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A new method to simultaneously analyze various glucosinolates (GSLs) and isothiocyanates (ITCs) by reversed-phase ultra-high-performance liquid chromatography-electron spray ionization-tandem mass spectrometry has been developed and validated for 14 GSLs and 15 ITCs. It involved derivatization of ITCs with N-acetyl-l-cysteine (NAC). The limits of detection were 0.4-1.6 μM for GSLs and 0.9-2.6 μM for NAC-ITCs. The analysis of Sinapis alba, Brassica napus, and Brassica juncea extracts spiked with 14 GSLs and 15 ITCs indicated that the me