RP-HPLC METHOD FOR SIMULTANEOUS DETERMINATION OF METRONIDAZOLE AND OFLOXACIN IN SYNTHETIC MIXTURE

Development and validation of a high-performance liquid chromatographic analytical procedure for simultaneously determining metronidazole and ofloxacin in a synthetic mixture is described in this paper. The separation was made with a LiChrosorb RP-18 (250 × 4.6 mm) column, at 30 °C temperature, with isocratic mode and mobile phase, containing triethylamine, acetonitrile, and 0.3% o-phosphoric acid solution (0.02:20:80 v/v/v). Eluent was monitored at 290 nm and the flow rate was 1.0 ml/min. Metronidazole and ofloxacin were effectively separated with retention time (tr) of 3.42 min and 6.15 min, respectively, within the selected chromatographic conditions. The method was validated for analytical parameters: specificity, linearity, precision, accuracy, and limits of detection and quantitation. The calibration curves were linear in the concentration range of 12.5 to 100.0 μg/ml for metronidazole and ofloxacin, and the regression coefficients were more than 0.999. For metronidazole and ofloxacin the recovery was 100.01% and 100.04%, respectively. This analytical procedure is applicable for the quality control of drug formulations. UDC Classification: 543.6, DOI: http://dx.doi.org/10.12955/cbup.v4.871


HPLC Analysis
The HPLC analysis was performed by isocratic elution with a flow rate 1.0 ml/min. For the analysis, a HPLC system (SHIMADZU Corporation, LC-20 AD quaternary pump) was used with a Shimadzu SPD-20A ultraviolet-visible (UV/VIS) detector. LabSolutions (Shimadzu Corporation) software was used for recording the data. Separation was carried out at 30 ºC, using LiChrosorb ® RP-18 (250 × 4.6 mm) column, packed with octadecylsilyl silica gel 5-µm. The mobile phase, containing triethylamine, acetonitrile, and 0.3% o-phosphoric acid solution (0.02:20:80 v/v/v) was sonicated for 10 min and then filtered through a 0.45-µm filter paper. The analysis was performed at 290 nm with 20 µl aliquots of the solution were injected. The chromatogram is shown in Figure 1. A bulk mixture of both drugs was prepared using 500 mg MET, 500 mg OFL, and excipients of 100 mg microcrystalline cellulose (type Vivapur 101 ® , JRS Pharma-Germany), 140 mg lactose monohydrate (Meggle Pharma-Germany), 60 mg cross-linked sodium carboxymethylcelulose (Vivasol ® JRS Pharma-Germany), 10 mg povidone (Kollidone ® K30, BASF-Germany), 14 mg magnesium stearate, and 6 mg silica colloidal anhydrous (Aerosil ® 200, Evonik Ind.), which were used in the tablet formulation and well triturated. A powder equivalent to 250 mg of MET and 250 mg of OFL was weighed accurately and transferred into a 50 ml volumetric flask, with 25 ml of solvent A added, and then sonicated for 10 minutes. The suspension was then made up to volume with solvent A and filtrated. The final concentrations were 50 µg/ml of MET and 50 µg/ml of OFL.

Results and Discussion
Several mobile phase combinations were trialed for optimization of the RP-HPLC conditions. Taking into account the protolysis constants (K a ) and solubility of both the compounds, several important parameters, such as, percentage and type of organic modifier, pH of the mobile phase, and concentration of the acid, were studied. Resolution was the most important criterion for the method and it was imperative to achieve adequate separation. The trials showed that a mobile phase, consisting of triethylamine, acetonitrile, and 0.3% o-phosphoric acid solution, in the proportion of 0.02:20:80 v/v/v, with reverse phase LiChrosorb ® RP-18 (250 x 4.6 mm) column, yielded symmetric and sharp peaks. Greatly improved detector responses for both drugs were obtained at the optimum wavelength of 290 nm.
As shown in Figure 1, the retention times were 3.42 min for MET and 6.15 min for OFL. The method was simple and had a shorter run time (7 min) in comparison with that reported by Patel (2011) and Bind et al. (2015). The proposed method was validated in terms of specificity, linearity, accuracy, precision, and limits of detection and quantitation, according to guidelines of the International Council on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH guidelines, 2005).

Method Validation
The selectivity of the current method demonstrated adequate separation of the two active ingredients (MET and OFL). The matrix components, e.g., excipients, did not interfere with the two analytes.
For examining linearity, standard solutions containing MET (12.50-100.0 µg/ml) and OFL (12.50-100.0 µg/ml) were prepared in the solvent A. Each concentration level was triplicated in chromatography, using 20 µl injections for each standard solution.
In addition, studies were prepared with five different concentrations of mixtures for both drugs. The responses, measured as peak areas, are shown in Table 1. The calibration curves showed linearity in the selected concentration range for both drugs. The linear regression equations for MET and OFL were as follows: y = 11827.3 × −31.6; and y = 14929.5 × −10454.4, respectively. The regression coefficients (r) were greater than 0.999, which indicated a high degree of linearity for both drugs (Figures 2 & 3).  Source: Author For determining the limit of detection (LOD) and limit of quantitation (LOQ), the method based on signal to noise ratio (3:1 for LOD and 10:1 for LOQ) was adopted (ICH guidelines (2005)). The limit of detection for MET was 0.125 µg/ml and for OFL 0.250 µg/ml, while the limit of quantitation for MET was 0.250 µg/ml and for OFL, 0.300 µg/ml.
Intra-daily precision was evaluated by calculating the standard deviation (SD) of six replicate determinations using the standard solutions. The SD values revealed the high precision of the method (values were in the range of 0.77 to 0.98 for both drugs). For inter-daily reproducibility, a series was run, in which the standard drug solutions were analyzed over five consecutive days. The SD values were in the range of 0.98 to 1.90.
The accuracy of the method was specified by preparing samples of 25%, 50%, 100%, 150%, and 200% of the target concentration. Each concentration level was injected three times. The results showed perfect recoveries (Table 2).

Conclusion
The high performance liquid chromatographic method described in this paper was developed for quantitative control and determination of the contents of MET and OFL. It can be applied in clinical pharmacy, toxicology, and for routine analysis of tablet formulations. The method has high degree of accuracy and precision (less than to 2% Relative SD) and requires a markedly short performance time (7 min). The parameters for validation of the method (linearity, accuracy, and precision) meet all requirements of the International Council for Harmonization (ICH). One advantage of this method is that it does not use buffers, which are onerous to prepare. The procedure is also simple and easy for implementation.