The proposed method permits the separation of MEB and SUL from degradation product and impurity. Figure 3 shows a chromatogram indicating good resolution of MEB (tR = 5.0 min.) and SUL (tR = 2.4) in pure form and in tablet extract, whereas Figure 4 show good resolution of MEB, MEB metabolite (veratic acid), SUL and SUL impurity (sules).
In an attempt to optimize the separation of MEB and SUL and their impurities and degradation products in bulk and plasma, the effects of some chromatographic parameters such as mobile phase composition and pH of the buffer solution were investigated.
Effect of the mobile phase composition
Type of Organic Modifier
Acetonitrile was replaced by methanol but it did not give good resolved peaks. Acetonitrile was the organic modifier of choice giving symmetrical narrow peaks.
Ratio of Organic Modifier
The effect of changing the ratio of organic modifier on the selectivity and
retention times of the test solutes was investigated using mobile phases containing concentrations of 40-75% for acetonitrile. Table 1 shows that 45%
acetonitrile was the best one giving well resolved peaks and highest number of theoretical plates. Ratios less than 45% resulted in peaks with very long unacceptable retention times, whereas ratios higher than 75% resulted in precipitation in the mobile phase.
Effect of pH
The effect of changing the pH of the mobile phase on the selectivity and retention times of the test solutes was investigated using mobile phases of pH ranging from 3.0-5. Table 1 illustrated that a pH of 4.0 was the most appropriate one giving well-resolved peaks and highest number of theoretical plates. pHs higher than 5.0 produced precipitation in the mobile phase.
Ionic Strength of Buffer
The effect of changing the ionic strength of phosphate buffer on the selectivity and retention times of the test solutes was investigated using mobile phases containing concentration of 0.0075- 0.04 M of phosphate buffer. Table 1 shows that 0.01 M phosphate buffer was found to be the most suitable giving best resolution and highest number of theoretical plates.
Effect of Flow Rate
The effect of flow rate on the formation and separation of peaks of the studied compounds was studied and a flow rate of 1 mL/min was optimal for good separation in a reasonable time (Table 1).
Validation of the Method
Concentration Ranges and Calibration Graphs
Under the above described experimental conditions, a linear relationships was established by plotting peak area ratio for MEB to 0.1 μg/mL SUL as internal standard against MEB concentrations and on the other hand, plot peak area ratio for SUL to 0.1 μg/mL MEB as internal standard against SUL concentrations,. The concentration range was found to be 10- 100 ng/mL for each drug. Linear regression analysis of the data gave the following equations:
where: C is the concentration of MEB or SUL in ng/mL and P is the peak area ratio. The high value of the correlation coefficient (r-values > 0.999) with small intercept indicate the good linearity of the calibration graphs. Statistical analysis of the data gave small values of the standard deviation of the residuals, and the% relative error,  as shown in table 2.
Limit of Quantitation and Limit of Detection
The limits of quantification (LOQ) was calculated according to ICH Q2B recommendations . The limits of detection (LOD) was also calculated according to ICH Q2B recommendations . The results of LOD and LOQ of MEB and SUL respectively are abridged in Table 2.
LOQ and LOD were calculated according to the following equations :LOQ = 10 σ/S
LOD = 3.3 σ/S
Where, σ is the standard deviation of the intercept of regression line and S is the slope of regression line of the calibration curve. Statistical analysis  of the results, obtained by the proposed and the reference method  using Student's t-test and variance ratio F-test, shows no significant difference between the performance of the two methods regarding the accuracy and precision, respectively (Table 3).
The reference method is based on HPLC separation of the two drugs on a reversed phase, Bondapak CN column and UV detection was done at 243 nm using buclizine hydrochloride as internal standard .
Accuracy and Precision
The repeatability was evaluated by applying the proposed method for the determination of three concentrations of MEB and SUL in pure forms on three successive times, and the results are abridged in Table 4. The low %Error and low % RSD indicates high accuracy and high precision of the proposed method respectively.
Intermediate precision was performed through replicate analysis of MEB and SUL in pure form. The results are shown in Table 4, for a period of 3 successive days.
Application of the method
The proposed method was applied for the determination of the studied drugs in their co-formulated tablets. The specificity of the method was investigated by observing any interference encountered from the common tablet excepients, such as lactose, gelatin, magnesium stearate and starch. These excepients did not interfere with the proposed method (Table 5). The average percent recoveries of different concentrations were based on the average of three replicate determinations. The results shown in Table 4 are in good agreement with those obtained with the reference method . Figure 3 shows a chromatogram indicating good resolved peaks of MEB and SUL.
Degradation product of MEB (veratic acid) and impurity of SUL (sules) of is easily detectable and can be determined quantitatively as shown Figure 4. Therefore, the proposed method can be used for the quality control of the tablets.
The high sensitivity of the proposed method allowed the determination of MEB metabolite and SUL in biological fluids by HPLC method. The method was further applied to the in-vivo determination of both drugs in real human plasma. Sulpiride is absorbed from gastro-intestinal tract. Following oral ingestion of a single oral dose of 50 mg of SUL, a peak plasma concentration of 0.03-0.60 mg/L (mean 0.18 mg/L) is attained in about 4-7 hours . This value lies within the working concentration range of the proposed method.
After oral administration of mebeverine HCI (270 mg) to fasted human volunteers, measurable concentrations of the drug were not found in plasma. By contrast, the metabolite veratric acid achieved considerable concentrations (mean peak plasma concentration of 13.5 μg/mL at (40-80 min) . Veratic acid (MEB metabolite) is also highly fluorescent compound and the second metabolite is mebeverine alcohol which is non-fluorescent at the studied pH 4 . However, after oral administration of mebeverine HCI (2 mg), only traces of mebeverine were found in plasma with simultaneous appearance of veratic acid. Veratric acid was achieved with considerable concentrations (mean peak plasma concentration of 0.90 μg/mL at (15 min-4 h). The mean percentage recoveries, % Error and % RSD are abridged in Table 6. The results show that mebeverine undergoes rapid and extensive first-pass metabolism involving hydrolysis of the ester function, as shown in Figure 5 and that very trace circulating concentrations of the parent drug are found in humans .
The typical chromatograms for the separation of the MEB, veratic acid and SUL in real plasma are illustrated in Figure 6. The plasma samples obtained from the volunteers were investigated using the previously obtained calibration graph or regression equations and the results obtained are shown in Figure 7 showing maximum plasma levels are reached after 3.5 hours for SUL and veratic acid. Hence, the proposed method allows for the therapeutic drug monitoring for MEB and SUL mixture level in plasma.