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Profiling chicory sesquiterpene lactones by high resolution mass spectrometry
Food Research International 67 (2015) 193–198
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Profiling chicory sesquiterpene lactones by high resolution mass spectrometry Giulia Graziani a,⁎, Rosalia Ferracane a, Paolo Sambo b, Silvia Santagata b, Carlo Nicoletto b, Vincenzo Fogliano c a b c
Department of Agricultural and Food Science, University of Napoli “Federico II”, Parco Gussone, 80055 Portici, Napoli, Italy Department of Agronomy, Food, Natural resources, Animals and Environment (DAFNAE), Agripolis — University of Padova, Viale dell'Università 16, 35020 Legnaro, PD, Italy Food Quality & Design Group, Wageningen University, PO Box 8129, 6700 EV Wageningen, The Netherlands
a r t i c l e
i n f o
Article history: Received 31 July 2014 Accepted 13 November 2014 Available online 20 November 2014 Keywords: Chicory Radicchio Bitterness Sesquiterpene lactones High resolution mass spectrometry Tandem mass spectrometry
a b s t r a c t Sesquiterpene lactones (SLs) are the main determinants of radicchio bitterness and the control of their concentrations is a key point for the market value of this product. An innovative analytical approach based on two complementary mass spectrometers, Orbitrap-HRMS and MS/MS, was used for quantitative analysis of SLs in aerial part of four different varieties of chicory. Data highlighted the presence of eight SLs: 11β,13-dihydrolactucin, lactucin, 8-deoxy-lactucin, dihydro-8-deoxylactucin, dihydrolactucopicrin, lactucopicrin, lactuside C (jaquinellin glucoside) and dihydro-lactucopicrin oxalate. Significant varietal differences were found. The highest amount of SLs was found in the radicchio “Treviso Precoce” variety (189.71 μg/g), the lowest amount in “Treviso Tardivo” variety (45.78 μg/g). Lactucopicrin was the most abundant compound with concentration ranged between 99.36 in “Treviso Precoce” and 13.50 μg/g in “Treviso Tardivo” while dihydro-lactucopicrin oxalate was the less abundant in all analyzed varieties with an average concentration of about 1% on the total amount of SLs. Published by Elsevier Ltd.
1. Introduction Diet rich in vegetables and fruit has been linked with lower rates of cancer and coronary disease (Drewnowski & Gomez-Carneros, 2000; Ou, Huang, Hampsch-Woodill, Flanagan, & Deemer, 2002; Vinson, Hao, Su, & Zubik, 1998; Yan‐Hwa, Chang, & Hsu, 2000). Plant-based phenols, flavonoids, isoflavones, glucosinolates, terpenes and other compounds that are present in the everyday diet have antioxidant and anticarcinogenic properties and a wide spectrum of tumor-blocking activities (Drewnowski & Gomez-Carneros, 2000; Higdon, Delage, Williams, & Dashwood, 2007). These compounds are known as phytochemicals or phytonutrients and were often bitter, acrid or astringent (Craig, 1997; Drewnowski & Gomez-Carneros, 2000; Lesschaeve & Noble, 2005). Unfortunately, bitter taste lowered consumer acceptability despite the fact that bitter compounds were often potentially helpful to human health (Ames, Profet, & Gold, 1990). Bitter taste also posed considerable problems at industrial level because de-bittering processes are complex and can reduce the overall quality, especially for vegetable food (Rousseff, 1990; Roy, 1990). Therefore, the control and modulation of the bitter taste in food played a pivotal role in order to match health quality and marketing needs. Genetic improvement techniques or innovative farming practices can be used to handle the bitter taste (Martínez-Ballesta et al., 2008). However, rapid and precise analytical methods to identify and quantify the chemical components responsible ⁎ Corresponding author. Fax: +39 81 7762580. E-mail address: [email protected] (G. Graziani).
http://dx.doi.org/10.1016/j.foodres.2014.11.021 0963-9969/Published by Elsevier Ltd.
