A rapid LC-MS method for qualitative and quantitative profiling of plant apocarotenoids

Carotenoid cleavage products (apocarotenoids; APOs) exert important biological functions in light perception and as vitamin A source, signaling molecules, hormone precursors, pigments and volatiles. However, an analytical method that allows simultaneous profiling of these diverse compounds is still missing. We developed an efficient method to analyze APOs present in plant tissues, which is based on ultra-high performance liquid chromatographic separation and high-resolution hybrid quadrupole-Orbitrap (Q-Orbitrap) mass spectrometry (MS). Our approach allowed unambiguous identification and quantification of volatile and non-volatile APOs in a single run. Modified sample preparation and optimized ultra-high performance liquid chromatography (UHPLC)-MS parameters permitted the measurement of APOs in Oryza sativa seedlings and Spinacia oleracea leaves, unraveling 20 endogenous APOs with chain lengths ranging from C10 to C30, confirmed by high-resolution MS, MS/MS data and using synthetic standards. Our experimentation demonstrates that the usage of methanol with 0.1% butylated hydroxytoluene facilitates the extraction of both short-chain and long-chain APOs from plant materials. In addition, our validated analytical method allows the quantitative analysis of APOs with a wide content range from 2.5 pg/mg to 10 ng/mg dried weight. The adoption of the analytical protocol, as described in this study, realizes the measurement of volatile APOs by using a LC-MS method, hence, allowing informative and reliable profiling of APOs, which is important for determining the content of these compounds in food and crucial for understanding their function and metabolism in plants.