Qualitative analysis of catechins from green tea GMB-4 clone using HPLC and LC-MS/MS

ABSTRACT

Objective: To identify the bioactive compounds in catechins isolation and its compo-nents from green tea GMB-4 clone. Methods: Green tea GMB-4 clones were extracted with distilled water at 90 ? C. Samples were eluted into the column with 10%ethanol. Subsequently, the column was eluted with 95% ethanol and evaporated separately. Green tea extract was identified by thin layer chromatography. Catechins were separated by the stationary phase in column chroma-tography using polyamide with 10% ethanol eluent and 95% ethanol. The results of isolations were analyzed by high performance liquid chromatographic (HPLC) and LC-MS/MS. Analysis of catechins by HPLC was done by external standard. Results: Fraction from 10% ethanol showed that four major peaks at retention time of 1.663, 2.367, 2.950 and 4.890, indicated the presence of four catechins components including catechin, epicatechins, gallocatechin and epigallocatechin. Whereas, fraction from 95% ethanol showed two main peaks at retention time of 5.167 and 9.82, which indicated the presence of epigallocatechin gallate (EGCG) and epicatechin gallate (ECG). EGCG (m/z 459), epigallocatechin (m/z 307), ECG (m/z 443), and epicatechin (m/z 291) were isolated and separated successfully using HPLC and LC-MS/MS. Conclusions: The HPLC and LC-MS/MS methods were successfully tuned for the qualitative analysis of green tea extract with EGCG and ECG. Four major catechins were separated and identified by LC-MS/MS, such as EGCG, epigallocatechin, ECG and epicatechin. The result of HPLC analysis showed that EGCG and ECG were main components from catechins isolation of green tea GMB-4 clone.