffer (pH 6.0): methanol (50:50, v/v) with 1.0 ml/ min of flow rate. The analytes detection was done at wavelength 225 nm.
Other equipment used was Digital pH meter; Ultrasonicator (Enertech Electronics Pvt. Ltd), Balance (Shimadzu AUX-120).
Methods:
Preparation of in-house Piracetam and Vinpocetine Bilayer tablet:
The bilayer tablet of Piracetam and Vinpocetine was prepared using the wet granulation method as reported2 by R. T. Jadhav et al. The bilayer tablet contains 400 mg of Piracetam in one layer and 5 mg Vinpocetine in another layer. The excipients used for bilayer tablet includes maize starch, P.V.P K-30, sodium methyl paraben, sodium propyl paraben, talc, sodium starch glycolate, magnesium stearate, aerosil, lactose, microcrystalline cellulose. The prepared bilayer tablets 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
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.
System suitability parameter
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.
Table 1 System Suitability Parameters
|
Parameters |
PIRA |
VINP |
|
Retention time (tR) (min) |
3.52 ± 0.02 |
7.41 ± 0.01 |
|
Peak Area |
247127±1585 |
53467 ± 29.24 |
|
Theoretical plate (N) |
130771 ± 206 |
110689 ± 273 |
|
Capacity factor (k’) |
0.4 |
1.96 |
|
Asymmetry |
1.05 ± 0.015 |
1.09 ± 0.034 |
|
Resolution |
24.67 |
|
|
Separation factor |
4.9 |
|
Linearity and Range
Initially, 100 μg/mL working standard solutions was prepared. From this working standard, solution mixtures in the range of 80–480 and 2–12 μg/ml for PIRA and VINP respectively were acquired by diluting with mobile phase. The samples were analysed thrice under optimized chromatographic conditions. The calibration curves were plotted as the concentration of drug versus the corresponding peak areas. The slope and Y intercept value of the calibration curve were calculated. 16
Application of the developed RP-HPLC method for evaluation of bilayered tablet
Analysis of PIRA and VINP from bilayered tablets
For quantification of PIRA and VINP in a bilayered tablet (label claim: 400 mg PIRA and 5 mg VINP per 600 mg of the tablet) 20 tablets were taken and triturated. An amount of triturated powder corresponding to 400 mg PIRA and 5 mg VINP was added to volumetric flask and dilute up to 50 mL with mobile phase. The solution in volumetric flask was subjected to sonication for 15 min to confirm thorough drug extraction from tablet powder and then the volume was made up to 100 mL with mobile phase. The resultant solution (4000 μg/mL of PIRA and 50 μg/mL of VINP) was passed through 0.45 µm filter paper. To obtain sample solution containing 400 μg/mL of PIRA and 5 μg/mL of VINP, 1 mL of above prepared solution mixture was pipetted in a volumetric flask and diluted upto 10 mL with mobile phase. This assay procedure was repeated for five times.
Method validation
Validation of this proposed method was carried as per ICH Q2 (R1) guidelines considering different parameters like system suitability, linearity, range, precision, accuracy, LOD, LOQ, Ruggedness and Robustness.15, 17, 18
Accuracy
For PIRA and VINP, recovery study was carried out at 80 %, 100 % and 120 % of label claim using the standard addition method. 19
Sample Solution
Twenty tablets had been taken (each tablet containing 400 mg of PIRA and 5 mg of VINP) weighed separately and its average weight was calculated. All the tablets were triturated and an accurately weighed tablet powder equivalent to about 40 mg of PIRA and 0.5 mg of VINP was shaken with 10 mL diluents and sonicated them for 30 min. The volume was adjusted up to the mark using the diluents. The resulting solution was then filtered through the Whatman's filter paper; discarded first 5-6 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.
Standard solution (4000 μg/mL of PIRA and 50 μg/mL of VINP)
Accurately weighed quantity of 0.5 mg VINP, 40 mg of PIRA was dissolved in 100 mL mobile phase in a volumetric flask.
Working standard solution mixture (400 μg/mL of PIRA and 5 μg/mL of VINP)
From above standard solution (400 μg/mL of PIRA, 5 μg/mL of VINP), 1.0 mL was pipetted and diluted with mobile phase up to 10 mL.
Working solution
Three sets of 10.0 mL volumetric flask were taken. To each set, the sample solution (0.8 mL) was pipetted. The first set of 80 % of 3 volumetric flasks, to which 0.64 mL of standard solution and working standard mixture was added and the volume was adjusted to the mark. The second set 100 % of 3 volumetric flasks, to which 0.8 mL of standard solution and working standard mixture was added and the volume was attuned up to the mark. The third set of 120 % of 3 volumetric flasks, to which 0.96 mL of standard solution and Working standard solution mixture was added and the volume was attuned up to the mark.
Procedure
A fixed quantity 20.0 µl was injected as per optimized chromatographic conditions for HPLC analysis and the peak area was noted for each analysis for further calculations.
Precision
Both the intraday and interday variation study was performed thrice. The results obtained were expressed in terms of % RSD (% relative standard deviation).
