Anal Bioanal Chem (2009) 394:1661–1669 DOI 10.1007/s00216-009-2823-8
ORIGINAL PAPER
Identification and quantification of glucosinolates in rapeseed using liquid chromatography–ion trap mass spectrometry Silvia Millán & M. Carmen Sampedro & Patricia Gallejones & Ander Castellón & Maria L. Ibargoitia & M. Aranzazu Goicolea & Ramón J. Barrio
Received: 27 February 2009 / Revised: 20 April 2009 / Accepted: 22 April 2009 / Published online: 12 May 2009 # Springer-Verlag 2009
Abstract A rapid and sensitive method for the speciation and quantification of glucosinolates in rapeseed is described. The method combines liquid chromatography (LC) with ion trap mass spectrometry (ITMS) detection. Electrospray ionization (ESI) has been chosen as the ionization technique for the online coupling of LC with ITMS. Glucosinolates are extracted from different rapeseeds with MeOH and the extracts are cleaned-up by solid phase extraction with Florisil cartridges. Aqueous extracts are injected into LC system coupled to an ITMS, leading to accurately quantify eight of the most important glucosinolates in rapeseed, by MS2 mode and confirming their structure by MS3 acquisition. All the glucosinolates found in rapeseeds provide good signals corresponding to the deprotonated precursor ion [M-H]−. The method is reliable and reproducible, and detection limits range from 0.5 nmol g−1 to 3.7 nmol g−1 when 200 mg of dried seeds of certified reference material are analyzed. Within-day and between-day RSD percentages range between 2.4–14.1% and 3.9–16.9%, respectively. The LC-ESI-ITMS-MS method described here allows for a rapid assessment of these metabolites in rapeseed without a desulfatation step. The overall process has been successfully applied to identify and quantify glucosinolates in rapeseed samples.
S. Millán : M. C. Sampedro : M. A. Goicolea : R. J. Barrio (*) Department of Analytical Chemistry, Faculty of Pharmacy, University of the Basque Country, 01006 Vitoria, Spain e-mail:
[email protected] P. Gallejones : A. Castellón : M. L. Ibargoitia Neiker Tecnalia, c/ Berreaga, 1, 48160 Derio, Biscay, Spain
Keywords Oily seeds . Glucosinolates . Liquid chromatography . Ion trap mass spectrometry
Introduction Rape is extensively cultivated as a valuable source of edible oil, the seed having an oil content of about 40%. Furthermore, the cake which remains after the oil is expelled from seed is widely used as feed for farm animals and poultry. Although it is an important source of protein and fiber, the presence of a number of antinutritional factors [1, 2] the most important of which are glucosinolates, limit their use. Not only do these compounds reduce animal intake (because they reduce the palatability of the cake), but they also interfere with the thyroid function, damage vital organs or interfere with metabolic processes [3]. Therefore there is a move towards rapeseed that is low in glucosinolates and this has prompted the need for modern analytical methods for the rapid identification and assessment of glucosinolates in rapeseed. The general structure of glucosinolates is characterized by a β-D-thioglucose group, a sulfonated oxime moiety and a variable side-chain derived from methionine, tryptophan or phenylalanine [4]: aliphatic glucosinolates derived from mainly methionine, but also alanine, leucine or valine. Indolic glucosinolates are derived from tryptophan, while aromatic ones come mainly from phenylalanine and tyrosine. More than 120 glucosinolates are known to occur naturally [5] with different side-chains (aliphatic, aromatic or indolic) which result in a wide range of biological activity and polarity of these compounds. Glucosinolates are hydrolyzed by endogenous thioglucosidases, called myrosinases, to produce a wide range of degradation products (isothiocyanates, nitriles,
10
1.20 1.20
10 10
1.30 1.30
10 10
1.05 1.20
10 10
1.30 1.30
10
1.20
10 Frag. width (m/z)
4.0
100–450
114 121 110 104 125 100 109 105 105
4.0
100–470 90–430
4.0 4.0
90–400 100–500
4.0 4.0
90–400 90–420
4.0 4.0 4.0
100
100 100
100 100
100 100
100 100
100 100
100 100
100 100
422
259 259
447 408
259 259
386 463
285 259
372 402
259
15.05–16.5
15.6±0.1 14.9±0.1
14–15.05 11–14
12.8±0.1 9.6±0.2
8.4–11 6.0–8.4
8.3±0.2 4.9±0.1
