Phenomenex Kinetex C₁₈ column (4.6 × 100 mm 2.6 µm) with binary pump and UV detector (USA). Vortex mixer (Model: VM-300P, Gemmy Industrial Corp., Taiwan region) was used for human plasma samples preparation.Centrifuge (Model: 2-16P, Sigma Laborzentrifugen, Germany) was used for human plasma samples preparation.Sonicator (Thermo Scientific, Waltham, USA).

Pure Febuxostat: B. No. OP-FAB/06/16/001 was kindly provided by Mash Premiere with purity 100.61% according to supplier. Pure diclofenac potassium: B. No. DK/1808/0080B was kindly provided by The Arab Company for Gelatin & Pharmaceutical products with purity 99.85% according to supplier. Ethanol and methanol (Sigma - Aldrich, USA) analytical grade.

Xanfeb DSR^® tablets, it is labelled to contain 40 mg FEB and 100 mg DIC manufactured by Indoco Remedies, India) were purchased from pharmacies.

Stock standard solutions of the drug (1.0 mg/mL) were prepared in methanol. Working solution (0.1 mg/mL) was prepared by dilution with the mobile phase.

Mobile phase: consisted of water: ethanol (85:15 v/v) The mobile phase was filtered through 0.45-µm Millipore membrane filter and degassed by sonication for 30 min before use flow rate: 1 ml/min Detection: UV at 280 nmTemperature: ambient temperature

Accurately measured aliquots of the working standard solutions (100 µg/mL) were transferred into two separate sets of 10-mL volumetric flasks and adjusted to volume with the mobile phase to obtain final concentrations of (0.4 - 4.0 µg/mL) or (0.5 - 5.0 µg/mL), respectively for FEB and DIC. The standard solutions were then analyzed by injecting 10 µL of each solution under the above chromatographic conditions. The calibration graphs were constructed by plotting the peak area against the corresponding drugs concentration in µg/mL and the corresponding regression equations were computed.

Mixtures of different ratios for FEB and DIC were prepared by transferring aliquots from the corresponding working solutions (100 µg/mL), mixed well and completed to volume with mobile phase to be analyzed by the proposed method.

Ten tablets of Xanfeb DSR^® were accurately weighed and grounded. An amount of the powder equivalent to 80 mg FEB and 200 mg DIC was transferred to a 50-mL volumetric flask. A volume of 25 mL of methanol was added, and the flask was sonicated for 30 min, then completed to volume with methanol followed by filtration. One milliliter of this solution was diluted to 100 mL with methanol to get the final solution of 16 µg/mL of FEB and 40 µg/mL of DIC and filtered through a 0.45-μm membrane filter. Further dilution with the mobile phase was done to obtain the working standard solution to be analyzed as described above. The recovered concentration of each analyte was calculated from the corresponding regression equation.

The calibration graphs were constructed using spiked human plasma as follows: in a stoppered centrifuge tube, an aliquot quantity of 300 µL plasma was added and spiked with 100 µL of working solutions of (1.25, 2.5, 3.75, 5 and 7.5 µg/mL) for FEB or (2, 5, 7.5, 10 and 12.5 µg/mL) DIC, 600 µL methanol was added and the samples were vortex mixed for 5 minutes and centrifugated at 3000 rpm for 15 minutes. 400 µL of the supernatant was diluted with 600 µL mobile phase to obtain final concentration in the range 0.05 - 0.3 µg/mL and 0.08 - 0.5 µg/mL for FEB and DIC; respectively, all samples were filtered through a 0.45-μm membrane filter and directly injected into the chromatographic system under the above described conditions. The linear regression equations relating the peak areas to the concentration were derived for each analyte. A blank plasma experiment was performed simultaneously.

4.1.1. System Suitability Testing

System suitability test (SST) parameters were performed during the development and optimization of the method to ensure that the system is working correctly during the analysis. The test was performed by injecting the standard drug solution in triplicate and the parameters were calculated according to the BP [11] and USP [13] Guidelines. The final SST parameters including tailing factor (T), column efficiency (number of theoretical plates N) and Resolution (Rs) are summarized in Table 1.

