Simple RP-HPLC method for Aceclofenac quantitative analysis in pharmaceutical tablets

A reverse phase liquid chromatographic method for estimation of Aceclofenac in bulk drug and tablet dosage form was developed and validated. The chromatographic conditions to achieve the highest performance parameters using octylsilyl column with guard filter were optimized. The separation was carried out using a mobile phase containing 10 mM Phosphate Buffer, pH 2.1 and methanol (30:70% v/v) pumped at a flow rate of 1.0 mL/min with detection at 272 nm. The method was shown to be linear in 19.8–148.5 μg/ mL concentration range (regression coefficient of 0.999). The limit of detection (LOD) and limit of quantification (LOQ) was found to be 0.0692 μg/mL and 0.2076 μg/mL, respectively. The accuracy of the method was assessed by adding fixed amount of pre-analyzed sample to different standard solutions (80%, 100%, and 120% of the tested concentration) in triplicate. The percentage mean recoveries were 97.91% to 100.39% with %RSD values of 0.64–0.79. The method was found to be precise with %RSD value of 1.13 and 1.60 for intraday and interday precision study, respectively. The method specificity and robustness were also established. New and sensitive HPLC method for estimation of Aceclofenac has been developed, in respect to the reviewed analytical methods.


Aceclofenac
(2-[(2,6-dichlorophenyl)amino]phenylacetoxyacetic acid) is a nonsteroidal anti-inflammatory drug (NSAID) of the phenylacetic acid group. Aceclofenac (ACF) [ Fig. 1] selectively inhibit Cyclooxygenase (COX)-2 enzyme to inhibit generation of inflammatory mediators and in turn suppress pro-inflammatory prostaglandins and cytokines production resulting in analgesic, antipyretic activity by both central and peripheral actions (Arslan and Tirnaksiz 2010). Ratio-spectra first-derivative (RSD1) spectrophotometry The absorption spectra of Aceclofenac in the linearity range were divided by that of diclofenac sodium (25 mg/mL), and the ratio spectra were differentiated with respect to wavelength. Calibration curve was obtained by plotting the first-derivative values at 252 nm against the corresponding concentration.

nm 4-32 μg/mL
Spectrodensitometric method of Thin-layer chromatogram Chloroform, methanol and ammonia in the ratio of 48:11.5:0.5 (%, v/v/v) were used for TLC development. Calibration curve was constructed by plotting the area under the peak against the corresponding concentrations to develop the regression equation.
274 nm 2-10 µg/spot 12 Third derivative spectrophotometry UV-spectrum of Aceclofenac solution was measured against absolute ethanol as a blank.
The peak height at 242 nm was measured. The calibration curve was constructed with the measured peak height against the corresponding concentration.
242 nm 5-40 μg/mL Hasan et al. 2003 Ratio-spectra first-derivative (RSD1) spectrophotometry Absorption spectra of Aceclofenac solutions were divided by the absorption spectrum of 5 mg/ml of the degradate. The ratio spectra thus obtained were smoothed and differentiated to determine first derivatives of the ratio spectra. The calibration curve was constructed between the measured first derivative values at 245 nm against the corresponding concentration.

Reagents
The 0.25-µm PTFE filters were obtained from Chromafil Xtra (Macherey Nagel GmbH & Co. AG., Germany). Working standards of pharmaceutical grade ACF (batch no. 28296) was supplied as a gift sample by Beximco Pharmaceutical Ltd (Dhaka, Bangladesh). Marketed ACF 100 mg tablets (Square Pharmaceuticals Ltd.) were purchased from local drug store. All chemicals and reagents of analytical grade were purchased from Active Fine Chemicals, Dhaka, Bangladesh.

Buffer Preparation
10 mM phosphate buffer was prepared by dissolving 0.64 gm potassium dihydrogen phosphate and 0.4 mL phosphoric acid to 900 mL deionized water. pH of the solution was adjusted to 2.1 with dilute phosphoric acid solution if necessary. The final volume was then adjusted to 1000 mL with deionized water.

Placebo preparation
Commonly used excipients were mixed at appropriate amount to obtain the placebo mixture as Table 2. Placebo stock solution was prepared by mixing average tablet placebo content with mobile phase to obtain concentration of approximately 1.5 mg/mL.

Method Optimization
Stock solution of ACF was prepared in methanol (≈50 µg/ mL) and the UV spectrum was taken in the range of 200-400 nm to obtain the wavelength for maximum absorbance (λ max ) of Aceclofenac. As Aceclofenac (pKa≈3.4) is a strongly acidic drug, pH 2.1 was chosen as the buffer pH for mobile phase preparation. Mobile phase was taken as 10 mM phosphate buffer of pH 2.1 and methanol in the ratio of 30:70 (%, v/v). Solvent stability was checked at approximately 100 µg/mL concentration for consecutive five days in the mobile phase by determining the relative standard deviation (%RSD) of response and peak purity of the drug. The described method has been validated for response function, accuracy, repeatability and intermediate precision.

Standard and Sample preparation
Standard stock solution at 1 mg/mL concentration was prepared in mobile phase by dissolving it first on not more than 5% of methanol. Sample stock solution was prepared by crushing randomly selected 10 tablets. Average weight equivalent sample was taken in volumetric flask to obtain concentration of approximately 1 mg/mL. Sample was also first dissolved in not more than 5% of methanol. The final volume was adjusted with mobile phase. The concentration for assay preparation was approximately 100 µg/mL. Drug content was determined using Equation 1.