4.4–6.0
259
17.9±0.1
16.5–19
NAS GBC TROP GBN 4OH GNA GNL EPRO
388 388
Frag. ampl. (V)
23.25±0.50
Cut-off (m/z)
total GSL
90–400
0.51±0.08
90–400
0.20±0.01
Gluconasturtin
Scan (m/z)
Glucobrassicin
NAS
Isol. width (m/z)
GBC
100
1.37±0.03
100
3.96±0.23
Glucobrassicanapin
100
4-hidroxiglucobrassicin
GBN
Trap drive level (%)
4OH
Compound stability (%)
3.89±0.08
259
Gluconapin
To waste
0.53±0.04
GNA
0.24±0.02
Product ion (m/z)
Gluconapoleiferin
Target mass (m/z)
GNL
4.0±0.2
12.56±0.25
3.5–4.4
Epiprogoitrin
0–3.5
Progoitrin
EPRO
Time segment (min)
PRO
Retention time (min)
mmol kg−1
BCR-190R
PRO
Table 1 Individual and total GSL contents in mmol kg−1 in the BCR190R certified reference material (200 mg)
Table 2 Data acquisition parameters and MS2 transitions used in LC/MSMS for the quantification of glucosinolates
epithionitriles, oxazolidine-2-thiones, and thiocyanates) with diverse biological activities [6, 7]. Several methods have been applied for the determination of total glucosinolates including gas chromatography [8, 9], X-ray fluorescence [10], colorimetric method [11], capillary electrophoresis [12, 13], amperometric flow analyser, [14] and high performance liquid chromatography [15, 16]. In 1992 the International Organization for Standardization (ISO) published an official method [17] for their determination and quantification involving HPLC of desulfoglucosinolates obtained after enzymatic desulfatation. However, the sample preparation required before chromatographic analysis was complex and tedious. Some methods currently feature in the published bibliography which determine intact glucosinolates by HPLC-UV [18, 19] or LC-MS [20–22] with different types of analyzers eliminating the drawbacks mentioned above, but they are basically semiquantitative methods. To date, LC/MS-MS analyses of glucosinolates have been carried out on a quadrupole ion trap [23], quadrupole time-of-flight [24] or on triple-quadrupole mass spectrometers [25]. Direct analysis of intact glucosinolates is important, because it can reflect the specificity of each glucosinolates. Therefore, in this paper, we offer an alternative for simultaneous identification/confirmation and quantification in a single analysis of eight of the predominant intact glucosinolates in rapeseed: progoitrin (PRO), epiprogoitrin (EPRO), gluconapoleiferin (GNL), gluconapin (GNA), 4-hydroxyglucobrassicin (4OH), glucobrassicanapin (GBN), glucobrassicin (GBC), and gluconasturtiin (NAS) using an ion trap mass spectrometer which enables MS n experiments. It is difficult and timeconsuming to identify and accurately quantify several components such as glucosinolates in complex sample matrices. To date, most of the applied methods with this purpose tend to carry out purification and desulfation processes [26, 27]. Measurement of desulfoglucosinolates
20.0±0.2
S. Millán et al.
19–21
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Glucosinolates in rapeseed
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Fig. 1 Typical LC-ITMS chromatogram of glucosinolates in an extract of rapeseed CRM (ERM-BC190). Peak numbers correspond to (1) PRO, (2) EPRO, (3) GNL, (4) GNA, (5) 4OH, (6) GBN, (7) TROP (internal standard), (8) GBC and (9) NAS. The ions monitored are displayed in the right side of each trace
do not require the use of ion-pair reagents but the time required for analysis is significantly increased because of additional sample processing, including extraction, binding to Sephadex A-25, enzymatic desulfation, and finally elution of the desulfoglucosinolates. The aim of our work is therefore to develop an accurate and sensitive method for the determination of these compounds in different rapeseed samples based on LCMS-MS technique without any desulfation step. The combination of HPLC and ion trap mass spectrometry has provided a good solution for the accomplishment of a secure identification of the target compounds. ESI is a powerful tool for identifying highly polar, heat-labile
compounds such as glucosinolates, present as ions in solution. Therefore, due to the polar nature of the target compounds, ESI was chosen as the ionization technique for the on-line coupling of LC with MS, and was found to be particularly sensitive.
