The method’s selectivity was determined by assessing the interference of adjuvants at the wavelength employed for quantitatively determining the combined drugs in the commercial sample. As the list of adjuvants in the commercial sample was confidential, a placebo solution was created, comprising a mixture of adjuvants: Sodium lactate (19%), sorbitol (60%), sodium chloride (6%), calcium chloride (0.1%), magnesium chloride (0.2%), and potassium chloride (0.3%). Both the sample and placebo solution were prepared in water (q.s.p. 100 mL), following a procedure similar to the one described earlier for sample solution preparation. The chromatogram of the commercial sample was then compared with that of the placebo solution.

The evaluation involved constructing three analytical curves for each drug, derived from dilutions of the respective standard stock solutions. Specifically, the analytical curve for Sodium Lactate (SL) was established within the concentration range of 0.79 to 3.8 mg∙mL⁻¹, for Sodium chloride (SC) in the range of 0.16 to 0.79 mg∙mL⁻¹, and for Sorbitol in the range of 1.5 to 7.2 mg∙mL⁻¹. These determinations were performed in triplicate, and the obtained results underwent linear regression analysis to derive analytical curves, line equations, and correlation coefficients for each drug.

The LD and LQ of SL, SC, and Sorbitol were determined from three analytical curves obtained for each drug, using the standard deviation of the intercept (SD) and the mean slope ( a ). Equations (1) were used to calculate LD and LQ:

L D = 3.3 × S D a     L Q = 10 × S D a (1)

The precision of the method was evaluated through intra-day (repeatability) and inter-day (intermediate precision) tests. Repeatability involved analyzing six determinations of the stock solution of the commercial sample at concentrations of 3.15, 0.65, and 6.0 mg∙mL⁻¹ for SL, SC, and Sorbitol, respectively. This analysis was conducted under the same chromatographic conditions and by the same analyst. Intermediate precision was assessed by preparing new sample-stock solutions, with two analysts performing the analysis on three different days, again at the concentrations mentioned above. Relative standard deviations (%RSD) were then calculated based on the test results.

The accuracy of the method was determined through recovery tests, involving the addition of known amounts from standard solutions to commercial sample solutions. Separate assays were conducted for each drug to cover the linear concentration range of the method. In the recovery tests, standard solutions and the previously described stock samples were subjected to recovery assays at three concentration levels (80% to 120% of the target concentration) for each drug, performed in triplicate. Quantities of the solution prepared with the raw materials of the drugs were added to the stock solutions to obtain concentration solutions of 150, 190, and 230 mg∙mL⁻¹ for SL; 32, 36, and 46 mg∙mL⁻¹ for SC; and 480, 600, and 720 mg∙mL⁻¹ for Sorbitol.

The recovery percentages of each drug were calculated using equation 3 (AOAC, 2005): (3) %R = [(Ca-Cna)/Ctp] × 100 Where: R: recovery, Ca: drug concentration found in the standard added sample (mg∙mL⁻¹), Cna: drug concentration found in the standard non-added sample (mg∙mL⁻¹) and Ctp: theoretical standard concentration added to the sample (µg∙mL⁻¹).

The method’s robustness was deliberately evaluated by introducing changes to the chromatographic conditions. Variations were made in the column temperature (±5˚C), specifically at 35˚C and 45˚C, and the flow of the mobile phase was adjusted (±0.1 mL/minute) within the range of 0.5 to 0.7 mL/min. For each condition, only one parameter was modified, keeping all other factors constant. The robustness assessment was conducted in triplicate by injecting sample solutions containing 3.1, 0.65, and 6.0 mg∙mL⁻¹ of SL, SC, and Sorbitol, respectively.

The impact of variations in each parameter on the final results was assessed by calculating the average of the results obtained with normal parameters and comparing it with the average corresponding to altered parameters. The effect generated by each variable was determined as the difference between the results obtained under normal conditions and those obtained with changed parameters.

To assess solution stability, both test and standard preparations were carried out following the specified procedure and stored under two conditions: at ambient temperature (20˚C - 25˚C) and in the refrigerator (2˚C - 8˚C). On the initial day, day 1, day 2, and day 3, these solutions were injected into the HPLC system. The assay percentage for the test preparation was calculated in comparison to a freshly prepared standard solution. The calculated % limits were found to be within acceptable ranges for solutions stored for 3 days in the refrigerator and 24 hours at ambient temperature (20˚C - 25˚C), indicating that the assay of SL, SC, and sorbitol remained stable under both ambient and refrigerated conditions.

