ABSTRACT Distribution of glucosinolates within plant families of the order Brassicales was investigated using qualitative and quantitative UHPLC-DAD-MS/MS analysis of desulfoglucosinolates in different plant tissues of forty-eight (48) plants from five (5) plant families (Brassicaceae, Capparidaceceae, Moringaceae, Resedaceae, Tropaeolaceae) growing in the Republic of Croatia and worldwide. A database of MS2 spectra of desulfoglucosinolates was developed using commercially available standards as well as glucosinolates isolated from natural sources. The Brassicaceae plant family contained a wide variety of glucosinolates derived from seven (7) different amino acid precursors, most commonly from methionine, followed by glucosinolates derived from tryptophan, phenylalanine and/or tyrosine, while branched aliphatic glucosinolates deriving from valine, leucine and isoleucine were scarce. Glucocapparin, the only known glucosinolate derived from the amino acid alanine, was identified solely in the Capparidaceae family as the main one in all the species studied, while the other glucosinolates largely derived from tryptophan. Moringa oleifera (family Moringaceae) contained 4-(α-L-rhamnopyranosyloxy)benzyl glucosinolate and, in less amount, three O-acetylated isomers of the same glucosinolate derived from phenylalanine and/or tyrosine. In the species Reseda lutea and R. phyteuma (family Resedaceae) an isomer of 4-(α-L-rhamnopyranosyloxy)benzyl glucosinolate, 2- (α-L-rhamnopyranosyloxy)benzyl glucosinolate, was identified characteristic for the plant family Resedaceae, originating from phenylalanine. On the other hand, 2-hydroxy- 2-methylpropyl glucosinolate derived from isoleucine was the major glucosinolate identified in R. alba. The main glucosinolate in Tropaeolum majus was arylaliphatic benzyl glucosinolate followed by 3-methoxybenzyl glucosinolate, both derived from phenylalanine, the only known amino acid precursor of glucosinolates in the Tropaeolaceae family. Insight into the quantitative composition of glucosinolates enabled the selection of the plant species as the sources of glucosinolates for their isolation in the form of desulfoglucosinolates and confirmation by spectroscopic techniques (MS2, 1 H, 13 C, COSY and HSQC NMR). 3-(Methylsulfinyl)propyl glucosinolate was isolated from Anastatica hierochuntica (family Brassicaceae), 4-hydroxybenzyl glucosinolate from Sinapis alba (family Brassicaceae), methyl glucosinolate from Capparis orientalis (family Capparidaceae), 2-hydroxy-2-methylpropyl glucosinolate from Reseda alba (family Resedaceae) and benzyl glucosinolate from Tropaeolum majus (family Tropaeolaceae). Also, based on MS2 spectra and biosynthetic characteristics, four (4) new glucosinolates were identified: ortho or meta hydroxy-4-(α-L- rhamnopyranosyloxy)benzyl glucosinolate (Moringa oleifera), 4´-O-(β-D- apiofuranosyloxy)benzyl glucosinolate (Hesperis laciniata and Thlaspi perfoliatum), 4- hydroxy-1-methoxyindol-3-ylmethyl glucosinolate (Thlaspi perfoliatum) and 3-hydroxy- (4-methylsulfonyl)butyl glucosinolate (Barbarea vulgaris) whose structures need further confirmation by NMR. The stability of desulfo-4-(methylsulfinyl)but-3-enyl glucosinolate isolated from Matthiola incana was studied over a one-month period and its instability in aqueous solution was observed, showing a significant drop in the content (up to 80%) followed by spontaneous conversion to cyclic thioimidate N-oxides. The enzymatic and thermal degradation of glucosinolates was studied by using conventional (enzymatic hydrolysis and hydrodistillation) and modern methods (microwave distillation and hydrodiffusion and gravity). Under microwave-assisted thermal decomposition conditions, lower yields of decomposition products were obtained compared to thermal decomposition during hydrodistillation and enzymatic hydrolysis. Isothiocyanates as degradation products of glucosinolates showed to be unstable at GC-MS conditions for ω-methylsulfinyl C3, C5 and C6 isothiocyanates, which decomposition resulted in the formation of the corresponding olefinic isothiocyanates. Additionally, due to the presence of an electron- donating hydroxyl group in the para position on the benzene ring, in 4-hydroxybenzyl isothiocyanate and 4-hydroxy-3,5-dimethoxybenzyl isothiocyanate, instability lead to formation of artifacts found during GC-MS analysis. Also, degradation products of indole glucosinolates have not been identified. This highlights the disadvantage of an indirect method for the identification of glucosinolates via their degradation products in plant species. In the Eruca vesicaria biofortified by selenium from sodium selenate, 4- (methylseleno)butyl glucosinolate derived from selenomethionine was identified based on the MS2 spectrum shown for the first time. Hydroponically grown sprouts of the five (5) selected plants of Brassicaceae family were found to have a high content of characteristic glucosinolates presenting a good source for their isolation.