Beyond the wall: High-throughput quantification of plant soluble and cell-wall bound phenolics by liquid...

Plants accumulate several thousand of phenolic compounds, including lignins and flavonoids, which are mainly synthesized through the phenylpropanoid pathway, and play important roles in plant growth and adaptation. A novel high-throughput ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method was established to quantify the levels of 19 flavonoids and 15 other phenolic compounds, including acids, aldehydes, and alcohols. The chromatographic separation was performed in 10 min, allowing for the resolution of isomers such as 3-, 4-, and 5-chlorogenic acids, 4-hydroxybenzoic and salicylic acids, isoorientin and orientin, and luteolin and kaempferol. The linearity range for each compound was found to be in the low fmol to the high pmol. Furthermore, this UHPLC-MS/MS approach was shown to be very sensitive with limits of detection between 1.5 amol to 300 fmol, and limits of quantification between 5 amol to 1000 fmol. Extracts from maize seedlings were used to assess the robustness of the method in terms of recovery efficiency, matrix effect, and accuracy. The biological matrix did not suppress the signal for 32 out of the 34 metabolites under investigation. Additionally, the majority of the analytes were recovered from the biological samples with an efficiency above 75%. All flavonoids and other phenolic compounds had an intra- and inter-day accuracy within a ±20% range, except for coniferyl alcohol and vanillic acid. Finally, the quantification of flavonoids, free and cell wall-bound phenolics in seedlings from two maize lines with contrasting phenolic content was successfully achieved using this methodology.