2.1. Reagents and chemicals
Oxcarbazepine OXC, Methyl paraben (M.P.), Propyl paraben (P.P.) and potassium sorbate (P.ST.) Were obtained from Simco pharma. Oxaleptal 60 mg/ml® oral suspension were purchased from the Egypt market. Plasma samples were taken from Egyptian blood bank. Methanol (HPLC grade optained from Scharlau), KH2PO4 and Sodium hydroxide were obtained from Panreac, Sample solution filter were purchased from Whatman for syringe filter. Ultrapure water (Milli-Q) (Millipore Corporation, Billerica, MA, USA) was used.
2.3. Preparation of standard solutions
(Solution A): To 33 mg of OXC working standard in 50 ml volumetric flask, 25 ml acetonitrile was added, stirred till complete dissolution then mobile phase was used to complete to the final volume. (Solution B): Transfer methyl paraben (19.8 mg) of working standard into volumetric flask (100 ml), add mobile phase to complete to a required volume and the mixture shake well to dissolve. (Solution C): 5 ml from solution B was transferred to volumetric flask (50 ml) then completed by mobile phase to required volume. (Solution D): Propyl paraben (22 mg) working standard transferred into volumetric flask (100 ml), then final volume obtained by mobile phase and shake well to dissolve. (Solution E): 1 ml from solution D was taken into 100 ml volumetric flask then completed to volume by mobile phase. (Solution F): 22 mg of Potassium sorbate working standard was transferred into volumetric flask (100 ml), and then completed to volume by mobile phase and shake well to dissolve. (Solution G): 5 ml from solution F was transferred into volumetric flask (50 ml) then completed to volume by mobile phase.
Standard solution
5 ml from each of solutions A, C, E and G was transferred into volumetric flask (100 ml) then completed to volume by mobile phase. (conc. Of OXC, Methyl paraben, Propyl paraben and Potassium sorbate is 33, 0.99, 0.11 and 1.1 µg/ml)
2.4. Preparation of sample solutions
Oxaleptal oral suspension (11 ml) was accurately measured and transferred to a volumetric flask (200 ml), added water (10 ml), stirred for 5 min; then acetonitrile (50 ml) was added, stirred for 30 min then the volume was completed by mobile phase.
1 ml was transferred from the previous solution into volumetric flask (100 ml), the volume was completed by mobile phase, mixed and centrifuged. (conc. of OXC, Methyl paraben, Propyl paraben and Potassium sorbate is 33, 0.99, 0.11 and 1.1 µg/ml).
Forced degradation conditions
Acid degradation: the mixture of OXZ., M.P., P.P. and P.ST: was prepared as previously described in the preparation of working standard solution but 5 ml of 5N HCl were added to the analytes. The mixture was shaken for 5 minutes and left in the dark at room temperature for 1 hour, then neutralized by 5 ml of 5N NaOH before completing volume into 100 ml with the mobile phase.
Alkaline degradation: prepared by adding 5 ml of 5N NaOH to the mixture of OXZ., M.P., P.P. and P.ST: standard solution, containing the same amounts of working standard solution, and shaken for 5 minutes and left in the dark at room temperature for 1 hour, then neutralized with 5 ml of 5N HCl. The volume was completed to 100 ml with the mobile phase.
Oxidative degradation: prepared by adding 5 ml of 30% H2O2 to the same concentration of a mixture of OXZ., M.P., P.P. and P.ST: standard solution used in acid and alkaline degradation. The mixture was shaken for 5 minutes and left in the dark at room temperature for 1 hour, then the volume was completed to 100 ml with the mobile phase.
Heating Hydrolysis of of OXZ., M.P., P.P. and P.ST: 11 gm of Oxaleptal oral suspension was accurately weighed and transferred to 100 ml volumetric. 10 ml of purified water was added and shaked well. 50 ml Methanol was added, Stirred for 30 min put in water path at 60 C° for 3 hours, cooled to room temperature then the volume completed to volume with Mobile phase mixed and filtered. 5 ml was transferred into 50 ml volumetric and then completed to volume with by Mobile phase, and then 5 ml was transferred into 100 ml volumetric and then completed to volume with Mobile phase.
Day light Hydrolysis of OXZ., M.P., P.P. and P.ST Suspension:
Carried out as mentioned in heat hydrolysis & put in day light for 6 hours instead of heating.
2.5. Method validation
2.5.1. Accuracy:
Accuracy estimated by applying of the proposed study to standard solution of OXC, M.P., P.P. and P.ST to which known quantities of analyte have been spiked within the range of the calibration curve. Accuracy should be assessed using at least three concentrations (80, 100 and 120%) with average recovery percents ranging from 98–102% of spiked drug in plasma.
2.5.2. Precision:
Precision of the proposed method is the degree of agreement among results of individual test when it applied repeatedly and it is expressed as the relative standard deviation RSD of a series of measurements. It should be assessed using at least six quantitations at 100% of the test concentration. Obtained Relative Standard Deviation Percents (RSD %) are within the accepted range (NMT 2%) indicating that the method is Precise and repeatable.
2.5.3. Linearity, LOD and LOQ
The linearity represents the ability to get results that are directly proportional to the concentration of drug in samples within specific range. A minimum of five concentrations should be used. If appears to be a linear relationship, calculate regression coefficient and y-intercept which should be higher than 0.99 and near to zero respectively.
Linearity of the method represents its ability to get a direct relationship between the obtained results and the given concentration of the drug. At the optimized HPLC conditions, linearity was obtained by injection of standard solution series of OXC, M.P., P.P. and P.ST at five different levels; 60, 80, 100, 120 and 140% of the selected concentration range. Plotting the peak area against the selected concentration was used estimate the calibration curves of each sample. The Slope (b), intercept (a) and correlation R2 were estimated. LOD was determined from the formula; LOD = 3 σ /SD, where (σ) is SD of the intercept and (S) is the slope of regression equation. LOQ was calculated similarly from the equation LOQ = 10 σ/S.
2.5.4. Robustness :
The robustness of a method is its ability to be not affected by small variations in its parameters as temperature, wavelength, and change in mobile phase. This reproducibility under the normal parameters compared to the precision to get a measure of the robustness of the analytical method.
2.6. Analysis in human plasma
Into 10 ml centrifuge tube, serial working solutions were prepared then an accurate volume of 200 µl OXC, M.P., P.P. and P.ST standard solution was quantitatively added to 500 µl of blank plasma (plasma samples taken from Egyptian blood bank), then 2.0 ml acetonitrile were added as a protein precipitation, and vortex mixed for two minutes followed by centrifugation at 5000 rpm for 20 min. Further supernatant filtration was done using membrane filter of cellulose acetate (0.45-m), then 20 µL was applied to the HPLC-UV system.