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

Abstract

Introduction. The functional food Cruciferous vegetables contain glucosinolates which are decomposed by the myrosinase enzyme upon tissue damage. The isothiocyanates are the most frequent decomposition products. Because of their various bioactivities, these compounds and the myrosinase is of high interest to many scientific fields.Objective. Development of a capillary electrophoresis method capable of myrosinasecompatible, simultaneous quantification of glucosinolates and isothiocyanates.Methods. Capillary electrochromatography parameters were optimized, followed by optimization of a myrosinase-compatible derivatization procedure for isothiocyanates. Vegetable extracts (Brussels sprouts, horseradish, radish and watercress) were tested for myrosinase activity, glucosinolate content and isothiocyanate conversion rate. Allyl isothiocyanate was quantified in some food products.Results. The method allows quantification of sinigrin, gluonasturtiin and allyl isothiocyanate after myrosinase compatible derivatization in-vial by mercaptoacetic acid. The chromatograhpic separation takes 2.5 minutes (short end injection) or 15 minutes (long end injection). For the tested vegetables, measured myrosinase activity was between 0.960 -27.694 and 0.461 -26.322 µmol min -1 mg -1 protein, glucosinolate content was between 0 -2291.8 and 0 -248.5 µg g -1 fresh weight for sinigrin and gluconastrutiin, respectively. The possible specificity of plants to different glucosinolates was also shown. Allyl isothiocyanate release rate was different in different vegetables (73.13 to 102.13 %). The method could also be used for quantification of allyl isothiocyanate from food products.Conclusions. The presented capillary electrophoresis method requires a minimal amount of sample and contains only a few sample preparation steps, and can be used in several applications (glucosinolate determination, myrosinase activity measurement, isothiocyanate release estimation). Short abstractA fast myrosinase-compatible capillary electrophoresis -micellar electrokinetic chromatography method for simultaneous determination of glucosinolates and allyl isothiocyanate was developed. The method was successfully used to determine glucosinolates (sinigrin and gluconasturtiin) from plant matrices (radish, horseradish, Brussels sprouts and watercress), to determine myrosinase activity using either sinigrin or gluconasturtiin as substrates as well as to determine allyl isothiocyanate from food products, and glucosinolate -isothiocyanate conversion rate.

A Simple Method for On-Gel Detection of Myrosinase Activity

“…LC-ESI-MS has the ability to detect desulfosinigrin with high sensitivity and specificity, while ITCs are usually detected by GC-MS, however, we wanted a single distinguishing measurement. Therefore, given our experience with ITC derivatization with thiols [ 24 ], we tested several thiols for ITC derivatization and LC-MS detection.…”

“…Several thiols with various side-chains that can be expected to ionize well under ESI-MS conditions were purchased and tested to react with 1 µg mL −1 allyl isothiocyanate in 50 mM NH 4 OAc buffer (pH 9.0). The amount of ITC-thiol adduct (dithiocarbamate) produced is a function of pH [ 24 ], which is controlled by addition of the buffering agent at 40-fold excess compared to the reagent.…”

“…Thereafter, the active bands were cut out and incubated in a derivatization solution containing 0.24 mM sinigrin, 100 mM NaH 2 PO 4 buffer (final pH 7.5), 1 mM ascorbic acid and 46.1 mM cysteamine. Our previous study has shown that small aliphatic thiols are compatible with the myrosinase reaction, no enzyme inhibition was observed with mercaptoacetic acid in the reaction medium [ 24 ].…”

“…The enzyme containing crude extracts were prepared as in our recent study [ 24 ]. Briefly, the raw plant material was mixed in a blender with cold 25 mM phosphate buffer (pH 6.5), centrifuged (13,000 rpm, 5 min), filtered on PES 0.22 μm membrane if necessary, and used directly for gel electrophoresis after protein content determination by a Bradford assay [ 38 ] as described earlier.…”

“…Myrosinase assays usually detect either the decomposition of the substrate (glucosinolates), or release of one of the product compounds. The possibilities include direct detection of volatile products by GC-MS [ 20 , 21 ], measurement of the released acid (pH stat assay) [ 22 ], a decrease of concentration of the glucosinolate substrate via spectrophotometry or chromatographic techniques [ 23 , 24 ], or derivatization of the released glucose ( Figure 1 ) via coupled enzyme reactions [ 25 ]. The detection of the enzyme by immunological methods [ 26 , 27 , 28 ] is also a viable option.…”