High-performance liquid-phase separation of glycosides analytical and micropreparative HPLC combined with spectroscopic and enzymatic methods for generating a glucosinolate library

• Published in: Journal of AOAC International Vol. 82; no. 5; pp. 1115 - 1127
• Main Authors: Arguello, L.G, Sensharma, D.K, Qiu, F, Nurtaeva, A, El Rassi, Z
• Format: Journal Article
• Language: English
• Published: 01.09.1999
• Subjects: cabbage; determination; food analysis; food composition; glucosinolates; high performance liquid chromatography; quantitative analysis
• ISSN: 1060-3271; 1944-7922
• DOI: 10.1093/jaoac/82.5.1115

Analytical and micropreparative high-performance liquid chromatographic (HPLC) methods assisted by spectroscopic and enzymatic tools were developed for generating a glucosinolate library from white cabbage. This involved the study of the chromatographic behavior of glucosinolates by reversed-phase chromatography (RPC) using octadecyl-silica (ODS) stationary phases and ion-exchange chromatography with strong anion-exchange (SAX) columns. The glucosinolate extract from white cabbage was fractionated on a micropreparative Zorbax ODS column of 250 x 6.2 mm id. The collected fractions from the ODS column effluent were further purified by rechromatographing the early eluting fractions on a SAX column and the late-eluting peaks on the same ODS column under different elution conditions. These studies demonstrated the benefits of using more than one HPLC method in the isolation of pure glucosinolates. These chromatographic separation schemes combined with efficient sample cleanup provided pure glucosinolate fractions that were readily identified by high-field nuclear magnetic resonance (NMR) and high-resolution mass spectrometry (MS), namely negative-ion liquid secondary ionization mass spectrometry (LSIMS). The structural determination of a given glucosinolate by 1- and 2-dimensional NMR was further confirmed by LSIMS, which gives the mass of the molecular anion of glucosinolates. In addition, the structure of a given glucosinolate fraction was readily assessed by performing RPC of the myrosinase digest of the particular glucosinolate and that of the standard degradation product that has a structure that is reflective of the parent glucosinolate. Also, through the myrosinase degradation products (e.g., isothiocyanates) under controlled conditions, the quantitative determination by RPC of the corresponding glucosinolates in the white cabbage extract was reliably achieved. By using the ability of myrosinase to hydrolyze the glucosinolates, it was possible to profile the glucosinolates in the cabbage extract quite rapidly. For the glucosinolates present at very low amounts in white cabbage, and also for the HPLC fractions containing more than one glucosinolate, the isothiocyanate degradation products resulting from the hydrolyzing effect of myrosinase were analyzed by gas chromatography/MS, and their structures were determined. This in turn allowed the determination of the structure of the parent glucosinolates. In summary, white cabbage seems to contain at least 8 glucosinolates.