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.
Max F. Perutz Laboratories, University of Vienna, Vienna Biocenter, Dr. Bohrgasse 9, 1030, Vienna, Austria. wilfried.rozhon@univie.ac.at
Wilfried
W
Rozhon
Elena
E
Petutschnig
Michael
M
Wrzaczek
Claudia
C
Jonak
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