3.2.1. Selectivity

The selectivity of the proposed HILIC-PDA method is demonstrated in Figure 4, where a representative chromatogram obtained from the analysis of standard solution containing risedronate at 3.5 μg mL−<sup>1</sup> is presented overlaid with a chromatogram obtained from the analysis of risedronate commercial tablets and a blank sample (dilution solvent). No significant interfering peaks have been observed at the retention time of risedronate, which is eluted at 4.59 min.

**Figure 4.** Typical HILIC chromatograms of a blank sample (green line) overlaid with a 3.5 μg mL−<sup>1</sup> risedronate standard solution (red line) and a solution obtained from the analysis of risedronate commercial tablets (blue line). Chromatographic conditions: ZIC®-pHILIC analytical column, mobile phase: 38% 9 mM ammonium acetate and 1 mM sodium pyrophosphate aqueous solution pH 8.8 in acetonitrile, flow rate of 0.15 mL min−<sup>1</sup> and UV detection at 262 nm.

#### 3.2.2. Statistical Analysis of Data

The calibration curves of the peak area signals of risedronate versus the corresponding concentrations were linear, as shown by the results presented in Table 1. Back-calculated concentrations in the calibration curves were within 2.1% of the nominal values, which are in agreemen<sup>t</sup> with international guidelines.


**Table 1.** Statistical analysis of the calibration curves of risedronate.

a Peak areas signal of risedronate, SRsd vs the corresponding concentration of risedronate, CRsd; b correlation coefficient; c standard error of the estimate.

The limit of detection (LOD) and the limit of quantitation (LOQ) were estimated experimentally by analyzing risedronate samples spiked at low concentrations. These limits were defined according to a signal-to-noise ratio (S/N) corresponding to 3:1 for the LOD and to at least 10:1 until a %CV of less than 2.5% was obtained for the LOQ. The LOD was found to be at 0.3 μg mL−1, and the LOQ at 1.5 μg mL−1.

Risedronate can be determined by appropriate precision and accuracy as is indicated by the intraand inter-assay precision data that are presented in Table 2. Precision was evaluated by one-way

analysis of variance. Intra-assay relative standard deviation values, %RSD, were between 0.3% and 0.6%, while inter-assay %RSD was no more than 0.6%. The overall accuracy of the method was expressed by the relative percentage error, Er%, that ranged from 2.3% to 1.8%.


**Table 2.** Accuracy and precision data.

a (n = 3 runs; 5 replicates per run).

During the method development, it was observed that both the volume fraction of water (ΦH2O) and the concentration of ammonium acetate affected the chromatography of risedronate. To assess method robustness, small deliberate variations were performed in the aforementioned parameters and in the wavelength of detection. Each parameter was changed at two levels (0 and 1) using a univariate approach and robustness was estimated by measuring the peak area signal. Ammonium acetate concentration was altered by 1.0 mM (range 8 to 9 mM), volume fraction of water Φwater was altered by 2% (range 38% to 36%) and the wavelength of detection was altered by of 2 nm (range 262 to 264). A standard solution of risedronate at 3.5 μg mL−<sup>1</sup> was injected in three replicates under changes of the parameters mentioned above. The proposed method could be considered as robust, since %RSD values of the peak area signal of risedronate does not exceed 3.2% (acceptance criteria < 10%) in all of the tested conditions.

#### 3.2.3. Accelerated and Long-Term Stability Studies

The results of the accelerated and the long-term stability studies are presented in Table 3. In the acid stressed samples and after 10 days, a 21.9% of risedronate was degraded using 1.0 M HCl at 50 ◦C, while no degradation products have been detected. In the base stressed samples and after 1 day, a 21.2% of risedronate was degraded using 1.0 M NaOH at 50 ◦C, while no degradation products were detected. In the acid stressed samples and after 3 h, a 17.3% of risedronate was degraded using 3.0% (*v*/*v*) H2O2 at 25 ◦C while the degradation products could not be detected. After the degradation of risedronate blistered tablets under low (15%) and high (75%) humidity conditions for three months, the percentage recovery of the analyte was 95.9% and 78.5%, respectively.




**Table 3.** *Cont*.

Based on the results presented in Table 3, the proposed HILIC-PDA method is stability-indicating, since it is able to quantitate risedronate in commercial formulations without any interference from degradation peaks.

#### *3.3. Analysis of Commercial Tablets*

The applicability of the proposed method was evaluated through the analysis of commercially available tablets containing 35 mg of risedronate sodium (equivalent to 32.5 mg risedronic acid). The analysis was performed on accurately weighted amount of the pulverized tablets. Percentage recovery was found to be 99.3 ± 0.7% of the label claim, or 32.3 ± 0.2 mg of risedronic acid per tablet (n = 10, RSD = 0.6%). Additionally, this method was used for the content-uniformity testing, in which many assays on the individual tablets are required. Percentage recovery was found to be 100.2 ± 1.2% of the label claim, or 32.6 ± 0.4 mg of risedronic acid per tablet (n = 10, RSD = 1.1%).

Table 4 presents data obtained from the analysis of real samples and indicate that the proposed HILIC-PDA method is applicable to the accurately quantitation of risedronate in commercially available tablets.

**Table 4.** Risedronate quantitation in a commercial formulation.

