Quantization of Aceclofenac in Pharmaceutical Formulations by RP-HPLC

 

Santanu Ghosh and BB Barik

University Department of Pharmaceutical Sciences, Utkal University, Bhubaneswar, Orissa.-751004, India.

 

 

ABSTRACT:

An isocratic reversed phase high-performance liquid chromatographic (HPLC) method with ultraviolet detection at 281 nm has been developed for the determination of aceclofenac in dosage formulation. Good chromatographic separation aceclofenac was achieved by using a stainless steel analytical column Inertsil ODS, C18, 250 x 4.6 mm, 5 µ. The system was operated at (30±2°C) using a mobile phase consisting of buffer: acetonitrile (600:400) at a flow rate of 1.5 ml/min. The calibration curve for aceclofenac was linear over the tested concentration range of 50%, 75%, 100%, 125% and 150% with reference to the label claim and a correlation coefficient of 1.00. The intra- and inter-run precision and accuracy results were 99.07 to 100.20 with the %RSD of 0.45% and tailings factor 1.16. The proposed method was validated for its selectivity, linearity, accuracy, and precision. The method was found to be suitable for the quality control of aceclofenac in bulk drug as well as in formulation.

 

KEYWORDS: Aceclofenac, UV detection, RP-HPLC, dosage formulation, method validation.

 

 

INTRODUCTION:

Aceclofenac or 2-[2-[2-[(2,6-dichlorophenyl) amino]phenyl] acetyl]oxy]acetic acid, is a white or off white crystalline, odourless and bitter taste powder melts at 149 to 150°C 1. Aceclofenac is one of the emerging NSAID molecules for arthritis treatment. It is a newer derivative of diclofenac and has less gastrointestinal complications 2,3  used to management of rheumatoid arthiritis, Osteoarthiritis and ankylosing spondylatis 4,5,6.

 

It is official in British Pharmacopoeia7 Several analytical techniques like titrimetric7,8 , colourimetric9, spectroflurimetric10, densitometric11,12, HPLC12-14, RP– HPLC15,16, spectrophotometer17,18 and stripping voltametric19 have been reported for assay of aceclofenac. However some of these methods are costlier and time consuming.

 

In literature some liquid chromatography method and a visible spectrophotometric method for the analysis of aceclofenac were reported for the determination of aceclofenac and its related substances in biological fluids like plasma, blood, urine and in formulation were reported but not a single method has been reported for its determination in bulk and solid dosage forms by RP-HPLC method. The main purpose of this study was to develop a simple and reliable method to quantitate aceclofenac in a relatively short time with high linearity. Therefore, this study focused on the development of simple and rapid isocratic RP-HPLC method which can be employed for the routine analysis of aceclofenac in bulk drug and formulations. The established method was validated with respect to specificity, linearity, precision, accuracy, and ruggedness.

 

MATERIALS AND METHODS:

Materials and reagents:

All chemicals and reagents were of HPLC grade quality. Aceclofenac was kind gift from Mepro Pharmaceuticals Pvt. Ltd. Surendranagar, Gujarat.

A stock solution of 0.5 mg/ml aceclofenac was prepared in Methanol. More dilute solutions were made daily with same solvents just before use.

 

Pharmaceutical dosage form:

Tablet label claim of 200 mg aceclofenac sustained release tablet was taken for analysis.

 

Chemicals:

HPLC grade acetonitrile, glacial acetic acid and triethyl amine were purchased from Ranbaxy India Ltd., India. Other chemicals used were HPLC grade from Qualigens, Bombay, India. Tablet sample were trial sample.

 

Instrumentation and chromatographic conditions:

The quantitative determination of aceclofenac was performed by HPLC. A gradient HPLC (Dionex HPLC Ultimate 3000 Chromeleon series, Dionex corporation, USA) With a variable wavelength programmable UV/VIS Detector, column oven and Inertsil ODS, C18, 250 x 4.6 mm, 5µ column. The HPLC system was equipped with the software Chromeleon.