4.1.2. Detection Wavelength

The UV absorption spectra of the methanolic solution of the studied drugs exhibited maxima at 316 for FEB and 283 nm for DIC as shown in Figure 2. Both drugs showed reasonable absorbance at about 280 nm. However, three wavelengths (230, 254, 280) were tried to select the most suitable one where 280 nm showed the highest sensitivity.

4.1.3. Column

Three different columns were used for performance investigations, including phenomenex kinetex C₁₈ column (4.6-mm × 100-mm, 2.6 μm particle size), BDS hypersil C₁₈ column (150 mm × 4.6 mm, 5 μm particle size) and Intersil C₁₈ (4.6-mm × 250-mm, 5 μm particle size). Experimental studies revealed that, the first column showed better results where the peaks of both analytes were more symmetrical and well-defined with a total run time less than 5 min. The second column was not suitable as it couldn’t separate the two drugs. In addition, the third column resulted in asymmetrical peaks with delayed retention time (Table 1).

Figure 2. Zero order spectra of (8 μg/mL) of each FEB (red) & (20 μg/mL) DIC (blue) in methanol.

Table 1. Optimization of the chromatographic conditions for determination of FEB and DIC by the proposed UHPLC method.

4.1.4. Mobile Phase Composition

Different mobile phases were tried as methanol: phosphate buffer, acetonitrile: phosphate buffer and ethanol:water. Initial experiments showed good separation with ethanol: water compared with the other mobile phases. Several modifications in the mobile phase composition were performed in order to study the possibilities of improving the performance of the chromatographic system which provide satisfactory, selectivity and sensitivity in a short separation time. Different % of ethanol were studied (5% - 30%) where 15.0% ethanol was found to be the optimum concentration regarding separation efficiency and resolution (Table 1).

4.1.5. Flow Rate

(Table 1) shows the effect of different flow rates (0.6 - 1.2 mL/min) on the chromatographic separation. A flow rate was optimized at 1 mL/min due to the highest efficiency in a short analysis time. Although lower flow rates showed higher resolution, but they were not selected as they led to an increase in the total run time, in addition to a decrease in the number of theoretical plates for both analytes. Optimum chromatographic conditions for the UHPLC determination of the studied drugs are summarized in Table 1. The proposed method permitted the separation of the two drugs with good resolution in a reasonable time, less than 5.0 min. Figure 3 shows typical chromatograms for laboratory prepared mixtures of FEB and DIC under the described chromatographic conditions where well-separated symmetrical peaks were observed. The retention times for FEB and DIC were 2.5 and 4.2 min, respectively.

The linearity of the developed method was confirmed by plotting the peak area against the analyte concentration in µg/mL. The graphs were linear over the concentration range of (0.4 - 4.0 µg/mL) and (0.5 - 5.0 µg/mL) for FEB and DIC; respectively. Linear regression analysis of the obtained data was presented in Table 2.

LODs were determined by evaluating the lowest concentration of the analytes which could be detected but not necessarily quantitated as exact values. LOQs were determined by establishing the lowest concentration of the analytes which could be quantitatively measured with suitable precision and accuracy. The results are shown in Table 2.

The accuracy of the results was checked by applying the proposed method for determination of different samples of FEB and DIC. The concentrations were obtained from the corresponding regression equations of the calibration graphs. The % recoveries for the proposed method were 99.76% and 100.63% for FEB and DIC as shown in Table 2.

Three different concentrations (0.8, 2.4 and 3.6 µg/mL) of FEB and (1, 3 and 4.5 µg/mL) of DIC solution were analyzed three times each, intra-daily and on three successive days using the previous mentioned procedure described for linearity. The relative standard deviations for the two drugs were calculated using the corresponding regression equations. The results are shown in Table 2.

Table 2. The regression parameters and validation results for determination of FEB and DIC by the proposed method.

