Determination of Cephalexin Monohydrate in Pharmaceutical Dosage Form by Stability-Indicating RP-UFLC and UV Spectroscopic Methods

An ultra-fast liquid chromatographic method and two UV spectroscopic methods were developed for the determination of cephalexin monohydrate in pharmaceutical dosage forms. Isocratic separation was performed on an Enable C18G column (250 mm × 4.6 mm i.d., 5 μm) using methanol:0.01 M TBAHS (50:50, v/v) as the mobile phase at a flow rate of 1.0 ml/min. The PDA detection wavelength was set at 254 nm. The UV spectroscopic method was performed at 261 nm and at 256–266 nm for the AUC method using a phosphate buffer (pH=5.5). The linearity was observed over a concentration range of 1.0–120 μg/ml for UFLC and both of the UV spectroscopic methods (correlation coefficient=0.999). The developed methods were validated according to ICH guidelines. The relative standard deviation values for the intraday and interday precision studies were < 2%, and the accuracy was > 99% for all of the three methods. The developed methods were used successfully for the determination of cephalexin in dry syrup formulation.


Chemicals and Reagents
Cephalexin monohydrate (purity > 99.8%) was obtained as a gift sample from Cadilla Pharmaceuticals Ltd., India. Methanol (Merck Ltd., Mumbai, India) was of HPLC grade. Analytical grade sodium hydroxide, potassium dihydrogen phosphate, hydrochloric acid, and hydrogen peroxide were procured from S.D. Fine Chem. Ltd., Mumbai, India. The water for HPLC was obtained by using the TKA Water Purification System, Germany. Tetrabutylammonium hydrogen sulfate (TBAHS; Hi-Media Laboratories Ltd., Mumbai, India) was of AR grade. The dry syrup formulation containing 125 mg/5 ml of CEM was bought from the local market.

UFLC (Method-I)
Quantitative UFLC was performed on a binary gradient UFLC with two Shimadzu Prominence UFLC LC-20AD pumps, with a 20 μl sample injection loop (manual) and SPD M20A PDA detector. The signal was recorded and integrated using Shimadzu LC Solution Software. An Enable C18G, (250 mm × 4.6 mm i.d., particle size 5 µm) was used for separation. Chromatographic analysis was carried out at ambient temperature on the column using the methanol: 0.01 M TBAHS (50:50, v/v) as the mobile phase at a flow rate of 1.0 ml/min in isocratic mode. The 0.01 M TBAHS solution was prepared by accurately weighing 3.3954 g of TBAHS salt and dissolving it in 1000 ml of HPLC grade water. Afterwards, both the methanol and TBAHS were ultrasonicated (Enertech, India) up to 20 minutes for degassing before use. The PDA detection was carried out at 254 nm. A water bath (Thermolab, India) and UV chamber (Jain Scientific Glass Works, Ambala, India) were used for the forced degradation study of the drug.

UV Spectroscopy (Method-II & III)
A Shimadzu UV Visible Model 1800 double beam spectrophotometer with 10 mm matched quartz cuvettes was used for the spectral measurements. The spectrophotometer was controlled by UV Probe software which was also used to transform the UV spectra obtained. A potassium dihydrogen phosphate buffer of pH 5.5 was used as the solvent.

Preparation of Standard and Sample Solution
Standard stock solutions of CEM were prepared by transferring 25 mg of the drug into two separate 25 ml volumetric flasks having 10 ml of diluents and were ultrasonicated for 5 minutes. Finally the volume was made up with suitable diluents, which gave 1000 µg/ml solutions. The chromatographic mobile phase and buffer solution were used as diluents for UFLC and UV spectroscopic methods, respectively.
Powder equivalent to 25 mg of CEM was accurately measured and transferred into two separate 25 ml volumetric flasks, containing 10 ml of diluents and ultrasonicated for 20 minutes; the volume was made up and mixed well. Solutions were filtered by a 0.2 μm filter to remove particulate matter, if any. The filtered solutions were properly diluted for analysis as already described. The drug present in the sample solutions was calculated by using the calibration curves. All the solutions were stored at 2-8 ºC for future use.

Specificity
The specificity of the UFLC method was determined by checking the interference of any of the possible degradation products produced during the forced degradation study of CEM. The forced degradation of the drug was carried out with 0. For the UV spectroscopic method, the specificity of the method was checked for any possible interference because of the commonly used excipients in the syrup formulation.

Linearity
An eight-point (1.0, 5.0, 10, 20, 40, 80, 100, and 120 µg/ml) and two eleven-point (1.0, 2.0, 5.0, 10, 20, 30, 40, 50, 70, 100, and 120 µg/ml) calibration curves were prepared for the UFLC and UV spectroscopic methods, respectively. The peak area for the UFLC (Method-I) was obtained by injecting 20 µl of the drug solution into the column. For UV spectroscopic determination, the absorbance (Method-II) and AUC (area under the curve; Method-III) were measured at 261 nm and 256-266 nm, respectively. Calibration curves were plotted by taking the peak area, absorbance, and area under the curve on the y-axis and the concentration (µg /ml) on the x-axis.

