Objective: To develop a certified reference material (CRM) of pedunculoside according to the Directives for CRM General and Statistical Principle for Certification(GB/T 15000.3-2008) based on the CRM project approved by Standardization Administration of the People's Republic of China (SAC). Method: Pedunculoside samples were obtained by means of macroporous resin enrichment

high speed counter-current chromatography

and recrystalization from the dried bark of Ilex rotunda

and their structures were confirmed by element analysis

UV

IR

HR-MS

NMR

and XRD. In addition

thin layer chromatography was carried out for identification

and high performance liquid chromatography ultraviolet detection and analysis technology was established. The pedunculoside samples divided into 600 bottles with 5 mg per bottle for homogeneity test

stability test

and cooperative certification by 8 laboratories. Result: The homogeneity of pedunculoside samples was excellent in 95% confidence interval

with good stability within 24 months at 0-8℃. The certified purity value of the pedunculoside samples was 99.60% with an expanded uncertainty of 0.10%(k=2.00). They were approved by the National Standardization Management Committee

and consistent with the technical requirements of the national standard samples. Conclusion: National certified reference materials for pedunculoside were successfully studied and developed for the first time

and their purity was higher than that of samples purchased from markets

so this method can be used in the content determination

testing method validation

as well as testing and quality control of pedunculoside products.