 

The mobile phase used was a mixture of Buffer and Acetonitrile in the ratio of 600:400(Buffer was prepared by 1.2 ml of glacial acetic acid in 1000 ml of water and pH was adjusted to 5.2 with triethyl amine). The filtered mobile phase components were pumped from the respective reservoirs at a flow rate of 1.5 ml min-1. The column temperature was maintained at 300C. The eluent was detected by UV detector at 281 nm and the data were acquired, stored and analyzed. A standard curve was constructed for Aceclofenac in the range 50% to 150%.

 

Preparation of standard and sample solutions Standard preparation:

A standard stock solution of 50mg of aceclofenac in methanol was prepared in volumetric flask. Working solutions of following concentrations 25%, 50%, 100%, 125% and 150% of the labeled amount aceclofenac 200 mg Sustained release tablets were prepared by diluting the stock solutions with the same solvent.

 

Procedure for tablets:

Twenty tablets were weighed, and crush to powder. Weigh accurately powder equivalent to 50 mg into 100ml volumetric flask then 50 mg of aceclofenac  into a 100ml of volumetric flask then 50 ml methanol was added and sonicate to dissolved. From that a portion was filter through 0.45 µ filter, which was used for the estimation. Then diluted with methanol. 5 ml of this solution taken in a 50 ml volumetric flask and diluted to volume with methanol and mixed well.

 

RESULTS DISCUSSION:

Development and optimization of isocratic HPLC conditions:

A HPLC attached PDA scan of aceclofenac showed a maximal area at or near 281 nm. Initial method development was conducted on a Inertsil ODS, C18, 250 x 4.6 mm, 5 µ column was used for separation at ambient temperature. This column provides efficient and reproducible separations of non polar compounds. Consequently, it was selected for method development and validation of assay. Preliminary method development of suitable isocratic conditions to resolve aceclofenac on the C18 column was conducted with Acetonitrile: Buffer at various ratios as the mobile phase for initial method development. A mobile phase of acetonitrile: buffer 600: 400 with 300C column temperature was found to provide a reproducible, baseline resolved peak (figs. 1-2). These conditions allowed for separation of aceclofenac from sustained release tablet formulation. The chromatographic conditions were optimized with respect to specificity, resolution and time of analysis. The specificity of the method was established through the placebo study on common additives used for tablets which shows no peak with placebo. For validation of analytical methods, the guidelines of the International Conference on the Harmonization of Technical Requirements for the Registration of Pharmaceuticals for Human Use20 have recommended the accomplishment of accuracy tests, precision, specificity, linearity, and robustness of the method. The calibration curve of   aceclofenac was 25%, 50%, 100%, 125% and 150% were linear in the range as mentioned above of the labeled amount aceclofenac 200 mg sustained release tablets (fig. 3).

 

Fig. 1: Blank Chromatogram

 

Fig. 2: Standard graph of aceclofenac.

 

Fig. 3: Chromatogram of aceclofenac standard.

 

System suitability:

The HPLC system was equilibrated with the initial mobile phase composition, followed by 5 injections of the same standard. These 5 consecutive injections were used to evaluate the system suitability on each day of method validation. The system suitability parameters including percentage RSD (Relative Standard Deviation) of peak areas (0.80 %) <2 and tailing factor (1.16) <2.

 

 

Table 1: Linearity, precision and accuracy (recovery) characteristics of aceclofenac

Linearity

Precision of the method

Accuracy of the test Method (Recovery studies)

Concentration

Inter day*

Intra day**(3days)

Spike level

% Recovered†

25 %

 

 

0.45%

 

 

0.79%

50 %

98.44%

50%

75%

98.17%

100%

100%

99.20%

125%

125%

99.60%

150%

150%

99.70%

Correlation coefficient(r)

1.00

Intercept

0.1172

Slope

11719.47

* %RSD of 10 determinations,  †RSD of six determinations at each level.