for the bitter taste are needed. In this framework, chicory species and in particular radicchio (Cichorium intybus L., group rubifolium) represented a peculiar case: in fact, they are well-known for their bitter taste which is differently desired for consumers. Radicchio is one of the most popular and expensive type of chicory also due to the smart red colored leaves, which makes it very eye-catching among leafy vegetables. The bitter taste of chicory originates from sesquiterpene lactones (SLs) mainly present in the latex (Poli et al., 2002). SLs are C-15 terpenoids which occur as hydrocarbons or in oxygenated forms such as alcohols, ketones, aldehydes, acids or lactones in nature. The principal SLs found in species of Lactuca and chicory were lactucin, lactucopicrin, 8-deoxylactucin and derivatives such as 11,13 dihydro-analogs. The SLs lactucin, 8-deoxylactucin, and lactucopicrin were also intensely bitter, so their presence within salad lettuce and chicory should be carefully controlled to avoid product depreciation (Poli et al., 2002; Price, Dupont, Shepherd, Chan, & Fenwick, 1990). Previous studies also reported the presence of SL glycosides such as 15-glycosyl conjugate of 11,13-dihydrolactucopicrin that has been identified in roots of Lactuca tatarica, Lactuca aculeata and C. intybus (Beharav et al., 2010; Kisiel & Zielińska, 2001; Kisiel & Barszcz, 1997). This glycoside and others such as picriside A (lactucin 15-glycoside) and crepidiaside A (8-deoxylactucin-15-glycoside), have also been identified in other members of the Lactuceae species (Adegawa et al., 1985; Nishimura et al., 1986). The presence of 15-oxalates and 8sulfates as additional major conjugates of lactucin, 8-deoxylactucin, and lactucopicrin in lettuce and chicory has also been reported by Sessa, Bennett, Lewisi, Mansfield, and Bealei (2000). The most frequently used
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analytical techniques for the separation and analysis of SLs are reversed-phase HPLC (RP-HPLC) with UV detection, supercritical fluid chromatography, micellar electrokinetic chromatography (MEKC), gas chromatography and thin-layer chromatography (Merfort, 2002). Among these, HPLC-UV, mainly on reversed phase HPLC was the most applied analytical method using mostly acetonitrile–water or methanol–water gradients, less numerous were data reporting SL analysis performed by mass spectrometry. Sessa et al. (2000) studied chicory SL profile by HPLC-UV and electrospray mass spectral data for each HPLC peak were obtained from analysis of components of individual peaks collected from preparative HPLC. Ferioli and D'Antuono (2012), reported the identification of chicory SLs by HPLC-mass spectrometry using a single quadrupole mass spectrometer and Selected Ion Monitoring (SIM) acquisition. The use of two complementary mass spectrometry techniques such as high resolution mass spectrometry (HRMS) and ion spray triple quadrupole could be very useful for qualitative analysis of target compounds and can be adopted for the identification and quantification of the maximum number of SLs. LC/MS/MS in MRM (Multiple Reaction Monitoring) acquisition allows one to obtain high specificity for each selected analyte, even in complex matrices. The measured compounds are limited to those targeted by MRM events programmed in the method. In addition, quantitative performance decreases with an increasing number of MRM scan events (Nagy, Redeuil, Bertholet, Steiling, & Kussmann, 2009; Sanchez-Rabaneda et al., 2003; Xie, Zhang, Kong, & Rexit, 2011). Moreover, with Orbitrap a generic full scan method can be used, looking for every compound in the selected scan range. Ion-specific chromatograms are generated for all observed ion signals, using a 5 ppm m/z window. The number of compounds that can be detected is virtually unlimited (Scigelova & Makarov, 2006). In this work, we adopted an innovative analytical technique based on combination of mass spectrometry (MS) techniques, liquid chromatography/electrospray ionization HRMS (LC/ESI-HRMS) and liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ ESI-MS/MS) on a triple quadrupole, for the identification and quantification of SLs in four different chicory varieties. 2. Materials and methods 2.1. Plant material and chemicals The marketable aerial part of four different varieties of chicory “Cicoria Catalogna” (C. intybus L. var dentatum), radicchio “Rosso di Chioggia”, radicchio “Rosso di Treviso Precoce” and radicchio “Rosso di Treviso Tardivo” (C. intybus L. Rubifolium Group) was considered. Plants were cultivated in Northern-East of Italy (Veneto Region), transplanted on loamy soil in the second half of July following normal agronomical practices. For each chicory type three plots 25 m2 (5 × 5 m) were singled out and 35 representative plants were uprooted from each of these at harvest time. The harvest was carried out at marketable maturity in November for “Cicoria Catalogna” and in December for radicchio cultivars. After harvest radicchio “Rosso di Treviso Tardivo” was subjected to the usual forcing process before commercialization as reported by Nicoletto and Pimpini (2009, 2010). For all varieties, the marketable material of each plot was cut in pieces, immediately frozen at − 80 °C overnight, freeze-dried and ground before extraction. All reagents and solvents of HPLC grade were purchased from Merck (Darmstadt, Germany). Santonin used to quantify SLs was obtained from Sigma (Milano, Italy). 