Development and validation of demethylwedelolactone and wedelolactone by HPLC-PDA
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Abstract
This research aimed to develop and validate the method for the analysis of demethylwedelolactone and wedelolactone in extracted Kameng samples by HPLC – PDA. Various types of Kameng extractions were examined. The optimum condition for separation of demethylwedelolactone and wedelolactone was 99.9% (v/v) methanol and 0.05% (v/v) formic acid at the ratio 95:5 (v/v) as mobile phase. The mode of operation was gradient elution. The results of method validation to analyze demethylwedelolactone and wedelolactone were found as follows: the limit of detection (LOD) 0.41 µg L-1 and 0.004 µg L-1, respectively, and the limit of quantification (LOQ) 1.37 µg L-1 and 0.013 µg L-1 respectively. The linearity of demethylwedelolactone in the working range was 5.0 – 25 µg L-1 (r2=0.998), and wedelolactone in the working range was 1.0 – 25 µg L-1 (r2=0.999). The method validation of precision for intraday precision of demethylwedelolactone and wedelolactone were found as 0.38 – 1.61 and 1.18 – 1.46, respectively. The intraday precision of demethylwedelolactone and wedelolactone were found as 0.34 – 0.47 and 0.59 – 1.20 respectively. The percentage of relative standard deviation (%RSD) and accuracy was performed by the spiked standard method. The recovery of demethylwedelolactone and wedelolactone were found as 89.21 – 97.00% and 100.52 – 100.81% respectively. For standard addition method, the recovery of demethylwedelolactone and wedelolactone were found as 103.01 – 104.93% and 89.69 – 105.94% respectively. The recovery of demethylwedelolactone and wedelolactone from both methods was acceptable. Optimum extraction condition of demethylwedelolactone and wedelolactone from Kameng were investigated using maceration, ultrasound-assisted extraction and decoction. The results show that contents of demethylwedelolactone obtained from the three extraction methods were not significantly different, whereas wedelolactone was found only in maceration and ultrasound-assisted methods.
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