INTRODUCTION:

Aceclofenac (ACF), {[2-(2', 6'-dichlorophenyl) amino] phenyl acetoxyacetic acid} is a new phenyl acetic acid derivative with potent analgesic and anti-inflammatory properties and improved gastric tolerance. It should not be given to people with porphyria or breast-feeding mothers, and is not recommended for children. Aceclofenac has higher anti-inflammatory action than conventional NSAIDs. It is a cytokine inhibitor. Aceclofenac works by blocking the action of a substance in the body called cyclo-oxygenase. Cyclo-oxygenase is involved in the production of prostaglandins (chemicals in the body) which cause pain, swelling and inflammation. Aceclofenac is the glycolic acid ester of Diclofenac.

Paracetamol (PCM), chemically 4-hydroxy acetanilide, is a centrally and peripherally acting analgesic and antipyretic agent. Paracetamol is classified as a mild analgesic. It is commonly used for the relief of headaches and other minor aches and pains and is a major ingredient in numerous cold and flu remedies. In combination with opioid analgesics, paracetamol can also be used in the management of more severe pain such as post-surgical pain and providing palliative care in advanced cancer patients. Though paracetamol is used to treat inflammatory pain, it is not generally classified as an NSAID because it exhibits only weak anti-inflammatory activity.

Several combination dosage forms of these two drugs containing ACF (100 mg) and PCM (500 mg) are available commercially. This combination is used for pain relief and management of rheumatoid arthritis. Only a few methods have been reported for determination of ACF individually, whereas many methods [1-5] have been described in literature for determination of PCM alone or in combination with other drugs. However, there is no HPLC method reported for the simultaneous estimation of these drugs in pharmaceutical dosage forms. The present work describes a simple, precise, and accurate reverse phase HPLC method for simultaneous estimation of ACF and PCM in combined dosage form.

Fig. No.1 Chemical Structure of Aceclofenac

Fig. No.2 Chemical Structure of Paracetamol

MATERIALS AND METHOD

Chemicals and Reagents Used:

The following chemicals were procured for the process Potassium Dihydrogen Phosphate, Sodium Hydroxide, Water [HPLC Grade], Acetonitrile [HPLC Grade], Aceclofenac and Paracetamol [Working standards] all the chemicals were procured from MERCK manufacturer and the tablets were collected from the Local market.

Apparatus and Chromatographic Conditions:

Equipment : High performance liquid chromatography equipped with Auto Sampler and DAD or UV detector.

Column : Cosmicsil Adze C18 (150 x 4.6nm x 5 μm)

Flow rate : 1.0 mL per min

Wavelength : 265 nm

Injection volume : 10 ml

Column oven : Ambient

Run time : 10min

Detector : Photo diode array

Soft ware : Empower 2

Retention time : 3.31min (Paracetamol), 6.66min (Aceclofenac)

Preparation of Phosphate buffer [6-9]:

The Phosphate buffer was prepared by mixing 50.0 mL of 0.2M Potassium Dihydrogen Phosphate in a 200 mL volumetric flask and added 29.1 mL of 0.2 M sodium hydroxide further the volume was made upto the mark with water [HPLC Grade]. The pH of the solution was adjusted to 7.0. The resultant buffer solution was filtered through 0.45 micrometer membrane filter and degas.

Preparation of mobile phase:

The Mobile phase was prepared by mixing the above buffer 750 mL (75%) and 250 mL of acetonitrile (25%) [HPLC Grade] and degas in ultrasonic water bath for 5 minutes. The resultant solution was filtered through 0.45 µ filter under vacuum filtration.

Diluent Preparation: The Mobile phase was used as Diluent.

Preparation of standard stock solution:

The Standard stock solution was prepared by dissolving 50 mg of each individual drug in 50 mL of methanol to get concentration of 1 mg/mL for both drugs separately in a 50ml of clean dry volumetric flask. Transferred 10 mL of stock solutions into 100ml of volumetric flask and the volume was made upto the mark with the diluent.

