Corresponding author: Md. Hossain Sohrab (
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 (
Aceclofenac (2-[(2,6-dichlorophenyl)amino]phenylacetoxyacetic acid) is a nonsteroidal anti-inflammatory drug (
Aceclofenac.
Several methods utilized techniques like HPLC, UV-VIS Spectroscopy, GC, TLC etc. for quantification of
Analytical methods of Aceclofenac.
Sl No | Analytical Method | Method Condition/Mobile Phase/ Stationary Phase/Retention Time (Approximately) | Wavelength/Detector | Linearity Range | Reference |
---|---|---|---|---|---|
1 | HPLC | Methanol and 0.02% of orthophosphoric acid in the ratio of 70:30 (%, v/v); C18; 10 min | 275 nm | 1–100 µg/mL |
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2 | UV spectroscopy | Phosphate buffer saline of pH 7.4 as diluent | 273 nm | 0–20 µg/mL |
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3 | HPLC | Acetonitrile, methanol and water in the ratio of 60:28:12 (%, v/v) and pH of 7.0 adjusted with either glacial acetic acid and sodium hydroxide; C18; 6 min | 274 nm | 0.0138–0.370 µg/mL |
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4 | HPLC | Acetonitrile, methanol and phosphate buffer of pH 7.0 in the ratio 30:17:53 (%, v/v); C18; 13.8 min | 280 nm | 2–10 μg/mL |
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5 | GC | Injector temperature: 260 °C; Detector temperature: 300 °C; N2 flow rate: 5.0 mL/min; Make up flow: 30 mL/min; Split ratio: 10:1. Caffeine was used as internal standard (IS) | FID Detector (30 m X 0.53 mm; 1.5 µm) | 10–110 μg/mL |
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6 | Spectrophotometric method | Aceclofenac was reacted with 0.25% w/v solution of |
665.5 nm | 20–100 μg/mL |
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HPLC | Methanol, acetonitrile and acetic acid (2% solution in deionized water) in the ratio of 100:150:250 (%, v/v/v) containing 0.3 ml triethylamine; C18; 8.8 min | 275 nm | 20–70 μg/mL | ||
Densitometric method | Chloroform, ethyl acetate and acetic acid in the ratio of 75:25:5, (% v/v/v). Calibration curve is obtained from area under the peak against the concentrations. | 254 nm and 275 nm | 1–10 μg/spot | ||
7 | Spectrophotometric method | Aceclofenac was reacted with p-dimethylamino-cinnamaldehyde and 1% perchloric acid at 90 °C for 10 min and then diluted with methanol. | 658 nm | 1–200 μg/mL |
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Aceclofenac was reacted with 3-Methyl-2-benzothiazolinone hydrazine hydrochloride and 0.1% ferric chloride for 20 min and then diluted with water. | 592 nm | 1–100 μg/mL | |||
8 | HPLC | Sodium phosphate buffer pH 5.0 and Acetonitrile in the ratio of 60:40 (%, v/v); C18; IS: Etoricoxib (ETC); 8 min | 275 nm. | 25–125 μg/mL |
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9 | HPLC | Mixed phosphate buffer pH 6.8 and acetonitrile in the ratio of 50:50 (%, v/v); C18; 8.5 min | 278 nm | 2–10 µg/mL |
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10 | UV spectrophotometry | Diluent: Methanol | 276 nm | 0–120 μg/mL |
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11 | Third-derivative spectrophotometry (D3) | Calibration curve was constructed from peak amplitude (height) against corresponding concentration for the linearity range of Aceclofenac solution in methanol. | 283 nm | 4–24 μg/mL |
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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. | 252 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 |
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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. | 245 nm | 10–40 μg/mL | ||
pH-induced difference (ΔA) spectrophotometry | The difference in absorbance was observed between 0.1 N sodium hydroxide and 0.1 N hydrochloric acid solution of Aceclofenac. Calibration curve was constructed by plotting the difference in absorbance against respective concentration. | 273 nm | 15–50 μg/mL | ||
Quantitative densitometric evaluation of thin layer chromatogram | Tetrahydrofuran and methanol (90:10, % v/v) was used for TLC development. Calibration curve was constructed by plotting the area under the peak against the corresponding concentrations. | 275 nm | 50–200 μg/mL | ||
HPLC | Methanol and water in the ratio of 60:40 (%, v/v); C18; 8 min | 230 nm | 1–50 μg/mL | ||
13 | HPLC | 0.01 M ammonium acetate buffer with 2 ml (%, v/v) triethylamine and acetonitrile in the ratio of 68:32 (%, v/v) and pH was adjusted to 6.5 with glacial acetic acid; C8; 6.5 min | 270 nm | 8–16 µg/mL |
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14 | HPLC | 0.07% of orthophosphoric acid and acetonitrile in the ratio of 68:32 (%, v/v) at pH 7.0 ± 0.05; C18; 6 min | 275 nm | 160–240 µg/mL |
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15 | Microwave assisted spectrophotometry | Aceclofenac was reacted with ammonium molybdate in presence of sulfuric acid under microwave irradiation for 5 min. | 740 nm | 50–250 µg/mL |
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All weighing were done on Electronic balance (A & D Company Ltd, Japan). Digital pH meter (SENSION+, Spain), bath sonicator (Wisd Laboratory Instrument, Germany) were also used in this study. UV-Vis spectra were recorded on a Specord 250 plus PC double beam spectrophotometer using 1.0 cm quartz cells. High purity deionized water was obtained from Millipore, Milli-Q (Merck KGaA, Darmstadt, Germany) water purification system. Assay test was performed with a HPLC (Hitachi High – Tech Science Corporation, Tokyo, Japan) machine with pump (Hitachi chromaster 5110), autosampler (Hitachi chromaster 5210) and PDA Detector (Hitachi chromaster 5430). LC separations were performed on a C8 column (250 × 4.6 mm i.d., 5 μm particle size), LaChrom, Hitachi, Japan with C8 guard column (23 mm X 4 mm; 3 µm), LaChrom, Hitachi, Japan. Data was integrated using Agilent open lab control panel CDS software. The mobile phase consisted of 10 mM Phosphate Buffer, pH 2.1 and methanol in 40:60%, v/v. The flow rate was set to 1.0 mL/min and UV detection was carried out at 272 nm at 25 °C.