Experimental Chemicals and reagents Certified reference materials (ERM-BC366, ERM-BC190, ERM-BC367) from the European Community Bureau of
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Table 3 Linearity and detection limits of LC-ESI-ITMS-MS method for rapeseed certified reference material BCR-190R Compound
Regression equation
Progoitrin
ye ¼ e0:162x þ 0:066 ye ¼ e0:099x 0:000 ye ¼ e0:235x 0:000 ye ¼ e0:549x 0:033 ye ¼ e0:232x 0:027 ye ¼ e0:930x 0:029 ye ¼ e0:068x þ 0:002 ye ¼ e0:133x 0:000
Epiprogoitrin Gluconapoleiferin Gluconapin 4-hidroxiglucobrassicin Glucobrassicanapin Glucobrassicin Gluconasturtin
Regression coefficient
LOQs (µ mol g−1) MS/MS mode
LODs (µ mol g−1) MS/MS mode
0.9950
0.0044
0.0013
0.9979
0.0018
0.0005
0.9983
0.0038
0.0011
0.9949
0.0123
0.0037
0.9953
0.0025
0.0008
0.9945
0.0043
0.0013
0.9916
0.0020
0.0006
0.9992
0.0048
0.0014
y: ratio of the peak area of glucosinolates to the peak area of the surrogate standard; x: glucosinolates to surrogate concentration ratio.
Reference (BCR, Brussels, Belgium) were purchased from LGC Standards (Wesel, Germany) in an aluminum plasticlaminated sachet sealed under nitrogen. For the analysis of glucosinolates, certified reference material (CRM) with a medium content of total glucosinolates (ERM–BC190), which is 23 mmol kg−1, with certified individual glucosinolate content was used to construct the calibration curves. Commercially available glucotropaeolin (TROP) as potassium salt from ChromaDex Inc. (Santa Ana, CA, USA) was used as an internal standard. The gradient HPLC grade organic solvents methanol, dichloromethane, n-hexane, and ethyl acetate were purchased from Scharlau Chemie SA (Barcelona, Spain). Ammonium acetate was obtained from Panreac (Barcelona, Spain) and formic acid 99% was purchased from Across Organics (New Jersey, USA). The ultra-high-purity water (UHP) was prepared from tap water pre-treated by reverse osmosis (Elix, Millipore, Bedford, MA, USA) prior to filtration by a Millipore Milli-Q system. For the clean-up process Bond elute Florisil (1 g, 6 ml) cartridges were purchased from Varian, Inc. (Palo Alto, CA, USA) and all the aqueous extracts were filtered prior to injection into LC-ITMS system with Nylon syringe filters 13 mm, 0.45 μm from Scharlau Chemie S.A. (Barcelona, Spain). Preparation of standards and samples Prior to use, seeds were dried overnight in an oven at 60°C. The dried seeds (1 g) were crushed with a mortar to a fine powder and an aliquot of 200 mg transferred to screwcapped centrifuge tubes. Immediately after crushing, 2 ml of methanol at 70°C was added into each tube and then 20 μl of 10 mg ml−1 (22.3 mmol l−1) TROP solution in methanol was spiked to the matrix and samples, while being kept in magnetic agitation for 15 min and then centrifuged at 4,400 rpm for 5 min. The extraction process was repeated twice and the supernatants were finally
collected and were evaporated to dryness under a stream of nitrogen. It was reconstituted with 500 μl of methanol. A Florisil cartridge was activated before use with 5 ml 30% (v/v) dichloromethane in hexane. The supernatant (500 μl) was mixed with 5 ml 30% (v/v) dichloromethane in hexane and applied to the cartridge. Interferences were washed with 5 ml 30% (v/v) dichloromethane in hexane and the cartridge was aspirated to dryness. The glucosinolates were then eluted with 5 ml 30% (v/v) ethyl acetate in methanol. The extract was evaporated again to dryness and reconstituted with 500 μl of Milli-Q water. The final extract was filtered through Nylon 0.45 μm filters prior to its injection in the LC-ITMS system. Samples were prepared to developed calibrations from seeds with certified individual glucosinolate content (certified reference material ERM-BC190, Table 1). Seven aliquots of 200 mg of the CRM were extracted as described above (corresponding to seven levels of calibration), and the volumes of the final extracts were reconstituted in the range of 0.1–10 ml. The values were converted into mmol l−1 of the sample extracts and the calibration curves were constructed. Chromatographic conditions A liquid chromatograph model