Anthocyanin fingerprinting and dynamics in differentially pigmented exotic soybean genotypes using modified HPLC–DAD method

Abstract

More than as a phenotypic marker for breeding, seed coat colour of soybean is gaining momentum as a nutraceutical marker owing to the multitude of medicinal effects provided by anthocyanins. The acute obstacle for large scale phenotyping is a rapid, reliable and accurate quantification which simultaneously determines various anthocyanins and hence, in this study, the modified method efficiently separated cyanidin-3-glucoside (C3G), delphinidin-3-glucoside (D3G) and petunidin-3-glucoside (Pt3G) forms by eluting through a RP-C18 column with an optimized isocratic mobile phase containing 18% solvent B (0.4% trifluoro acetic acid in acetonitrile) in solvent A (0.4% trifluoro acetic acid in water). The elution profile of anthocyanins were C3G > D3G > Pt3G, with C3G as the predominant (~ 85%) form. The modified method was validated in terms of linearity (R2 = 0.998), low limit of detection (LOD = 5.8 μg ml−1), limit of quantification (LOQ = 22.25 μg ml−1), precision, repeatability, stability and recovery. C3G dynamics was found increased in a linear trend from 30DAF to later developing stages until maturity. The investigation on characterization of exotic soybean genotypes revealed that, maximum C3G content of 4.9 mg g−1 was in UPSL496 and the least in EC471921 (3.56 mg g−1). There was a positive correlation observed among all the variables, like monomeric anthocyanin content (MAC), C3G, D3G and Pt3G. Clustering and heat map analysis information on this efficient method can be used for future research for germ plasm evaluation and for developing nutritionally C3G enriched high yielding varieties.