molecules Article Extraction and Quantification of Sulforaphane and Indole-3-Carbinol from Rapeseed Tissues Using QuEChERS Coupled with UHPLC-MS/MS Xu Yu 1,2,3, Fei Ma 1,2,3,4,*, Liangxiao Zhang 1,2,4,5 and Peiwu Li 1,2,3,4,5 1 Oil Crops Research Institute, Chinese Academy of Agricultural Sciences, Wuhan 430062, China; [email protected] (X.Y.); [email protected] (L.Z.); [email protected] (P.L.) 2 Key Laboratory of Biology and Genetic Improvement of Oil Crops, ministry of Agriculture and Rural Affairs, Wuhan 430062, China 3 Key Laboratory of Detection for Mycotoxins, ministry of Agriculture and Rural Affairs, Wuhan 430062, China 4 Laboratory of Quality and Safety Risk Assessment for Oilseeds Products (Wuhan), ministry of Agriculture and Rural Affairs, Wuhan 430062, China 5 Quality Inspection and Test Center for Oilseeds Products, ministry of Agriculture and Rural Affairs, Wuhan 430062, China * Correspondence: [email protected]; Tel.: +86-27-86711839 Academic Editors: Monika Waksmundzka-Hajnos and Miroslaw Hawryl Received: 22 March 2020; Accepted: 30 April 2020; Published: 4 May 2020 Abstract: Rapeseed (Brassica napus L.) is rich in phenols, vitamins, carotenoids, and mineral elements, such as selenium. Additionally, it contains the active ingredients sulforaphane and indole-3-carbinol, which have been demonstrated to have pharmacological effects. In this study, sulforaphane and indole-3-carbinol were extracted and quantified from rapeseeds using quick, easy, cheap, effective, rugged and safe (QuEChERS) method coupled with ultra high performance liquid chromarography tandem mass spectrometry (UHPLC-MS/MS). The major parameters for extraction and purification efficiency were optimized, including the hydrolysis reaction, extraction condition and type and amount of purification adsorbents. The limit of detection (LOD) and the limit of quantification (LOQ) for sulforaphane were 0.05 µg/kg and 0.15 µg/kg, and for indole-3-carbinol were 5 µg/kg and 15 µg/kg, respectively. The developed method was used to successfully analyze fifty rapeseed samples. The QuEChERS coupled with UHPLC-MS/MS simultaneously detect sulforaphane and indole-3-carbinol in vegetable matrix and evaluate the quality and nutrition of rapeseed samples. Keywords: sulforaphane; indole-3-carbinol; QuEChERS; quantification; UHPLC-MS/MS 1. Introduction Rapeseed (Brassica napus L.) is a major oilseed crop in China. China generates approximately 20% of the world’s yield [1]. Rapeseeds not only produce edible oils, but their sprouts, seedlings, and leaves have nutritional value for human consumption [2]. Vegetables such as Brassicaceae (Cruciferae) are vital nutritional sources throughout the world. They contain several chemopreventive compounds, such as glucosinolates, polyphenols, and selenium [3–5]. During plant tissue damage, glucosinolates decompose into isothiocyanates and nitriles in the presence of the endogenous enzyme myrosinase (EC 3.2.1.147), then undergo immediate hydrolysis of the β-thioglucoside group [6]. More than 300 glucosinolates and their hydrolytic products have been identified and evaluated for their beneficial effects [7,8]. The phytochemicals classified as isothiocyanates have been widely utilized to investigate their anti-tumor, anti-cancer and anti-inflammatory effects. Sulforaphane (SFN) has been demonstrated to be the most potent compound. It is the activator of the Nrf2 pathway, which regulates the cellular defense system and promotes various detoxifying and antioxidative effects [9,10]. Molecules 2020, 25, 2149; doi:10.3390/molecules25092149 www.mdpi.com/journal/molecules Molecules 2020, 25, x 2 of 12 utilized to investigate their anti-tumor, anti-cancer and anti-inflammatory effects. Sulforaphane (SFN)Molecules has2020 been, 25, 2149 demonstrated to be the most potent compound. It is the activator of the 2Nrf2 of 11 pathway, which regulates the cellular defense system and promotes various detoxifying and antioxidative effects [9,10]. Sulforaphane has beneficial effects for diabetic complications by effectivelySulforaphane reducing has beneficial fasting blood effects glucose for diabetic and glycat complicationsed hemoglobin by eff ectivelylevels in reducing obese type fasting 2 diabetic blood patientsglucose andwith glycated poor glycemic hemoglobin control levels [11]. in obeseThe bioavailability type 2 diabetic of patients sulforaphane with poor in glycemicvegetables control has also [11]. beenThe bioavailabilitystudied, and could of sulforaphane serve as a guide in vegetables for the desi hasgn also and been implementation studied, and of could SFN servein clinical as a guidetrials [12,13].for the designIndole-3-carbinol and implementation (I3C) is another of SFN important in clinical trialsanti-cancer [12,13]. chemopreventive Indole-3-carbinol