A liquid chromatography-mass spectrometry approach to study “glucosinoloma” in broccoli sprouts

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Research article Received: 25 January 2012

Revised: 9 May 2012

Accepted: 15 May 2012

Published online in Wiley Online Library

(wileyonlinelibrary.com) DOI 10.1002/jms.3028

A liquid chromatography-mass spectrometry approach to study “glucosinoloma” in broccoli sprouts† Mariateresa Maldini,* Simona Baima, Giorgio Morelli, Cristina Scaccini and Fausta Natella Glucosinolates are an important class of secondary plant metabolites, possessing health-promoting properties. Young broccoli plants are a very good source of glucosinolates with concentrations several times greater than in mature plants. The aim of our study was to develop a liquid chromatography–mass spectrometry and liquid chromatography/tandem mass spectrometry qualitative and quantitative method for the measure of glucosinolates in broccoli sprouts. The described method provides high sensitivity and specificity, allowing a rapid and simultaneous determination of 14 glucosinolates. The proposed method has been validated for eight glucosinolates: glucobrassicin, glucoraphanin, glucoiberin, glucoerucin, progoitrin, gluconapin, sinigrin and glucocheirolin. The linear range was 1–150 mg ml 1, the intra-day and inter-day precision values are within 6% and 8% at the lower limit of quantification, while the overall recovery of the eight glucosinolates was 99  9%. This validated method was used successfully for analysis of glucosinolates content of broccoli sprouts grown in different conditions. Copyright © 2012 John Wiley & Sons, Ltd. Supporting information may be found in the online version of this article. Keywords: broccoli sprouts; glucosinolates; brassicacee; growth condition; LC–MS



Recent studies have demonstrated that the regular consumption of fruits and vegetables is correlated with a decreased risk of chronic diseases, including cardiovascular disease and different types of cancer.[1–3] Vegetables belonging to the Brassicaceae family (e.g. broccoli, cabbage, kale and Brussels sprouts), widely consumed in the world, are considered to have a significant function in human nutrition. In fact, they provide bioactive phytochemicals, such as vitamins, minerals, phenolic compounds and glucosinolates, all molecules endowed with a variety of biological activities, including antioxidant activity, enzymes regulation and control of apoptosis and cell cycle.[4–7] Brassica vegetables represent, in particular, a rich source of glucosinolates, secondary plant metabolites, structurally characterized by a b-D-thioglucose unit, a sulfate oxime group and a variable side chain derived from different aminoacids.[8,9] Glucosinolates are produced almost exclusively in Brassica plants, where they are thought to play a role in microbe and insect defence.[10] When plant cells are disrupted (e.g. during cutting, chewing, cooking and freezing), glucosinolates are hydrolyzed by a b-thioglucosidase enzyme (myrosinase) to various bioactive breakdown products (isothiocyanates, nitriles, thiocyanates, epithicyanates, epithionitriles and oxazolidines).[11] Glucosinolates and isothiocyanates are known to possess anti-carcinogenic and antioxidant effects and have attracted great interest from both toxicological and pharmacological points of view, as they are able to induce phase 2 enzymes, to inhibit phase 1 activation enzymes and protect animals against chemically induced cancer.[12–17]

J. Mass Spectrom. 2012, 47, 1198–1206

Young broccoli seedlings (also known as broccoli sprouts) are an especially good source of glucosinolates, with concentrations several times greater than those of mature plants; then, broccoli sprouts could have an important role in diet and human health.[18,19] For this reason, it is necessary to accurately study and collect information on profiles and levels of glucosinolates in broccoli sprouts. In the past years, several methods (gas chromatography, colorimetry, capillary electrophoresis, high performance liquid chromatography (HPLC)) have been applied to detect and determinate glucosinolates in complex mixtures, but most of them are elaborate and require a complex and tedious sample preparation.[20–26] Moreover, all these methods have been developed without reference standard compounds. Most recently, the introduction and the rapid development in LC–mass specrometry (MS) has eliminated some difficulties in the analysis of glucosinolates. HPLC/tandem mass spectrometry (MS/MS) has been used with considerable success being a technique that frequently provide specific, selective and sensitive qualitative and quantitative results often with reduced sample preparation and analysis time, if compared with other commonly employed techniques.[14] The majority of these quantitative analyses of

