QUANTITY BASED QUALITY ESTIMATION OF DICLOFENAC SODIUM BY REVERSED PHASE-HIGH PERFORMANCE LIQUID CHROMATOGRAPHY SEPARATION METHOD FROM FORMULATED TABLETS

Abstract

A simple, cheap and reliable reversed phase-high performance liquid chromatographic (RP-HPLC) method has been developed for the quantitative determination of diclofenac sodium in pharmaceutical samples of tablets. The present isocratic method was carried out on C ^sub 18^-column with pH 3.4 adjusted mobile phase acetonitrile and water in the ratio of 80:20 (v/v) at the flow rate 1.0 mL/min. The detection was carried out at wave length (λ ^sub max^) 225 nm. The average retention time of diclofenac sodium was 4.25 (±0.5) min. Regression analysis showed good linearity R ^sup 2^=0.999 over the concentration 3.125-50 µg/mL. The limit of detection (LOD) and limit of quantification (LOQ) were calculated 0.03 and 0.09 µg/mL respectively. The average recoveries percentage was calculated 100.69 %, with 2.24 % coefficient variation (<10). Hence, the developed method was successfully validated to quantitative determination of diclofenac sodium in its pharmaceutical formulated tablets. [PUBLICATION ABSTRACT]

Headnote

ABSTRACT

A simple, cheap and reliable reversed phase-high performance liquid chromatographic (RP-HPLC) method has been developed for the quantitative determination of diclofenac sodium in pharmaceutical samples of tablets. The present isocratic method was carried out on C ^sub 18^-column with pH 3.4 adjusted mobile phase acetonitrile and water in the ratio of 80:20 (v/v) at the flow rate 1.0 mL/min. The detection was carried out at wave length (λ ^sub max^) 225 nm. The average retention time of diclofenac sodium was 4.25 (±0.5) min. Regression analysis showed good linearity R ^sup 2^=0.999 over the concentration 3.125-50 µg/mL. The limit of detection (LOD) and limit of quantification (LOQ) were calculated 0.03 and 0.09 µg/mL respectively. The average recoveries percentage was calculated 100.69 %, with 2.24 % coefficient variation (<10). Hence, the developed method was successfully validated to quantitative determination of diclofenac sodium in its pharmaceutical formulated tablets.

Keywords: Diclofenac sodium; RP-HPLC.

INTRODUCTION

Diclofenac is a salt derivative of phenyl acetic acid (Figure 1). The IUPAC name is sodium [2-[(2,6-dichlorophenyl) amino] phenyl] acetate.1 It is a non steroidal anti-inflammatory drug (NSAID).2 Diclofenac sodium also exhibits analgesic and anti-pyretic activities in both animals and human beings.3 It is used for the relief of signs and symptoms of osteoarthritis, rheumatoid arthritis and ankylosing spondylities. It is also used to treat chronic pain associated with cancer.4 It is inhibiting prostaglandin synthesis by inhibition cyclooxygenase (COX), inhibiting DNA synthesis. Commercially, it is available in the various formulations such as injections, tablets, gel, suppositories and powdered form. Several methods have been reported for the simultaneous determination of diclofenac in single, binary and tertiary combined form in other drugs5-7 by HPLC with UV Detector.8,9 The main purpose of the present study is to develop a cheap and reliable method for the quality control. In the present study is to develop a new reversed phase-high performance liquid chromatography (RP-HPLC) method.

MATERIALS AND METHODS

Chemicals and reagents

The 99.99 percent standard drug of Diclofenac sodium

was supplied by Ranbaxy Laboratories Ltd., HPLC grade acetonitrile, HPLC grade distilled water and 0.45 nylon filter membrane was purchased from Merck India Ltd. Mumbai.

Instrumentation

A binary pump CYBERLABTM HPLC chromatograph was used for analytical work. The separation was on non polar C18-column. The analyte were monitored with UV detector at wave length 225 nm. The flow rate was 1.0 mL/min. The high performance liquid chromatography instrument was operated at isocratic mode with eluation pH 3.4 acetonitrile/water in the ratio of 80:20 (v/v) mobile phase. The injection volume was 20 µL. An ultrasonic sonicator was used for the sonication of mobile phase, standard solution and sample solution.

Preparation of mobile phase

An Acetonitrile/water mobile phase was prepared. The ratio of acetonitrile and water was 80:20 (v/v). The pH was adjusted 3.4. The mobile phase was filtered through a 0.45 µm nylon membrane followed degassing.

Preparation of stock solution

10 mg standard drug of diclofenac sodium was well mixed with mobile phase and make up it to 100 mL and prepared 100 µg/mL stock solutions. The stock solution was filtered with 0.45 µm nylon filter membrane followed by degassing and sonication.

