Intact Glucosinolate Determination in Brassica Vegetables Using Anion Exchange Cleanup and HILIC (Hydrophilic Interaction Liquid Chromatography) HPLC Chromatography
2020
Felker, Peter | Bunch, Ronald A.
In this work an HPLC/hydrophilic interaction liquid chromatography (HILIC) system for intact glucosinolates was optimized using a weak anion exchange resin to remove carboxylic phenolics that absorbed at the same wavelength (229 nm) as glucosinolates, by selectively protonating contaminating phenolics with pKas from 2.98 to 4 while leaving the second OH of the sulfate of the thioglycosidic linkage (pKa = 1.92) ionized. Glucosinolates bound to the anion exchange resin, while the contaminating phenolics washed through. The extraction was performed in 2-ml snap top centrifuge tubes with 50 to 80 mg of fresh tissue in 0.3 ml of extractant. The corresponding spin tubes contained 20–22 mg of anion exchange resin and were loaded with 100 μl of extractant. A 48-well pressure plate was used to load, wash, and finally elute the glucosinolates without a centrifuge into autosampler vials. Seventeen glucosinolates found in common vegetables were separated in a 16-min isocratic run vs 50 min for the C18 gradient used in the desulfoglucosinolate method. The recovery of a spiked sample of phenethyl glucosinolate was greater than 90%, and the limit of detection (LOD) and limit of quantitation (LOQ) were 6.85 and 20.78 nmol respectively. The time from sample weighing to the end of the first chromatographic run was approximately 2 h.
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