Agilent 1100 series was equipped with a binary pump, vacuum degasser, an autosampler, and a thermostatted column compartment. The analytes were separated by reversed-phase LC injecting 20 μl aliquot of glucosinolates extract into a Tracer Extrasil ODS2 column (25 cm×4.6 mm, 5 μm) heated at 25°C. The mobile phase was prepared from 30 mmol l−1 ammonium acetate adjusted with formic acid at pH 5.0 (component A) and methanol (component B). All solutions were filtered through 0.22-µm filters and sonicated before their use. The gradient program was: 100%A–0%B for 5 min; increased to 80%A–20%B from 5 to 17 min; and achieving initial conditions at 20 min. The flow rate was held constant at 0.9 ml min−1, and after each
Glucosinolates in rapeseed
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Fig. 2 MS/MS product ion spectra of 1. PRO, 2. EPRO, 3. GNL, 4. GNA, 5. 4OH, 6. GBN, 7. TROP (internal standard), 8. GBC, 9. NAS
run the system allowed to equilibrate. Under these chromatographic conditions progoitrin (PRO), epiprogoitrin (EPRO), gluconapoleiferin (GNL), gluconapin (GNA), 4-hydroxyglucobrassicin (4OH), glucobrassicanapin (GBN), glucotropaeolin (TROP), glucobrassicin (GBC), and gluconasturtiin (NAS) were eluted at retention times of 4.0±0.2, 4.9±0.1, 8.3±0.2, 9.6±0.2, 12.8±0.1, 14.9±0.1, 15.6±0.1, 17.9±0.1, and 20.0±0.2 min, respectively. Ion trap MS analysis The MS analyses of glucosinolates were carried out on an MS n system consisting of a MSD Trap XCT Plus
spectrometer (Agilent Technologies, Palo Alto, CA, USA), equipped with a G1948A ESI source. System control and data analysis was provided by the Agilent LC Chemstation and by Bruker Daltonics Trap Control and QuantAnalysis. The ESI source was used and operated in negative ionization mode. Typical operating conditions were as follows: drying gas (N2) temperature of 365°C, 12 L min−1 drying gas flow, 50 psi nebulizer gas (N2) pressure, and 3,000 V of capillary voltage. Data were acquired with a smart target of 70,000 and a max accumulation time of 200 ms. First, full-scan MS spectra were obtained by scanning from 50–500m/z. Then, the chromatogram was divided into ten time segments as
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Fig. 3 MS3 spectra of 1. PRO, 2. EPRO, 3. GNL, 4. GNA, 5. 4OH, 6. GBN, 7. TROP (internal standard), 8. GBC, 9. NAS
shown in Table 2, and MS2 acquisition of the most abundant ions in the full-scan MS mode was carried out. Finally, MS3 acquisition was used to confirm the identity of the analytes in the rapeseeds.
Results and discussion Initially, the composition of the mobile phase was the primary target for a correct separation and ionization of the analytes. The use of a volatile buffer, compatible with an LC-MS system, increased the retention of the glucosinolates on the column and provided a good separation and
resolution of the compounds in less than 25 min. A gradient method using aqueous and methanol channels, the first one containing 30 mM ammonium acetate adjusted with formic acid at pH5, was found to give the best separation, a good chromatography peak shape and sensitivity for this analysis (Fig. 1). The glucosinolates were successfully separated and quantified by LC-ESI-ITMS-MS using internal standard calibration. Glucotropaeolin (TROP) formulated as potassium salt was chosen as a suitable internal standard because of its structural similarity to other analytes and due to the lack of deuterated standards. Otherwise, it has a retention time that does not correspond to the other eluted components and it does not occur naturally in oily seeds. On the
Glucosinolates in rapeseed
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Table 4 Quantification of glucosinolates in rapeseed samples (n=3) Sample
Glucosinolates (µ mol g−1) PRO
EPRO
GNL
GNA
4OH
GBN
GBC
NAS
Total
1
5.08±0.97
0.07±0.03
0.46±0.18
2.14±0.61
5.72±0.56
1.18±0.40
0.40±0.15
0.61±0.10
15.66±0.79
2
2.96±0.09
0.05±0.02
0.29±0.01
1.13±0.07
3.02±0.57
0.59±0.02
0.21±0.02
0.49±0.07
8.74±0.34
3
4.90±0.77
0.11±0.05
0.47±0.22
2.59±0.60
5.01±0.32
0.98±0.32
0.30±0.10
0.54±0.12
14.90±0.64
4
5.74±0.92
0.06±0.02
0.36±0.02