Preparation of sample stock solution:

The Sample stock solution was prepared weighing accurately the individual 20 tablets and powdered. Powder equivalent to 50 mg of paracetamol was weighed and transferred to 50 mL volumetric flask. In the same flask 50mg of pure Aceclofenac drug was added and dissolved in methanol [HPLC Grade] by shaking the flask for 10 min. The solution was filtered through Whatman filter paper no. 41 and first few mL were rejected. 2 mL of this filtrate was transferred into 200ml of volumetric flask and the volume was made upto the mark with the diluent.

Separately injected about 10microlitres of diluents as blank, standard preparation and sample preparations into chromatographic system and recorded the chromatograms and measure the peak area responses for the analyte peaks. The percentage content of Aceclofenac and Paracetamol was calculated by using suitable formula.

Assay calculation- :

System suitability [10-17]:

1. The Tailing factor for the peak due to Aceclofenac and Paracetamol in standard solution should not be more than 1.5.

2. The Theoretical plates for Aceclofenac and Paracetamol peaks in standard solution should not be less than 2000.

Fig. no. 3 The Chromatograph Represents for the Blank

Fig. no. 4 The Chromatograph Represents for the Standard

Fig. no.5 The Chromatograph Represents for the Sample

Table No. 1 Peak area results for calculation of Assay.

S.No	Peak areas
S.No	Paracetamol	Aceclofenac
1	559945	266376
2	565169	273456
Mean	562557	269916

Table no.2 Assay results for the Drugs

Parameters	Paracetamol	Aceclofenac
Standard peak area	562533	278614
Test peak area (mean)	562557	269916
Average Weight	615mg
Label claim	500mg	100mg
% Purity of Standard	99.1%	99.6%
Amt obtained	40mg	10mg
% Assay	100.6%	98.5%

VALIDATION DEVELOPMENT [18-22]

1. Precision: The precision of an analytical procedure expresses the closeness of measurements obtained from multiple sampling of the same homogenous sample under the prescribed conditions. Precision may be considered at three levels: repeatability, intermediate precision and reproducibility. The precision of an analytical procedure is usually expressed as the variance, standard deviation or coefficient of variation of a series of measurements.

A. System Precision:

The system precision was checked by using standard Paracetamol and Aceclofenac to ensure that the analytical system was precise. The retention time and area of six determinations was measured and RSD was calculated. % RSD of the assay value for six determinations should not be more than 2.0%. The standard solution was prepared as per the proposed assay method in six determinations and was injected into HPLC system. The retention time and peak area of six determinations was measured and RSD was calculated. The data was represented in Table no. 3 and 4 respectively for the drugs.

Standard deviation =

Table no. 3 System Precision results for Paracetamol

S.No	Retention time (Rt)	Area
1	2.80	564847
2	2.805	563890
3	2.810	579708
4	2.803	566988
5	2.802	563568
6	2.806	573725
AVG	_	568787.7
%RSD	_	1.4

Table no. 4 System Precision data for Aceclofenac

S.No	Retention time (Rt)	Area
1	6.033	265655
2	6.065	268324
3	6.055	264039
4	6.470	276490
5	6.033	265123
6	6.065	269802
AVG	_	268238.8
%RSD	_	1.7

B. Method Precision:

In method precision, a homogenous sample of a single batch was analyzed six times. This indicates whether a method was giving consistent results for a single batch. The method precision was performed on Paracetamol and Aceclofenac formulation. The % RSD of the assay value for six determinations should not be more than 2.0%. Method precision indicates whether a method is giving consistent results for a single material. The sample solution was prepared as per the proposed assay method in six determinations and was injected into HPLC system. The retention time and peak area of six determinations was measured and RSD was calculated. The data was represented in Table no. 5 and 6 respectively for the drugs.

Table no. 5 Method Precision data for Paracetamol

S.No	Retention time(Rt)	Area
1	2.815	592957
2	2.810	574148
3	2.806	567516
4	2.804	557545
5	2.807	573800
6	2.805	561221
AVG	_	566197.8
%RSD	_	1.2

Table no. 6 Method Precision data for Aceclofenac

S.No	Retention time (Rt)	Area
1	6.052	265455
2	6.102	273023
3	6.073	266539
4	6.150	276492
5	6.064	265673
6	6.031	263802
AVG	_	268497.3
%RSD	_	1.8

2. Intermediate Precision/Ruggedness:

Ruggedness is a measure of reproducibility of test results under the variation in conditions normally expected from laboratory to laboratory, from column to column and from analyst to analyst. All the system suitability parameters should be met as per the method. The data was represented in the Table no. 7 and 8.

