Validated HPLC Method for Simultaneous Quantitation of Diclofenac ...

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Sunil R. Dhaneshwar et al Der Chemica Sinica, 2010, 1 (2): 110-118 ______________________________________________________________________________ intercept were 80433 ± 1.18 and 187960 ± 1.82 for Diclofenac Sodium and 862734 ± 1.21 and 4750.9 ± 1.09 for Misoprostol respectively. Precision The results of the repeatability and intermediate precision experiments are shown in Table I. The developed method was found to be precise as the RSD values for repeatability and intermediate precision studies were < 2 %, respectively as recommended by ICH guidelines. LOD and LOQ Signal-to-noise ratios of 3:1 and 10:1 were obtained for the LOD and LOQ respectively. The LOD and LOQ were found to be 1 µg/mL and 2 µg/mL for Diclofenac Sodium and 0.03 µg/mL and 0.1 µg/mL Misoprostol respectively. Robustness of the method Each factor selected (except columns from different manufacturers) was changed at three levels (−1, 0 and 1). One factor at the time was changed to estimate the effect. Thus, replicate injections (n = 6) of mixed standard solution at three concentration levels were performed under small changes of three chromatographic parameters (factors). Insignificant differences in peak areas and less variability in retention time were observed (Table II). Table II: Robustness testing a (n = 3) Factor a Level Retention time Diclofenac Sodium Retention factor Asymmetry A: Flow rate (mL/min) 0.9 -1 1.94 0.22 1.19 1.0 0 1.87 0.25 1.15 1.1 +1 1.80 0.28 1.11 Mean ± SD (n = 3) 1.87 ± 0.07 0.25 ± 0.03 1.15 ± 0.04 B: % of acetonitrile in the mobile phase (v/v) 84 -1 1.92 0.23 1.18 85 0 1.87 0.25 1.15 86 +1 1.82 0.27 1.12 Mean ± SD (n = 3) 1.87 ± 0.05 0.25 ± 0.02 1.15 ± 0.03 C: Solvents of different lots First lot 1.87 0.25 1.15 Second lot 1.85 0.26 1.17 Mean ± SD (n = 3) 1.86 ± 0.01 0.25 ± 0.01 1.16 ± 0.01 Misoprostol Pelagia Research Library 115
Sunil R. Dhaneshwar et al Der Chemica Sinica, 2010, 1 (2): 110-118 ______________________________________________________________________________ A: Flow rate (mL/min) 0.9 -1 3.52 0.40 1.15 1.0 0 3.48 0.39 1.11 1.1 +1 3.44 0.37 1.07 Mean ± SD (n = 3) 3.48 ± 0.05 0.39 ± 0.01 1.11 ± 0.04 B: % of methanol in the mobile phase (v/v) 84 -1 3.54 0.41 1.16 85 0 3.48 0.39 1.11 86 +1 3.42 0.36 1.06 Mean ± SD (n = 3) 3.48 ± 0.06 0.38 ± 0.02 1.11 ± 0.05 C: Solvents of different lots First lot 3.48 0.39 1.11 Second lot 3.50 0.40 1.10 Mean ± SD (n = 3) 3.49 ± 0.01 0.39 ± 0.01 1.10 ± 0.01 a Three factors were slightly changed at three levels (-1, 0, 1) Specificity The peak purity of Diclofenac Sodium and Misoprostol was assessed by comparing their respective spectra at the peak start, apex and peak end positions i.e., r (S, M) = 0.9989 and r (M, E) = 0.9989. A good correlation (r = 0.9997) was also obtained between the standard and sample spectra of Diclofenac Sodium and Misoprostol respectively. Also, excipients from formulation were not interfering with the assay. Recovery Studies As shown from the data in Table III good recoveries of the Diclofenac Sodium and Misoprostol in the range from 99 to 101 % were obtained at various added concentrations. Label claim (mg/tablet) Amount added (mg) Table III : Recovery studies (n = 6) Total amount (mg) Amount Recovered (mg) ± % RSD % Recovery Diclofenac Sodium 50 40 (80%) 90 90.87 ± 0.96 100.96 50 50 (100%) 100 99.31 ± 1.01 99.31 50 60 (120%) 110 111.26 ± 0.78 101.14 Misoprostol 0.2 0.16 (80%) 0.36 0.363 ± 1.16 100.83 0.2 0.20 (100%) 0.40 0.406 ± 1.40 101.50 0.2 0.24 (120%) 0.44 0.441 ± 0.98 100.22 Pelagia Research Library 116
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Validated HPLC Method for Simultaneous Quantitation of Diclofenac ...

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