Validated stability indicating RP-HPLC method for simultaneous determination and in vitro dissolution studies of thiocolchicoside and diclofenac potassium from tablet dosage form

2.4.1 For assay method

Mixed Standard stock solution of 80 μg mL⁻¹ of TH and 50 μg mL⁻¹ of DP was prepared by dissolving about 80 mg of TH reference standard and about 50 mg DP reference standard in 30 mL of diluent which was a mixture of water:methanol (50:50, v/v) in 100 mL volumetric flask and stirred in an ultrasonic bath for 10 min. The volume was completed with the same diluent. Further diluted 5 mL of mixed standard stock to 50 mL with the same diluent.

To study the linearity range of each component, the serial dilutions of mixed standard stock were made in the range of 40.48–121.43 μg mL⁻¹ of TH and 24.91–74.72 μg mL⁻¹ of DP (i.e. 50–150% of test concentration). A graph was plotted as concentration of drugs versus peak area response. It was found to be linear for both the analytes. The system suitability test was performed from five replicate injections of mixed standard solution.

2.4.2 For in vitro dissolution studies

Standard stock of TH (8.8 μg mL⁻¹) was prepared by dissolving about 88 mg of reference standard of TH in 100 mL of diluent which was respective dissolution media, this 5 mL solution to 50 mL was diluted with same diluent. Standard solution of DP (55 μg mL⁻¹) was prepared by dissolving about 55 mg reference standard of DP into 100 mL of same diluent. The mixed standard was prepared by mixing 5 mL of standard stock of TH and 5 mL of DP standard stock solution.

To study linearity of each component, the serial dilution of TH and DP stocks were made in the range of 0.4–13.19 μg mL⁻¹ of TH and 2.7–82.50 μg mL⁻¹ of DP (i.e. 1–150% test concentration). A graph was plotted as concentration of drugs versus peak area response. It was found to be linear for both the analytes. The system suitability test was performed from five replicate injections of mixed standard solution.

2.5.1 For assay method

Twenty tablets each containing 8 mg of TH and 50 mg DP were weighed, averaged and finely powdered. A portion of powder equivalent to the weight of one tablet was accurately weighed into 100 mL A-grade volumetric flasks and 70 mL diluent was added. The volumetric flasks were sonicated for 20 min to effect complete dissolution of the TH and DP in the solutions, and then made up to volume with diluent. The solution was filtered through 0.45 μm nylon filter.

TH sample (80 μg mL⁻¹) – aliquot portion of filtrate use as such.

DP sample (50 μg mL⁻¹) – further dilute aliquot portion of the filtrate to 50 mL with diluent.

Twenty microliters of test solution was injected and chromatogram was recorded for the same and amount of drug calculated.

2.5.2 For in vitro dissolution studies

A calibrated dissolution tester was used with paddle (USP-II) at 50 rpm and bath temperature maintained at 37 ± 5 °C. Nine hundred milliliters of freshly prepared and degassed pH 6.8 phosphate buffers was used as dissolution medium. Six tablets were evaluated. Dissolution samples were collected at 5, 10, 15, 30, 45, 60 min. At each time point, a 10 mL sample was removed from each vessel using an auto-sampler and filtered through a 0.45 μm nylon filter into labeled glass tubes and analyzed by HPLC.

2.6.1 Hydrolytic conditions: acid, base induced degradation

Solutions containing 80 μL of TH and 50 μL of DP were treated with different conc. of HCl and NaOH which was 0.1 N, 0.5 N and 1 N for 30 min at 80 °C. The solutions were neutralized as needed.

2.6.2 Oxidative condition: hydrogen peroxide-induced degradation

Sample was treated with 5 mL of 5% and 10% w/v H₂O₂ in dark under the condition 80 °C for 30 min.

2.6.3 Thermal degradation

To investigate the stability of the drugs under thermal stress conditions, sample was spread in a thin layer on a petri plate and subjected to the conditions indicated at 60 °C for 12 h.

2.6.4 Photolytic degradation

Photolytic studies were performed after exposition of sample in solid state and diluted in purified water, subjected in a photo stability chamber up to 22 h.

Similarly the placebo of tablet was treated as same like above in each condition separately and tested. All these stressed samples and placebos were periodically analyzed by HPLC for the appearance of degradant impurity, calculated against main drug component.