2.2. Extraction of sesquiterpene lactones (SLs) 0.5 g of freeze-dried sample was exactly weighed in a screw-cap plastic centrifuge tube to which was added 100 μg of santonin (internal standard, methanolic solution) and 15 mL of 2% (v/v) formic acid in methanol/water 4/1 (v/v). The sample was then shaken on a vortex
stirrer for 1 min, sonicated at room temperature for 10 min and then centrifuged at 4000 g for 10 min. After collection of the supernatant fraction, the extraction procedure was repeated. The extracts were pooled, dried under reduced pressure at 35 °C, subjected to nitrogen flux for 5 min, and recovered with 2 mL of methanol. Before SPE, the SL fraction was centrifuged at 4000 g for 10 min to remove solid particles. SLs were purified from phenols and other interfering compounds by SPE, employing silica cartridges (3 mL reservoir, 500 mg sorbent mass). The cartridges were conditioned with 6 mL of dichloromethane/ isopropanol 1/1 (v/v), equilibrated with 6 mL of dichloromethane and, after sample loading, eluted with 6 mL of dichloromethane/ethyl acetate 3/2 (v/v). Both the loading and elution fractions were collected, dried under reduced pressure at 35 °C, recovered with 1 mL of methanol/water 1/1 (v/v), filtered in a HPLC glass vial through a nylon syringe filter (diameter: 13 mm; pore dimension: 0.45 μm) and stored at −18 °C until mass spectrometry analyses.
2.3. LC/MS/MS analysis Chromatographic separation was performed using an HPLC apparatus equipped with two micropumps Series 200 (Perkin Elmer, CanadaShelton, USA) and Luna C18, 250 × 4.6 mm, 5 μm (Phenomenex, Torrance, CA, USA) (Phenomenex, CA, USA). The solvent system consisted of (A) water and (B) acetonitrile. The gradient program was as follows: 10–42% B (30 min), 10% B (5 min), constant to B (5 min), at a constant flow of 1 mL/min. Injection volume was 20 μL. These conditions were also used for preparative HPLC. MS/MS analyses of SLs were performed on an API 3000 triple quadrupole mass spectrometer (Applied Biosystems, Canada) equipped with a Turbo Ion Spray source. The LC flow was split and 0.2 mL/min was sent to the mass spectrometer. The declustering potential (DP) and the collision energy (CE) were optimized for each compound infusing directly into the mass spectrometer standard solutions (10 μg/mL) at a constant flow rate of 10 μL/min using a model 11 syringe pump (Harvard Apparatus, Holliston, MA, USA). Analysis was performed in the positive and negative ion mode in MRM (Multiple Reaction Monitoring). Drying gas (air) was heated to 350 °C and the capillary voltage (IS) was set to + 5000 V in positive ion mode and −4000 V in negative ion mode. Data acquisition and processing were performed using Analyst software version 1.4.2.
2.4. High resolution mass spectrometry (HRMS) analysis LC–MS data were acquired on an Accela U-HPLC system coupled to an Exactive mass spectrometer (Thermo Fisher Scientific, San Jose, CA). The Accela system consisted of a quaternary pump, an autosampler and a column oven. The analysis was performed using the same column and chromatographic condition reported in LC/MS/MS section. The injection volume was 10 μL and the partial loop was used as injection technique. The Exactive Orbitrap MS equipped with a heated electrospray interface (HESI) was operated in the full spectral acquisition mode, in the positive and negative ionization mode in the mass range of m/z 150–900. The resolving power was set to 25,000 full width at half-maximum resulting in a scan time of 0.5 s. An automatic gain control target was set into high dynamic range, and the maximum injection time was 250 ms. The interface parameters were as follows: the spray voltage was + 3500 V and − 3000 V, respectively in positive and negative ion mode, the tube lens was at 20 V, the capillary voltage was 30 V, the capillary temperature was 275 °C, and a sheath and auxiliary gas flow of 45 and 15 arbitrary units were used. Chromatographic data acquisition, peak integration, and quantification were performed using Xcalibur software (Thermo Fisher Scientific, San Jose, USA). Xcalibur software (Thermo Fisher Scientific, San Jose, USA), with a specifically modified Excel macro was used. Quantitative results were obtained using santonin as internal standard.
