Reverse phase-HPLC method for determination of marker compounds in NP-1, an anti-osteoporotic plant product from Butea monosperma.

An isocratic, reversed phase, high-performance liquid chromatographic (HPLC) method was developed for the determination of the marker compounds K052 (iso-formononetin), K054 (methoxy derivative) and K080 (formononetin) in NP-1, an anti-osteoporotic plant product from Butea monosperma. The separation was achieved on a C18 column with a mobile phase consisted of a mixture of 0.05M potassium dihydrogen phosphate containing 0.1% v/v triethyl amine (pH adjusted to 2.5 with phosphoric acid) buffer and acetonitrile (70:30 v/v), at a flow rate of 1.5 mL/min. The retention times of K054, K080 and K052 were about 13, 21 and 23 mins, respectively. The effluents were monitored at 254 nm. The calibration curves were linear over the concentration ranges of 2.7-21.3, 2.1-33.6 and 2.8-22.4 microg/mL for K054, K052 and K080, respectively. The limits of detection were 0.42, 0.53 and 0.56 microg/mL, respectively. The accuracies and precisions in all cases were less than 5% in the calibration range.