*2.2. Instrumentation*

Experiments were performed on an HPLC-PDA system consisting of an auto sampler model Waters 717 plus, a constant temperature oven, an isocratic pump model Waters 1515 and a photodiode array detector model Waters 996 (Milford, MA, USA). Data acquisition and analysis was attained by the use of the Empower software (Milford, MA, USA). The analytes were detected over the wavelength range of 200 to 400 nm and the chromatograms were extracted at λ = 262 nm. Chromatography was performed by using a polymeric zwitterionic ZIC ®-pHILIC analytical column (150.0 × 2.1 mm i.d., 200 Å, particle size 3.5 μm) (Merck Millipore, Darmstadt, Germany). Moreover, a guard column (20 × 2.1 mm, 3.5 μm) of the same packing material was used to prolong the column lifetime. During the method development, various mobile phases consisting of mixtures of acetonitrile and ammonium formate or ammonium acetate aqueous solutions were usedand the flow rate was set at 0.25 mL min−1. Aqueous solutions of ammonium acetate were prepared freshly every day. For the quantitation of risedronate, the mobile phase consisted of 38% 9 mM ammonium acetate and 1 mM sodium pyrophosphate aqueous solution pH 8.8 in acetonitrile and pumped at a flow rate of 0.15 mL min−1. It was filtered through a 0.22 μm Nylon-membrane filter, Membrane Solutions (Kent, WA, USA) and degassed under vacuum prior to use. Chromatography was performed at 40 ± 2 ◦C, with a chromatographic run time of 6 min; a 60 μL volume was injected into a 10 μL loop.

## *2.3. Statistical Analysis*

Regression analysis was performed using IBM SPSS Statistics ver. 22, IBM software. The ionization state of each compound was estimated using ADME boxes ver. 3.0, Pharma Algorithms software.

#### *2.4. Stock and Working Standard Solutions*

Stock standard solutions of risedronate, zoledronate and tiludronate were prepared at 500 μg mL−<sup>1</sup> in acetonitrile-water mixture (60:40, *v*/*v*). Stock standard solution of risedronate was prepared in duplicate for the calibration standards and the quality control samples. These solutions were stable for several weeks when stored at −17 ◦C for several months. The stock standard solutions were further diluted in acetonitrile to prepare working standard solutions at two concentration levels 5 and 10 μg mL−<sup>1</sup> for each analyte. These solutions were used for the method development and were stored under refrigeration at 4 ◦C for two months.

Calibration standard solutions of risedronate were prepared in acetonitrile over the concentration range of 1.5 to 5 μg mL−1. Quality control samples of risedronate were also prepared in acetonitrile at three concentration levels (1.5, 3.5 and 5 μg mL−1). Calibration standard solutions and quality control samples were prepared freshly every day and remained stable throughout the analysis.

#### *2.5. Assay Procedure for the Pharmaceutical Samples*

To calculate the tablet weight, 10 tablets containing 35 mg of risedronate sodium were weighted and then pulverized. A portion of this powder, equivalent to 35 mg of risedronate sodium, was transferred into a 100 mL volumetric flask and diluted to volume with acetonitrile/water mixture (10:90, *v*/*v*). The mixture was sonicated for 10 min and then transferred into a 2 mL Eppendorf tube for centrifugation at 4.000× *g* and 25 ◦C for 10 min. The supernatant was then sonicated in an ultrasonic bath for additional 10 min and filtered through a PTFE hydrophilic syringe filter. A 100 μL aliquot of the filtrate was then transferred into a 10 mL volumetric flask and diluted to volume with acetonitrile prior to HILIC-PDA analysis.

#### *2.6. Accelerated and Long-Term Stability Studies*

Degradation studies were performed in risedronate under various stress conditions where degradation was stimulated by acidic or basic hydrolysis, oxidation and thermal degradation. Risedronate bulk substance was stressed under accelerated degradation conditions with 1.0 M HCl at 50 ◦C ( ± 2) for 10 days, 1.0 M potassium hydroxide (NaOH) at 50 ◦C ( ± 2) for 24 h and 3.0% *v*/*v* hydrogen peroxide (H2O2) at 25 ◦C ( ± 2) for 3 h. The concentration of risedronate bulk substance in the accelerated stability samples was 0.35 mg mL−1. During each degradation experiment and at predetermined time intervals, appropriate aliquots were neutralized with base or acid, and analyzed according to the proposed method. The concentration of risedronate in the analyzed sample solution was 3.5 μg mL−1.