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Table 1 MS/MS conditions used for the quantification of sesquiterpene lactones present in the chicory extracts: precursor ion, product ion, collision energy (CE), declustering potential (DP). Compound
Peak ID nr.
Precursor ion [M + H]+ (m/z)
Product ions (m/z)
DP
CE
11-β,13-Dihydro-lactucin Lactucin 8-Deoxy-lactucin Dihydro-8-deoxy-lactucin (jaquinellin) Santonin
1 2 3 5 8
279 277 261 263 247
215–243–187 213–241-185 243–215–187 245–217–189 201
40 30 45 40 60
20–16–26 19–17–23 18–20–24 17–21–25 21
151–215–259 151–213–257
35 37
18–23–14 18–24–16
Precursor ion [M − H]− (m/z) Dihydro-lactucopicrin Lactucopicrin
6 7
411 409
2.5. Method validation The method was validated for linearity, accuracy, precision (repeatability and reproducibility) and sensitivity. Linearity was evaluated using the calibration curve of santonin standard at five concentration levels from 0.3 to 50 ppm. The accuracy of the extraction method was evaluated with the recovery test as the ratio of the mean observed concentration and the known spiked concentration in the matrix and was expressed as [(mean observed concentration) / (added concentration)] × 100. In particular, the performance of the extraction was evaluated spiking six replicates of freeze dried Cicoria Catalogna with known quantities of santonin standard (1, 10 and 50 ppm). The spiked samples were then extracted and analyzed as described above. Intraday precision (repeatability) was assessed by calculating the relative standard deviation (RSDr), calculated from results generated under repeatability conditions of three determinations for concentration in a single day. Interday precision (reproducibility) was calculated by the relative standard deviation (RSDR) calculated from results generated under reproducibility conditions by one determination per concentration on three days. Sensitivity was evaluated by limit of detection (LOD) and limit of quantification (LOQ) values. The LOD was calculated as the quantity of analyte able to produce a chromatographic peak three times higher than the noise of the baseline in a chromatogram (S/N = 3) of a nonspiked sample, after having estimated the endogenous amount. The LOQ was set at ten times higher than the noise of the baseline in a chromatogram (S/N = 10). Five replicates were carried out for LOD and LOQ determination. 2.6. Statistical analysis We performed analysis of variance (ANOVA) in order to test the significance of the observed differences. When the effects of variety were
significant (P ≤ 0.05), the mean values for each value were compared by a multiple comparison Duncan test to look for grouping (at P = 0.05). 3. Results and discussion The first part of the work was focused at developing a new analytical method to study SLs in chicory applying an innovative approach based on a combined use of tandem mass spectrometry (LC/MS/MS) and highresolution mass spectrometry (Orbitrap MS). The SL extracts were initially analyzed by preparative HPLC, afterwards each peak on the chromatogram was collected, freeze-dried and used for the optimization of the analytical parameters to the mass spectrometer. The optimum LC/MS/ MS conditions were set up using different SL solutions of 10 μg/mL by infusion experiments, in particular declustering potential (DP) and collision energy (CE) were optimized for each compound. In Table 1 the LC/MS/MS data for each chicory SL were summarized, while in Fig. 1 the total ion chromatogram (TIC) of chicory SL extract was reported. LC/MS/MS highlighted the presence of 11β,13-dihydrolactucin, lactucin, 8-deoxylactucin, dihydro-8-deoxylactucin (jaquinellin), dihydrolactucopicrin, and lactucopicrin. LC/MS/MS data showed that positive ESI was more sensitive for the determination of the different SLs except for dihydrolactucopicrin and lactucopicrin which were detected at higher sensitivity in the negative ion mode. Dihydro-lactucin and lactucin showed [M + H]+ ions at m/z 279 and 277, respectively. The ion fragments were in accord to those reported in literature (Wulfkuehler, Gras, & Carle, 2013). The most abundant fragments (m/z 215 and 243 for dihydro-lactucin, m/z 213 and 241 for lactucin), corresponded to the loss of formic acid and water (64 amu) and two molecules of water, respectively. Deoxy-lactucin and dihydro-deoxy-lactucin showed [M + H]+ ions at 261 and 263, respectively. The main ion fragments were formed by
Fig. 1. Total ion chromatogram (TIC) of chicory SL extracts obtained by MS/MS working in the positive and negative ion mode in MRM (Multiple Reaction Monitoring).
