Development and Validation of Stability Indicating RP-HPLC Method for Piracetam

 

Sushil D. Patil¹*, Prajkta Varpe², Sayali Chaure², Siddharth B. Bhalerao², Sanjay Kshirsagar²

^1*Department of Pharmaceutical Chemistry, MET's Institute of Pharmacy, Bhujbal Knowledge City, Adgaon Nashik, Maharashtra, India.

²Savitribai Phule Pune University, Pune, Maharshtra state, India

 

ABSTRACT:

A simple, rapid and validated HPLC method was developed for determination of Piracetam in film coated tablets. A Grace C18 (250mm x 4.6ID, Particle size: 5 micron) RP-18 column with a mobile phase consisting of Methanol: Water (20:80v/v) was used. Quantitative evaluation was performed at 205 nm. The HPLC method is selective, precise and accurate and can be used for routine analysis of preparations in pharmaceutical industry quality control laboratories.

 

 

 

INTRODUCTION:

Piracetam (2-oxo-pyrrolidin-1-yl)-acetamide is a nootropic psychopharmacological agent having a variety of physiological effects that may result from the restoration of cell membrane fluidity. It is used for treatment of patients suffering from pathological, neurosensitive and cognitive deficits, brain-organic psychosyndromes (e.g., primary degenerative dementia), vertigo, and myoclonus of cortical origin. Piracetam is a white powder and is freely solu­ble in water, soluble in alcohol, and slightly soluble in methylene chloride. Literature survey revealed HPLC methods were developed for the estimation of Piracetam in biological fluids. Capillary electrophoresis, thin layer densi­tometric determination, micelle electro kinetic chromatography methods were also developed for the estimation of piracetam in biological fluids.[1,3-4]

 

Structure of Piracetam.

 

EXPERIMENTAL:

Material and Reagents:

Methanol  (HPLC grade), Water (HPLC grade), Concentrated Hydrochloric acid (AR grade), Sodium Hydroxide (AR grade), Hydrogen Peroxide (AR grade), All the chemicals were purchased from Thomas Baker (Chemicals), Mumbai (India).Membrane filter paper was used of 0.45 m purchased from Milipore Pvt. Ltd., Peenya, Bangalore, India.

 

Instrumentation and Chromatographic Condition:

JASCO, Binary high pressure gradient RP-HPLC Equipped with UV-3000-M.detector was employed in this method development, force degradation study, and method validation. PumpP-3000-M Reciprocating (40MPa), Rheodyne manual loop injector (20 µL), Analytical column –Grace C18 (250mm x 4.6ID, Particle size: 5 micron), Separation was perform by using Methanol: Water (20:80) (pH 7). Mobile Phase filtered through 0.45 μm membrane filter (0.45 μ, Millipore) and degassed in ultrasonic bath. Injection volume 20 µL, Flow rate 0.8 ml/min. UV detection carried out at 205 nm.

 

Preparation of Standard Solution:

An accurately weighed quantity of 10 mg Piracetam was transferred to 10 mL volumetric flask, dissolved with sufficient quantity of methanol and volume was then  made up to  the mark with same solvent and sonicated for 15 min. From the resulting solution 0.1mL was transferred to 10 mL volumetric flask and the volume was made up to the mark with same solvent. The resulting 10 µg/mL of solution was subjected to chromatographic analyses using mobile phases of different strengths.

 

Sample Solution Preparation:

To determine the content of Piracetam in marketed tablets (label claim 800 mg per tablet), twenty tablets were weighed, and average weight was calculated. Tablets were triturated and powder equivalent to 10 mg of Piracetam was weighed. The drug was extracted from the tablet powder with 100 ml methanol. To ensure complete extraction it was sonicated for 15 min. 0.1mL of supernatant was then diluted up to 10 ml with mobile phase.

