A Five-Minute UHPLC–MS/MS Method Facilitates Anthocyanin Biofortification in Maize (Zea mays L.)

Anthocyanin-rich maize offers opportunities for nutritional biofortification and natural food colorants, but conventional chromatographic methods limit the throughput needed for large-scale breeding programs. We developed a 5-minute ultra-high-performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS) method for quantifying maize anthocyanins and compared its performance with a 25-minute method. Anthocyanins from five commercial products and one breeding line were analyzed using optimized chromatographic gradients and multiple-reaction monitoring. Anthocyanin concentration and composition varied substantially among varieties, with differing contributions from cyanidin-, peonidin-, and pelargonidin-derived compounds. The 5-minute method reduced analytical runtime fivefold while preserving the relative ranking of varieties by total anthocyanin concentration, although absolute concentrations differed slightly between methods for most varieties. Random-effects modeling indicated that the methodology used to phenotype maize represented less than 1% of the total variation, further reinforcing that our rapid method is suitable for profiling breeding populations. This rapid UHPLC–MS/MS method provides a high-throughput analytical platform for quantifying predominant maize anthocyanins, facilitating phenotyping and selection for anthocyanin biofortification in maize breeding programs.