Precision
The intraday and interday precision study was carried out to check the reproducibility of the results. A concentration of 40 µg/ml and 30 µg/ml of CEM (n=6) were analyzed to find out relative standard deviation (RSD) for UFLC and UV Spectroscopic methods, respectively.

Accuracy
To check the accuracy of the proposed methods, recovery studies were carried out at 80, 100, and 120% of the test concentration. The recovery study was performed three times at each level. The amount of CEM present in the sample was calculated using the calibration curves.

Robustness
The robustness of the UFLC method was studied by deliberately changing the method parameters like flow rate of the mobile phase, detection wavelength, and organic phase composition. A series of system suitability parameters like retention time, theoretical plates, and tailing factor were determined for each changed condition according to ICH [33]. The robustness of the UV spectroscopic method was determined by changing the slit width and carrying out a solution stability study of CEM. The sample solutions were kept at room temperature on a benchtop for 24 h and the amount of drug recovered by the developed methods was calculated.

Limit of Detection and Limit of Quantitation
The LOD and LOQ were determined separately according to the ICH guidelines. For the UFLC method, concentrations providing a signal-to-noise ratio 3:1 and 10:1 were considered as the LOD and LOQ, respectively. In the case of the UV-Spectroscopic method, the LOD and LOQ were determined based on 3.3 and 10 times the standard deviation of the response, respectively, divided by the slope of the calibration curves.

Results and Discussion
Optimization

UV Spectroscopy (Method-II & Method-III)
The CEM in the phosphate buffer of pH 5.5 shows maximum absorbance at 261 nm (Method-II) as shown in Fig.3. Another novel approach called the AUC method was undertaken for the calculation of the integrated value of absorbance between the two selected wavelengths λ 1 = 256 nm and λ 2 = 266 nm (Method-III) as shown in Fig.4.   Fig. 3.

Specificity
To evaluate the specificity, a PDA detector was applied to find out the peak purity of the chromatographic peaks obtained for the stress-treated drug solution. Peak purity results are indicative for finding out the peak homogeneity. CEM underwent severe degradation under the alkaline stress conditions by using 0.1 M NaOH and UV radiation exposure for 45 min. So the stress conditions were optimized to get moderate degradation of CEM. The modified alkaline stress was applied by using 0.01 M NaOH solution. In the case of photolysis degradation, the exposure time was decreased to 25 min. CEM showed degradation in the order of H 2 O 2 > thermal > alkali > photolysis > acid. Fig.5 represents typical chromatograms obtained for CEM after being subjected to thermal, alkali, and photolysis degradation conditions, respectively. The run time for each stressed drug solution was increased from 6 min to 10 min in order to find out the presence of any extra peak because of the possible degradation of CEM. But no such extra peaks were found in the chromatogram. Also, the obtained peak purity values (>0.999) suggested that there were no co-eluting or hidden peaks with the drug peak, which shows specificity and the stability-indicating nature of the method. The results for the forced degradation study are summarized in Table 1. The UV spectrums (Fig. 6) obtained for the blank and placebo show no interference due to the solvent used and presence of the commonly used excipients suggesting the specificity of the two methods.

Linearity
The calibration curves were found to be linear over a concentration range of 1-120 µg/ml for all three methods (correlation coefficient 0.999 for all the methods). The method parameters and regression data are shown in Table 2.

Tab. 2.
Analysis of method parameters and regression data

Precision
The methods were found to be precise as the RSD (%) values for the precision studies were well below 2%. The results are shown in Table 3.

Accuracy
The accuracy of the developed methods was found out by the standard addition method.
High recovery values suggest that all three methods are accurate. The results are shown in Table 3.

Limit of Detection and Limit of Quantitation
The LOD and LOQ values shown in Table 3 suggest that the developed methods are sensitive to determine CEM.

Robustness
The UFLC method was found to be robust under deliberate changes in the mobile phase flow rate (±0.1 mL/min), detection wavelength (±5 nm), and organic phase composition (±2%). The results of system suitability for the robustness study are shown in Table 4. For the UV spectroscopic methods, changing the slit width shows no significant effect on absorbance, indicating the robustness of the developed methods. No significant changes were obtained in the content of CEM during the solution stability studies by the developed methods. The recoveries for the solution stability by Method-I, Method-II, and Method-III were found to be 100.27%, 101.12%, and 100.65%, respectively.

Analysis of Commercial Dry Syrup Formulation
The developed methods were successfully applied for the determination of CEM in the dry syrup formulation. The result for the assay of CEM is shown in Table 5. The assay results obtained for CEM in the syrup formulation using the UFLC and UV spectroscopic methods were compared by applying the ANOVA test, which revealed no significant differences between the values obtained by all three methods:

Conclusion
Three novel analytical methods were developed for the determination of cephalexin monohydrate (CEM). The validation study shows the methods are specific, linear, precise, accurate, and sensitive in the proposed working range. The methods were found to be fast, simple, accurate, precise, and sensitive. The excipients present in the commercial formulation were found to be non-interfering in the assay results. The methods were successfully applied for the determination of the drug in dry syrup formulation. Furthermore, the developed methods may be applied for the routine analysis of the drug in API, formulations, and dissolution medium.