3.1.1 Selection of stationary phase

As both drugs were polar in nature they can readily ionize. Hence we started the development activity with different stationary phases starting from non-polar to polar, i.e. C_18, C_8, Phenyl, and Cyano of various manufacturers using different mobile phases. Columns with C₈ and C₁₈ stationary phases were tried for better peak shape and resolution, but TH was eluting at about void volume due to the highly polar nature indicating that it is not retaining on the column surface but eluting with mobile phase. Hence to retain the peaks the polar stationary phase needs to be used. The poor resolution between TH and DP and distorted peak shape on phenyl column implies that this column is also not suitable for this application. Hence column with Cyano stationary phase was chosen to improve the peak shape and resolution among the peaks. The peak shape and system suit parameters among all components were improved with Zorbax SB CN (250 mm × 4.6 mm, 5 μm) column (Fig. 2 and Table 2). But the stationary phase is not only the parameter, which can give better chromatography. Mobile phase pH and organic modifiers also play very important role which leads to the best chromatographic condition.

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Figure 2 A typical chromatogram of a tablet sample solution containing 80 μg mL⁻¹ of thiocolchicoside and 50 μg mL⁻¹ of diclofenac potassium.

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3.1.2 Selection of mobile phase

Highly polar substances, especially those which take ionized form in water, have peak tailing and bad peak symmetry when separated by applying the RP-HPLC method, which affects resolution, sensitivity and reproducibility. Hydrogen bonding and ion exchange interaction can occur between the chromatographic support and the basic compounds. It can be prevented by addition of modifier to the mobile phase such as acid, base and ion pairing agents. More accepted approach is to adjusted pH value of the mobile phase (Swadesh, 1997).

The elution of TH and DP was done using ion pair agent 1-pentane sulfonic acid in mobile phase containing water and water:acetonitrile:triethylamine (75:15:10 v/v/v) but elution of TH occurred at void volume, while DP does not elute up to 60 min on different stationary phases given in Table 2. The buffer KH₂PO₄, K₂HPO₄, Na₂HPO₄, NaH₂PO₄ and sodium acetate were tested. The good performance and better separation was achieved in NaH₂PO₄ buffer and methanol using Cyano stationary phase (Fig. 2). We optimized the elution by setting gradient program at flow rate 1.0 mL min⁻¹ (Table 1).

3.1.3 Influence of pH and organic modifier

The molecular structure of both components implies that TH is having amide group and DP is having carboxylic acid functional group, which makes separation pH sensitive. For the method to be robust, pH of mobile phase must not be at pK_a of compounds. The basic pH deteriorates the silica in HPLC column. We tried different mobile phase buffers pH ranging from 2.5 to 5 and monitored the resolution and performance of column. The best separation was achieved with pH 2.5. DP was not eluted on polar (Cyano) stationary phase as it retains due to polar likes polar theory. To elute within our satisfactory run time, organic modifier composition needs to be increased. The satisfactory elutions were observed at gradient program mentioned in Table 1.

3.2.1 Solubility determination and sink condition verification

Drug solubility and solution state stability need to be determined before selecting the dissolution medium. The dissolution characteristics of an oral formulation should be evaluated in the physiologic pH range of 1.2–6.8 (1.2–7.5 for modified-release formulations). USP <711>. Solubility data were used as the basis for the selection of a dissolution medium for TH and DP. Drugs solubility was determined at 37 °C in 0.1 N HCl, pH 4.5 acetate buffer and pH 6.8 phosphate buffers.

DP is practically insoluble in 0.1 N HCl. The quantitative solubility of drugs was determined in pH 4.5 acetate buffer and pH 6.8 phosphate buffer. Based on solubility results for TH and DP in each dissolution medium tested (Table 3) and considering a test volume of 900 mL per vessel, sink conditions were verified. The term sink conditions is defined as the volume of medium at least greater than three times of that required to form a saturated solution of drug substance (United State Pharmacopoeia, xxxx; Tracqui et al., 1996). A pH 6.8 phosphate buffer was used as dissolution media for TH and DP tablet (Table 3).

3.2.2 Development of the dissolution test condition

From pre-selected, pH 6.8 phosphate buffer media, dissolution testing was performed on tablet (n = 6) in compliance with USP <711> using paddle (USP-II). A paddle speed of 50 rpm was selected to minimize coning. The media volume used 900 mL. The medium which was vacuumed and degassed under house vacuum was maintained at 37 ± 0.5 °C. The 1-L glass dissolution vessels were covered to minimize evaporation. Samples were drawn at 5, 10, 20, 30, 45, 60 min. The tablet was immediate releasing, the earlier time points provided more discriminating ability (Fig. 3). Sampling was performed using auto sampler of 10 mL aliquots. This solution was immediately filtered using a syringe of 0.45 μm nylon filters. The first 2–3 mL of sample was discarded prior to collecting the sample for analysis.

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Figure 3 Drug release of TH and DP.