The 0.25-µm PTFE filters were obtained from Chromafil Xtra (Macherey Nagel GmbH & Co. AG., Germany). Working standards of pharmaceutical grade
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.
Commonly used excipients were mixed at appropriate amount to obtain the placebo mixture as Table
Placebo Constituents.
Lactose | : | 480 mg |
Microcrystalline cellulose | : | 500 mg |
Sodium Starch Glycolate | : | 200 mg |
Povidone K-30 | : | 125 mg |
Magnesium Stearate | : | 100 mg |
Talc | : | 100 mg |
Stock solution of
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.
The linearity of the method was established by determining linear regression equation from the calibration curve of
Accuracy of the method was determined by performing the recovery experiment of standard addition method at three concentration levels in triplicate (
The precision of the instrument (
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 (1st, 3rd, and 6th 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 (%
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 response (
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.
Six replicate injection of
The wavelength for maximum absorbance of
Absorbance maxima of Aceclofenac.
Marketed tablets were analyzed through the developed method which showed 101.78% of
Evaluation of linearity, Limit of Detection (
λmax. (nm) | Regression equation (y = mx + c) | Linearity range (µg/mL) | Residual sum of squares | Correlation coefficient | Baseline Noise in rms | S/N for |
S/N for |
---|---|---|---|---|---|---|---|
275 | 74894x + 94097 | 19.8 to 148.5 | 2.213 | 0.999 | 218.7 | 3.005 | 9.36 |
Calibration curve of linearity Study.
The baseline noise value was obtained from the software (Table
Sensitivity Study.
The mean recoveries were 97.91% to 100.39% substantiated the method as accurate (Table
Evaluation data of accuracy study.
Level | Theoretical Conc. (µg/mL) | Peak area | Actual Conc. (µg/mL) | % Recovery | % |
---|---|---|---|---|---|
80% | 101.16 | 7553140 | 100.64 | 99.48 | 0.64 |
101.16 | 7618998 | 101.52 | 100.36 | ||
101.16 | 7525643 | 100.28 | 99.13 | ||
100% | 121.36 | 8984771 | 119.72 | 98.65 | 0.66 |
121.36 | 8917608 | 118.83 | 97.91 | ||
121.36 | 9034943 | 120.39 | 99.2 | ||
120% | 141.55 | 10664687 | 142.11 | 100.39 | 0.79 |
141.55 | 10737296 | 143.07 | 98.94 | ||
141.55 | 10644064 | 141.83 | 100.2 |
Evaluation data of precision study.
Sample No. | Repeatability | Intermediate Precision | |||
---|---|---|---|---|---|
Intra – day | Inter – day | ||||
1 | 102.83 | 1st hour | 102.57 | 1st day | 103.70 |
2 | 99.86 | 3rd hour | 101.40 | 2nd day | 101.22 |
3 | 101.73 | 8th hour | 102.68 | 3rd day | 102.53 |
4 | 102.48 | – | – | 4th day | 99.86 |
5 | 101.73 | – | – | 5th day | 103.59 |
6 | 101.69 | – | – | – | – |
Mean | 101.721 | 102.216 | 102.181 | ||
SD | 1.025 | 0.579 | 1.464 | ||
% |
1.007 | 0.567 | 1.432 |
The developed analytical method should be specific for
Assay at Accelerated State of 80 °C.
Concentration (%) | |||||
---|---|---|---|---|---|
Initial | 8 hour (80 °C) | 24 hour (80 °C) | 48 hour (80 °C) | ||
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: | 100.16 | 100.72 | 103.28 | 103.13 |
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: | 96.18 | 100.49 | 97.81 | 97.48 |
Specifity study of Aceclofenac-standard spiked with excipients.
Fixed drug substance spiked with different concentration of excipient | Fixed excipient spiked with different concentration of drug substance | ||||
---|---|---|---|---|---|
Nominal drug substance concentration (µg/mL) | Excipient concentration (µg/mL) | Absorbance | Nominal excipient concentration (µg/mL) | Drug substance concentration (µg/mL) | Absorbance |
87.693 | 128.72 | 6705872 | 160.9 | 70.154 | 5428265 |
144.81 | 6664433 | 78.923 | 5898186 | ||
160.9 | 6707371 | 87.693 | 6873075 | ||
176.99 | 6784787 | 96.462 | 7506926 | ||
193.08 | 6805847 | 105.231 | 8074567 | ||
Regression equation, y = 78699x - 145122 | |||||
R² = 0.9892 |
Specifity study-response of the standard, sample, excipient and mobile phase.
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 %
Robustness Study (a) Variance of peak area for change in different method parameters with %
In this study a simple RP-HPLC-DAD method for use in routine estimation of
Table S1. HPLC Analytical methods for simultaneous estimation of Aceclofenac with other constituents
HPLC Analytical methods
Comparision of published simultaneous HPLC analytical method of Aceclofenac with other drugs.