Development of a solid-phase extraction--spectrofluorimetric method for trace glutathione determination

A simple and efficient solid-phase extraction--spectrofluorimetric method has been developed to determine glutathione (GSH). Fluorescent probe N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3- yl)methyl)iodoacetamide (BODIPY Fl-[C.sub.1]-IA) was used as the derivatization reagent. The procedure was based on a BODIPY Fl-[C.sub.1]-IA selective reaction with GSH to form the highly fluorescent product BODIPY Fl-[C.sub.1]-IA-GSH, using a solid-phase extraction column and spectrofluorimetric determination. The variables affecting analytical performance were studied and optimized. The calibration graph using the preconcentration system for GSH was linear over the range of 1-200 nmol/L with a limit of detection of 0.05 nmol/L (signal-to-noise ratio = 3). The relative standard deviation for six replicate determinations of GSH at the 100 nmol/L concentration level was 3.9%. The method was applied to water samples and average recoveries between 87.5% and 111.5% were obtained for spiked samples. Key words: spectrofluorimetry, glutathione, solid-phase extraction, N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s- indacene-3-yl)methyl)iodoacetami-de. On a developpe une methode simple et efficace d'extractoin spectrofluorimeetrique en phase solide pour determiner le glutathion (GSH). On a utilise la sonde fluorescente N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3- yl)methyl)iodoacetamide (BODIPY F1-[C.sub.1]-1A) comme reactif pour obtenir des deriveis. La methode repose sur le fait que le BODIPY F1-[C.sub.1]-1A donne une reaction selective avec le GSH conduisant a la formation du produit hautement fluorescent BODIPY F1-[C.sub.1]-1A-GSH qui peut etre isole par extraction en phase solide sur colonne et soumis a une determination spectrofluorimetrique. On a etudie et optimise les variables qui affectent la performance analytique. Le graphique de calibration a l'aide du systeme de preconcentration du GSH est lineiaire sur la plage de 1 a 200 nmol/L, avec une limite de detection de 0,05 nmol/L (rapport signal a bruit = 3). La deviation relative standard pour six determinations de GSH en duplicata, a un niveau de concentration de 100 nmol/L, est de 3,9 %. On a applique la methode a des echantillons d'eau et on a obtenu des taux de recuperations moyens variant de 87,5 % a 111,5 % pour les echantillons ensemences. Mots-cles: spectrofluorimeitrie, glutathion, extraction en phase solide, N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a- diaza-s-indacene-3-yl)methyl)iodoacetamide. [Traduit par la Redaction]
Introduction The tripeptide glutathione (L-γ-glutamyl-L-cysteineglycine, GSH) is the most abundant low molecular mass thiol in the cells of many organisms. (1) It is the major compound containing the -SH group [...]