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Intact Glucosinolate Determination in Brassica Vegetables Using Anion Exchange Cleanup and HILIC (Hydrophilic Interaction Liquid Chromatography) HPLC Chromatography

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Abstract

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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Abbreviations

HILIC column:

Hydrophilic interaction liquid chromatography

DEAE:

Diethylaminoethyl

PTFE:

Polytetrafluoroethylene

PolyWAXLP:

Weak anion exchange resin with linear polyethyleneimine

LOD:

Limit of detection

LOQ:

Limit of quantitation

DAD:

diode array detector

PEEK:

Polyetheretherketone

mAU:

Milli-absorbance unit

pKa:

Negative log of the acid dissociation constant

HPLC:

High pressure liquid chromatography

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Acknowledgments

The authors gratefully acknowledge the advice and support of K. Wade and J. Fahey of Johns Hopkins in getting started, Niels Agerbirk for assistance with the relative absorbances at 229 nm, and Amos Heckendorf of the Nest Group for many helpful suggestions.

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  1. D’Arrigo Bros of California, 21777 Harris Road, Salinas, CA, 93908, USA

    Peter Felker & Ronald A. Bunch

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  1. Peter Felker
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  2. Ronald A. Bunch
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Correspondence to Peter Felker.

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All the funding for this research was D’Arrigo Bros in-house funding.

Conflict of Interest

The authors are employees of a major agricultural company growing brassica vegetables and would like to see more of these vegetables consumed and their beneficial properties reported in the literature. Peter Felker declares that he has no conflict of interest. Ronald Bunch declares that he has no conflict of interest.

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Felker, P., Bunch, R.A. Intact Glucosinolate Determination in Brassica Vegetables Using Anion Exchange Cleanup and HILIC (Hydrophilic Interaction Liquid Chromatography) HPLC Chromatography. Food Anal. Methods 13, 1817–1828 (2020). https://doi.org/10.1007/s12161-020-01798-0

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  • DOI: https://doi.org/10.1007/s12161-020-01798-0

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