Analysis of homoisoflavonoids in Caesalpinia digyna by HPLC-ESI-MS, HPLC and method validation

The roots of Caesalpinia digyna have been reported to contain gallic acid derivatives and minor homoisoflavonoids, but HPLC-ESI-MS and HPLC analyses of the homoisoflavonoids were challenging due to their low concentration in the roots. Separation and identification was accomplished by HPLC-ESI-MS and further elaborated for quantification using a C18 column with detection at 330 nm. A gradient mobile phase consisting of methanol and water (0.1% acetic acid) was used. The developed HPLC method showed good linearity (r2 > or = 0.998), high precision (RSD < 5%) and a good recovery (99.3-104.5%) of the compounds. The lowest detection limit was 0.75 ng and the method was found to be robust. All the validation parameters were found to be within the permissible limits and, therefore, the developed method is accurate and reliable for the quality control of C digyna and other Caesalpinia species. This is the first report of sample preparation on Diaion HP-20 resin and characterization of homoisoflavonoids by HPLC-ESI-MS, extended by extensive quantitative HPLC analysis of homoisoflavonoids in C. digyna roots and method validation.