Linearity
The linearity of the method was established by determining linear regression equation from the calibration curve of ACF. The calibration curve was prepared using six different concentration levels in triplicate ranging from 20% to 150% of the assay preparation of analyte. For this, a stock solution (1 mg/mL) of the drug was prepared in mobile phase using not more than 5% of methanol initially. A range of concentrations (19.8-148.5 µg/mL) were then prepared after suitable dilution of the prepared stock with mobile phase.

LOD and LOQ
LOD and LOQ were separately determined based on the signal to noise ratio as per ICH guidelines (ICH Q2 R1. 2005).

Accuracy
Accuracy of the method was determined by performing the recovery experiment of standard addition method at three concentration levels in triplicate (Chakraborty et al. 2018). Different amount of standard stock solution was taken at 80% to 120% of assay concentration and mixed with fixed amount of previously analyzed sample stock solution of 1 mg/mL concentration to obtain final concentration of approximately 101-140 µg/mL.

Repeatability
The precision of the instrument (RSD) was checked by repeated scanning of samples (n = 6) for ACF standard without changing the parameter of the proposed method.

Intermediate precision
To determine the intra-day and inter-day precision of the method, the drug solution at assay concentration (100 µg/ mL) was prepared (n = 6) in one laboratory on the same day (1 st , 3 rd , and 6 th hour) and also on five different days from the same standard stock solution. The concentration was calculated from the areas obtained and the results were expressed as relative standard deviation (%RSD).

Specificity
Specificity was determined by checking the chromatograms of blank, placebo, standard and sample solution for interference with analyte peak, as well as through determination of peak purity for the drug in the presence of degradation products. At first different placebo concentration were spiked with nominal concentration of drug substance and then different concentration level of drug were spiked with fixed placebo concentration to determine the peak res-ponse (Chakraborty et al. 2018). The responses of the standard ACF, marketed product and excipient of stressed condition (kept at 80 °C for 48 hrs) were also compared with the response of the same samples of unstressed condition at the assay concentrations to establish the stability indicating nature of the developed method as part of the forced degradation studies.

Robustness
Robustness was determined by changing the different method parameters like mobile phase composition, pH of buffer, column temperature, mobile phase flow rate and detector wavelength.

System Suitability
Six replicate injection of ACF standard solution at assay concentration was checked for tailing factor, theoretical plate, retention time, capacity factor and relative standard deviation of response to establish suitability of the method in the instrument.

Results and discussion
The wavelength for maximum absorbance of ACF was found to be at approximately 275 nm (Fig. 2).
Marketed tablets were analyzed through the developed method which showed 101.78% of ACF with 0.186 of %RSD. The proposed method was found to be linear with a correlation coefficient of 0.999 (Table 3 and Fig. 3).
The baseline noise value was obtained from the software (Table 3). The minimum concentration levels at which ACF can be reliably detected (LOD) and quantified (LOQ) were found to be 0.0692 µg/mL and 0.2076 µg/mL respectively ( Fig. 4 and Table 3) which was more sensitive      than some of the other published methods (Ravisankar et al. 2013, Balan andKannappan 2014). Signal to noise (S/N) ratio for LOD and LOQ was shown in Table 3 which was also within acceptable limits. The mean recoveries were 97.91% to 100.39% substantiated the method as accurate (Table 4). The method was also found to be precise with <2% of RSD value (Table 5)  The developed analytical method should be specific for ACF assay in presence of all the potential matrix components which was checked by evaluating the peak responses and peak purity of the standard, sample, excipient and mobile phase (blank) solutions in assay concentration (Fig. 5). Specificity of the proposed method is also evaluated by the stress study of the samples. The % assays of ACF standard and marketed product were unaffected by the stressed condition as compared to the initial result at different observation days (Table 6). So the placebo effect was checked in peak response as described in the method. The response obtained with the mixture showed no interference with the standard response (Table 7).
The change in organic solvent of ±2% [Buffer/68-72(%, v/v) Methanol] significantly changed the peak retention time from 22.40-12.41 min, although the %RSD of the peak response was found to be 0.83. The method was found to be robust for pH variation of the buffer solution from 1.7 to 2.5 (%RSD of 0.45 for the peak response), although the retention time (R t ) changed from 11.09 to 12.52 min. For the change in column temperature from 20-25 °C the retention time changed from 14.23-12.27 min with acceptable %RSD (0.63) of the peak response.  Flow rate of mobile phase was changed from 0.8 to 1.2 mL/min. The R t and peak response of ACF both changed significantly for change of flow rate from 0.9 to 1.1 mL/ min. Robustness of the method was checked for change in wavelength from 270-280 nm. The method was found to be robust for 272-278 nm with %RSD of 1.39 for peak response. The method conditions at which response were most stable was shown in Fig. 6.

Conclusion
In this study a simple RP-HPLC-DAD method for use in routine estimation of ACF in bulk drug and tablet dosage form has been developed and validated. The accuracy, precision and specificity of the method have been established, with determination of the method parameters up to which the method was found to be stable.