LOD and LOQ
The quantitation limit is a parameter of a quantitative assay for low levels of compounds in sample matrices and is used particularly for the determination of impurities and/or degradation products. The limit of detection (LOD) and limit of quantitation (LOQ) were determined using following formulae. 20, 21
Specificity Study
The specificity study of PIRA and VINP was performed to check interference due to excipients like Maize starch, 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 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
Robustness study
It is a measure of ability of developed method to stay unaffected by small, but deliberate variation in method parameter and provide an assurance of its consistency during usage. To determine the robustness of a method the experimental condition was deliberately changed. The results for intraday and interday precision for PIRA and VINP were compared for estimation of ruggedness of the method. To perform this study evaluation was carried out on two different HPLC systems by two different analysts in same laboratory conditions. Along with this for proving the robustness of the method study was carried out under diverse situations including alterations of analyst, flow rate and detector wavelength, etc. 24, 25
RESULT AND DISCUSSION:
The primary purpose of this research was to develop an validated and optimised RP-HPLC technique for simultaneous estimation of piracetam and vinpocetine in bulk and pharmaceutical formulation.
Method optimization
The chromatographic conditions were optimized as stationary phase (C18 Princeton SPHE-100 C-18 100 A (250 mm × 4.6 mm x 5 μm particle size). The mobile phase comprising of potassium dihydrogen phosphate buffer (0.05 M, pH 6.0): methanol (50:50, v/v) with flow rate 1.0 ml/min, detection wavelength 225 nm at ambient temperature. The VINP has a higher absorbance at 200-230 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, reproducible and precise RP-HPLC method was developed and optimized for simultaneous estimation of PIRA and VINP.
Figure 2. HPLC graph of PIRA AND VINP
System suitability
The parameters for a drug combination were evaluated and outcomes are stated in Table No. 1. From the results obtained it was observed that good peak area (above 10000) with a symmetric peak (asymmetry value more than 1) was obtained. The resolution obtained using this method was also good. Therefore the developed technique was found appropriate for simultaneous estimation of both drugs with reproducible retention time.
Linearity and Range
Least squares linear regression was used to establish linearity of the standard curve. From results obtained it was observed that the established standard curve was linear over 80–480 μg/mL concentration range for PIRA (n=5) and 2 – 12 μg/mL concentration range for VINP (n=5).
Application of the established RP-HPLC method for tablet formulation assessment
Analysis of PIRA and VINP from bilayered tablets
The RP-HPLC chromatogram of tablet sample analysis as depicted in Fig. No. 2. No sign of interference was observed from the commonly used tablet excipients that were added to the formulations. The result for % drug content of tablet was found to be 100.92 ± 0.68 for PIRA and 103.63 ± 0.02 and VINP.
Method validation
Accuracy
The analytical method recovery is the propinquity of the test outcomes to the true value. It has been estimated by applying recovery studies to the analytical procedure where low, medium and high concentrations were analyzed.
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 percent. The mean percent recovery for PIRA was 102.04 ± 0.72 with mean % RSD value 0.70. The percent drug recovered for VINP was 100.57 ± 0.30, 101.25 ± 0.22 and 102.32 ± 0.08 at 80, 100 and 120 percent. The mean percent recovery for VINP was 101.38 ± 0.88 with mean % RSD value 0.87. The measurement was repeated thrice for samples at each level. The value of percent recovered was within limit and % RSD value less than 1 show the developed method is an accurate one.
Precision
It articulates the degree of scattering between successive quantifications acquired from numerous sampling of the similar homogeneous sample under the recommended conditions. The results of interday precision for PIRA achieved were 100.34 ± 0.2535 with 0.0025 %RSD and intraday precision results obtained were 100.98 ± 1.0227 with 1.0128 % RSD. On the hand, For VINP the interday precision results obtained were 101.2 ± 1.333 with 1.317 % RSD and intraday precision results obtained were 100.7 ± 0.847 with 0.841 % RSD.
Limit of Detection (LOD) and Limit of Quantification (LOQ)
Limit of Detection (LOD) and Limit of Quantitation (LOQ) estimations were performed to check the sensitivity of proposed method, by using formula. The results for Limit of Detection and Limit of Quantification for PIRA and VINP were 0.00456 & 0.013068 µg/mL and 0.04531& 0.1256 µg/mL respectively.
Specificity Study
There was no any extract peak or interference was observed in the analysis of PIRA and VINP. The peak purity value was determined for PIRA and VINP peak using photodiode array detector. The up slope and down slope similarity for the peak of PIRA was found 0.9999 and 1.0000 with threshold value 0.9900. The 3 point peak purity for PIRA was 0.9999 (threshold value 0.9900). The up slope and down slope similarity for the peak of VINP was found 0.9999 and 0.9996 with threshold value 0.9900. The 3 point peak purity for VINP was 0.9997 (threshold value 0.9900). The total peak purity value 1.00 was found for both drugs. There was no mark of interference due to the existence of tablet excipients. The proposed method is capable of quantifying PIRA and VINP in tablet formulation even in presence of its excipients.
Robustness study
It was confirmed that the developed method was robust with respect to measured variations made in flow rate, wavelength, and analyst. The % RSD on the peak area was less than 1 suggests the method is a robust one.
CONCLUSION:
It was established that the RP-HPLC method suggested for quantitative analysis of PIRA and VINP combination is simple, inexpensive and reproducible. It has short retention time (less than 10 min) that allows the analysis of many samples in less time. The method was precise, accurate, linear, robust and sensitive. From the results of % recovery and relative standard deviation it was reflected that that the developed technique is highly precise and accurate. Furthermore, the technique could easily be applicable to wide range of concentration, besides being less time consuming. The recommended RP-HPLC method is applicable for analysis of formulation like a tablet.
ACKNOWLEDGEMENTS:
The authors are thankful to M. J. Biopharma Pvt. Ltd, India for providing a gift sample of standard PIRA and VINP. The authors also thankful to Principal, H. R. Patel Institute of Pharmaceutical Education and Research, Shirpur for providing necessary facilities to carry out the work.
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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