 

Method validation:

The developed stability indicating method is then validated according to ICH guideline for linearity, accuracy, precision, specificity, limit of quantification, limit of detection, ruggedness, robustness of the method.[5-7,11]

 

Force Degradation Studies:

Stress studies are performing according to ICH Guidelines Drug was subjected to variety of stress conditions to effect degradation up to about 5-20 %. The stress testing was performed using heating mantle with temperature controller. Drug was stressed under variety of stress conditions like acid, alkali, wet heat, effect by oxidation, light and dry heat. Further, the stressed samples were subjected to chromatographic separation using mobile phase to resolve the drug from potential degradation products. [2,8-10]

 

Acid degradation study:

Stressed sample- In a round-bottom flask (RBF), 10 mg of Piracetam was taken, to it 10 mL of x N HCl was added, it was then heated under reflux on a heating mantle at y°C for z hrs .diluted up to 50 mL with methanol and sonicated for 15 min.

 

Alkali degradation study:

In a round-bottom flask (RBF) 10 mg of Piracetam was taken, to it 10 mL of x N NaOH was added, it was then heated under reflux on a heating mantle at y°C for z hrs diluted up to 50 mL with methanol and sonicated for 15 min..

 

Wet heat degradation:

In around bottom flask (RBF) 10 mg of Piracetam was taken, to it 10 mL of water was added, it was then heated under reflux on a heating mantle at y°C for z hrs .diluted up to 50 mL with methanol and sonicated for 15 min.

 

Dry heat degradation:

Piracetam drug was spread as thin film layer in Petri plate kept in a hot air oven at x°C for y hrs. From the above stressed sample, 10 mg was weighed accurately and diluted with methanol to make up the volume to 10 mL and sonicated for 15 min.

 

Photolytic Condition:

Piracetam drug was taken in petri plate, spread as thin layer and exposed to sunlight for x hrs. From the above stressed sample, 10 mg was weighed accurately and diluted with methanol to make up the volume to 50 mL and sonicated for 15 min.[2,8-11]

 

Stress testing under oxidative condition:

10 mg of Piracetam was taken in a 10 ml stopper volumetric flask, to it 10 ml x % v/v solution of hydrogen peroxide was added, it was sonicated for 10 min to ensure even mixing of drug in solution and then kept in dark at room temperature for y hrs. After y hrs the solution was heated to boiling temperature for 5 min to remove the excess of hydrogen peroxide. [2,8-11]

 

RESULT AND DISCUSSION:

Optimized Chromatographic condition:

The main aim of development of RP-HPLC method was to get reliable method for the quantification of Piracetamfrom bulk and pharmaceutical dosage form and which will be applicable for degradation product also. Different chromatographic condition where employed for the analysis of Piracetam. Finally the analysis was perform by using Methanol: Water (pH 7) (20:80) Sample where analyse at 205 nm. at an  Injection volume 20 µl, Flow rate 0.8 ml/min. the proposed method was optimized to give a sharp peak with minimum tailing for  Piraceatm.

 

Fig:1 Representative Chromatogram of Piracetam.

 

Assay of Piracetam: assay of Piracetam formulation was carried out by using developed method sample solution where prepared and injected into HPLC System. The sample solution scanned at 205 nm the % drug was found to be 97.76%a single peak where observed with retention time 4.844.

 

Accuracy and Precision:

The result of intra and interday variation of Piracetam at three different concentration levels (80%, 100% and 120%) are depicted in TABLE.1

 

Table No.1Accuracy and precision

Amount added	Amount Found (mg)
	Day1	Day 2	Day 3
600 mg 80%	605.44	601.63	599.29
	603.66	606.62	604.41
	605.72	607.75	605.38
Mean	405.27333	606	604.69333
S.D	0.491664	0.549454	0.516946
%RSD	1.062521	1.194466	1.156652
800 mg 100%	800	799.94	792.60
	807.5	800.17	798.59
	800.5	793.28	800.10
Mean	802.66	797.79	797.09
S.D	4.19	3.91	3.96
%RSD	0.52	0.49	0.496
1000 mg 120%	992.02	990.99	995.83
	991.00	980.04	978.03
	992.40	994.49	990.45
Mean	991.80	988.50	988.10
S.D	0.72	7.53	9.12
%RSD	0.07	0.76	0.92