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3.3.1 System suitability test

The system suitability test performed according to USP 29 (United State Pharmacopoeia, xxxx) and BP 2007 (British Pharmacopoeia, 2004) indications. The observed RSD values at 1% level of analyte concentration were well within the usually accepted values (⩽2%). Theoretical plates, USP tailing factor (T_f) and USP resolution (R_s) between TH and DP were determined for both assay and dissolution. The results are all within acceptable limits summarized in Table 4.

3.3.2 Specificity

The specificity of Assay HPLC method carried out by the peak purity indices for the analytes in stressed solutions were determined with PDA detector (Young and Gorenstein, 1994) under optimized chromatographic conditions found to be better (purity angle < purity threshold) indicating that no additional peaks were co-eluting with the analytes and evidencing the ability of the method to assess unequivocally the analyte of interest in the presence of potential interference. Baseline resolution was achieved for all investigated compounds. The FDA guidelines indicated that well separated peaks, with resolution, R_s > 2 between the peak of interest and the closest eluting peak, are reliable for the quantification (FDA, 1994). All stressed samples peak of TH and DP meet this specification. Major degradation impurity formed by both drug in hydrolytic stressed condition at 1 N HCl and NaOH and in oxidative condition at 10% H₂O₂, visibly confirmed in Figs. 4–6.

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Figure 4 Typical HPLC chromatogram of 1 N HCl stressed sample of TH (a) and DP (b).

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Figure 5 Typical HPLC chromatogram of 1 N NaOH stressed sample of TH (a) and DP (b).

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Figure 6 Typical HPLC chromatogram of 10% H₂O₂ stressed sample of TH (a) and DP (b).

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Dissolution method specificity was examined by analyzing a solution of a placebo containing the excipients mixture for the TH and DP tablet dissolved in the dissolution medium. Absence of interference was demonstrated.

3.3.2.1

3.3.2.1 Degradation kinetics of drugs in stressed condition

Sample solutions were treated with HCl, NaOH of different normality and H₂O₂ at different concentration. The chromatogram obtained revealed that the peak area for TH was degraded in all stressed conditions while DP peak area degraded only in acidic condition. The percentage degradation of each compound was calculated with respect to the controlled sample. The plot of respective component % degradation against concentration of HCl, NaOH and percentage of H₂O₂ indicated an apparent first order degradation of TH in all conditions and DP only in HCl conditions (Fig. 7). In thermal and photolytic conditions, the solution was stable.

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Figure 7 Degradation trends of TH and DP in acidic (a), basic (b) and peroxide (c) stressed condition.

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3.3.3 Accuracy

Accuracy of the assay method as well as dissolution method was calculated by recovery studies at three concentrations of 50%, 100%, and 150% levels by standard addition method (Table 5). The mean percentage recoveries obtained for TH were 100.25% for assay and 99.43% for dissolution, while 99.81%, 99.29% for DP assay and dissolution, respectively.

3.3.4 Linearity and range

The linearity of the HPLC detector response for TH and DF was determined for their assay method and dissolution method by analyzing series of different concentrations of each compound.

The plots of area under the curve (AUC) of the peak responses of the analytes against their corresponding concentrations, they exhibit linear responses with r > 0.999 for both methods. All the validation parameter such as a correlation coefficient, concentration ranges are summarized in Table 6.

3.3.5 Robustness

In order to demonstrate the robustness of the assay method, system suitability parameters were evaluated in the study as shown in Table 7 and include pH, flow rate, column temperature. The parameters and criteria used to define robustness were retention time (>4.5 and ⩽6.5 min for TH and >10.8 and ⩽12.8 min for DP), peak symmetry (⩽2.0) and efficiency (>2000).

The most important factors that affect peak asymmetry are % of organic, flow rate and temperature. The method, however, is robust with respect to peak asymmetry; this value was consistent <1.2 across all value of these parameters. Method suitability, therefore, will not be adversely affected with respect to peak asymmetry.

Flow rate is the parameter that significantly affects peak efficiency. The method, however, is robust with respect to peak efficiency; this value was consistent >4000 across all value of these parameters. Method suitability, therefore, will not be adversely affected with respect to peak efficiency. The mobile pH, in the study 1.5 and 3.5 has negligible affect on suitability criteria.

To evaluate robustness of the dissolution test, the variable selected mention in Table 7 includes paddle type, sampling device and degassing procedure. The variables were chosen as they were deemed the most significant factors that can potentially affect the rate of dissolution. The degassing procedure parameter involves three conditions. The media was either left spinning in an open flask overnight, left vacuum degassing overnight or vacuum degassing for 15 min as per the method. The first two options chosen to maximize and minimize, respectively, the amount of dissolved oxygen in the media. The all variable not significantly affect on % drug release.