1.91±0.15
5.85±0.45
0.87±0.02
0.18±0.02
0.37±0.17
15.34±0.61
5
3.59±0.19
0.05±0.02
0.32±0.07
0.71±0.02
6.16±0.40
0.52±0.17
0.15±0.07
0.29±0.01
11.80±0.28
6
1.38±0.20
0.02±0.01
0.11±0.01
0.25±0.02
4.23±0.04
0.24±0.02
0.09±0.01
0.19±0.04
6.49±0.12
7
1.90±0.01
0.03±0.01
0.19±0.02
0.65±0.04
3.08±0.10
0.59±0.02
0.11±0.01
0.38±0.02
6.93±0.07
8
1.28±0.15
0.03±0.02
0.14±0.04
0.68±0.04
0.65±0.02
0.45±0.02
0.14±0.01
0.23±0.02
3.59±0.10
9
0.31±0.02
0.02±0.01
0.14±0.02
0.25±0.02
0.30±0.04
0.46±0.12
0.01±0.01
0.34±0.01
1.83±0.08
10
2.24±0.04
0.03±0.01
0.27±0.07
0.84±0.10
0.83±0.10
0.60±0.02
0.10±0.04
0.30±0.01
5.22±0.10
other hand, the coelution with other components is not important since none of the studied glucosinolates display the same molecular mass, i.e. quasimolecular ion, as glucotropaeolin. The chromatograms were segmented into nine windows to select the optimum parameters for each compound and to enhance sensitivity. Flow was diverted to waste initially and data acquisition triggered 3.50 minutes after the run begun, with the aim of reducing the contamination of the system. Identification of the major glucosinolates was established through the use of negative ion electrospray MS2 mode with secondary confirmation by MS3 acquisition. The presence of a sulfonate moiety categorizes glucosinolates as hydrophilic compounds, which occur in nature in the anionic form. Consecutively, they are readily ionized in ESI mode, forming deprotonated molecular ions, [M-H]−, which were selected to generate MSn spectra. As mentioned above, once the [M-H]− ion has been identified by negative ion ESI-ITMS, the strategy to confirm the identity of unknown glucosinolates involves examination of their fragmentation behavior by multistage MS. The MS/MS fragmentation enables structure elucidation of the glucosinolates. The optimum fragmentation amplitude for each analyte was determined infusing the extract of the certified reference material isolating each compound and increasing the fragmentation amplitude until the precursor ion intensity was reduced to 5–15% of its major product ion response. The cut-off values (the minimal value of m/z ratio, so that the ions with smaller values than these quantities are not trapped by the IT) were set to the default value (27%) from the precursor ions m/z ratio. The observed fragment ion at m/z 259 was formed through rearrangements and loss of R-CNS [28] from the glucosinolates molecule [M-H-R-CNS]− for the majority of the analytes except for 4OH, the main product ions of
which resulted in m/z 267 and 285m/z which were due to the loss of (C6H10O5–H2S) and (C6H10O5–OH) from the precursor [M-H]− ion, respectively. The most abundant product ions (259m/z for PRO, EPRO, GNL, GNA, GBN, TROP, GBC, NAS, and 285m/z for 4OH) were chosen for the quantitative analyses, although other characteristic ions can be observed in the MS/MS spectra (Table 3). The ions with m/z 195 [C6H11O5S]−, m/z 259 [M-H-R-CNS]−, and m/z 275 [M-H-R-CNO]−, which correspond to the fragment ions from the glycone side chain, were found in all the examined glucosinolates as they share a common structure (Fig. 2). The MS3 spectrum from m/z 259 and 285 ions, selected as precursors, displayed a common fragment ion m/z 97 corresponding to the sulfate group HSO4−. Additional typical ions are m/z 139, 169, 199, and 241 for all compounds except for 4OH. The ions m/z 139, m/z 169, and m/z 199 obtained from the m/z 259 fragment ion correspond to a loss of C4H8O4, C3H6O3, and C2H4O2 from the sugar, respectively. The latter ion m/z 241 was identified as the fragment C6H9O8S− (Fig. 3). Therefore, the fragment product ions mentioned above were considered as diagnostic product ions of the common moiety of glucosinolates. Evaluation of the analytical method Evaluation of the analytical method was carried out with aqueous extracts at different dilutions of rapeseed certified reference material (BCR-190R), resulting in good chromatographic profiles. Glucotropaeolin (TROP) was selected as surrogate standard and calibration curves were generated by plotting the ratio of the peak area of glucosinolates to that of the surrogate standard as a function of their concentration ratios. Seven-point calibration plots were applied over the following ranges: from 0.15–7.7 mmol