 

The mean values of amount estimated of the drug was found to be very close to the amount added and the % RSD values of intra-day were found to be very low indicating acceptable accuracy and precision of the method. The intra and inter-day results at each level were subjected to one way ANOVA and F values at each level were obtained as a ratio of Between Mean Square to the Within Mean Square (F = BMS/WMS). The obtained values were found to be less than the tabulated F (2, 6) at α = 0.05 (Tabulated F value = 5.14). These indicated that there was no significant difference between intra and inter-day variability, suggested good intermediate precision of the method. [2,5-11]

 

Linearity:

Linearity of proposed method was evaluated according to ICH guidelines.

The data obtained in the calibration experiments when subjected to linear – regression analysis showed a linear relationship between peak areas and concentrations in the range 2-14 µg/mL.Table no 2 depicts the calibration data of Piracetam. The respective linear equation was y =36041x-22433 where x is the concentration and y is area of peak. The correlation coefficient was 0.998. [2]The calibration curve of Piracetam is depicted in Fig

 

Fig. 2.Calibration curve of Piracetam

 

Table No.2 Linearity

Sr. No.	Conc µg/ml	Peak Area			Mean Peak Area	Standard Deviation	% RSD
1	2	662686	672580	673840	669702	6108.6280	0.912138
2	4	1389536	1384545	1431710	1401930.3	25910.402	1.848195
3	6	2123501	2163521	2133856	2133856.67	20771.93	0.975018
4	8	3063017	296323	2963634	2996629.3	57493.748	1.918614
5	10	3521342	3528345	3628167	3559284.7	59756.5259	1.678891
6	12	4159642	4157265	4157265	4197567.3	67757.5708	1.614221
7	14	5065438	5016589	509988	5099886.1	41855.465	0.827079
Equation		Y=36041x-22433
R2 Value		0.9978

Table No.3 Detection Wavelength

Sr. No	Detection Wavelength	Retention Time	Area	Plates	Asymmetry
1	207	4.3	796579	5475	1.34
2	207	4.4	787237	5591	1.36
3	207	4.4	786718	5633	1.35

 

Table No.4 Mobile Phase Concentration changes

Sr. No	Mobile Phase concentration	Retention Time	Area	Plates	Asymmetry
1	40:60	4.104	674384	4956	1.34
2	70:30	3.961	752551	5376	1.33
3	15:85	4.552	855763	5623	1.13

 

Robustness:

The robustness was evaluated by making small changes in validation parameters such as mobile phase concentration and detection wavelength, the result where given in table 3 and 4.

 

Specificity:

The HPLC chromatograms recorded for the blank solution and blank solution exposed to the degradation conditions showed no peaks at the retention time of Piracetam and also the representative chromatograms of stressed samples under various stress conditions showed that Piracetam was well resolved from its degradation products, indicating the specificity of the method. The HPLC chromatograms recorded for blank, placebo, standard and sample solution showed that Piracetam peak was noted by diluents and placebo.

 

 

Fig. 3 Representative chromatogram for specificity

 

System Suitability:

System suitability tests are an integral part of method development and are used to ensure adequate performance of the chromatographic system. Retention time (t_R), number of theoretical plates (N), tailing factor (T) and peak asymmetry (A_f) were evaluated for five replicate injections of the drug at a concentration of 10µg/mL. The result are given in table were within acceptable limits. Atypical chromatogram of Piracetam is presented in table.