* Correspondence to: Mariateresa Maldini, INRAN - Via Ardeatina 546 – 00178 Roma, Italy. E-mail: [email protected]

This article is part of the Journal of Mass Spectrometry special issue entitled “2nd MS Food Day” edited by Gianluca Giorgi. National Research Institute for Food and Nutrition – INRAN – Via Ardeatina 546, 00178 Roma, Italy

Copyright © 2012 John Wiley & Sons, Ltd.

LC–MS determination of glucosinoloma glucosinolates has been performed by LC–MS multiple reaction monitoring methods (MRM), using the parent glucosinolate anion and the product ion at m/z value of 97 as the key ions.[27,28] However, the selection of more specific transitions from parent ions to product ions would be more beneficial. In the present study, a qualitative and quantitative method for the measure of glucosinolates in broccoli sprouts extracts has been set on the basis of electrospray ionization (ESI)–MS and ESI–MS/MS profiles. Direct flow injection/electrospray ionization/ion trap MS/MS has been used to screen the natural occurring glucosinolates. In a second stage, a selective LC–MS/MS (MRM) method was developed to monitor and determinate the variation of the ‘glucosinoloma’ of broccoli sprouts as modulated by modifications of the growth conditions. The described method provides high sensitivity and specificity, allowing a fast and simultaneous determination of 14 glucosinolates with a rapid and simple sample preparation. The quantitative method, performed by using external standards, was validated for eight glucosinolates (glucobrassicin, glucoraphanin, glucoiberin, glucoerucin, progoitrin, gluconapin, sinigrin and glucocheirolin) in agreement with European Medicines Agency (EMEA) note guidance on validation of analytical methods.[29] For the other glucosinolates (4-methoxyneoglucobrassicin, neoglucobrassicin, 4-hydroxyglucobrassicin, glucoiberverin, gluconapoleiferin, glucoalysin), just a relative quantification was performed. The method was able to evidence quantitative differences in the glucosinolates content of broccoli sprouts grown in different conditions.


allowed to grow for 3, 5, 7 and 10 days after sowing. Sprouts samples were rapidly and gently collected from the surface of the germination cylinder at midday, weighed (fresh mass) and immediately frozen in liquid nitrogen and stored at –80  C for further analysis. Frozen sprouts were ground to a fine powder in a Waring blendor cooled with liquid nitrogen, and aliquots of sprouts powder were used for humidity content determination. Extraction and sample preparation Each sample of broccoli sprouts was extracted with methanol: water (70:30 v/v; sample to solvent ratio 1:25 w/v) at 70  C for 30 min under vortex mixing to facilitate the extraction.[8] The samples were successively centrifuged (4000 rpm, 30 min, 4  C), the supernatants were collected and the solvent was completely removed using a rotary evaporator under vacuum at 40  C. The dried samples were dissolved in ultrapure water with the same volume of extraction and filtered through 0.20-mm syringe PVDF filters (Whatmann International Ltd., UK). ESI-MS and ESI-MS/MS analyses Full-scan ESI-MS and collision-induced dissociation ESI-MS/MS (by using both Product Ion and Enhanced Product Ion Scan modes) analyses of standards and samples were performed on an Applied Biosystems API3200 Q-Trap (Foster City, CA, USA) spectrometer. The analytical parameters were optimised by infusing a standard solution of Glucoraphanin (1 mg ml 1 in methanol 50%) into the source at a flow rate of 10 mL min 1. The optimised parameters were: declustering potential 52 eV, entrance potential 4.7 eV, collision energy 38 eV and collision cell exit potential 4 eV. Data were acquired in the negative ion MS and MS/MS modes. HPLC–ESI-MS and HPLC–ESI-MS/MS analyses

Solvents used for extraction were of high purity (Carlo Erba, Milano, Italy). HPLC grade methanol, acetonitrile and formic acid were from Sigma-Aldrich Chemical Company (St Louis, MO). HPLC grade water (18 mΩ) was prepared using a Millipore (Bedford, MA, USA) Milli-Q purification system. Glucobrassicin potassium salt, glucoraphanin potassium salt, glucoiberin potassium salt, glucoerucin potassium salt, progoitrin potassium salt, gluconapin potassium salt, sinigrin potassium salt and glucocheirolin potassium salt were purchased from PhytoLab GmbH & Co. KG (Vestenbergsgreuth, Germany).

