The pharmaceutical grade (purity, ≥97%) of all the actives was obtained from different local pharmaceutical industries.
The tablets of all actives were purchased from the pharmacy.
Chemicals and reagents
HPLC grade methanol was purchased from Merck Germany. Orthophosphoric acid and other chemicals used were of analytical grade.
Shimadzu HPLC system equipped with LC-10 AT VP pump, SPD-10A VP UV–vis detector utilizing a Purospher® STAR RP-18 end-capped (5 μm, 25 × 0.46 cm) column was used. The integrated chromatographic data were recorded using Shimadzu Class-GC 10 software (version 2) for data acquisition and mathematical calculations.
Mobile phase preparations
The mobile phase consisted of a mixture of methanol, acetonitrile, and water in a ratio of 50:20:30 with a pH adjusted to 3.1 using orthophosphoric acid.
Preparation of solutions
Solutions of cetirizine and quinolones (ofloxacin, levofloxacin, ciprofloxacin, enoxacin, sparfloxacin, and norfloxacin) were prepared by accurately weighing 10 mg of standards and transferred to 100-mL volumetric flask, separately, and volumes were completed with the unbuffered mobile phase. The resulting solutions of 100 μg mL−1 were sonicated and then filtered. From the stock solutions, different dilutions, i.e., 2.5, 5, 15, 20, 25, and 50 mL, were pipetted out in different 100-mL volumetric flasks and made up to the mark with the same solvent to have the required concentrations of 2.5, 5, 15, 20, 25, and 50, respectively.
Linearity studies were performed by preparing a solution at six different concentration levels, i.e., 2.5, 5, 15, 20, 25, and 50 μg mL−1 for all the drugs assayed. The standard calibration curves were evaluated by linear regression.
The method’s precision was performed by injecting four representative samples on each of the 2 days (n=18).
The method’s accuracy was performed at three different concentrations, i.e., 8, 10, 12 μg mL−1 (n=6), by adding known quantities of the analyte to the drug product.
Procedure for formulations
Twenty tablets of each drug were individually weighed and triturated to obtain a homogeneous mixture. Amount of powder equivalent to 10 mg of drug was transferred to a 100-mL volumetric flask. The flask’s content was shaken, and volumes were completed with the mobile phase. This solution was filtered and diluted to obtain five different concentration levels in the range of 2.5–25 μg mL−1. The % recovery and %RSD were calculated in each case.
System suitability requirement
The %RSD of the standard peak area should be less than 2, and the tailing factor of the standard peak should not be more than 2. The number of theoretical plates should not be less than 2000.
Serum drug analysis
The stated chromatographic conditions determined cetirizine and quinolones’ recoveries in human serum. Blood was deproteinated by acetonitrile. The supernatant obtained was filtered through a 0.45-μm pore size membrane filter. The serum thus obtained was mixed to different aliquots of the stock standard solution to produce desired concentrations. These were then stored at – 20 °C, and 10 μL volume of each sample was injected and chromatographed.