 

Table 2: Ruggedness and bench top stability data for aceclofenac

Bench top stability

Deviation from Initial Assay

Ruggedness

Initial assay

24 hours

48 hours

 

0.7 to 1.4

Analyst*

System*

1

2

1

2

100.4%

101.1%

101.8%

100.4%

100.2%

99.95%

99.42%

%RSD (limit NMT 2.0%)

0.68%

0.77%

0.59%

0.66%

* Mean of six determinations.

 

 

Fig. 4: Chromatogram of aceclofenac test.

 

All parameters were satisfactory with good specificity for the stability assessment of aceclofenac. Theoretical plates of the column were > 4700.

 

Intraday and inter-day accuracy and precision:

The precision of the method was investigated with respect to repeatability. For intra-day precision, standard solution of fixed concentration was injected at various time interval and %RSD was noted for that injections (0.45% limit %RSD<2.0%). And the day-to-day precision was studied by injecting the same concentration of standard solution at various days and the %RSD was calculate (0.79 % limit %RSD<2.0%) as shown in table 1. The recovery of aceclofenac  in dosage formulation was performed at five concentration levels (50, 75, 100, 125 and 150%), accuracy of the method in dosage formulation ranged from 98.17 % to 99.70 % as shown in table 1.

 

Limit of quantitaition and detection:

The detection limit (LOD), taken as the lowest absolute concentration of analyte in a sample, which can be detected but not necessary quantified under the stated experimental condition, was, 5%. The limit of quantitation (LOQ), taken as the lowest concentration of analyte in a sample, which can be determined with acceptable precision and accuracy under the stated conditions, was 25% to the labeled amount aceclofenac 200 mg sustained release tablets.

 

Specificity:

The specificity of the chromatographic method was determined to ensure separation of aceclofenac by placebo (fig. 5) study and by blank run of mobile phase (fig. 1) which were free from interference of solvent and commonly used tablet excipients. This is evidenced by the lack of interfering peaks in the chromatograms of placebo and blank run.

 

Fig. 5: Placebo study.

 

Ruggedness:

The ruggedness was established by determining aceclofenac in dosage formulation using the different chromatographic system and the same column by two analysts. The assay result indicated that the method was capable with high precision (table 2).

 

Bench top stability:

Stability of standard solution and sample solutions were determined by assay after 24 and 48 hours at room temperature against fresh standard solutions. It shows that the drug is stable and does not show much variation in the time span up to 48 hours (table 2).

 

DISCUSSION:

The maximal area   aceclofenac by HPLC  showed at 281 nm. No effect of dilution was observed on the maxima, which confirmed the maxima at 281nm.

 

 The statistical analysis of data obtained for the linearity curve of aceclofenac in pure solution indicated a high level of precision for the proposed method, as evidenced by low value of coefficient of variation. The coefficient of correlation was highly significant. The linearity range was observed between 0 – 75 mcg/mL. The plot clearly showed a straight line passing through origin (Y = M X + C ).

 

The assay method was validated by low values of % RSD and standard error, indicating accuracy and precision of the methods. Excellent recovery studies further proves the accuracy of the method. 

 

Robustness of the method was studied by varying the instrument, time of study and analyst. Reproducibility of the results confirmed the robustness of the method.

 

CONCLUSION:

A simple and reliable HPLC method for measuring aceclofenac in bulk pharmaceutical dosage formulation has been developed. A fully validated RP-HPLC procedure for the assay of aceclofenac drug in bulk and tablet formulation is described for the first time. Hence, it can be recommended for the routine quality control of this drug. The simplicity of the HPLC procedure, the short run time and the low volume of injection make this method suitable for quick and routine analysis. The intraday run and inter day run variability and accuracy results were with in the acceptable limit.

 

ACKNOWLEDGEMENT:

The authors are thankful to Mepro Pharmaceuticals and Medo Pharm Pvt. Ltd., for providing Reference standard and the raw materials of Aceclofenac

 

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Received on 01.09.2009

Accepted on 12.11.2009     

© A&V Publication all right reserved

Research Journal of Pharmaceutical Dosage Forms and Technology. 2(1): Jan. –Feb. 2010, 52-55