 

Table No.5.System Suitability Parameter

Sr. No.	Parameter	Value
1	Theoretical plates	5419
2	Retention time (min)	4.844
3	Asymmetry	1.21

 

Analysis of the marketed formulation:

The chromatograms of the drug samples extracted did not show any change in the retention time. There was no interference from excipient, which are commonly present in the tablets. The drug content was found to be 101.21% with a % RSD of 0.0069% as shown in Table 6. Therefore it was concluded that, degradation of Piracetam had not occurred in marketed formulations. The % RSD value indicated the suitability of the method for the routine analysis of Piracetam in marketed formulation.

 

Force degradation study of Piracetam:

Stress testing under acidic condition

When the drug was refluxed with 0.1N HCl at 60^oC for 30min. When stressed sample was analyzed, there was one additional peak at the retention time 4.84 min. There was no additional peak at the same retention time when zero time, stressed blank and blank sample were analyzed confirming the formation of one degradation product. Comparison of the peak area of Piracetam in stressed condition with that of the zero time sample give 24.85% degradation.

 

 

Table No.6 Drug content found

Amount per tablet(mg)	Amount Found (mg/mL)	% Found	Average	±SD	%RSD
800	798.488	98.7045	99.31446	0.84563	0.008619
800	798.475	99.80826
800	797.991	99.62069

 

 

 

Fig.4 Representative Chromatogram of acid treated Piracetam (0.1N HCL for 30min reflux at 60°C).

 

 

Fig.5 Representative Chromatogram of base treated Piracetam (0.1N NaOH for 15 hr reflux at 60°C).

 

Alkali degradation:

There were two degradants found when the drug was refluxed with 0.1N NaOH for 15 hr. at 60°C. When this stressed sample was analyzed, there was one additional peak at the retention of 4.9. There was no additional peak at the same retention time when blank, zero and stressed blank samples were analyzed confirming the formation of one degradation product. Comparison of the peak area of Piracetam in stressed condition with that of the zero time samples gave 16.11% degradation.

 

Wet heat degradation:

There were two degradation products found when the drug was refluxed with Water for 15 minutes at 60°C. When stressed sample was analyzed, there were two additional peak at the retention time 2.3 min and 4.8 min. There was no additional peak at the same retention time when zero time, stressed blank and blank sample were analyzed confirming the formation of one degradation product. Comparison of the peak area of Piracetam in stressed condition with that of the zero time samples gave 15.23 %degradation.

 

Fig.6 Representative Chromatogram of wet heat degradation for 45 min 80°C reflux

 

Dry heat degradation:

Stability of Piracetam in dry heat was studied by keeping it for 1 hr at 60°C. When the stressed sample was analyzed, no degradation was found and hence it was decided to extended the heating time for 2hrs, 3hrs, 4hrs and 5hrs with increased temperature of 60°C.When the stressed sample was analyzed, there was no additional peak found. Also the comparison between the peak areas of stressed sample of Piracetam with that of zero time samples showed no difference, indicating that there was no degradation. Hence it was concluded that the drug was stable under the conditions tested.

 

 

 

Photolytic degradation: There was no degradation products found when the powdered drug was exposed to sunlight for 36hrs. When stressed sample was analyzed, there was one additional peak of degradation. There was no additional peak at the same retention time when zero time,  Comparison of the peak area of Piracetam in stressed condition with that of the zero time sample were near about same, indicating that there was no degradation.

 

Oxidative degradation:

Oxidative degradation of Piracetam was studied using 3 % H₂O₂ for 24 hrs when the samples were analyzed 24.45% drug degradation found. Hence it was concluded that drug was oxidative under the conditions tested.

 

 

 

Fig: 7 Representative Chromatogram of oxidative degradation studies of Piracetam (3% H₂O₂ for 24 hrs).