Blistered tablets containing risedronate were stressed in long-term stability studies. Blistered tablets have been stored for 3 months at 50 ◦C ( ± 2) and 75% ( ± 2) relative humidity, and at 50 ◦C ± 2 ◦C and 15% ( ± 2) relative humidity. After the completion of each degradation treatment the samples were analyzed as described in the sample preparation procedure (Section 2.5).

### **3. Results and Discussion**

## *3.1. Method Development*

Bisphosphonates contain two phosphoric acid groups and are strongly polar and ionic compounds. The three bisphosphonates drugs studied in this work are divided into two groups: two nitrogen-containing compounds (risedronate and zoledronate) and one acidic compound (tiludronate). These compounds are poorly retained in the classical reversed phase analytical columns and their chromatographic analysis is challenging. The ZIC ®-pHILIC analytical column used is a polymeric and zwitterionic sulfoalkylbetaine stationary phase. The functional group of this column consists of a sulfonic acid group (acidic), which was separated with a short alkyl spacer from a quaternary ammonium group (basic). In this zwitterionic stationary phase, the electrostatic forces of each charge were partly counterbalanced by the proximity of an ion with opposite charge. Though the accessibility to the positively charged quaternary ammonium groups was limited, the negatively charged sulfonic acid groups might be responsible for weak, but important, electrostatic interactions [25]. The studied bisphosphonates were retained adequately in this analytical column through hydrophilic interactions, even if they had the same charge with the sulfonic acid groups of the stationary phase. Electrostatic repulsions in HILIC were first described by Alpert [26] as electrostatic repulsion hydrophilic interaction chromatography (ERLIC). These kinds of interactions were of grea<sup>t</sup> interest and can be used to selectively antagonize the retention of analytes that normally would be best retained [27].

#### 3.1.1. Effect of Chromatographic Parameters on the Bisphosphonates Retention

A one-variable-at-a-time approach was used to study the chromatography of bisphosphonates in the zwitterionic stationary phase. Mobile phases in HILIC typically contain high percentages of acetonitrile mixed with an aqueous salt solution. In this work, the mobile phase salts were limited to ammonium formate and ammonium acetate due to their good solubility in acetonitrile. The sulfonic acid groups of the stationary phase are responsible for weak electrostatic interactions that can be reduced by the addition of an aqueous salt solution. In preliminary experiments with a mobile phase containing 35% 10 mM ammonium formate water solution in acetonitrile, both nitrogen-containing bisphosphonates (risedronate and zoledronate) were not eluted, while tiludronate exhibited a broad asymmetrical peak. On the other hand, ammonium acetate improved the chromatography for all compounds. Consequently, ammonium acetate concentration was varied from 1 to 40 mM in mobile phases containing 35% Φwater. The logarithm of retention factor (k) was used to evaluate retention of the analytes. Retention factor (k) is independent of column geometry and flow rate and was often used for reproducibility evaluation, and method validation [28].

Typical HILIC chromatograms illustrating the effect of the concentration of ammonium acetate on the retention time and the peak shape bisphosphonates are presented in Figure 1. In all of the ammonium acetate concentrations tested tiludronate exhibits good peak symmetry while tailing peaks are observed for both risedronate and zoledronate. Bisphosphonates as strong chelators are capable to interact with the metals of the liquid chromatographic (LC) system [10,29]. This binding affinity is greater in nitrogen-containing bisphosphonates where the one side chain of the molecule is a primary amino-group and allows a tridentate interaction [30]. By increasing ammonium acetate concentration, the elution of the analytes was delayed, leaving them more time to interact with the metals of the LC system; hence, peak tailing of nitrogen-containing bisphosphonates increased.

**Figure 1.** Typical HILIC chromatograms displaying the effect of ammonium acetate concentration on the peak shape and the retention of bisphosphonates, accompanied by diagrams of their ionization state at pH 6.8 as calculated ADME boxes ver. 3.0, Pharma Algorithms software. Chromatographic conditions: ZIC®-pHILIC analytical column, mobile phase: aqueous solution of ammonium acetate pH 6.8/acetonitrile (35:65, *v*/*v*), 0.25 mL min−<sup>1</sup> flow rate and wavelength of detection at 262 nm.

