RP-HPLC method with indirect UV detection for determination of sodium ibandronate in pharmaceuticals

Ibandronate sodium (IBN) [(1-hydroxy-3- (methyl pentyl amino) propylidene bisphosphonic acid monosodium monohydrate)] is the sodium salt of ibandronic acid, a synthetic nitrogen-containing bisphosphonate drug. The aim of this study was to develop a sensitive and accurate RP-HPLC method with indirect UV detection for determination of IBN in pharmaceutical formulations. Chromatographic separation was performed on a Waters Bridge C18 reversed-phase column (250 x 4.6 mm I.D.; particle size 5 µm), in an isocratic mode with a mobile phase constituted of 90% buffer: 10% acetonitrile (V/V). The buffer was made using 1.5 mL ortho-phosphoric acid, 990 mg 1-Hexanesulfonic acid sodium salt 98%, 140 mg EDTA in 1000 mL flask diluted with HPLC grade water. The elution was carried out at a flow rate of 1.0 mL minˉ1. A diode array detector measured the UV absorbance at 198 nm, in inverse mode. The method was validated for specificity/selectivity, linearity, LOD, LOQ, accuracy, precision and robustness according to ICH validation guidelines. The limits of detection and quantification were calculated at 0.0163 µg/mL and 0.0495 µg/mL, respectively. The method was effectively used for determination of IBN from commercial tablets and provided good results without any interference from commonly used excipients.

Keywords: RP-HPLC with indirect UV detection, Ibandronate sodium, validation, pharmaceuticals


Introduction
Ibandronate sodium (IBN) is the sodium salt form of ibandronic acid, a synthetic nitrogen-containing bisphosphonate (Fig. 1). Ibandronic acid inhibits farnesyl pyrophosphate synthase, resulting in a reduction in geranylgeranyl GTPase signaling proteins and apoptosis of osteoclasts. This agent increases bone mineral density, decreases bone remodeling, inhibits osteoclast-mediated bone resorption, and reduces metastases-related and corticosteroidrelated bone pain (Bausset al., 2002;Black et al., 2007).
For quantification of IBN in pharmaceutical formulations, few analytical methods have been reported. Determination of bisphosphonates by high-performance liquid chromatography (HPLC) with UV or fluorescence detection is hindered because of the lack of chromophores groups in their structures. Several analytical methods using refractive index or electrochemical detection were described in the literature (Brooks et al., 1992;Denhartigh et al., 1993;Han et al., 1996;Higuchiet al., 1992). However, these detection systems are not common in most of the routine analytical laboratories. Analytical methods for quantification of bisphosphonates using UV/VIS or fluorescence detectors in the indirect mode, or after pre-or post-column derivatization, have also been described (Brezovska et al., 2010;Biffar et al., 1989;Dansereau et al., 2001;Hartigh et al., 1997;Lovdahl et al., 1999;Kosonen, 1992). However, the derivatization step is time and solvent consuming, making this process inappropriate for routine analysis. Professional paper *Corresponding author: zharko.tanturovski@gmail.com 4 Therefore, the aim of this study was to develop a RP-HPLC method with indirect UV detection which would replace the existing commercial methods with RI detector.

Instruments and reagents
HPLC analyses were performed using a Schimadzu LC-2010 chromatographic system (Schimadzu, Kyoto, Japan) consisting of a LC-20AT Prominence liquid chromatograph pump with DGU-20A5 Prominence degasser, a SPD-M20A Prominence Diode Array Detector, RF 10AXI fluorescence detector and a SIL-20 AC Prominence auto sampler. Data analyses were done using Class VP 7.3 Software. The UV spectra of IBN dissolved in mobile phase were recorded on the Shimadzu UV-Visible spectrophotometer (UV-1800).
IBN working standard was obtained from Maprimed SA. HPLC-grade acetonitrile was from Merck (Darmstadt, Germany). Double-distilled water was used to prepare mobile phase solutions. All solvents and solutions for HPLC analysis were filtered through a membrane filter (0.45 µm pore size) and vacuum degassed before use.

