Development of a Forced Degradation Profile of Alosetron by Single Mode Reversed-Phase HPLC, LC-MS, and its Validation

Yamjala Karthik

doi:10.3797/scipharm.1411-07

Abstract

Determination of alosetron in the presence of its degradation products was studied and validated by a novel HPLC method. The separation of the drug and its degradation products was achieved with the Jones Chromatography C18 analytical column (150 mm x 4.6 mm; 3 µm) with a stationary phase in isocratic elution mode. The mobile phase used was 0.01 M ammonium acetate, pH-adjusted to 3.5 with glacial acetic acid and acetonitrile in the ratio of 75:25 (V/V) at a flow rate of 1 ml/min and UV detection was carried out at 217 nm. Further, the drug was subjected to stress studies for acidic, basic, neutral, oxidative, and thermal degradations as per ICH guidelines and the drug was found to be labile in base hydrolysis and oxidation, while stable in acid, neutral, thermal, and photolytic degradation conditions. An MS study has been performed on the major degradation products to predict the degradation pathway of alosetron. The method provided linear responses over the concentration range of 100–1500 ng/ml and regression analysis showed a correlation coefficient value (r2) of 0.994. The LOD and LOQ were found to be 1 ng/ml and 3 ng/ml, respectively. The developed LC method was validated as per ICH guidelines with respect to accuracy, selectivity, precision, linearity, and robustness.

Highlights

Alosetron (Figure 1) is a 5-HT3 antagonist used in the prophylaxis treatment for women with predominant irritable bowel syndrome (IBS) [1, 2]
For each forced degradation condition, the sample was dissolved in 25 ml of water and degradation samples were prepared with acid, alkali, hydrogen peroxide, and water to get a concentration of 1000 μg/ml and it was subjected to different stress conditions by refluxing for hr with each ml of 0.1 N HCl, 0.1 N NaOH, 3% H2O2, and water at 60oC
The mobile phase solvents were filtered through a 0.22 μm polytetrafluoroethylene membrane filter before injecting into the HPLC system and the chromatograms were recorded using Class VP software

Summary

Introduction

Alosetron (Figure 1) is a 5-HT3 antagonist used in the prophylaxis treatment for women with predominant irritable bowel syndrome (IBS) [1, 2]. Review of the literature revealed that only a few studies have been performed for the estimation of alosetron. Lloyd et al have reported the estimation of alosetron in human plasma or serum by high-performance liquid chromatography employing robotic sample preparation, cyanopropyl stationary phase, and fluorescence detection [5]. Karthik et al have proposed an HPLC method for the estimation of alosetron in bulk drug employing a UV detector for the first time [7]. There is no reported stability-indicating assay method for the estimation of alosetron by reversedphase high-performance liquid chromatography, we determined to develop a validated stability-indicating analytical assay method. LC–MS studies were performed by the Shimadzu LC-2020 quadrapole mass spectrometer with an ESI source in positive mode equipped with LC-10AD gradient pumps, a DGU-14AM degasser, SCL-10A system controller, CTO10A column oven, diode array detector (SPD-M10A), and an autoinjector (SIL-10AD) (all from Shimadzu, Kyoto, Japan). The samples were withdrawn and stored at 4 ̊C

Results

Discussion

Conclusion