The determination of glucoraphanin in broccoli seeds and florets by solid phase extraction and micellar electrokinetic capillary chromatography

Abstract

A robust method for the determination of glucoraphanin in broccoli ( brassica oleracea ssp. italica ‘Marathon’) seeds and florets has been developed using solid phase extraction (SPE) and micellar electrokinetic capillary chromatography (MECC) as the determinative step. Glucosinolates were extracted from the broccoli seeds and florets with hot water. Unwanted impurities were removed by passing the extracts through C18 and protonated amino propyl SPE cartridges connected in series. The glucosinolate fraction was removed from the protonated amino propyl cartridge with 2% v/v ammonia solution in methanol. The solvent was removed with a stream of nitrogen, the residue dissolved in water and the level of glucoraphanin determined by MECC using a 77 cm × 75-μm id bare fused silica capillary column (effective length 69.4 cm) and a buffer consisting of 18 mM sodium tetraborate, 30 mM sodium dihydrogen orthophosphate and 30 mM cetyltrimethylammonium bromide, pH 7. MECC parameters and capillary conditioning procedures were optimised with respect to reducing the analysis time without compromising peak integrity. The level of glucoraphanin in broccoli seeds and florets compared favorably with the levels determined by a validated high performance liquid chromatography (HPLC) literature procedure; broccoli seeds MECC 2.1 gm/100 g, HPLC 2.0 gm/100 g; broccoli florets, MECC 71 mg/100 g, HPLC 70 mg/100 g. The MECC instrument reproducibility data ( n = 7) for glucoraphanin in broccoli seed and floret extracts for migration time (CV; seeds 1.2%, florets 2%) and area calculation (CV; seeds 3.7%, florets 7%) relative to the internal standard were suitable.