Quantification of free and total salicylic acid in plants by solid-phase extraction and isocratic high-performance anion-exchange chromatography.
Salicylic acid (SA) is an important signaling compound in plants and is involved in various defense responses. Here we report a new method for quantification of free and total soluble SA in Arabidopsis thaliana with 5-fluorosalicylic acid (5-FSA) as internal standard. The SA was isolated from leaf extracts by solid-phase extraction with phenyl-phase cartridges and selectively eluted as the cationic iron(III)-complex. Recoveries of SA and 5-FSA were equal and exceeded 90%. Free SA was subsequently released from the iron(III)-complex by addition of 2,2'-bipyridyl and high-performance anion-exchange chromatography was performed on an NH2 column. The SA appeared as last peak with a retention time of 15 min, baseline-separated from other substances. On-line detection was performed fluorimetrically for both SA and 5-FSA at an excitation wavelength of 300 nm and an emission wavelength of 410 nm, because both substances give similar fluorescence spectra. The detection limit for SA was 5 ng g(-1) FW for a sample size of 100 mg. Thus the main advantages of the method are highly selective sample preparation, increased sensitivity, reduced analysis time compared with reversed-phase HPLC, and use of a novel internal standard detectable under the same conditions as SA. The techniques described are applicable to other plant materials.
Rozhon
Wilfried
W
Max F. Perutz Laboratories, University of Vienna, Vienna Biocenter, Dr. Bohrgasse 9, 1030, Vienna, Austria. wilfried.rozhon@univie.ac.at
Petutschnig
Elena
E
Wrzaczek
Michael
M
Jonak
Claudia
C
D017360:Arabidopsis / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D012015:Reference Standards; D012459:Salicylates / Q000737:chemistry; D020156:Salicylic Acid / Q000032:analysis*; D012680:Sensitivity and Specificity; D052616:Solid Phase Extraction; D013050:Spectrometry, Fluorescence
D012459:Salicylates; C075234:5-fluorosalicylic acid; D020156:Salicylic Acid