The commercial sample RHEOPAR-S® (consisting of Sodium Lactate 19%, Sodium Chloride 6.0%, and Sorbitol 60.0%) underwent analysis using the proposed analytical method. Sample-stock solutions were prepared as outlined earlier, resulting in theoretical concentrations of SL 3.1 mg∙mL⁻¹, SC 0.65 mg∙mL⁻¹, and Sorbitol 6.0 mg∙mL⁻¹. The content of each drug in the commercial sample was then calculated using the established analytical curves.

The HPLC technique presented in this study is applicable for the analysis of pharmaceutical product formulations, drug substances, routine and in-process samples, as well as the measurement of pharmaceutical formulations and infusion solutions. Notably, this technique offers simplicity, user-friendliness, and cost-effectiveness, making it suitable for routine quality control (QC) analysis.

The study followed ICH Q1 stability studies, subjecting the commercial sample RHEOPAR-S® to exposure at 40˚C ± 2˚C for six months using a drying oven (NI 1521, NOVA Instruments®) equipped with a temperature controller. Organoleptic evaluation was conducted at 0, 3, and 6 months. The assay was performed using high-performance liquid chromatography (HPLC) with the previously validated method. The chromatographic conditions included a Shimadzu Shim-pack SRC-102(H) ion exclusion analytical column (300 mm × 7.9 mm, 8μm), a mobile phase of 0.1% phosphoric acid in isocratic mode, a flow rate of 0.6 mL∙min⁻¹, an injection volume of 10 µL, and a diode array detector set at 200 nm. Sample solutions were prepared in water, yielding theoretical concentrations of 0.31 mg∙mL⁻¹ for SL, 0.65 mg∙mL⁻¹ for SC, and 6.0 mg∙mL⁻¹ for Sorbitol. Areas were recorded in triplicate, and drug contents were calculated using the equations of the straight lines obtained from analytical curves.

The system suitability parameters were examined and validated to meet the acceptance criteria. Refer to Table 2 for a detailed presentation of the results.

Specificity, defined as the ability to accurately assess an analyte in the presence of anticipated components, was evaluated in the method. This was accomplished by analyzing solutions of blank, placebo, and control samples in the HPLC system and recording chromatograms. Additional details can be found in Figure 2, Figure 3, and Table 3.

In the investigation of linearity, preparations were made for five different concentrations of Sodium Lactate (SL), Sodium Chloride (SC), and Sorbitol, covering a range from 25% to 120% of the specification level. The correlation coefficient was determined by plotting concentration (X-axis) against peak area (Y-axis) for SL, SC, and Sorbitol. The concentration ranges for linearity solutions were 0.9 mg/mL to 2.1 mg/mL, 0.2 mg/mL to 0.45 mg/mL, and 3.0 mg/mL to 7.2 mg/mL, respectively. Regression equations of SL, SC, and Sorbitol were. The regressioncoefficient values (r) were all greater than 0.999 for each component, indicating linearity. The linearity of the area response versus concentration met the criteriareferred to in Table 4, and the figures are depicted in Figure 4.

The precision and intermediate precision tests for the analyzed drugs using the developed method are summarized in Table 5. The results, expressed as Percent Relative Standard Deviations (%RSD) for Sodium Lactate (SL), Sodium Chloride (SC), and Sorbitol, were observed to be below the maximum recommended limit of 5.0% (ICH, 2005). This outcome affirms the accuracy of the developed analytical method.

The accuracy of the method was assessed through recovery tests, and the results for the recovery of standard solution amounts for each component are detailed in Table 6. The recovered amounts of Sodium Lactate (SL), Sodium Chloride (SC), and Sorbitol fell within the acceptable limits of 98% - 102% for all three concentration levels analyzed. The %RSD of the average of these levels was below 2.0%. The mean recovery percentages for SL, SC, and Sorbitol were 99.3%, 99.1%, and 99.8%, respectively. Consequently, the developed method demonstrates satisfactory accuracy for the simultaneous determination of SL, SC, and Sorbitol.

%RSD-Percentage Relative standard deviation.

The System suitability parameters were evaluated, and it was determined that the results were within acceptable limits.