Table no.7The Standard Chromatogram values of Paracetamol for Ruggedness

S.No.	Analyst-1		Analyst-2
S.No.	R_t	Area	Rt	Area
1	2.80	564847	2.805	566858
2	2.805	563890	2.815	578708
3	2.810	579708	2.801	563525
4	2.802	566988	2.812	563768
5	2.816	563538	2.808	572827
6	2.806	573725	2.810	574842
Avg	_	568787.7	_	570088
%RSD	_	1.14	_	1.10

Table no.8The Standard Chromatogram values of Aceclofenac for Ruggedness

S.No.	Analyst-1		Analyst-2
S.No.	Rt	Area	Rt	Area
1	6.033	265655	6.070	268390
2	6.065	268324	6.051	266451
3	6.055	264039	6.142	275803
4	6.470	276490	6.064	265313
5	6.455	265123	6.132	273821
6	6.491	269802	6.083	264092
AVG	_	268238.8	_	268978.3
%RSD	_	1.7	_	1.77

3. Accuracy: The accuracy of an analytical procedure expresses the closeness of agreement between the value which is accepted either as a conventional true value or an accepted reference value and value found. The data was represented in the Table no. 9 and 10.

Table no. 9 Accuracy results for Paracetamol

Sample Id	Pure Drug Conc. (µg/ml)	Formulation Conc. (µg/ml)	%Recovery of pure drug	Statistical Analysis
50%	150	100	99.8	%RSD= 1.3
50%	150	100	98.3
50%	150	100	99.38
100%	400	100	99.97	%RSD= 1.6
100%	400	100	99.6
100%	400	100	99.61
150%	650	100	99.4	%RSD= 1.4
150%	650	100	99.5
150%	650	100	99.15

Table no. 10 Accuracy results for Aceclofenac

Sample Id	Pure Drug Conc. (µg/ml)	Formulation Conc. (µg/ml)	%Recovery of pure drug	Statistical Analysis
50%	60	40	99.1	%RSD= 0.9
50%	60	40	98.3
50%	60	40	98.5
100%	160	40	100.1	%RSD= 1.5
100%	160	40	100.2
100%	160	40	99.8
150%	260	40	100.1	%RSD= 1.3
150%	260	40	100.2
150%	260	40	99.93

From the above results, it was concluded that the recovery was within the limits. Hence, the method was accurate.

4. Linearity: The linearity of the analytical procedure is its ability (within a given range) to obtain the test results which are directly proportional to the concentration (amount) of analyte in the sample. The data was represented in Table no. 11.

Table no. 11The Linearity Data for Paracetamol and Aceclofenac

Linearity Levels	Paracetamol		Aceclofenac
Linearity Levels	Concentration (µg/ml)	Peak Areas	Concentration (µg/ml)	Peak Areas
L1-50%	250	265317	100	120031
L2-75%	375	429032	150	195338
L3-100%	500	569968	200	278243
L4-125%	625	750374	250	353390
L5-150%	750	925363	300	443708

Figure no. 6 Graphical Representation of Linearity range for Paracetamol

Figure no.7 Graphical Representation of Linearity range for Aceclofenac

5. Limit of Detection:

The detection limit of an individual analytical procedure is the lowest amount of analyte in a sample which can be detected but not necessarily quantities as an exact value. Several approaches for determining the detection limit are possible, depending on whether the procedure is a non instrumental or instrumental.

6. Limit of Quantification:

The Quantification limit of an individual analytical procedure is the lowest amount of analyte in a sample which can be quantitatively determined with suitable precision and accuracy. The Quantification limit is a parameter of quantitative assays for low levels of compounds in sample matrices, and is used particularly for the determination of impurities and/ or degradation products. Several approaches for determining the Quantification limit are possible, depending on whether the procedure is a non- instrumental or instrumental.

The limit of detection and limit of quantitation of the present method were established based on the standard deviation of the response and slope. The slopes were calculated from the respective calibration. The data was represented in Table no. 12.