As illustrated in Figure 2A, the retention of both the nitrogen-containing bisphosphonates (risedronate and zoledronate) and the negatively charged tiludronate increased by increasing ammonium acetate concentration due to the reduction of the electrostatic repulsions between the analytes and the stationary phase. From these experiments, we concluded that by using a 10 mM

ammonium acetate concentration all bisphosphonates are adequately retained and well separated fromthesolventfront.

**Figure 2. (A**) Impact of the concentration of ammonium acetate (mM) on the log k. ZIC®-pHILIC column; mobile phase: acetonitrile/ammonium acetate aqueous solution pH 6.6 (65:35, *v*/*v*), (**B**) Impact of the percentage of water, Φwater, on the log k. ZIC®-pHILIC column; mobile phase: acetonitrile/ammonium acetate aqueous solution pH 6.8 containing 3.5 mM ammonium acetate in whole monile phase.

In HILIC a minimum percentage of water, Φwater, at 2% to 3% in the mobile phase is crucial for the creation of the water layer around the stationary phase. In mobile phases with high percentages of acetonitrile, the elution of polar compounds was increased, since the water interacts strongly with the polar stationary phase. To study the effect of Φwater on the retention of bisphosphonates, the concentration of ammonium acetate in whole mobile phase stayed constant at 3.5 mM, while Φwater varied from 30% to 40 %. As shown in Figure 2B the retention of all analytes decreases linearly with increasing Φwater, implying partition as the dominant retention mechanism for bisphosphonates in HILIC.

The studies presented above indicate that both hydrophilic partition and secondary electrostatic interactions contribute to the retention of bisphosphonates on the ZIC®-pHILIC analytical column. Bisphosphonates are strong chelators and their interaction with the metals of the LC system causes serious peak tailing [29]. This binding affinity of bisphosphonates is greater in nitrogen-containing bisphosphonates, where the one side chain of the molecule is a primary amino-group and allows a tridentate interaction [30]. The presence of phosphate groups in the mobile phase can be critical for the analysis of bisphosphonates on a standard stainless steel LC system [31]. To overcome peak tailing for nitrogen-containing bisphosphonates, sodium pyrophosphate was added to the aqueous content of the mobile phase. As can be seen in Figure 3, risedronate and zoledronate peak tailing is seriously reduced in the presence of sodium pyrophosphate, since pyrophosphate anions interact selectively with the metals of the LC system [10]. Tiludronate retention is not seriously affected by the presence of sodium pyrophosphate in the mobile phase.

**Figure 3.** Typical HILIC chromatograms showing the effect of sodium pyrophosphate concentration (mM) on the on the retention time and the peak shape of bisphosphonates. Chromatographic conditions: ZIC®-pHILIC analytical column, mobile phase: acetonitrile–10 mM ammonium acetate aqueous solution pH 6.8 (65:35, *v*/*v*), flow rate of 0.25 mL min−<sup>1</sup> and UV detection at 262 nm.

#### 3.1.2. Optimization of the Chromatographic Parameters for the Quantitation of Risedronate

It was observed that the back pressure of the chromatographic system was increased by increasing sodium pyrophosphate concentration; thus, it was decided to reduce its concentration to 1 mM and to decrease the flow rate to 0.15 mL min−1. Moreover, the addition of sodium pyrophosphate salt in the aqueous content of the mobile phase resulted in alkaline pH that was adjusted to 8.8 using acetic acid. By keeping sodium pyrophosphate concentration constant at 1 mM, a one-variable-at-a-time approach was used to identify the optimal mobile phase composition for the quantitation of risedronate in tablets. The parameters selected to study were the percentage of water, Φwater and the concentration of ammonium acetate (mM). It was found that an increase in the percentage of water from 35% to 39% reduced the retention factor of risedronate. Moreover, it was observed that an increase in the concentration of ammonium acetate from 6 to 10 mM increased the retention of the negatively charged risedronate due to the disruption of the electrostatic repulsions between this and the negatively charged sulfonic acid groups of the stationary phase. Thus, a mobile phase consisting of 38% 9 mM ammonium acetate and 1 mM sodium pyrophosphate aqueous solution pH 8.8 in acetonitrile was finally used. At the beginning of each experiment, the column was equilibrated for 1.5 h and column temperature was set at 40 ◦C. Due to the isocratic separation, there was no need for time-consuming re-equilibration of the analytical column.

## *3.2. Method Validation*