Qualitative on-line HPLC-ESI-MS/MS analysis of extracts were performed using HPLC system interfaced to an Applied Biosystems (Foster City, CA, USA) API3200 Q-Trap instrument (Q1). LC analyses were conducted using a system equipped with a 200 binary pump (Perkin-Elmer, USA). Samples were injected (10 ml) into a Luna C18 column (Phenomenex, USA) (150  2.1 mm i.d., 5 mm d) and eluted at flow rate of 0.3 ml min 1. Mobile phase A was H2O containing 0.1% formic acid, while mobile phase B was acetonitrile containing 0.1% formic acid. Elution was carried out using a gradient commencing at 94% A and changing to 88 % A in 15 min, then from 88% A to 75% A in 6 min, then from 75% A to 40% A in 9 min and finally to 0% A in 1 min. The column was kept at 25  C, using a Peltier Column Oven Series 200 (Perkin Elmer). The flow from the chromatograph was injected directly into the ESI source. Qualitative analysis of the compounds was performed using information-dependent acquisition (IDA). The IDA method created included an IDA criteria (specify the charge state, mass range), enhanced MS scan (EMS), enhanced resolution (ER), enhanced product ion scan (EPI) or MS/MS scan. The source temperature was held at 450 C, and MS parameters were those optimised for the ESI-MS and ESI-MS/MS analyses with ion spray voltage at 4300. MS data were acquired using the software provided by the manufacturer (Analyst software 1.5.1), and extracted ion chromatogram were elaborated in order to identify glucosinolates from their deprotonated molecule and retention time. Quantitative on-line HPLC-ESI-MS/MS analyses were performed using the same LC-ESI-MS/MS equipment and the same chromatographic conditions described above, but the mass

Growth conditions of broccoli sprouts Broccoli seeds (Brassica oleracea L. var. botrytis subvar. cymosa) were purchased from SUBA&UNICO (Longiano, FC, Italy). Seeds were surface sterilized by incubating for 15 min in 40% v/v commercial bleach (2% sodium hypochlorite) with shaking, then drained and rinsed ten times with distilled water. After soaking in distilled water for 16–18 h at 21  C, seeds were rinsed in distilled water and transferred in the germination cylinder of Vitaseed sprouter (Vitaseed AG, Switzerland). In this system, sprouts were immersed in distilled water for 5 min every 2 h. Sprouts were grown at 21 C in a plant growth chamber (Clf Plant Climatics, Wertingen, Germany) equipped with PHILIPS Master TL-D 36W/840 cool-white fluorescent tubes providing a photosynthetic photon flux density of 110 mmol m 2 s 1, under three different light regimes: ‘continuous light’, ‘light/dark cycle’ (L/D cycle) (16-h light/8-h dark cycle) and ‘dark’ (achieved by covering the sprouting device with a cardboard box). The sprouts were

J. Mass Spectrom. 2012, 47, 1198–1206

Copyright © 2012 John Wiley & Sons, Ltd.




M. Maldini et al. spectrometer worked in MRM mode. The API 3200 ES source was tuned by infusing solutions of standards (1 mg ml 1 in methanol 50%) into the source at a flow rate of 10 ml min 1. The optimised parameters, fragmentation reactions selected for each compound and retention times were reported in Table 1. For all compounds, dwell time was 60 ms, and the voltage applied was 4500. Data acquisition and processing were performed using Analyst software 1.5.1.

