Development of RP-HPLC method for the estimation of β-amyrin and syringic acid from Marigold flower

Marigold (Tagetes erecta L.; Family: Asteraceae) has been reported for several pharmacological activities. This experiment was designed to develop a reverse phase HPLC method according to ICH and FDA guidelines to assure the quality standard of marigold flower for medicinal uses and to find out the content of its biomarkers. β-amyrin (yield 0.3% w/w) and syringic acid (yield 4% w/w) were isolated from methanol extract of flower; dissolved in HPLC mobile phase, subsequently diluted to 200–1000µg/mL and used as external standards. Methanol and water acidified with 1% acetic acid (20:80 v/v) was optimized as mobile phase. The Waters Spherisorb 5µm ODS2, 250×4.6mm column was used along with isocratic elution for optimum separation and detection at 280nm. Retention time of β-amyrin and syringic acid were found to be 12.95 and 23.01min. The response was linear with good correlation between concentration and mean peak area with the coefficient of determinants (r² ) >0.99. β-amyrin and syringic acid contents in methanol extract were found to be 0.06 and 2.30% w/w and mean % recoveries were calculated to be 99.88±0.07 and 99.60±0.20 respectively. The % RSD of intra and inter-day precision were <2.0%. The LOD & LOQ were 30.23µg/mL and 94.75µg/mL for β-amyrin and 44.13µg/mL and 176.59µg/mL for syringic acid. However, it can be concluded that HPLC standardization of marigold with respect to β-amyrin and syringic acid is properly validated as simple, accurate, specific, precise and reproducible method.