Table no. 12The LOD and LOQ Data for Paracetamol and Aceclofenac

PARACETAMOL
Conc. (x) (µg/ml)	Peak Areas (y)	Statistical Analysis
2	5315	S = 2555.9 c = 366.4 s = 505.102 LOD: 0.652153µg/ml LOQ: 1.976221µg/ml
4	10542
6	15792
8	21450
10	25420

ACECLOFENAC
Conc. (x) (µg/ml)	Peak Areas (y)	Statistical Analysis
2	6421	S = 2196.35 c = 2247.3 s = 242.3878 LOD: 0.364186µg/ ml LOQ: 1.103594µg/ml
4	11245
6	15462
8	19984
10	24015

Where,

S: Slope of respective calibration curves

c: y-intercept

s: Standard deviation of y-intercepts

The lowest possible concentrations of Paracetamol and Aceclofenac that can be detected by the present method were found to be 0.652153µg/ml and 0.364186µg/ml respectively and that can be Quantitated were found to be 1.976221µg/ml and 1.103594µg/ml respectively.

7. Robustness:

The robustness of an analytical method is a measure of its capacity to remain unaffected by small but deliberate variations in method parameters and provides an indication of its reliability during normal usage.

Robustness was done by changing the mobile phase (±1ml), flow rate (±1%), changing the wavelength (±5nm). All the system suitability parameters must be met as per the method. The data were represented in Table no. 13 and 14.

The drug solutions were found to be stable for two days (48 hours) from the time of preparation. On the third day, a significant degradation (nearly 5%) of the drugs was observed.

9. System suitability: Five replicate injections of standard solution were injected and the chromatograms were recorded. The system was suitable for analysis if the % relative standard deviation (%RSD) of area counts in five replicate injections should be not more than 2.0%. USP tailing factor for Paracetamol and Aceclofenac peak should be not more than 2.0. USP resolution factor between the peaks corresponding to Paracetamol and Aceclofenac should be more than 2.0. The standard solution was prepared as per the proposed assay method and was injected into the HPLC system. The tailing factor and theoretical plate count of Paracetamol and Aceclofenac peak from fifth injection and % RSD on replicate injections were recorded. The data were represented in Table no. 17 and 18.

Table no. 17 The System Suitability Parameters for Paracetamol

Injection	R_t(min)	Area	USP Plate Count	USP Tailing
1	2.80	553412	2873	1.8
2	2.805	560133	2749	1.8
3	2.810	554051	2707	1.8
4	2.802	567359	2832	1.7
5	2.816	552598	2722	1.8
Mean	-	55750.6	-
%RSD	-	1.4	-	-

Table no.18 The System Suitability Parameters for Aceclofenac

Injection	R_t (min)	Area	USP Plate Count	USP Tailing
1	6.033	266022	3964	1.08
2	6.065	272182	3995	1.13
3	6.055	265101	3927	1.08
4	6.470	272938	3940	1.18
5	6.455	263458	3970	1.09
Mean	-	267940.2	-	-
%RSD	-	1.6	-	-

RESULT AND DISCUSSION:

The present study was carried out to develop a sensitive, precise and accurate RP-HPLC method for the analysis of Paracetamol and Aceclofenac in bulk as well as in pharmaceutical dosage form. In order to method development under isocratic conditions, mixtures of Phosphate Buffer (pH 7) and Acetonitrile [HPLC Grade] in different combinations were tested as mobile phase on a Cosmicsil Adze C18 (150 X 4.6nm X 5micrometer) column. A binary mixture of Phosphate Buffer (pH 7) and Acetonitrile [HPLC Grade] in 75:25 v/v proportion was proved to be the most suitable of all combinations since the chromatographic peaks were better defined and resolved and almost free from tailing. The retention time obtained for Aceclofenac and Paracetamol was 6.03 and 2.80 minutes respectively. A model chromatogram was shown in Fig. no.5.