Method validation LC–MS/MS method was validated according to the EMEA guidelines related to the validation of analytical methods.[29] Precision was evaluated at three concentration levels for each compound through triplicate intra-day assays and inter-day assays over 3 days; the intra-day precision (coefficient of variance) was within 6%, while the inter-day was within 8% for all analytes (Table 3). Specificity was defined as the non-interference by other analytes detected in the region of interest. For the LC–MS/MS method, which was developed on the basis of the characteristic fragmentation of compounds 1–14, no other peaks interfered with the analytes in the MS/MS detection mode. Accuracy of the analytical procedure was evaluated using the recovery test. Sprout samples were added with three different amounts of the eight standards, and recoveries were calculated from the difference between the amount of analytes measured before and after standards addition. The mean recoveries for each standard and each concentration level are reported in Table 3. The calibration graphs, obtained by plotting the area of ES against the known concentration of each compound, were linear in the range used for the analysis for all glucosinolates. The sensitivity of the method was determined with respect to limit of quantification (LOD) and limit of detection (LOQ). The LOQ (equivalent to sensitivity of the

Calibration and quantification of glucosinolates In order to prepare the calibration plot, a sample (1 mg) of each compound was weighted accurately into a 1-ml volumetric flask, dissolved in methanol 50% (v/v) and the volume made up to the mark with methanol 50%. The resulting stock solution was diluted with methanol in order to obtain reference solutions containing 1, 2.5, 5, 10, 15, 25, 35, 50, 75, 100, 125 and 150 mg ml 1 of external standards. The calibration curves, for each compound, were made by linear regression by plotting the peak area of external standard against their known concentrations (Table 2). The result represents the average of curves performed by three injections of each concentration. All quantitative data were elaborated with the aid of Analyst software (Applied Biosystems).

Table 1. LC–MS/MS conditions for quantitation of glucosinolates by negative ion MRM Compound

Glucoiberin Glucocheirolin Glucoraphanin Progoitrin Sinigrin Glucoalysin Gluconapin Glucoiberverin 4-Hydroxyglucobrassicin Glucoerucin Glucobrassicin 4-Methoxyglucobrassicin Gluconapoleiferin Neoglucobrassicin

tR (min) 5.7 6.4 6.5 6.8 7.7 8.8 13.7 18.3 20.2 22.9 25.2 27.5 29.0 30.5






Collision Cell

ion [M-H]-

ion [A-H]-




Exit Potential

422 438 436 388 358 450 372 406 463 420 447 477 402 477

358 358 178 195 195 386 195 195 267 178 259 259 195 446

48.5 58 51 36 50 52 44 52 52 52 52 52 52 52

4 6 5 5 3 4.6 3 4.6 4.6 4.2 4.6 4.6 4.6 4.6

26 27 37.8 34 24 30 27 30 30 31.4 30 30 30 30

11.6 7 4 5 2 4 5 4 4 1 4 4 4 4

Table 2. Linearity, LOQ and LOD of LC-ESI-QqQ-MS/MS MRM method for the analysis of standard compounds Compound


Glucoiberin Glucocheirolin Glucoraphanin Progoitrin Sinigrin Gluconapin Glucoerucin Glucobrassicin

Calibration curve equation


LOQ (mg/100g fw)

LOD (mg/100g fw)

y = 2.03*105x-8.74*104 y = 7.61*103x-2.27*103 y = 1.92*105x-5.24*105 y = 6.66*103x-3.44*103 y = 6.63*104x-2.95*104 y = 4.98*104x-3.33*104 y = 7.45*104x-6.85*104 y = 5.34*104x-2.93*104

0.997 0.998 0.992 0.997 0.999 0.997 0.997 0.997

0.003 0.020 0.031 0.006 0.003 0.022 0.025 0.026

0.001 0.008 0.003 0.001 0.002 0.005 0.002 0.003


Copyright © 2012 John Wiley & Sons, Ltd.