compound (I3C) is another that actsimportant via further anti-cancer hydrolysis chemopreventive of isothiocyanate compound (indol-3-methyl that acts viaisothi furtherocyanate), hydrolysis which of is isothiocyanatederived from cruciferous(indol-3-methyl plants. isothiocyanate), The I3C compound which has is derived been show fromn cruciferousto regulate plants.the growth The I3Cof cancer compound cells hasby modulatingbeen shown genes to regulate involved the in growth growth, of signal cancer transd cells byuction modulating and carcinogenesis genes involved [14]. inIt can growth, inhibit signal the proliferationtransduction of and cancer carcinogenesis cells [15,16], [14 inhibit]. It can the inhibit expression the proliferation of drug resistance of cancer genes cells [ 15[17],,16 ],and inhibit induce the apoptosisexpression [18]. of drug Preliminary resistance clinical genes trials [17], andhave induce indicated apoptosis that indole-3-carbinol [18]. Preliminary clinicalcould be trials used have to protectindicated against that indole-3-carbinol hormone dependent/independent could be used to protect human against cancers hormone [19]. As dependent shown /inindependent Figure 1, sulforaphanehuman cancers and [19 indole-3-carbinol]. As shown in Figure are both1, sulforaphane non-toxic, “natural and indole-3-carbinol products” and are are both often non-toxic, used in combination“natural products” with conventional and are often chemotherapy used in combination to treat with human conventional malignancies chemotherapy to give a tolower treat toxicity human andmalignancies higher efficacy. to give a lower toxicity and higher efficacy. Figure 1. The molecular structures of sulforaphane and indole-3-carbinol. Figure 1. The molecular structures of sulforaphane and indole-3-carbinol. An appropriate sample pretreatment method is a key step to the quantification of sulforaphane andAn indole-3-carbinol appropriate sample in vegetables. pretreatment Current method pretreatment is a key extractionstep to the techniques quantification for sulforaphane of sulforaphane and andindole-3-carbinol indole-3-carbinol include in vegetables. liquid–liquid Current extraction pretre [20atment], solid-phase extraction extraction techniques (SPE) for [21 sulforaphane], dispersive andliquid–liquid indole-3-carbinol microextraction include (DLLME) liquid–liquid [22], high-speed extraction countercurrent [20], solid-phase chromatography extraction (HSCCC)(SPE) [21], [23] dispersiveand preparative liquid–liquid HPLC [microext24]. However,raction the(DLLME) majority [22], of high-speed the extraction countercurrent and purification chromatography methods are (HSCCC)tedious and [23] requireand preparative large volumes HPLC of[24]. organic However, solvents the majority or sophisticated of the extr equipment.action and purification QuEChERS methods(quick, easy, are tedious cheap, eandffective, require rugged large and volumes safe) isof a organic comprehensive solvents methodor sophisticated that has beenequipment. widely QuEChERSused to determine (quick, targeteasy, cheap, analytes effective, in foods, rugged agri-products, and safe) is environmental a comprehensive substances method andthat biologicalhas been widelyfluids [ 25used,26]. to The determine QuEChERS target method analytes constitutes in foods, two agri-products, key steps including environmental liquid–liquid substances extraction and and biologicaldispersive fluids solid-phase [25,26]. extraction The QuEChERS clean-up. method This method constitutes is simple, two key versatile, steps including eco-friendly, liquid–liquid eliminates extractionmatrix eff ectsand and dispersive has excellent solid-phase recovery. extraction The QuEChERS clean-up. is an This ideal method method foris thesimple, extraction versatile, and eco-friendly,purification ofeliminates sulforaphane matrix and effects indole-3-carbinol and has excellent in vegetable recovery. samples. The QuEChERS is an ideal method for theQuantitative extraction and methods purification for sulforaphane of sulforapha includene and indole-3-carbinol thin-layer chromatography in vegetable (TLC)samples. [27 ], gas chromatographyQuantitative (GC)methods [28], for and sulforaphane gas chromatography-mass include thin-layer spectrometry chromatography (GC-MS) (TLC) [29]. However,[27], gas chromatographyhigh temperatures (GC) at the [28], injection and gas ports chromatograp in the GC orhy-mass GC-MS spectrometry system could (GC-MS) result in the[29]. degradation However, highof sulforaphane temperatures and at the indole-3-carbinol injection ports [in30 the]. To GC measure or GC-MS the system stability could and solubilityresult in the of degradation the analytes, ofliquid sulforaphane chromatography and indole-3-carbinol coupled with various