Fig. no.8 A model Chromatograph showing the separation of the Drug

The Precision data was represented by Table no. 3, 4, 5 and 6. It was observed from the data tabulated above; the retention time and area responses are consistent as evidenced by the values of relative standard deviation. The retention time and area of six determinations was measured and RSD was calculated. % RSD of the assay value for six determinations should not be more than 2.0%. Hence, it was concluded that the system precision parameter meets the requirement of method validation.

The sample solution was prepared as per the proposed assay method and injected into HPLC system. The same solution was injected into same HPLC system using another column to check column variability. The same solution was injected into another system to check system variability. The sample solution was prepared by another analyst as per assay method and injected into first RP-HPLC system to check analyst variability. The retention time and peak area of all chromatograms was measured, %assay and RSD was calculated. The data were represented in Table no. 7 and 8.

The accuracy of an analytical method is the closeness of test results obtained by that method to the true value. The accuracy of an analytical method should be established across its range. Accuracy is performed in three different levels for Paracetamol and Aceclofenac at 50%, 100% and 150%. Samples analyzed at each level in triplicate. From the results, % recovery was calculated. Average % recovery at each spike level not less than 98.0 and not more than 102.0. The data were represented in Table no. 9 and 10. The chromatographs were represented in Fig. no. 9, 10 and 11.

Fig. no.9 The Chromatograms represents for 50%Accuracy level

Fig. no.10 The Chromatograms represents for 100%Accuracy level

Fig. no.11 The Chromatograms represents for 150%Accuracy level

For Linearity a graph was plotted, weight taken (%) versus chromatographic area. The regression line obtained was linear. From the data obtained, co-relation coefficient, slope and y-intercept were calculated. Ideally it was found that the co-relation coefficient was not less than 0.999. The data was represented in table no.11. The Linearity graph was represented in Fig. no. 6 and 7.

For Robustness study the standard solution was prepared as per the proposed assay method and was injected into HPLC system by changing chromatographic conditions. The actual mobile phase ratio (75:25) and the standard solution was injected and also injected at 74:26 and 76:24. The retention time and peak area of chromatograms was measured and %RSD was calculated. The actual flow rate was 1ml/min and the standard solution was injected and also injected at flow rate 0.9ml/min and 1.1ml/min. The retention time and peak area of chromatograms was measured and %RSD was calculated. The actual wavelength was 265nm and the standard solution was injected and also injected at wavelength 260nm and 270nm. The retention time and peak area of chromatograms was measured and %RSD was calculated. On evaluation of the above results, it was concluded that the variation in change in mobile phase composition, flow rate and wavelength does not affected the method significantly. Hence it indicates that the method was robust even by change in the Mobile phase ±1, flow rate ±0.1ml/min. and wavelength ±5nm. The data was represented in Table no. 13 and 14. The chromatographs were represented in Fig. no. 12 and 13.

Fig. no.12 The Chromatograms represents for Standard Flow Rate.

Fig. no.13 The Chromatograms represents for Change in Flow Rate.

Stability was estimated with standard (at 100% level) and sample solutions. The standard and sample solutions were injected after their preparation and the peak area values were recorded. After 24 hours, the solutions were prepared in the similar way and were injected thrice (in order to minimize errors) along with the solutions of the initial day and the peak areas were recorded. The same procedure was repeated at an interval of 24 hours until there was a significant change (due to degradation) in the peak area values. The fresh solutions were prepared in order to eliminate the effect of the environmental conditions on the stability study. The drug solutions were found to be stable for two days (48 hours) from the time of preparation. On the third day, a significant degradation (nearly 5%) of the drugs was observed. The data was represented in Table no. 17 and 18. The chromatographs were represented in Fig. no. 14 and15.

Fig. no.14 The Chromatogram recorded at 24^th hour

Fig. no.15 The Chromatogram recorded at 48^th hour

CONCLUSION:

It was concluded that the proposed RP-HPLC method developed for the quantitative determination of Paracetamol and Aceclofenac in bulk and in its formulations was simple, selective, sensitive, accurate, precise and rapid. The proposed HPLC method was sufficiently sensitive and reproducible for the analysis of Paracetamol and Aceclofenac Tablet formulation dosage forms within a short analysis time. The method was proved to be superior to most of the reported methods. The mobile phases was simple to prepare and economical. The sample recoveries in the formulation were in good agreement with their respective label claims and they suggested non-interference of formulation excipients in the estimation. Hence the proposed method was found to be rapid, accurate, precise, specific, robust and economical. The mobile phase is simple to prepare and economical. The method shows non - interference of formulation excipients in the estimation.