J. Mass Spectrom. 2012, 47, 1198–1206

LC–MS determination of glucosinoloma Table 3. Accuracy and precision of eight analytes at three concentration levels Compound

Concentration (mg/ml)

Accuracy (% recovery)

Precision Intra-day (CV%)

Precision Inter-day (CV%)


2.5 10 50 2.5 10 50 5 50 100 2.5 10 50 2.5 10 50 2.5 10 50 2.5 10 50 2.5 10 50

112 117 83 92 119 90 101 99 89 118 99 94 105 99 90 100 96 96 112 87 102 96 98 95

2.5 0.5 0.3 5.9 3.0 2.1 3.9 2.4 0.5 0.6 4.3 1.9 1.5 1.4 2.3 2.2 2.8 1.1 2.1 2.8 1.2 2.3 3.9 0.9

5.9 6.1 2.1 5.2 5.7 2.4 2.5 5.2 8.2 4.0 4.4 4.1 6.8 3.6 5.7 5.8 2.8 2.1 6.1 2.9 3.1 7.4 5.3 1.2








Precision and accuracy were evaluated at three concentration levels for each compound through triplicate intra-day assays and inter-day assays over 3 days

quantitative method), defined as the lowest concentration of analyte that could be quantified with acceptable accuracy and precision, was estimated by injecting a series of increasingly dilute standard solutions until the signal-to-noise ratio was reduced to 10. The LOD (equivalent to sensitivity of the qualitative method), defined as the lowest concentration of analyte that could be detected, was estimated by injecting a series of increasingly dilute standard solutions until the signal-to-noise ratio was reduced to 2. Linearity (calibration curves equations and regression), together with LOQ and LOD for each of the eight compounds analyzed, are reported in Table 2. Statistical analysis Data are expressed as mean  standard deviation and analyzed by two-way ANOVA (Kaleidagraph software version 3.6; Synergy Software, Reading, PA).

Results and discussion

J. Mass Spectrom. 2012, 47, 1198–1206

Copyright © 2012 John Wiley & Sons, Ltd.



The development of a suitable procedure for a rapid screening of ‘glucosinoloma’ profiles was achieved by a two-step procedure. First, we performed a qualitative and fast analysis in which extracts were infused directly into ESI source of the mass spectrometer and spectra were acquired using both Product Ion and Enhanced Product Ion Scan modes. In order to optimise MS conditions, a standard solution of glucoraphanin (1 mg ml 1) was infused as described in the experimental section.

Analyses of glucosinolates were performed in negative ion mode since it has been demonstrated for this class of compounds that negative ionization is more sensitive and selective than the positive one, because of the sulfate moiety in their molecular structure.[30] Glucosinolates-containing extracts gave very clear spectra with strong peaks generating a spectrum view of the parents ions detected. ESI-MS fingerprints obtained for broccoli sprouts grown under continuous light or dark conditions (Fig. 1) suggested the presence of the following 13 glucosinolates: m/z 477 4-methoxyglucobrassicin and/or neoglucobrassicin, m/z 463 4-hydorxyglucbrassicin, m/z 447 glucobrassicin, m/z 438 glucocheirolin, m/z 436 glucoraphanin, m/z 422 glucoiberin, m/z 420 glucoerucin, m/z 406 glucoiberverin, m/z 402 gluconapoleiferin, m/z 388 progoitrin/epiprogoitrin, m/z 372 gluconapin, m/z 358 sinigrin. As a first step, the identity of the revealed glucosinolates was verified by the comparison of the MS2 spectra recorded for each compound with those of the standards and/or with those reported in literature.[31,32] Then, an opportune IDA method with EMS survey scans, ER and EPI scans was developed to clearly identify the glucosinolates by comparison of both their MS2 and retention times with those observed for the analytical standards in LC-ESI-MS/MS analyses (data not shown). LC-ESI-MS/MS analyses allowed, also, to evidence the presence of another glucosinolate at m/z value of 450. MS2 spectra and retention time reported in literature suggested that this compound was glucoalysin.[11,33] Thus, this preliminary analysis allowed to identify 14 glucosinolates (Fig. 2) in broccoli sprouts. As observed in Fig. 1, ESI-MS fingerprints do not show qualitative differences