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l−1 for PRO, 0.003–0.15 mmol l−1 for EPRO, 0.006– 0.33 mmol l−1 for GNL, 0.05–2.4 mmol l−1 for GNA and 4OH, 0.02–0.84 mmol l−1 for GBN, 0.002–0.12 mmol l−1 for GBC, and 0.006–0.31 mmol l−1 for NAS. The ion trap mass analyzer revealed a linear response in the selected ranges with good correlation coefficients (Table 3). The LC-ESI-ITMS-MS quantification and detection limits were estimated with extracts at different dilutions of rapeseed certified reference material ERM-BC366, ERMBC190, and ERM-BC367, with an increasing content of individual glucosinolates. The selected quantification method for real samples was MS/MS because of its high selectivity, which allows unequivocal identification of target compounds. Detection limits were calculated for a signal-to-noise ratio as 3 and ranged from 0.5 nmol g−1 to 3.7 nmol g−1 for dried seeds. Limits of quantification, calculated on the basis of a signalto-noise ratio of 10, ranged from 1.8 to 12.3 nmol g−1 (Table 4). To determine the precision of the LC-ESI-ITMS-MS method relative standard deviations were calculated on ten extracts of rapeseed CRM at two different levels of concentration under the selected conditions. The same samples were also analyzed over a period of five successive days to determine the inter-day RSD. In most cases, the RSD was often less than 10%: for low concentration (calibration level 1) varying from 2.4 to 14.1% for intraday and from 3.9 to 16.9% for inter-day precision and for high concentration (calibration level 6) the RSD varied from 1.6 to 10.9% for intra-day and from 6.3 to 10.7% for inter-day precision. Real samples analysis The developed method can provide comprehensive glucosinolates profiles and concentration from a single sample and simultaneously collect confirmatory spectra of each analyte identified. Concentrations of the glucosinolates in ten different samples of rapeseeds were quantified on the basis of internal standard calibration plots. The samples come from a field experiment where different N and S fertilizer doses were applied. Results for the quantification of glucosinolates in oily seeds show that eight glucosinolates of interest were found in complex matrix samples at different concentration levels, the most abundant being progoitrin (PRO), gluconapin (GNA), 4-hydroxiglucobrassicin (4OH), and glucobrassicanapin (GBN). Table 5 displays the results of quantitative determination of the most important glucosinolates in oily seeds in these representative samples. It is noted that the total glucosinolates content varied considerably for different samples. The highest concentration of total compounds was found to be 16.08 μmol g−1 and the lowest corresponded to 1.83 μmol g−1.
S. Millán et al.
Conclusions The results demonstrate that the proposed method can determine the total glucosinolate content in different rapeseed samples. Eight intact glucosinolates, progoitrin, epiprogoitrin, gluconapoleiferin, gluconapin, 4-hydroyglucobrassicin, glucobrassicanapin, glucobrassicin, and gluconasturtiin were detected and quantified by direct analysis LC-ESI-IT MS-MS in negative ion mode on the basis of a reproducible fragmentation of glucosinolates to a sulfated glucose anion, except for 4OH the characteristic fragment ion of which is probably formed by cleavage of thio-glucose after OHrearrangement from the sugar moiety [24]. The m/z 259 fragment ion is also formed for 4OH, but this pathway is not favored for this compound. Indeed, the fragmentation process described for 4OH occurs for the other compounds but the resulting fragment ion intensities are weak. Sensitivity of the method is sufficient for the quantification of the analytes in the real samples. Acknowledgements The authors would like to thank the Central Service of Analysis—Araba Campus of the University of the Basque Country (SGiker) for its excellent technical assistance.
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