This method is also having an advantage that the retention time of both the drugs is below 8 min and both the drugs can be assayed with the short time. Thus the method is not time consuming and can be used in laboratories for the routine analysis of combination drugs and it can be used in pharmacological studies. Hence this method can easily be adopted as an alternative method to reported ones for the routine determination of Paracetamol and Aceclofenac depending upon the availability of chemicals and nature of other ingredients present in the sample.

FUTURE ASPECT:

The proposed method can be use in future for the clinical, biological and pharmacokinetic studies of Paracetamol and Aceclofenac.

REFERENCE:

1. Gopinath R, Rajan S, Meyyanathan S N, Krishnaveni. N, Suresh. B; A RP-HPLC method for simultaneous estimation of Paracetamol and Aceclofenac in tablets; Indian Journal of Pharmaceutical Sciences.2007; Vol 69; (1); 137-140.

2. G Garg, Swarnlata Saraf, S Saraf; Simultaneous estimation of Aceclofenac, Paracetamol and Chlorzoxazone in tablets; Indian Journal of Pharmaceutical Sciences; 2007; Vol 69 (5); 692-694.

3. K K Srinivasan, J Alex, A A Shirwaikar, S Jacob, M R Sunil Kumar, S L Prabu; Simultaneous derivative Spectrophotometric estimation of Aceclofenac and Tramadol with Paracetamol in combination solid dosage forms. Indian Journal of Pharmaceutical Sciences; 2007; Vol 69 (4); 540-545.

4. P R Mahaparale, J N Sangshetti, B S Kuchekar; Simultaneous Spectrophotometric estimation of Aceclofenac and Paracetamol. Indian Journal of Pharmaceutical Sciences; 2007; Vol 69 (2); 289-292.

5. N. Hari Krishnan, V. Gunasekaran, C. Roosewelt, K. Kalaivani, S. Chandrasekaran and V. Ravichandira; Simultaneous Estimation and Validation of Paracetamol, Aceclofenac and Chlorzoxazone by HPLC in Pure and Pharmaceutical Dosage Form. Asian Journal of Chemistry; 2008; Vol. 20, (4), 2557-2562.

6. Remington, The Science and Practice of Pharmacy, Printed by the Mackh Printing Company Eston, Pennsylvania, 19^th edn., 1995, Vol.-1, 537-5446.

7. British Pharmacopoeia, The Department of Health Sciences and Public safety, Vol II, 2003, 2544.

8. Indian Pharmacopoeia, Controller of Publication New Delhi, Vol II (2003)555-556.

9. United State Pharmacopoeia, The Official Compendia standard Asian Edn, 2003, 18.

10. B. K. Sharma, Instrumental Methods of Chemical Analysis, Goel Publishing House, Meerut, 19^th edn., 2000, 1-12.

11. H.H. Willard, L.L. Merritt, J. A. Dean, F. A. Settle, An Introduction to Instrumental Methods of Analysis, CBS Publisher Distributors, New Delhi, 8^th edn., 2002, 580-654.

12. P. Brown, K. Deanotonis, F. Settle, Handbook of Instrumental Techniques for Analytical Chemistry, A Simon and Schuster Company, New Jersey, 1997, 147-159.

13. R.I. Snyder, J.J. Kirkland, J.L. Glajch, Practical HPLC Method development, Published By John Wiley and Son, Inc, New York, 2ndEdn., 1997, 21-57.

14. Yuri Kazakevich and Rosario Lobrutto Seton. HPLC for Pharmaceutical Scientists. 1st ed. Published by Wiley-VCH; 2007; 369-382.

15. Stavros Kromidas. HPLC Made to Measure, A Practical Book for Optimization. Published by Wiley-VCH; 2006; 62-66.

16. Yuri Kazakevich and Rosario Lobrutto Seton. HPLC for Pharmaceutical Scientists. 1st ed. Published by Wiley-VCH; 2007; 389-391.