M. Maldini et al.

Figure 1. FIA-ESI-MS (Q1) (negative ion mode) fingerprints of extracts of broccoli sprouts grown under continuous light or dark conditions.


between broccoli sprouts samples grown under continuous light or dark conditions. In the second step of the study, the variation of glucosinolates content among broccoli sprouts was monitored by using MS/MS, coupled with HPLC. The quantitative analysis was performed using MRM. Fragmentation patterns were studied in ESI-MS/MS spectra with the aim to select a peculiar and specific transition from parent ion to daughter ion (Fig. 3 and Supplementary Fig. S1). Previous studies using negative ion MS/MS showed that MS/MS of deprotonated molecule [M-H]- of intact glucosinolates produce characteristic fragments at m/z values of 275, 259, 241, 195 and 97, and the majority of quantitative assays developed are based on monitoring the diagnostic ions at m/z values of 259, due to a sulfated glucose moiety,[32] and at 97 corresponding to the [SO4H]- ion.[14,34–37] The MS/MS spectra of 4-methoxyglucobrassicin and glucobrassicin showed the most intense peak at specific product ion at m/z value of 259. The MS/MS spectra of glucoiberverin, gluconapolieiferin, progoitrin, gluconapin and sinigrin showed the most intense ion peak at m/z value of 195 due to the thioglucose anion. The MS/MS spectra of glucoraphanin and glucoerucin showed a major ion peak at m/z value of 178, consequence of the combined loss of sulfur trioxide and the neutral loss of glucose unit. The MS/MS spectra of glucoiberin (Fig. 3A) and glucoalysin (Fig. 3B) showed a major product ion at m/z value of 358 and 386, respectively, due to the neutral loss of the CH3SOH. The MS/MS spectrum of glucocheirolin (Fig. 3C) showed a major product ion at m/z value of 358 ascribable to neutral loss of the SO3 group. The MS/MS spectrum of neoglucobrassicin


showed a product ion at m/z value of 446, not detected in that of 4-methoxyglucobrassicin, corresponding to the loss of methoxy radical. The MS/MS spectrum of 4-hydroxyglucobrassicin (Fig. 3D) showed a specific product ion at m/z value of 267 due to the loss of thioglucose. As a result, an MRM method was developed. The calibration curves obtained by plotting the area of ES against known concentration of compounds were linear in the range of 1–150 mg ml 1 (Table 2). Figure 4 shows representative MRM analysis of glucosinolates in broccoli sprouts grown under light conditions. The chromatographic profile contained all the peaks corresponding to the compounds under investigation, with appreciable intensity for quantitative purpose. The method based on the characteristic fragmentation reactions of glucosinolates was highly specific with no other peaks interfering at the retention times of the marker compounds in the MRM chromatograms and allowed the simultaneous determination of 14 compounds with high sensitivity and selectivity. Retention times and transitions for analyzed compounds are reported in Table 1. The MRM method was used to study the influence of light regime and developmental stage on the ‘glucosinoloma’ of broccoli sprouts (Table 4). Glucoraphanin was the most predominant glucosinolate in broccoli sprouts, accounting for about 50% of total glucosinolate content, followed by glucoiberin (9%), glucoerucin (8%), 4-hydroxyglucobrassicin (7%), gluconapoleiferin (6%) and 4-metoxyglucobrassicin (4%) (the remaining glucosinolates account for about 2%).

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J. Mass Spectrom. 2012, 47, 1198–1206

LC–MS determination of glucosinoloma

Figure 2. Molecular structure of glucosinolates.

Among glucosinolates, just 4-hydroxyglucobrassicin, glucoraphanin, glucoiberin, glucoerucin and glucoiberverin were not significantly affected by light regime. Generally, broccoli sprouts grown under light (continuous and L/D cycle) showed higher glucosinolates levels than those grown in the dark. Moreover, all glucosinolates (excluding gluconapin, sinigrin and glucoalysin) were strongly affected by seedling age. In fact, the total glucosinolates amount dropped dramatically after seed germination both in the light (continuous and L/D cycle) and dark condition. Just one glucosinolate (4-methoxyglucobrassicin) shows significant interaction between LD condition and seeding age (p = 0.007); this means that for almost all glucosinolates, different light conditions are not able to influence the age-induced glucosinolates decrease.


