Micellar electrokinetic capillary chromatography--synchronous monitoring of substrate and products in the myrosinase catalysed hydrolysis of glucosinolates.
A micellar electrokinetic capillary chromatography (MECC) method has been developed for monitoring the myrosinase catalysed hydrolysis of 2-hydroxy substituted glucosinolates and the simultaneous formation of the corresponding degradation products (oxazolidine-2-thiones (OZTs) and nitriles). Glucosibarin ((2R)-2-hydroxy-2-phenylethylglucosinolate) was chosen as the model glucosinolate owing to the difficulties in determining hydrolysis rates of this type of substrates in traditional UV-assays. The method was afterwards validated with glucobarbarin ((2S)-2-hydroxy-2-phenylethylglucosinolate) and progoitrin ((2R)-2-hydroxybut-3-enylglucosinolate). Aromatic glucosinolates without a 2-hydroxy group in their side chains, such as glucotropaeolin (benzylglucosinolate) and gluconasturtiin (phenethylglucosinolate) were also tested. Formation of the glucosinolate hydrolysis products was monitored simultaneously at 206 nm and 230 nm. This allowed estimation of the extinction coefficient of the OZT derived from glucosibarin, which was found to be 18,000 M(-1) cm(-1) and 12,000 M(-1) cm(-1) at 206 nm and 230 nm, respectively. The developed method has limit of detection of 0.04 mM and 0.06 mM and limit of quantification of 0.2 mM and 0.3 mM for the glucosibarin derived OZT and nitrile, respectively. Linearity of the glucosinolate concentration was examined at six concentration levels from 2.5 mM to 100 mM and at 206 nm a straight line (R(2)=0.9996) was obtained. The number of theoretical plates (N) at the optimal system conditions was 245,000 for the intact glucosibarin, 264,000 for the OZT and 252,000 for the nitrile.
Bellostas
Natalia
N
Department of Natural Sciences, Biochemistry and Natural Product Chemistry, The Royal Veterinary and Agricultural University, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark.
Sørensen
Jens C
JC
Sørensen
Hilmer
H
D002384:Catalysis; D020374:Chromatography, Micellar Electrokinetic Capillary; D005961:Glucosinolates / Q000378:metabolism*; D006026:Glycoside Hydrolases / Q000378:metabolism*; D006868:Hydrolysis; D008956:Models, Chemical; D015394:Molecular Structure; D009570:Nitriles / Q000378:metabolism; D015203:Reproducibility of Results
D005961:Glucosinolates; D009570:Nitriles; D006026:Glycoside Hydrolases; C022739:thioglucosidase