Micellar electrokinetic capillary chromatography—Synchronous monitoring of substrate and products in the myrosinase catalysed hydrolysis of glucosinolates

Abstract

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.

Ingestion of Broccoli Sprouts Does Not Improve Endothelial Function in Humans with Hypertension

“…Immediately after harvesting they were refrigerated and subsequently dried at 40°C.The dried sprouts were afterwards packaged in airtight plastic bags each containing 10 grams of dried sprouts, equivalent to 100 g fresh sprouts. The dried sprouts had the glucosinolate content measured using previously described methods[16], [17]. In accordance with the study by Wu et al [8] it was attempted to construct a placebo for the control group by freezing/thawing sprouts before drying.…”

Myrosinase Compatible Simultaneous Determination of Glucosinolates and Allyl Isothiocyanate by Capillary Electrophoresis Micellar Electrokinetic Chromatography (CE‐MEKC)

“…The presented method is similar in speed to the fastest available CE methods for glucosinolates (Karcher and El Rassi, ; Bellostas et al ., ), an analysis time of 15 to 25 min is typical. However, the separation of ITC adducts usually requires more time (Bjergegaard et al ., ).…”

“…Higher specificity can only be achieved by subjecting the reaction mixture to chromatographic separation. For this purpose, the CE methods of both (Bellostas et al ., ; Nehmé et al ., ) can be used. They operate with a minimal amount of sample and are able to study decomposition of different glucosinolates, but neither can give information on the ITC release rate that is of primal biological significance.…”

“…The derivatisation reaction was not affected significantly by ascorbic acid concentration in the range 0-10 mM tested (p > 0.05). Thus, the ascorbic acid concentration range usually used in assays of myrosinase (Kleinwachter and Selmar, 2004;Bellostas et al, 2006;Nehmé et al, 2014) worked without any experienced problems. Inhibiton by higher concentrations (Travers-Martin et al, 2008) could also be tested without major interferences.…”

“…However, these approaches are not compatible with myrosinase measurement. A study (Bellostas et al ., ) successfully utilised CE to simultaneously quantify glucosinolates and decomposition products, but used glucosinolates that do not form ITCs at all. The method of (Nehmé et al ., ) used CE to measure myrosinase activity, but as the released sulphate is quantified, no information on the decomposition products could be obtained.…”