title: Quantification of camalexin, a phytoalexin from Arabidopsis thaliana: a comparison of five analytical methods.
abstract: 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) C(18) 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 R(2) 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 μgg(-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.
authors.affiliation: Department of Biochemistry, University of Johannesburg, Auckland Park, Johannesburg, South Africa.
authors.forename: Caryn
authors.initials: C
authors.lastname: Beets
authors.forename: Ian
authors.initials: I
authors.lastname: Dubery
mesh_terms: D017360:Arabidopsis / Q000737:chemistry*; D001671:Biochemistry / Q000379:methods*; D002849:Chromatography, Gas; D002851:Chromatography, High Pressure Liquid; D002855:Chromatography, Thin Layer; D005410:Flame Ionization; D007211:Indoles / Q000032:analysis* / Q000737:chemistry; D013058:Mass Spectrometry; D012015:Reference Standards; D012717:Sesquiterpenes / Q000032:analysis* / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D013844:Thiazoles / Q000032:analysis* / Q000737:chemistry; D000094162:Phytoalexins
chemical_list: D007211:Indoles; D012717:Sesquiterpenes; D013844:Thiazoles; C102405:camalexin; D000094162:Phytoalexins