Validation of the Stability-Indicating HPLC Method for the Major Flavonoids in Spray- Dryed Leaf Extract of Aleurites moluccana L. Willd

Talita      Gesser Cesca; Luciana      Catia Block; Marina      Silva Machado; Carolina      Wittkowski; Christiane      Meyre-Silva; Marcia      Maria de Souza; Nara      Lins Meira Quintao; Ruth      Meri Lucinda Silva; Denise      Brentan da Silva; Eduardo      Fernandes; Leandro      de Santis Ferreira; Norberto      Peporine Lopes; Valdir      Cechinel Filho; Tania      Mari Belle Bresolin

Abstract

The HPLC-UV method previously developed for the analysis of the flavonoid 2”-O-rhamnosylswertisin (I), an active compound of dried leaf extracts of Aleurites moluccana L. Willd, was fully validated with the inclusion of another flavonoid, swertisin (II). The stability-indicating capability of the method was established by analyzing forced degradation extract samples (acid, alkali, neutral, oxidative and photolytic condition) in which the spectral purity of the markers was ascertained together with the separation of degradation products from the markers. The method proved be linear over a range of 5.89-117.8 and 1.38-27.68 μg.mL-1 for I and II, respectively. The recovery for I and II was 100.3 and 102.8%, respectively, at a level of 100%. A relative standard deviation (RSD%) < 1.0% (intra-day) and < 3.5% (inter-days) for area, and less than 0.5% for retention time indicated that the precision of the method is acceptable. Validation parameters such as selectivity and robustness were also determined. In addition, during the course of LC-MSn analysis three other flavonoids were discovered in the A. moluccana extract and their tentative structures identified.

Keywords: Aleurites moluccana, Analytical Validation, Flavonoids, Leaf Standardized Extract, HPLC-UV, LC-MS, Stability- Indicating Method, Spray Drying