Simultaneous liquid chromatography-fluorescence analysis of type A and type B trichothecenes as fluorescent derivatives via reaction with coumarin-3-carbonyl chloride.
A method for the simultaneous LC-fluorescence detection (FLD) determination of eight trichothecenes A and B by pre-column derivatization with coumarin-3-carbonyl chloride, a highly fluorescent fluorophore, has been developed. The reaction conditions (temperature, reaction time, reactant ratios) were optimized to give a reproducible quantitative conversion. All derivatives were characterized by LC-MS. The chromatographic parameters were optimized (column, eluent) to give a very good separation of three type A (diacetoxyscirpenol, T-2 toxin, HT-2 toxin) and five type B trichothecenes [deoxynivalenol (DON), nivalenol, fusarenon-X, 3-acetyldeoxynivalenol, 15-acetyldeoxynivalenol]. The best conditions were obtained on a narrow-bore C18 column with a water-methanol gradient. The detection limits (S/N = 3:1) in grain samples, with an injected volume of 5 microl, were 0.2-1 ng/g for all trichothecenes. These values are more than one order of magnitude lower than those of other LC-FLD and LC-MS methods and are similar to those obtained by GC-MS. The calibration curves were linear between 100 and 2500 ng/g. The method was successfully applied to the analysis of a certified wheat reference material, after solvent extraction and clean-up on a Mycosep column, obtaining a good recovery (89% for DON) and a high accuracy (z-score value: 0.67).
Dipartimento di Chimica Organica e Industriale, Università degli Studi di Parma, Parco Area delle Scienze 17/A, 1-43100 Parma, Italy.
C
C
Dall'Asta
G
G
Galaverna
A
A
Biancardi
M
M
Gasparini
S
S
Sforza
A
A
Dossena
R
R
Marchelli
D002138:Calibration; D002853:Chromatography, Liquid / Q000379:methods*; D003374:Coumarins / Q000737:chemistry*; D005456:Fluorescent Dyes / Q000737:chemistry*; D005504:Food Analysis; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence / Q000379:methods*; D014255:Trichothecenes / Q000032:analysis*
D003374:Coumarins; D005456:Fluorescent Dyes; D014255:Trichothecenes; C406451:coumarin-3-carbonyl chloride