Quantification of camalexin, a phytoalexin from Arabidopsis thaliana: A comparison of five analytical methods

Camalexin is a phytoalexin of Arabidopsis thaliana and an important component of inducible defenses. Accurate quantification of low concentrations suffers from interference by structurally related metabolites. A. thaliana plants were induced with silver nitrate and camalexin was extracted using methanol and identified and quantified by (i) TLC as a blue fluorescent band, (ii) microtiter plate-based fluorescence spectroscopy, (iii) GC on a midpolar column coupled to flame ionization detection, (iv) C18 HPLC coupled to a photodiode detector, and (v) UPLC coupled to a mass spectrometer detector. Standard curves over the range of 0.1–15 μg ml−1 gave R2 values from 0.996 to 0.999. The different methods were compared and evaluated for their ability to detect and quantify increasing concentrations (<0.4–8 μg g−1 FW) of camalexin. Each of the techniques presented advantages and disadvantages with regard to accuracy, precision, interference, analytical sensitivity, and limits of detection. TLC is a good qualitative technique for the identification of camalexin and fluorescence spectroscopy is subject to quenching when performed on crude extracts. Comparable results were obtained with GC–FID, HPLC–PDA, and UPLC–MS, with UPLC–MS having the added advantage of short analysis times and detection based on accurate mass.