Apparatus and software
The separations were performed on a Shimadzu Prominence high-performance liquid chromatography (HPLC) system consisted of degasser (Model DGU-20A5), pump (Model LC-20AD), Rheodyne manual injector fitted with 20-μL loop, variable wavelength UV–Vis detector (Model SPD-20A). The separation was carried out on a Zorbax CN column (250 mm × 4.6 mm, 5 μm). The mobile phase was pumped at a flow rate of 1.0 mL/min, and the analytes were monitored at 250 nm.
The experimental design data analysis and response surface was performed with Design Expert v. 8.1 (Stat-Ease Inc., Minneapolis, MN, USA). Other statistical analyses were carried out using Microsoft Excel 2013 software (Microsoft, USA).
Chemicals and reagents
Analytical-grade reagents ammonium acetate (BDH, Poole, England), glacial acetic acid acid (Sd fine Chem. Ltd., India), acetonitrile HPLC grade (Scharlau Chemie, Spain), and double-distilled water were used throughout the chromatographic analysis. Aqueous acetonitrile solution (75% v/v) was used as a diluent.
The reference standards of loratadine (99.5%), sodium benzoate (99.7%), and pseudoephedrine sulphate (98.8%) were used in this work.
Marketed formulations Lorinase syrup with label claim pseudoephedrine sulphate 60 mg and loratadine 5 mg per 5 mL, and Clarinase tablets labeled to contain 5 mg loratadine and 120 mg pseudoephedrine sulphate per tablet were purchased from the local market.
Preparation of standards and sample solutions
Optimization standard solution
Ten milliliters of a 100 μg/mL loratadine standard solution in acetonitrile were transferred into a 100-mL volumetric flask containing 5 mg sodium benzoate and 90 mg pseudoephedrine sulphate; the content was mixed and made to volume with the diluent.
Standards stock solution
Accurately weighed about 340 mg pseudoephedrine sulphate, 5 mg sodium benzoate, and 15 mg loratadine standards were transferred into a 25-mL volumetric flask; 10 mL of the diluent were added. The content of the flask was sonicated for 5 min and allowed to cool, and the volume was adjusted to the mark with the same diluent.
The sample density was first determined, and sample weight equivalent to 5 mL was accurately weighed into a 50-mL volumetric flask; 10 mL of the diluent were added, and the content was mixed and completed to mark with the same diluent. The sample was then filtered through 0.45-μm nylon filter and injected into the HPLC system. The concentrations of LOR and PSE were calculated from the linear regression equations of their calibration curves.
Ten tablets were finely powdered, and a portion equivalent to one tablet was suspended in 10 mL water in a 50-mL volumetric flask. The suspension was sonicated for 15 min, and the volume was completed to mark with acetoniltrile. The resulting solution was filtered through a 0.45-μm nylon filter and injected into the HPLC system. The concentrations of LOR and PSE were calculated from the linear regression equations of their calibration curves.
Aliquot volumes (1–5 mL) of the standard stock solution were transferred into a series of 25-mL volumetric flasks, and volumes were adjusted to the mark with the diluent.
Ammonium acetate buffers
The different concentrations of the buffer used were prepared by dissolving the required amount of the ammonium acetate in 500 mL distilled water. The pH of these buffers was adjusted using dilute glacial acetic acid.
The influence of the mobile phase components on chromatographic resolution between the analytes was studied using the following conditions: ionic strength (10, 15, and 20 mM), pH (3.5, 4.0, 4.5, and 5.0), and percent acetonitrile (65, 70, 75, and 80%). The experiments were carried by varying one parameter while keeping the other two constant. The resolutions between the adjacent peak pairs were calculated and plotted as a function of the experimental conditions.
Optimization of the mobile phase composition
The Box–Behnken experimental design  was used to optimize the mobile phase composition. The experiments were carried out using a series of mobile phases containing ammonium acetate buffer (10–20 mM) adjusted to pH = 3.0–5.0 and acetonitrile (74–80%),
The optimized method was validated in agreement with the ICH guidelines . The method linearity in the relevant working ranges, precision, accuracy, and specificity were evaluated. System suitability parameters were also determined.
The method’s linearity was evaluated using five standard mixtures prepared from their corresponding stock solution to obtain final concentrations of PSE in the range of 594.8–2974.0 μg/mL and LOR 24.6–123.2 μg/mL. The method of least squares was used to obtain the linearity parameters from calibration data of the analyte concentration versus their corresponding peak areas.
Six replicate determinations of the samples containing 100% of their corresponding expected concentrations in the pharmaceutical product were injected, and the method's precision was verified in its repeatability and intermediate precision aspects according to the ICH guidelines. For verification of the intermediate precision, the process was repeated on a different day using fresh reagents and samples.
The method accuracy was demonstrated using the standard addition method by evaluating analyte recoveries from a pre-assayed pharmaceutical formulation sample, containing 60% of the declared amount of the drugs, which was fortified with known amounts of the two analytes, to obtain concentration levels of 60–120% of the expected drug concentrations in the pharmaceutical dosage form.
Limits of detection LOD and quantification LOQ
In order to assess that the validated concentration ranges of the analytes were above their LOQ values, the LOD and LOQ were determined employing the ICH method based on the calibration curve .
System suitability parameters
The system suitability parameters: the column efficiencies (N), resolution between adjacent peeks (Rs), and asymmetry factor (As) were calculated from five replicate injections of the standard solution made under optimized conditions.