Roger J.W. Truscott and John T. Tholen, Bio-organic Chemistry Research Unit, University of Wollongong N.S.W.

Summary
A method for the separation and quantification of glucosinolates from plant tissue Is described.

Following enzymic desulfation, the corresponding desulfoglucosinolates are separated using reversed phase HPLC in less than 30 minutes. Accurate quantification of each glucosinolate can be obtained using this method.

Introduction
Glucosinolates are a group of secondary metabolites found in all members of the plant family Cruciferae. This family contains many representatives of economic importance to man including cabbage, cauliflower, broccoli, brussels sprouts, swede and mustard. The flavour of these species is to a large part governed by the content of glucosinolates present. These chemicals are also of interest since they act to protect the plants which contain them from attack by pathogens such as fungi and bacteria. At higher levels however, such as those found in oil seeds (e.g: rapeseed), glucosinolates and their breakdown products can have adverse effects on the growth rates of non, ruminant animals when the seed meal is used in the diet (1).
For this reason plant breeders are attempting to decrease the levels of glucosinolates in rape and mustard seeds.

Several methods have been used for monitoring glucosinotate Levels. These include gas chromatography of the isothiocyanate breakdown products resulting from myrosinase digestion of the glucosinolates and gas chromatography of the silyl derivatives of intact gluco- sinolates. There are a number of problems associated with the use of these G.C techniques, for example neither can measure the content of the indole gucosinolates.

For this reason HPLC has been developed fbr the analysis of glucosinolates in plant tissues. The procedure employs on-column enzymic desulfation of extracted glucosinolates followed by HPLC separation of the resulting desulfoglucosinolates (2).

HPLC
A powerful tool
for separating
and quantifying
complex plant
metabolites

Materials and Methods

Instrumentation
ICl/Knauer HPLC Pump 64(2) ICl/Knauer Gradient Programmer 50B ICl/Knauer Variable Wavelength UV- Vis Detector Model 87
ICI 5 micron ODS 2 SphensorbT" HPLC Column (4.6mm x 250mm)

Chemicals
HPLC grade water
HPLC grade Acetonitrile -Mallinckrodt Plant Material:
Rapeseed (Brassica napes)
Brussels Sprouts (Brassica oleracea)

Chromatography Conditions
A binary gradient of Water (A) and 20% Acetonitrile (B) was used.


Gradient Programme

Alternatively this technique can be used in order to collect sufficient of each desulfoglucosinolate for the preparation of standard curves versus an internal standard such as o-nitrophenyl galactoside (3) Thus all glucosinolates can be quantified with minimal detectable limits of the order of 30 ng.
These are not the only advantages which the HPLC method enjoys over the previously used GC methods( 6) .
1. No derivatisation of the desulfoglucosinolatesis necessary and the eluate arising from on-column arylsulptase digestion can be injected directly onto the HPLC.

2. The four indole glucosinolates (3-indolylmethyl-GS, 4-hydroxy-3-indotylmethyl-GS, 4-methoxy-3-indoylmethyl-GS, and 1-methoxy-3-indolytmethyl-GS) are well resolved.

Detector: 229 nm

Sample Preparation Summary

1. Seed or seed meal is extracted with boiling water. Vegetative tissue is extracted with boiling methanol.
2. Proteins are precipitated
3. Supernatent is applied to DEAE Sephadex A-25
4. Positively charged and neutral species are removed by washing with water.
5. Aryl sulfatase is added to the column.
6. Desulfoglucosinolates are eluted.
7. Internal standard can be added.
8. An aliquot is injected directly onto the HPLC.

The method is described in detail in reference 3.

In addition, the rapid and simple isolation and analytical methods ensure minimal degradation of labile glucosinolates such as 4-hydroxy-3-indolylmethyl-GS.

5. All of the major glucosinolates encountered so far in the various plant species are well resolved and there seems to be little or no interference from nonglucosinolate substances.

Conclusion
This example of the analysis of glucosinolates in plant tissues serves to illustrate the power of conventional gradient HPLC equipment for the separation and quantification of biological metabolites. Similar HPLC techniques are being employed for the analysis of other important plant chemicals.

The HPLC method described in this application note offers a great number of advantages over previously used GC methods and thus it is not surprising that it is currently being adopted worldwide for the analysis of glucosinolates in plants.