Comparison Study of Conventional and Microwave Assisted Force Degradation by RP-HPLC Method of Pharmaceutical Drug and Dosage form

 

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

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

2MET’s Institute of Pharmacy, MET League of Colleges, Bhujbal Knowledge City, Adgaon, Nashik, Maharashtra State 422003, India

*Corresponding Author E-mail: sushilpharma@rediffmail.com

 

ABSTRACT:

Comparison Study of conventional and Microwave assisted Force Degradation by using RP- HPLC was carried 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. Microwave assisted Force Degradation was performed acid, alkali, wet heat, oxidative and dry heat degradation study to prove that similar degradation product using microwave oven reduced time as compared to conventional. 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.

 

KEYWORDS: RP-HPLC, Force degradation, microwave and Piracetam.

 


 

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.

 

Structure of Piracetam

 

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] Aim of the present work was to comparison study of conventional and microwave assisted force degradation by RP-HPLC method of pharmaceutical drug and dosage form.

 

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, Banglore, 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.

 

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. [5-7,11]

 

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 hrsdiluted 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:

Piracetamdrug 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.

 

Stress testing under oxidative condition:

10 mg of Piracetamwas taken in a 10 mL stoppered 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.

 

RESULT AND DISCUSSION:

Optimized Chromatographic condition:

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 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. When force degradation experiment were performed under various conditiion and the resulting sample were analysed no degradation in drug peak area was observed in case of photolytic degradation and dry heat degradation  condition. Degradationa was obtained under acid,alkali oxidative and wet heat degradation condiation. The chromatogram of force deragadation sample conventional and microwave –assisted degratdation are shown in fig 1 and fig 2.


 

 

Fig1: Chromatography of Piracetam under various conditions by conventional degradation

TABLE 1 Summary of forced degradation studies on Piracetam.

Sr. No.

Stress Condition

Drug peak area at zero time sample(mcV.sec)

Drug peak area of stressed sample (mcV.sec)

Retention time(s) of degradation products(min)

% degradation

1.

Acid 0.1N HCL (Refluxed for 30 min)

781505

587227

2.8

 24.85%

2.

Base 0.1N NaOH (Refluxed for 15 min at 60oC)

781505

645531

2.5

16.11%

3.

Oxidative 3% v/v H2O2 (In Dark Room temperature)

781505

661748 

2.5

15.23%

4.

Photolysis(exposed to direct sunlight for 36 hrs)

781505

585496

2.3

24.45%

5.

Dry heat 60°C(Kept in oven for 24 hrs)

781505

-

No degradation

No degradation

6.

Neutral(Refluxed for 15 min)

781505

-

No degradation

No degradation

 

 

Fig 2: Chromatogram of microwave assisted force degradation

 


To prove that similar degradation products were obtained under conventional and microwave –assisted force degradation, dual wavelength analysis was performed in which, peak area of degradation product formed under conventional and microwave-assisted degradation were monition at 205nm and 210 nm. From III it was observed that the peak area ratio was near about same for conventional and microwave –assisted forced degradation confirmed that similar degradation products were formed under conventional and microwave-assisted acid and alkali degradation condition [5]


 

Table II: Summary of microwave assisted forced degradation studies on Piracetam

Sr. No.

Stress Condition

Drug peak area at zero time sample(mcV.sec)

Drug peak area of stressed sample (mcV.sec)

Retention time(s) of degradation products(min)

% degradation

1.

Acid 0.1N HCl (280 watt for 1min)

781505

594527

2.5

21.67%

2.

Base 0.1N NaOH (350 watt for 70 sec)

781505

647531

2.3

14.13%

 

 

 

Table III: Comparison of conventional and microwave assisted stress testing.

Sr. no.

Method of  Degradation

Conditions

Peak area

Peak  Area  Ratio  (205 / 207)

205nm

210nm

1

Conventional

Acid

587227

652580

1.11

2

Microwave

Acid

594527

678454

1.14

3

Conventional

Alkali

645531

704637 

1.12

4

Microwave

Alkali

647531

 706323

1.14

 


 

 


TABLE IV: Accuracy and precision studies

Amount added (mg)

Amount found (mg)

Mean

S.D.

% R.S.D.

Day 1

Day 2

Day 3

80% (600mg)

605.44

601.63

599.29

605.27

0.4916

1.062

603.66

606.62

604.41

606.56

0.5494

1.19

605.72

607.75

605.38

604.69

0.5169

1.15

100 % (800mg)

800

799.94

792.60

802.66

0.419

1.23

807.5

800.17

798.59

797.79

0.451

1.34

800.5

793.28

800.10

797.09

0.476

1.37

120 % (1000mg)

992.02

990.99

995.83

991.80

0.672

1.24

991.00

980.04

978.03

988.50

0.675

1.32

992.40

994.49

990.45

988.10

0.668

1.23

 


Accuracy and Precision:

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

 

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.

 

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 V 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.

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.

 

Table VI:  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 VII: Mobile Phase concentration

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

 


 

TABLE V: Linearity data of Piracetam

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

5099886

5099886.1

41855.465

0.827079

Equation

 Y=36041x-22433

R2 Value

0.9978

 

 

Fig.3: Calibration curve of Piracetam


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.

 

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 (tR), number of theoretical plates (N), tailing factor (T) and peak asymmetry (Af) 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 VIII.

 

TABLE VIII:Sytem suitability

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: IX 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.

 

 

Fig. 4 Representative chromatogram for specificity


 


 

TABLE IX: Analysis of marketed formulations

Amount per tablet(mg)

Amount Found (mg/mL)

% Found

Average

±SD

%RSD

800

798.488

98.7045

 99.31446

 

0.424

1.2619

 

800

798.475

99.80826

800

797.991

99.62069

 


CONCLUSION:

It was observed that microwave-assisted degradation takes seconds to complete as compared to conventional heating in case of acid and alkali degradation conditions. Also, similar degradation behavior was observed under acid and alkali degradation conditions. Hence, it can be concluded that microwave oven can be used to substitute conventional hydrolytic forced degradation. The developed method is precise, specific, accurate and stability-indicating.

 

Validation of the method proved that the method is suitable for analysis of Piracetam in tablet formulation without interference from common excipient or potential degradation products of Piracetam and excipients. The developed method can be used for routine analysis of Piracetam tablets.

 

REFERENCES:

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

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Received on 03.07.2017                Accepted on 16.10.2017               

© Asian Pharma Press All Right Reserved

Asian J. Pharm. Ana. 2017; 7(4): 218-224.

DOI: 10.5958/2231-5675.2017.00035.7