Abstract
Plant secondary metabolites regulate plant growth and serve as valuable pharmaceutical resources. Napier grass (Pennisetum purpureum Schumach.), a Poaceae species, shows potential as a functional food. In this study, we employed high-resolution mass spectrometry combined with a data-independent acquisition (DIA) strategy for the untargeted detection of anthocyanins, a group of secondary metabolites, in napier grass. Clear MS(2) fragmentation patterns were observed for anthocyanins, characterized by diagnostic aglycone signals and sequential losses of hexosyl (C(6)H(10)O(5)), deoxyhexosyl (C(6)H(10)O(4)), pentosyl (C(5)H(8)O(4)), and p-coumaroyl groups (C(9)H(8)O(3)). Based on matching with authentic standards and an in-house database, ten anthocyanins were identified, seven of which were newly reported in napier grass. In a single-laboratory validation analysis, both absolute and semi-quantitative results reliably reflected the specific distribution of metabolites across different cultivars and plant organs. The purple cultivar (TS5) exhibited the highest anthocyanin content, with the cyanidin 3-O-glucoside content reaching 5.0 ± 0.5 mg/g, whereas the green cultivar (TS2), despite its less pigmented appearance, contained substantial amounts of malvidin 3-O-arabinoside (0.7 ± <0.1 mg/g). Flavonoid profiling revealed that monoglycosylated anthocyanins were the dominant forms in floral tissues. These findings shed light on napier grass metabolism and support future Poaceae breeding and functional food development.