INTRODUCTION

Ciprofloxacin hydrochloride (Figure 1), the most frequently used quinolone [1], is available for oral use as hydrochloride monohydrate [2] and parenteral use as lactate [3]. It is slightly yellowish to light yellow crystals or crystalline powder, dissolved in water, and somewhat soluble in methanol [4]. It is thoroughly absorbed and is immediately excreted from the body under typical conditions with an elimination half-life of 3 to 5 hours. A polymer combination of ciprofloxacin showed more diffusivity and an extended-release rate connected to ciprofloxacin salt [5].

Several analytical techniques have been published to analyze ciprofloxacin in biological fluids and various pharmaceutical dosage forms (tablets, injectables, suppositories, gels, eye drops, etc.) [6-12]. A direct and sensitive HPLC method for determining ciprofloxacin in the influenza vaccine has been described [7] employing a mobile phase of acetonitrile/water/phosphoric acid. Its pH was adjusted to pH 3 with triethylamine. The separation was obtained at a flow rate of 0.6 mLmin^-1 at 280 nm. A method was mentioned for ciprofloxacin with other quinolones using a mobile phase consisting of water-acetonitrile (50:50, v/v) with a pH of 2.9 adjusted with phosphoric acid. Different wavelengths were applied at a flow rate of 1 mLmin^-1, while propylparaben was used as an internal standard [8]. A liquid chromatographic method was also reported for ciprofloxacin's concurrent determination with rosuvastatin in formulations and human serum. The separation was achieved using a mobile phase of methanol-water at a ratio of 90:10 v/v. The flow rate was 1 mLmin^-1, the pH of the mobile phase was pH 3, and drugs were monitored at 255 nm [9]. Similarly, an HPLC–UV method was described for the determination of ciprofloxacin in human plasma with a phosphate buffer (pH 2.7) and acetonitrile (77:23, v/v), and the drug was detected at 277 nm [10, 13-15].

The suggested task deals with the precise analysis of ciprofloxacin using a straightforward, moderate-cost, and less time-consuming HPLC method with significant accuracy and good LOQ (Limit of detection) value. The technique contains readily available chemicals, solvents, and an internal standard suitable for routine analysis.

MATERIALS AND METHODS

Material and reagents

Ciproxin^TM 250 mg tablets (Bayer Pakistan Pvt. Ltd. Karachi) were purchased from the local pharmacy, while mebeverine HCl (internal standard) was a kind gift from AGP (Private) Limited Karachi. De-ionized water was prepared in the lab from double distilled water, while all other solvents were HPLC grade (Merck Germany). Shimadzu Corporation Japan's chromatographic system consisted of an LC-10 AT VP pump and SPD-10 AV VP UV-visible detector. The separation was achieved on the µ Bondapak 125A C-18 10 µm column.

Analytical procedure

10 mg of ciprofloxacin hydrochloride and internal standard (mebeverine hydrochloride) were dissolved in water in a 100 mL volumetric flask separately, with the same solvent. The concentrations of these immediate solutions were 100 ppm. For the stock solution preparation, 10 mL of ciprofloxacin (100 ppm) was taken in a 100 mL volumetric flask with water. The concentrations of these stock solutions were 10 ppm, from which standard working solutions of desired concentrations were prepared.

Different dilutions ranging from 0.50, 1.0, 1.50,..3.0 ppm were prepared from the stock solution of ciprofloxacin. For this purpose, 5, 10, 15,…..30 mL of stock solutions were pipetted out in different 100 mL volumetric flasks and made up to the mark with the same solvent (water) to have the required concentrations of 0.50, 1.0, 1.50, . . . 3.0 ppm, respectively, and in all solutions, added 1 mL of the internal standard (primary solution) added as internal standard became 5 ppm of final concentration. Six different concentrations of each sample were analyzed. For intra-day precision, different concentrations of every compound were investigated on a similar date and injected four times. The mean value of each concentration was used to calculate %RSD. Generally, satisfactory repeatability of the outcome in one day and alternate days was observed. Lower RSD values showed that the method is reliable to analyze both drugs separately.

RESULTS AND DISCUSSION

Many methods have already been reported for estimations of ciprofloxacin [6-12] and but the present method is straightforward and is validated according to ICH (International Conference on Harmonization) guidelines [16]. All of the parameters were found under the limits of ICH guidelines, and the chromatogram of the analyte revealed that the method was specific and no peak of internal standard interfered, and can be considered selective.

The analysis was conceded in isocratic conditions using a mobile phase acetonitrile-water (80:20), while pH 2.7 was adjusted using phosphoric acid (85%). The flow rate of 1.7 mLminutes^-1 was used, and all experiments were performed at room temperature at 275.0 nm. The retention time of both the drug and internal standard were 1.52 and 2.7 minutes, respectively. The method was found linear (r²=0.9999), accurate (%RSD>2%), precise, specific, and sensitive (DL= 120 ngmL^-1 and QL=500 ngmL^-1).

Discussion

Linearity and regression analysis

The present method is linear over a range of 0.5 to 3.0 ppm, and all the calibration curves have a correlation coefficient value of at least 0.9999 (Table 1).

Table 1. Statistical regression characteristics of method

 Accuracy

Accuracy was performed according to the guidelines published by ICH [16]. The recovery data expressed in Table 2 showed that the method is accurate for determining and quantifying ciprofloxacin.

Table 2. Recovery of Ciprofloxacin in dosage form

*Concentration

Precision

Like accuracy, the method's precision was also performed using the same guidelines [16].

The precision was examined regarding repeatability, and intra-day precision was analyzed on the same day using six ciprofloxacin concentrations. Table 3 summarizes the relative standard deviation (RSD).

Table 3. Intra and inter day variations in the analysis of ciprofloxacin hydrochloride

System suitability

System suitability is the parameter that assists in checking the behavior of the system and was evaluated by analyzing the symmetry of the ciprofloxacin, internal standard (mebeverine hydrochloride), peaks, theoretical plates of the column (>2000), and the resolution between the peaks of internal standard and drugs.

Specificity

Specificity is ensured by the separation of the internal standard and ciprofloxacin. The technique demonstrated a resolution between the internal standard and ciprofloxacin. HPLC chromatogram showed that extraneous peaks by adding an internal standard are the baseline resolved from the parent analyte (Figure 2).