Prestera T, Fahey J W, Holtzclaw W D, Abeygunawardana C, Kachinski J L, Talalay P
Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland, 21205, USA.
Anal Biochem. 1996 Aug 1;239(2):168-79. doi: 10.1006/abio.1996.0312.
Much effort has been devoted to developing methods for the efficient isolation and identification of glucosinolates. Existing methods for separation involve ion exchange, GLC, and HPLC (mostly after chemical modification by enzymatic sulfate removal and/or silylation). We demonstrate a simple and direct strategy for analyzing the glucosinolate content of plant extracts, made possible by a new combination of widely available techniques: (a) reverse-phase paired-ion chromatography (PIC) of plant extracts, (b) hydrolysis of glucosinolates by myrosinase and quantitation of resulting isothiocyanates by cyclocondensation with 1, 2-benzenedithiol; (c) a novel method for replacing the PIC counterions by ammonium ions, permitting direct bioassay, mass, and 1H NMR spectrometry; (d) mass spectrometric analysis of ammonium salts by negative-ion fast atom bombardment (FAB) to determine m/z of the [M - H]- ion, and by chemical ionization (CI) in ammonia to obtain accurate masses of characteristic fragment ions, principally [R-CN:NH4]+, [R-CH=NOH:H]+ and [R-CH=NOH:NH4]+; and (e) high-resolution 1H NMR spectroscopy of intact glucosinolates. FAB and CI mass spectra, as well as high-resolution 1H NMR spectra were obtained for a variety of glucosinolate standards. The results provide guidance for the isolation and characterization of unknown glucosinolates from plants. These combined procedures were applied to a sample of broccoli (cultivar SAGA), in order to resolve and identify its major glucosinolates: 4-methylsulfinylbutyl glucosinolate (glucoraphanin) and 4-methylthiobutyl glucosinolate (glucoerucin). Thus, this analytical strategy provides a powerful technique for identifying and quantitating glucosinolates in plant extracts without resorting to derivatization.
人们已付出诸多努力来开发高效分离和鉴定硫代葡萄糖苷的方法。现有的分离方法包括离子交换、气相色谱（GLC）和高效液相色谱（HPLC）（大多是在通过酶促脱硫和/或硅烷化进行化学修饰之后）。我们展示了一种用于分析植物提取物中硫代葡萄糖苷含量的简单直接策略，这得益于广泛可用技术的新组合得以实现：（a）植物提取物的反相离子对色谱法（PIC）；（b）用黑芥子酶水解硫代葡萄糖苷，并通过与1,2 - 苯二硫醇进行环缩合对生成的异硫氰酸酯进行定量；（c）一种用铵离子取代PIC抗衡离子的新方法，从而允许直接进行生物测定、质谱分析和¹H NMR光谱分析；（d）通过负离子快原子轰击（FAB）对铵盐进行质谱分析以确定[M - H]⁻离子的m/z，并在氨中通过化学电离（CI）获得特征碎片离子的精确质量，主要是[R - CN:NH₄]⁺、[R - CH = NOH:H]⁺和[R - CH = NOH:NH₄]⁺；以及（e）完整硫代葡萄糖苷的高分辨率¹H NMR光谱。获得了多种硫代葡萄糖苷标准品的FAB和CI质谱以及高分辨率¹H NMR光谱。这些结果为从植物中分离和表征未知硫代葡萄糖苷提供了指导。这些组合程序被应用于西兰花（品种SAGA）的一个样品，以解析和鉴定其主要硫代葡萄糖苷：4 - 甲基亚磺酰基丁基硫代葡萄糖苷（萝卜硫苷）和4 - 甲基硫代丁基硫代葡萄糖苷（葡糖芥苷）。因此，这种分析策略提供了一种无需衍生化即可鉴定和定量植物提取物中硫代葡萄糖苷的强大技术。
