Quantitative Analysis of Glucosinolates in Oilseed Rape Based on HPLC of Desulfoglucosinolates and HPLC of Intact Glucosinolates

Abstract

High performance liquid chromatography (HPLC) of desulfo-glucosinolates and of intact glucosinolates are the only two methods of analysis known at present, which can fulfil the requirements for reliable determinations of individual glucosinolates in oilseed rape and other Brassica species or cultivars. Use of HPLC requires purification of the glucosinolate-containing extract to avoid serious problems from interfering compounds. Analysis of glucosinolates in double low oilseed rape or other kinds of materials with a low content of glucosinolates requires in addition a concentration of the sample. The experimental steps in performance of these two types of analysis are nearly identical.

The requirements for the extraction of glucosinolates for preparation of ‘crude glucosinolate extracts’ do not deviate for the two methods. It is recommended to use a glucosinolate of known purity as internal standard and added to the extraction solution before start of the homogenisation to control the myrosinase inactivation efficiency. It is also recommended to perform the analysis with and without internal standard or with different glucosinolate standards in the two determinations, e.g. glucotropaeolin and glucobarbarin. The desulfotechnique requires concentration of the extract to remove the methanol; an evaporation step is also acceptable, but not required, for the technique based on intact glucosinolates.

Purification and concentration are in both techniques based on use of mini-columns. The differences consist in the column material used and the elution step where desulfatation with sulfatase or elution with 0.1 M hydrogencarbonate (or 0.1 M hydrogenphosphate, pH 8.0) are used in the techniques based on desulfoglucosinolates or intact glucosinolates, respectively.

The HPLC steps are in both cases based on reversed phase columns of the same types. The mobile phases are for both methods acetonitrile - water mixtures which for the intact glucosinolates in addition require a counterion content. HPLC of desulfogluco-sinolates is a gradient technique which requires two pumps and a system controller. HPLC of intact glucosinolates can be performed both as a gradient technique and an isocratic technique which is based on a simple instrumentation with only one pump. A column heater is recommended for both techniques. The same types of auto-sampler and laboratory integrator or microcomputer can be used with advantage in both techniques.

Both of the HPLC techniques give a nearly equal and high resolution of the peaks with use of the methods now recommended. These consist in minor modifications of the original proposals for HPLC of glucosinolates. However, for the intact glucosinolates, the reduced viscosity of the mobile phase obtained by change of the mobile phases composition and especially by using 50° C or 70° C as column temperature has improved the technique appreciably.

Limitations and possibilities as well as advantages and disadvantages of these two techniques are presented and discussed briefly. These two HPLC methods supplement each other very well, the results obtained from both methods can be used either alone or in combination. It is recommendable to have both methods as accepted standard methods. Thereby, different laboratories according to their available instrumentation and HPLC experience can choose, without problems, the method or methods which fulfil their requirements.