Simultaneous direct determination of 15 glucosinolates in eight Brassica species by UHPLC-Q-Orbitrap-MS
Introduction
Vegetables of genus Brassica (e.g., cabbage, kale, cauliflower, broccoli, and mustard) are rich in health-promoting compounds such as soluble fiber, ascorbic acid, and glucosinolates (GLS), and are therefore classified as health foods. In particular, GLS and their derivatives are the main contributors to bitter food taste (Williams et al., 2013, Park et al., 2014) and have been intensively studied in the past several decades for their anti-oxidative, anti-fungal, anti-bacterial, and anti-cancer activities (Fahey et al., 2001, Traka and Mithen, 2009). According to the type of amino acid–derived side chain, GLS (>200 reported to date) can be divided into aliphatic, indolic, and aromatic ones (Sønderby et al., 2010, Clarke, 2010, Giamoustaris and Mithen, 1996), and their content in Brassica species depend on their growth stage and genotype (Bhandari, Jo, & Lee, 2015).
Among the large number of recent studies on the biological activity of GLS, most have been focused on the anti-cancer and fungicidal effects of several GLS breakdown products (Song and Thornalley, 2007, Conaway et al., 2002, McNaughton and Marks, 2003, Popova and Morra, 2014, Gupta et al., 2014, Wagner et al., 2013, Hanschen et al., 2014). Moreover, the European Food Safety Authority suggested that the intake of GLS with food enhances the efficiency of body defense and immune systems (Ares, Valverde, Nozal, Bernal, & Bernal, 2016). When the cells of Brassica species are disrupted by chewing or cutting, the contained GLS hydrolyze by myrosinase, an endogenous enzyme that is physically separated from GLS in intact cells by cell walls. This hydrolysis reaction furnishes bioactive breakdown products with therapeutic potential such as isothiocyanates, thiocyanates, nitriles, or indoles. Additionally, GLS in ingested food can be degraded to isothiocyanates by intestinal microorganisms (Shapiro, Fahey, Wade, Stephenson, & Talalay, 2001).
GLS can be extracted from Brassica species with methanol under water/ultrasonic bath conditions. Once extracted, GLS are separated using reverse-phase high-performance liquid chromatography and detected/quantified by UV absorption spectroscopy or mass spectrometry (Matthäus and Luftmann, 2000, Francisco et al., 2009, Ares et al., 2014, Crocoll et al., 2017). Also, some of quantitative analysis was accomplished using each GLS response factors (Brown et al., 2003, Toledo-Martín et al., 2017). However, despite the high number of available analytical procedures, data on the corresponding validation results and matrices do not exist extensively. Keeping in view this knowledge gap, the current study aimed to validate an analytical method for the simultaneous direct quantification of 15 GLS by UHPLC-Q-Orbitrap-MS; identify the difference in responsiveness of each GLS compounds; obtain and compare the GLS profiles of eight Brassica species; broccoli, cabbage, cauliflower, kale, mustard, kimchi cabbage, radish, young radish leaf, and young radish root, and identify the similar species based on GLS contents.
Section snippets
Reagents and instrumentation
The UPLC-HESI-MS system comprised of Dionex Ultimate 3000 UHPLC module and a heated electrospray ionization (HESI) quadrupole Orbitrap mass spectrometer (Thermo Scientific, Bremen, Germany) controlled by Xactive Tune 1.1 and Xcalibur 2.2 software (Thermo Fisher Scientific, San Jose, USA).
All GLS standards, namely glucoiberin (GIB), glucoraphanin (GRA), glucoerucin (GER), glucocheirolin (GOC), glucoberteroin (GOB), progoitrin (PRO), sinigrin (SIN), gluconapin (GNA), glucobrassicanapin (GBN),
Results and discussion
The quality assurance parameters of the instrument for GLS determination and the spike recovery (%) data for 15 GLS are listed in Table 2, Table 3, respectively. Table 4 summarizes the average and minimum – maximum concentrations values of the analyzed GLS reported on dry weight basis. Fig. 1 presents the calibration curves obtained for 15 GLS, and Fig. 2 presents the LDA plot for GLS concentrations in Brassica species.
Conclusions
In this study, a method for the simultaneous identification and quantitation of 15 GLS in eight Brassica species by UHPLC-Q-Orbitrap-MS was developed, optimized, and validated. Optimization of analytical conditions such as the extraction method and dilution factor confirmed that each standard compound was characterized by a different detection sensitivity, which implied that reliable quantitative analysis requires the signal intensities of standard compounds to be compared with those of
Declaration of interest statement
The authors declare no conflict of interest.
Acknowledgements
This research was supported by a grant from the World Institute of Kimchi (KE1803-3) funded by the Ministry of Science and ICT, Republic of Korea.
Conflict of interest
The authors declare no conflict of interest.
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Both authors contributed equally to this paper.