Chromatographic conditions
Chromatographic separation was performed on a Waters Bridge C18 reversed-phase column (250 X 4.6 mm I.D.; particle size 5 µm), in an isocratic mode with a mobile phase constituted of 90% buffer: 53% acetonitrile (V/V). The buffer was made using 1.5 mL ortho-phosphoric acid, 990 mg 1-Hexanesulfonic acid sodium salt 98%, 140 mg EDTA in 1000-mL flask diluted with HPLC grade water. The elution was carried out at a flow rate of 1.0 mL minˉ1. All analyses were performed at room temperature (25 °C+/-2 °C). A diode array detector measured the UV absorbance at 198 nm, in inverse mode.

Calibration curve
Stock solution was prepared by dissolving IBN standard substance (10 mg) with mobile phase in 10 mL volumetric flask (c=1 mg/mL) and stored at room temperature (25 °C +/-2 °C) during the study. Standard solutions were prepared by dilution of IBN stock solution with solvent (mobile phase) to obtain final concentrations ranging from 25-200 µg/mL (25, 30, 40, 45, 50, 55, 60, 75 and 200 µg/mL). Mobile phase was used as a blank.

Sample preparation
Twenty tablets containing IBN active substance were weighted, crushed, powdered and grinded finely. A portion of the powder equivalent to 10 mg of IBN was used and diluted with mobile phase to obtain a working concentration of 50 µg/mL. The sample solution was filtered through 0.45 nylon membrane filter before use. The amount of IBN per tablet was calculated using the standard calibration curve.

Results and discussion
Indirect UV detection is a method that is used typically when detecting analytes don't have chromophore present in their structure. Indirect UV detection allows measurement of non-UV-absorbing anions without derivatization. An ionic chromophore is placed in the mobile phase, and the decrease in absorbance observed when an analyte ion displacing the chromophore ion in the mobile phase is monitored (Brezovska et al., 2010). As there is no chromophore present in IBN, the detection of a UV transparent analyte is accomplished by adding light ionic (ion-pairing) species into the mobile phase. Ibandronate poses analytical challenges for reversed-phase HPLC due to the presence of two polar phosphonate groups.
This makes retention on commonly used RP columns difficult (Gawad et al., 2013). In addition, the metal chelation property of ibandronate can cause poor peak shape and analyte doi: Professional paper *Corresponding author: zharko.tanturovski@gmail.com 6 recovery in systems that are not metal-free. Chelating agents, such as ethylenediaminetetraacetic acid (EDTA), are added to the mobile phase to prevent metal contaminants from chelating with ibandronate (Furuta et al., 1993). EDTA was used as a complexing agent in combination with ion-pairing agent (1-hexanesulfonic acid sodium salt) that will separate the analyte and give good absorption, to enable retention and separation on a RP column.
In the preliminary research, the absorption spectra of IBN were studied using Shimadzu UV-Visible spectrophotometer. Solution of IBN substance was dissolved in mobile phase and was scanned within the wavelength region from 300-190 nm against the mobile phase as a blank. The UV spectrum of the IBN dissolved in mobile phase shows an absorption maximum at 198 nm.

Method validation
One of the most important steps in analytical determination is validation of the method for quantitative analysis. The method was validated for specificity/selectivity, linearity, LOD, LOQ, accuracy, precision and robustness. The ICH guideline was used to validate the proposed method (ICH, 2005).

Specificity and selectivity
Specificity is the ability to asses unequivocally the analyte in the presence of components which might be expected during the analysis. The analytes should have no interference from other components such as impurities and degradation products, and they should be remarkably separated from IBN (ICH, 2005;McMaster, 2007). This method showed that it is quite selective and showed great resolution between IBN and the given doi: Professional paper *Corresponding author: zharko.tanturovski@gmail.com impurities (the sample solution was spiked with ibanic acid). There was no other interfering peak around the retention time of IBN. Also, the baseline did not show any significant noise.

Limit of detection & quantitation limit
The detection limit in a single analytical procedure is the amount of analyte in a sample that can be detected, but not necessarily quantified as an accurate value. The quantitation limit of an individual analytical procedure is the amount of analyte in a sample that can be quantitatively determined by proper precision and accuracy. The LOD and LOQ were found using the ratio 3.3 σ/S and 10 σ/S respectively, where σ is the standard error of doi: Professional paper *Corresponding author: zharko.tanturovski@gmail.com 8 estimation and S is slope (ICH, 2005). The established LOD and LOQ for ibandronic acid sodium were 0.0163 µg/mL and 0.0495 µg/mL respectively.