 

Table No.7 Force degradation study

Sr. no	Stress Condition	Drug peak area at zero time sample	Drug peak area of stressed sample (mc.V.sec)	Retention time(s) of degradation products (min)	%Degradation
1	Acid 0.1N HCL (Refluxed for 30min) (600C)	781505	587227	2.8	24.85%
2	Alkali 0.1N NaOH (15min 600C)	781505	645531	2.5	16.11%
3	Wet heat  60°Cfor 15min	781505	661748	2.5	15.23%
4	Oxidative 30%v/v H2O2(in direct room temperature	781505	585496	2.3	24.45%
5	Dry heat 70°C(kept in oven for 48hrs)	781505	-	No degradation	No degradation
6	Photolytic (exposed to sunlight for 36 hrs)	781505	-	No degradation	No degradation

 

 

 

SUMMARY AND CONCLUSION:

Stress testing of Piracetam was carried out under acidic, alkaline, neutral, oxidative, photolytic and dry heat conditions.

The stressed samples and standard drug were subjected to chromatographic separations. The HPLC analysis was carried out using Grace C18 (250mm x 4.6ID, Particle size: 5 micron) RP-18 column.

The degradation products and in house tablet excipient in Piracetam assay were well resolved from the drug using the mobile phase Methanol: Water (20:80 % v/v) (pH 7).

The mobile phase flow rate was maintained at 0.8 ml/min and the detection was performed at wavelength 205 nm.

The average retention time for Piracetam was 4.844 min. Piracetam was found to be degraded under acidic, alkaline and wet heat photolytic conditions, while it was stable under dry heat, and oxidative conditions. Table No.7 summarizes the degradation behavior of Piracetam and the retention times of degradation products under tested conditions. Fig.4-7 gives the representative chromatograms of stressed samples of Piracetam showing well resolved peaks of degradation products under various stress conditions.

The developed method was validated as per ICH Guidelines Q2 (R1). The method was found to be economic, accurate, precise, specific, robust and linear in the range of 2-14μg/ml. The Detection limit was found to be 0.16 μg/ml. The Quantitation limit was found to be 0.46μg/ml. It can be concluded that the HPLC method developed for Piracetam is capable of discriminating between the drug and the degradation products.

Validation of the method is suitable for the analysis of Piracetam in tablet formulation without any interference from common excipient or potential degradation products of Piracetam and excipient.

The developed method can be used for routine analysis of Piracetam marketed formulation.

 

REFERENCES:

1.       V. Masarskal Analytical Method Development and Validation For Piracetam In Pharmaceutical Formulation Pharmacia, vol. 60, No. 4/2013

2.       Sushil D. Patil, Sunil V. Amurutkar and C. D. Upasani Development and Validation of Stability Indicating RP-HPLC Method for Empagliflozin Asian J. Pharm. Ana. 2016,Vol. 6; Issue 4

3.       Manutosh Acharya1, A.K Jain, et al  RP-HPLC Method Development And Validation For Simultaneous Determination of Citicoline And Piracetam In Pharmaceutical Dosage Form Indo American Journal of Pharmaceutical Research, 2015, Vol 5, Issue 08, 3639-3647.

4.       Bhowmick Anindita A., Khandelwal, et al Analytical Method Development and Validation for Piracetam as Bulk and in Pharmaceutical Formulation International Journal of PharmTech Research.2010,Vol.2, No.1, pp 201-204,

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8.       Government of India, Indian Pharmacopoeia Commission Ministry of Health and Welfare (2007) Indian Pharmacopoeia, Ghaziabad India

9.       Hong, Donald H., and Mumtaz Shah. "Development and Validation of HPLC Stability Indicating Assays." Drug Stability Principles and Practices. Ed. Christensen, Jens Thur. 3ed. New York: M. Dekker, 2002. 329-84.

10.     Q1BICH. "Photostability Testing of New Drug Substances and Products Q1B."International Conference on Harmonization. Geneva: ICH, 1996.

11.     Q2 (R1) ICH. "Validation of Analytical Procedures: Text and Methodology Q2 (R1)."International Conference on Harmonization. Geneva: ICH, 2005.

 

 

 

 

Received on 24.06.2017       Accepted on 11.08.2017     

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