J. Mass Spectrom. 2012, 47, 1198–1206

Copyright © 2012 John Wiley & Sons, Ltd.



In the present study, ESI-MS and ESI-MS/MS fingerprint techniques were used for a rapid determination of the ‘glucosinoloma’

profile in broccoli sprouts samples, as a profile of the samples can be obtained in a few minutes. It represents a quick and simple method for the analysis of glucosinolates requiring very little purification. LC-ESI MS/MS method, based on MRM technique was developed for the determination of intact glucosinolates in broccoli sprouts extracts. MRM detection allows direct and simultaneous quantification of 14 glucosinolates with improved sensitivity and selectivity. The method was validated according to EMEA Quality guidelines and found to be accurate, selective and precise in the applied range of concentration. The results demonstrate that the proposed method can determine and discriminate the glucosinolates content in different broccoli sprouts samples. Quantitative results on broccoli sprouts evidenced differences in the glucosinolates content: broccoli sprouts grown in light conditions showed higher glucosinolates level than sprouts grown in the dark, and 10-days old broccoli sprouts showed lower glucosinolates level than 3-days broccoli sprouts grown both in light and dark conditions.

M. Maldini et al.


Figure 3. MS/MS spectra of glucosinolates identified in extracts of broccoli sprouts. A: Glucoiberin, B: Glucoalysin, C: Glucocheirolin, D: 4-Hydroxyglucobrassicin.


Figure 4. LC/ESI(QqQ)/MS/MS XICs (extracted ion chromatograms) of MRM analysis of a sample of broccoli sprouts.

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J. Mass Spectrom. 2012, 47, 1198–1206

Copyright © 2012 John Wiley & Sons, Ltd.




Continuous Light

64 11 47  22 11 11 11 11 11 11 21 11 11 21 63 67  33




Light/Dark cycle 10d

40  16 24  4 15  5 14  3 82 61 51 51 207  85 116  47 75  45 76  45 18  5 13  2 10  4 10  4 93 84 75 75 72 62 62 62 93 84 74 74 71 51 41 51 34  27 12  1 73 73 45  34 16  9 83 12  9 10  1 73 64 64 17  4 93 73 91 30  7 16  2 11  3 12  2 83 83 10  2 11  1 449  173 255  41 181  33 187  34


Each data is the mean of three replicates per treatment and time point (meanSD). ns = not significant by two-way ANOVA. a =quantified as equivalent of glucoraphanin b =quantified as equivalent of glucoerucin c =quantified as equivalent of glucobrassicin d =quantified as equivalent of progoitrin

Glucoiberin 45  11 18  3 14  6 Glucocheirolin 62 21 21 Glucoraphanin 293  49 108  12 91  26 Progoitrin 13  1 31 31 Sinigrin 21 11 11 Glucoalysina 21 11 11 Gluconapin 11 11 11 Glucoiberverinb 51 11 11 4-hydroxyglucobrassicinc 33  17 63 11 Glucoerucin 38  1 94 62 Glucobrassicin 51 11 11 4-methoxyglucobrassicinc 20  8 2  12 21 Gluconapoleiferind 29  2 93 51 Neoglucobrassicinc 21 31 41 Total 497  83 164  15 131  41


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16  12 97 32 22 99  65 71  44 32 21 11 11 34 34 11 11 22 11 32 22 65 55 11 11 22 11 43 32 64 64 148  100 108  73


35  22 18  13 54 32 206  125 107  62 11  8 32 22 11 44 33 22 11 43 21 19  15 43 27  18 85 22 11 64 11 18  12 53 32 32 344  217 160  94


Table 4. Effect of light regime and seedling age on total and individual glucosinolates content (mg/100g ‘fresh’ weight) of Broccoli sprouts

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