Validation of spectrophotometric method for determination of esomeprazole and ciprofloxacin in their pure and dosage forms

A novel simple, accurate, sensitive and economical spectrophotometric method has been established and validated for the determination of esomeprazole and ciprofloxacin. The method is based on the oxidation of the studied drug by a known excess of potassium permanganate, followed by measuring the decrease in absorption (∆A) of KMnO4 in acidic medium at wavelength of 525nm. The detection limit is reported to be 1.01 and 1.06µg/mL showing a high degree of sensitivity. The proposed method was successfully validated according to ICH guidelines for the determination of esomeprazole and ciprofloxacin with a highly precise recovery and very low relative standard deviation. Finally, the method was compared statistically with a reference method showing equal accuracy, reproducibility and no significant difference with the reported one.

To our knowledge, no spectrophotometric method for the determination of ESM or CIP using potassium permenganate as an oxidant has yet been reported despite the versatility, simplicity and reliability of the technique in chemical analysis.
As such, in this paper, the method is based on an oxidation reaction of ESM and CIP with a known excess of KMnO 4 followed by measuring the decrease in absorption (∆A) of KMnO 4 in acidic medium at wavelength of 525nm.

Spectrophotometric procedures
Construction of the standard calibration curves: Aliquot portions of 200μg/mL ESM and CIP ranging from (0.40-2ml) were transferred into a series of 10mL measuring fl asks. To these, 1mL and 1.50mL of 2M sulfuric acid were added for both ESM and CIP respectively, then 1mL of 5×10 -3 M KMnO 4 were added to both drugs then the total volume was adjusted to 10mL with double distilled water. The absorbance of a reagent blank (similarly prepared without the drug) was measured against each drug concentration at 525nm either immediately in case of ESM or after 16minutes in case of CIP.
Procedure for pharmaceutical preparations: Accurately weights of Esmatac ® 40mg equal to 20mg of ESM or Ciprocin ® 250mg equal to 20mg of CIP were transferred to a 100mL measuring fl ask and completed to volume with double distilled water to give an equivalent fi nal concentration of 200μg/mL.
The procedures were then conducted as mentioned above under the general procedures applying standard addition techniques.

Optimization of the reaction conditions
The optimum conditions for the method development were established by varying each specifi c parameter and keeping the others constant and observing the effect produced on the absorbance of the colored species. The optimum parameters are reported in Table 1.
Absorption spectra: Absorption spectra of ESM and CIP with KMnO 4 was studied over a range of 400-800nm. Potassium permanganate reacts with ESM and CIP in acidic medium and the decrease in absorption can be measured at 525nm as depicted in Figure 2.

Effect of temperature
Effect of temperature was studied and results showed that there is no an evident effect of temperature on the reaction as increase in temperature is not accompanied with any increase in absorbance and so, optimum reaction was performed at room temperature.  Effect of addition sequence: Addition sequences were studied and results revealed that the most appropriate sequence was the drug then the added acid then the added KMnO 4 .

Effect of acidity :
To study the effect of sulfuric acid volume, the reaction was performed in a series of 10mL volumetric fl asks containing different volumes (0.5-4ml) of 2M sulfuric acid. It was found that the maximum absorbance was obtained when using 1mL of 2M sulfuric acid with ESM and 1.50mL with CIP as seen in Figure 3.
Effect of permenganate concentration: By studying the effect of KMnO 4 concentration referring to decrease of its color intensity, it was observed that the absorbance reached its maximum when 1mL of 5×10 -3 M KMnO 4 was used in case of both ESM and CIP (Figure 4).

Effect of time:
The effect of time on the oxidation reaction was studied to obtain the highest and most stable absorbance. As depicted in Figure 5, This absorbance can be achieved immediately after the reaction between the drug and KMnO 4 in case of ESM while it takes 16 minutes in case of CIP to complete the reaction.

Method validation
The method validation was performed according to International Conference of Harmonization (ICH) guidelines [19].
Linearity: Six different concentrations of ESM and CIP were prepared for linearity studies. The linearity ranges of absorbance as a function of drug concentration (Table 2) provided acceptable indication about sensitivity of reagents used. Linear regression equations of ESM and CIP were found to be y=0.0334x+0.0379 and y=0.0444x+0.2459, respectively and the regression coeffi cient values (R 2 ) were found to be 0.9994 and 0.9991, respectively indicating a high degree of linearity for both drugs ( Figure 6).

Accuracy:
The accuracy of the method was determined by investigating the recovery of ESM and CIP concentration levels covering the specifi ed range using the standard addition technique. It was performed by adding a fi xed standard drug concentration at different levels of the pharmaceutical products (Esmatac ® and Ciprocin ® ) and the proposed method was followed. From the amount of the drug estimated, the percentage recovery was calculated and the results are shown in Table 3.

Specifi city:
The specifi city studies revealed that the presence of the excipents in Esmatac ® 40mg and Ciprocin ® 250mg formulations didn't show any kind of impurity interference, since the recoveries lied in the range of 96.90-104.85% as reported in Table 3.

Limits of detection and limits of quantifi cation:
The calculation of limits of detection and quantitation was based on the following equations: LOD=3.3S/K and LOQ=10S/K, respectively, where S is the standard deviation of the seven replicate values under the same conditions as for the sample analysis in the absence of analyte and K is the sensitivity, namely, the slope of calibration graph. Limits of detection were calculated to be 1.01 and 1.06μg/mL while limits of quantifi cation were 3.35 and 3.55μg/mL, for ESM and CIP respectively (Table 2).

Robustness:
The robustness of the method was evaluated by making small changes (±0.05ml) in the volume of H 2 SO 4 and KMnO 4 keeping the other conditions constant where the effect of the changes was studied on the percent recovery and standard deviation of 20μg/mL ESM and CIP. The changes had negligible infl uence on the results where SD values were in the acceptable range as reported in Table 4.

Statistical analysis of the pharmaceutical formulation
Esmatac ® 40mg and Ciprocin ® 250mg have been successfully analyzed by the proposed method. Results obtained were compared to those obtained by applying reference methods [4,11]

Ethical approval
This manuscript does not include any studies on human or animals.