UHPLC-UV Analysis of Morin and Structurally Related Flavonoids with Potential Anticancer Activity

Background: Flavonoids as secondary metabolites of plants fulfill various functions in cell protection. They are of a considerable scientific interest because of their potentially medical use due to their anticancer, chemoprotective, antimicrobial, antiallergic, anti-inflammatory and antiviral activities. Objective: The study aimed to develop a new UHPLC-UV method for morin and 2 other structurally related flavonoids - naringenin and kaempferol as the structural similarity of huge numbers of flavonoids does not limit their various biological functions and activities. Methods: Separation of morin and 2 other structurally related flavonoids - naringenin and kaempferol - was achieved by using BEH C18 (1.7 μm, 2.1 x 50 mm) analytical column (Waters® Acquity UPLC) and a mobile phase composed of 0.05%v/v Formic acid in water and acetonitrile in proportion of 77:23 v/v and pumped at a flow rate of 0.4 ml/min. Column temperature was set at 25 ºC and samples were analyzed (3 μl injection volume) at a wavelength of 340 nm. Waters® Xevo G2-S QToF coupled with Waters® Acquity UPLC system with binary Solvent Manager (I-Class) via electrospray ionization (ESI) interface was used to confirm the identity of the peaks in biological samples. Results: A rapid and simple UHPLC-UV separation of morin, kaempferol and naringenin is documented including methods validation. The developed method was applied to measuring morin, kaempferol and naringenin in human plasma after a solid phase extraction. Additionally, stability of morin in tissue culture medium was verified. The extraction method and UHPLC-UV elution conditions described provide a practical means to analyze morin, kaempferol and naringenin in biological matrices. Conclusion: The developed method is fast and highly sensitive. Moreover, the flavonoids used were stable in human plasma for more than 10 days.