. 2004 Aug 7;812(1):135–148. doi: 10.1016/j.jchromb.2004.06.051

Table 6.

HPLC method for isolation and analysis of compounds in 50M and three fractions (M-flavonoids, M-saponins and MS-pa) that had been separated from M fraction

(1)	Each fraction was extracted with methanol under ultrasonication for 30 min, followed by centrifugation
(2)	Analysis of the supernatant solution was performed with an HPLC apparatus (Shimadzu, Kyoto, Japan) equipped with two pumps, a photodiode-array (PDA) detector and an ODS column placed in a column oven
(3)	The mobile phase was a mixture of (A) 20 mM H₃PO₄ aq. and (B) 100% CH₃CN with a linear gradient rate of 90% A and 10% B changing over 60 min to 100% B. Then 100% B was continued for 20 min
(4)	The flow rate of the mobile phase and the column temperature were 1.0 ml/min or 40 °C, respectively
(5)	The UV data of the effluent from the column ranging from 200 to 400 nm were collected, and the peak analysis and assignment were performed with the system analysis software (CLASS-LC10, Shimadzu, Kyoto, Japan)

This is a partial modification of the method reported by Nakai et al. [136].