Preparation of sample solution

The 10 tablets were weighed accurately and crushed with mortar pistol. The crushed tablets were mixed well, and then an equivalent amount of 10 mg was transferred into a small conical flask and extract with acetonitrile/water 80:20 (v/v) mobile phase. The extract was filtered into a 100 ml volumetric flask and the volume make up to 100 mL. Achieved aliquots was covered the working concentration range 100 ppm. The sample solution was filtered with 0.45 ìm nylon filter membrane followed by degassing and sonication.

Preparation of calibration curve

A calibration curve was constructed between average peak area and known concentrations by monitored different lower to highest concentration (3.125, 6.25, 12.5, 25 and 50 ìg/mL) dilutions of standard drug diclofenac sodium solution chromatograms. The concentrations of diclofenac sodium in replicates of different samples were calculated with the help of the calibration curve, and the mean values of three replicates were computed.

RESULTS AND DISCUSSION

Selection of wave length

The selection of wave length for Diclofenac sodium was investigated in order to determine a suitable wavelength for the assay. The suitable wave length was found 225 nm.

Selection of mobile phase

The mobile phase was select under reversed phase partition chromatographic conditions. The mobile phase developed was studied in order to achieve suitable system stability test. The different ratio of (10:90, 20:80, 30:70, 40:60 and 50:50) mobile phases compositions (Water/ acetonitrile) were tested with different pH value (3.0, 3.2 and 3.4). The mobile phase acetonitrile/water in the ratio of 80:20 (v/v) with pH 3.4 was given suitable retention time and better resolution. There is no interference with excipients (Figure 2).

Methods validation

The various method validation parameters were performed according to international conference harmonization guide line10 such as system suitability, Linearity, limit of detection (LOD) and limit of quantification (LOQ), selectivity specificity and accuracy.

System suitability test

The system suitability was checked to insure that, the system is working correctly. The system suitability parameters peak area, retention time, resolution factor, theoretical plates, and tailing factor were checked according to international conference harmonization guide line.9 This test was performed during development of the method. This test was performed by injecting the standard mixture in 6 replicates the standard deviation, coefficient of variation and standard errors were calculated. The standard error of each parameter is less than 1.0 % (Table 1). Hence, the system suitability test is statistically significant. Therefore the proposed method is suitable for assay analysis.

Linearity

The linearity was measured by regression analysis. The linearity range of Diclofenac sodium was 3.125-50 µg/mL. The results was showed good linearity with regression coefficient (R2) 0.999. The linear regression equation for Diclofenac sodium was described in Table 2.

Limit of detection (LOD) and limit of quantification (LOQ)

Limit of detection (LOD) and limit of quantification (LOQ) were calculated using following equations:-

Limit of detection (LOD) =3.3σ/s (i)

Limit of quantification (LOQ) =10.σ/s (ii)

Where σ is the standard deviation of y intercept and s is the slope of the curve. The limit of detection (LOD) and limit of quantification (LOQ) were reported 0.03 and0.09 µg/ml. The calculated result was presented in Table 2.

Selectivity

The selectivity was checked by analysis of standard stock dilution and sample dilution. The peak of Diclofenac sodium was identified by comparing their retention time in a chromatogram with those the standards.

Specificity

The specificity was assessed by comparing the chromatogram of standard drug with the chromatogram of tablet solutions. The retention time and peak area of standard drug and sample drug is same, so the method is specific. There was no interference of excipients with the drug analyte. Hence, the developed method is specific and selective.

Accuracy

Performance of the validated method is confirmed by performing inter-day and intra-day recovery study at three different concentration levels 15, 30 and 45 mg. The three different concentration diluted from the stock solution were added to an extract with a known content of Diclofenac sodium and the percentage recovery of the respective constituents were calculated. The recovery results showed good accuracy with <10 coefficient variations in both inter-day and intra-day study. The calculated result was presented in Table 3 and 4.

CONCLUSION

In the present study an isocratic reversed phase-high performance liquid chromatography (RP-HPLC) method was developed for checking the Quantity based quality in the formulated Diclofenac Sodium tablets. The linearity was calculated 0.999, which significant at the level p=0.1% error. The limit of detection and limit of quantification were calculated 0.03 and 0.09 µg/mL. The inter-day and intra-day average percentage recovery at three concentration levels were calculated 100.69 % with the coefficient variation percentage 2.240 (<10) and 0.8351 (<1.0). Hence, applied method validation was significant. Therefore this method is acceptable for checking the quantity based quality from formulated tablets.

ACKNOWLEDGEMENTS

Present study was supported by Dolphin Institute of Biomedical and Natural Sciences, Dehradun, Uttarakhand, India.

References

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