17. A. Backett, H. Stenlke, J. Davidson, Instrumental Methods in the Development and Use of Medicines Practical Pharmaceutical Chemistry , CBS Publishers and Distributors, New Delhi, 4thedn., 2002, Vol.-11, 85-174.

18. Sharma Ajay, Sharma Rohit: Validation of analytical procedures: a comparison of ICH VS Pharmacopoiea (USP) and FDA. International Research Journal of Pharmacy 2012; 3(6) 39-42.

19. Validation of analytical procedure: Methodology Q2B, ICH Harmonized Tripartite Guidelines 1996; 1-8.

20. International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use ICH Harmonized tripartite guideline Validation of analytical procedures: Text and Methodology Q2 (R1) 6 November 1996.

21. Ravichandran V, Shalini S, Sundram K. M and Harish Rajak: Validation of analytical methods – strategies and importance. International Journal of Pharmacy and Pharmaceutical Sciences 2010; Vol 2, Suppl 3, 340-345.

22. Tangri Pranshu, Rawat Prakash Singh, Jakhmola Vikash: Validation: A Critical Parameter for Quality Control of Pharmaceuticals. Journal of Drug Delivery and Therapeutics 2012; 2(3): 34-40.

Received on 22.11.2013 Modified on 02.01.2014

Asian J. Research Chem. 7(2): February 2014; Page 233-242

S. No.	Parameter	Condition	Rt	System suitability results
S. No.	Parameter	Condition	Rt	Peak Area	USP tailing	USP Plate Count
1	Flow rate by ± 1%	0.8 ml	2.7	554051	1.8	2459
		0.9 ml	2.5	551359	1.7	2668
		1.0 ml	2.6	552598	1.8	2860
2	Mobile phase by ± 1ml	74:26	2.4	552618	1.8	2776
		75:25	2.8	553639	1.8	2669
		76:24	1.9	551148	1.8	2654
3	Wavelength of analysis ± 5nm	260nm	2.4	553138	1.8	2687
		265nm	2.7	553068	1.7	2693
		270nm	2.0	553174	1.8	2643

S.No.	Parameter	Condition	Rt	System suitability results
S.No.	Parameter	Condition	Rt	Peak Area	USP tailing	USP Plate Count
1	Flow rate by ± 1%	0.9 ml	6.05	260101	1.09	10672
		1.0 ml	6.1	266938	1.1	10199
		1.1 ml	6.03	251458	1.07	9618
2	Mobile phase by ± 1ml	74:26	6.15	265715	1.0	9859
		75:25	6.03	26986	1.1	10199
		76:24	6.02	26606	1.08	10185
3	Wavelength of analysis ± 5nm	260nm	6.04	26817	1.1	10031
		265nm	6.08	26432	1.1	10199
		270nm	6.02	26038	1.0	9960

Data acquired time (hrs)	Data of Standard Solution (100%)				Data of Sample Solution
	Freshly prepared	Initial Day’s Solution			Freshly prepared	Initial Day’s Solution
	Mean Peak Area	Mean Peak Area	Mean Conc. (µg/ml)	Mean % Assay	Mean Peak Area	Mean Peak Area	Mean Conc. (µg/m)	Mean % Assay
0	569873	569873	500.3	100.2	569325	563364	500.00	100
24	569838	565688	499.36	99.73	568474	560573	498.45	99.01
48	569644	564837	498.90	97.53	569378	560147	499.47	98..70
72	570134	553980	473.83	95.33	569352	555968	473.06	96.29

Data acquired time (hrs)	Data of Standard Solution (100%)				Data of Sample Solution
	Freshly prepared	Initial Day’s Solution			Freshly prepared	Initial Day’s Solution
	Mean Peak Area	Mean Peak Area	Mean Conc. (µg/ml)	Mean % Assay	Mean Peak Area	Mean Peak Area	Mean Conc. (µg/ml)	Mean % Assay
0	268639	268639	199.30	100	266916	266916	198.37	100
24	268259	265239	197.93	98.90	264933	260839	198.81	98.45
48	268383	262313	196.95	97.76	260619	264323	197.63	97.67
72	268212	252131	176.13	94.0	258166	255035	178.04	95.13