The robustness of the proposed method was indicated by the constancy of the peak area with deliberate changes in the experimental parameters. These parameters included ethanol concentration (15% ± 0.5%, v/v) and UV detection (280 ± 1 nm). These minor changes did not affect the peak area of both drugs confirming robustness of the method (Table 3).

The selectivity of the proposed method was assured by applying it to laboratory prepared mixtures of FEB and DIC at different concentrations within the linearity range. Good recoveries of both drugs were obtained as shown in Table 4.

Evaluation of the stability of FEB and DIC standard solutions was achieved by quantification of each drug in this solution and comparing the results obtained to that obtained from freshly prepared standard solution. Similarly, the stability of the mobile phase was checked. No significant changes were observed in standard solution or mobile phase responses, relative to freshly prepared ones. The results obtained in both cases proved that the sample solution stable up to 7 days and mobile phase used during the assay were stable up to 5 days in refrigerator.

Table 3. Robustness of the proposed UHPLC method using FEB (2.0 µg/mL) and DIC (3.0 µg/mL).

Table 4. Determination of FEB and DIC in laboratory prepared mixtures by the proposed UHPLC method.

*Ratio of dosage form.

The proposed method was successfully applied to the simultaneous determination of FEB and DIC in laboratory prepared mixtures in different ratio and in their medicinally recommended ratios of 2:5; respectively as shown in Figure 3 and Table 4.

The proposed procedure was applied for the determination of FEB and DIC in their tablets. The obtained results were in good agreement with the label claim, indicating no interference from excipients and additives. The validity of the proposed method was further assessed by applying the standard addition technique, mean percentage recoveries of pure added were 100.28% ± 1.099 and 99.98% ± 1.871 for FEB and DIC; respectively (Table 5).

The obtained results were statistically compared to those obtained by the reported method [10]. No significant differences were found by applying t-test and F-test at 95% confidence level [24] indicating good accuracy and precision of the proposed method for the analysis of the studied drugs in its pharmaceutical dosage form as shown in Table 6.

FEB is reported to be rapidly absorbed after single or repeated doses with a maximum plasma concentration (C_max) of 1.9 ± 0.678 μg/mL reported for a 40 mg dose at t_max 1.0 - 1.5 h [25]. On the other hand, DIC is completely absorbed after oral administration. It exhibits a mean peak plasma concentration (C_max) 1.0 μg/ml after about two hours [26]. It is clear that UHPLC allows biological samples to be analyzed FEB and DIC a linear relationship was established by plotting the peak area against the drug concentration in µg/mL. Linear regression analysis of the data gave the following equation:

P = 6.665C – 0.2266 (r = 0.9995) for FEB

P = 5.6548C + 0.1135 (r = 0.9993) for DIC

Table 5. Determination of FEB and DIC in pharmaceutical dosage forms by the proposed UHPLC method and results obtained by standard addition technique.

*average of five concentrations.

Table 6. Statistical comparison for the results obtained by the proposed UHPLC method and the reported method (10) for the analysis of FEB and DIC.

*Average of three separate determinations. The values between parentheses are the tabulated t and F values at P = 0.05(²⁴).

where: P is the peak area, C is the concentration of the drug in µg/mL and r is the correlation coefficient. The high value of the correlation coefficient (r) indicated the good linearity of the calibration graph constructed in human plasma. The proposed method showed satisfying results for the determination of FEB and DIC in spiked human plasma over the concentration range of 0.05 - 0.3 µg/mL and 0.08 - 0.5 µg/mL for FEB and DIC; respectively, with a lower detection limit of 0.0106 µg/mL and 0.0231 µg/mL for FEB and DIC; respectively. The assay results using the proposed method are summarized in Table 7. Figure 4(a) and Figure 4(b) show representative chromatograms for blank and spiked plasma samples.

(a)

(b)

Figure 4. UHPLC chromatogram of blank plasma at 280 nm (a), UHPLC chromatogram of febuxostat (0.3 μg/mL) and diclofenac potassium (0.08 μg/mL) in spiked human plasma at 280 nm (b).

Table 7. Assay results for the determination of FEB and DIC in spiked human plasma by the proposed UHPLC method.