Troxerutin, a mixture of O-hydroxyethyl derivatives of the natural flavonoid rutin: Chemical stability and analytical aspects.

Troxerutin (TRX) is a mixture of semisynthetic hydroxyethylrutosides (Hers) arising from hydroxyethylation of rutin, a natural occurring flavonoid. TRX is commonly used for its anti-oxidant and anti-inflammatory properties in chronic venous insufficiency and other vascular disorders. In recent studies, the protective effects of TRX in Alzheimer's disease, colon carcinogenesis and hepatocellular carcinoma are emerged. However, the chemical stability of TRX has never been studied. Hence, the aims of the work were to study the TRX chemical stability through a forced degradation study and to develop and validate a new stability indicating LC-UV method for determination of TRX. In order to perform the study, TRX stability was tested in various stress conditions analysing the degradation samples by LC-MS. Three degradation products (DPs; D1, D2 and D3, 3',4',7-Tri-O-(β-hydroxyethyl)quercetin, 3',4',5,7-Tetra-O-(β-hydroxyethyl)quercetin and 3',4'-Di-O-(β-hydroxyethyl)quercetin respectively) arising from degradation in acidic conditions were identified and synthesized: among them, D1 resulted the stability indicator for hydrolytic degradation. Furthermore, a stability-indicating LC-UV method for simultaneous determination of triHer (3',4',7-Tri-O-(β-hydroxyethyl)rutin, the principal component of the mixture) and D1 was developed and validated. The LC-UV method consisted in a gradient elution on a Phenomenex Kinetex EVO C18 (150 × 3 mm, 5 μm) with acetonitrile and ammonium bicarbonate buffer (10 mM, pH 9.2). The method was linear for triHer (20-60 μg mL) and D1 (5.1-35 μg mL). The intraday and interday precision were determined and expressed as RSDs: all the values were ≤ 2% for both triHer and D1. The method demonstrated also to be accurate and robust and the average recoveries were 98.8 and 97.9% for triHer and D1, respectively. Moreover, the method resulted selective and specific for all of the components present in the degradation pattern of TRX (diHer (3',4'-Di-O-(β-hydroxyethyl)rutin), triHer, tetraHer (3',4',5,7-Tetra-O-(β-hydroxyethyl)rutin), D3, D1 and D2) and it was successfully applied for the stability studies of both drug substances and drug products.