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Fig. 2. Extracted ion chromatograms (XICs) of sesquiterpene lactones obtained using the high-resolution mass Orbitrap spectrometry in positive and negative ions.
the loss of water and formic acid (243 and 215 for deoxy-lactucin, 245 and 217 for dihydro-deoxy-lactucin). Santonin showed [M + H]+ ions at m/z 247 and a fragment ion at m/z 201 due to the loss of two molecules of water. Dihydro-lactucopicrin and lactucopicrin were analyzed in negative ion mode and showed [M − H]− ions at m/z 411 and 409. The main fragments, according to literature data (Ferioli & D'Antuono, 2012) derived from the loss of p-hydroxyphenylacetic moiety. Up to now, the SL concentration was measured mainly by HPLC and after treatment with enzymes or acidified methanol by which the glucosides, oxalates or sulfates were converted to their free forms (Mazijk-Bokslag, Cramwinckel, Essers, & Hollman, 1991; Price et al., 1990). In a recent study (Shah, Ali, Malik, Khan, & Saieda, 2011) two new compounds were identified in the aerial parts of C. intybus, 15hydroxytaraxacin and 6,8,11-epi-desacetylmatricarin along with three known compounds such as desacetylmatricarin, 11β,13-dihydrolactucin and 11β,13-dihydrolactucopicrin. Only few studies dealt with the SL analysis by MS: Ferioli and co-workers (Ferioli & D'Antuono, 2012) reported that methanol extract of chicory leaves contains lactucin, 8deoxylactucin, lactucopicrin and the corresponding 11,13-dihydro
derivatives. They performed single quadrupole MS analysis both in positive and negative atmospheric pressure ionization-electrospray source (API-ES). In this paper SL chicory extracts were analyzed using a triple quadrupole mass spectrometry in MRM acquisition (Multiple Reaction Monitoring) so results represented a significant improvement about specificity, sensitivity and quantitation. Sessa et al. (2000), reported in chicory the presence of lactucin-15-oxalate, lactucin, 8-deoxylactucin15-oxalate, lactucopicrin-15-oxalate and lactucopicrin using HPLC with UV absorption at 264 nm and diode array detection and confirming the peak identity by coupled HPLC-MS or by 1H and 13C NMR. In this paper for the first time the high-resolution mass spectrometry (HRMS) was combined with the MS/MS detection to study qualitative and quantitative profile of SLs in chicory. In Fig. 2 the chromatographic detection of SLs obtained using the HRMS is shown. The first result was that two further compounds namely lactuside C (jaquinellin-glucoside) and dihydro-lactucopicrin oxalate were found with respect to those found by MS/MS. As summarized in Table 2, a total of nine SLs could be detected with a mass accuracy b 5.0 ppm. Mass differences (delta mass error) between the theoretical and experimental m/z values
Table 2 High resolution mass spectral identification of sesquiterpene lactones (SLs) in chicory extracts: theoretical (exact) and experimental masses of SL ions with calculated absolute mass accuracies. Compound
RT (min)
Molecular formula
Theoretical
Experimental
Mass accuracy (ppm)
m/z+ 11-β,13-Dihydro-lactucin Lactucin 8-Deoxy-lactucin Dihydro-8-deoxy-lactucin (jaquinellin) Santonin
10.5 11.7 20.8 21.5 29.8
C15H18O5 C15H16O5 C15H16O4 C15H18O4 C15H18O3
279.12270 277.10705 261.11214 263.12779 247.13287
279.12390 277.10825 261.11301 263.12888 247.13364
4.30 4.33 3.33 4.14 3.12
Compound
RT (min)
Molecular formula
Theoretical
Experimental
Mass accuracy (ppm)
423.16577 483.12949 411.14490 409.12823
−0.69 −0.37 −0.07 −2.57
−
m/z Lactuside C (jaquinellin glucoside) Dihydro-lactucopicrin oxalate Dihydro-lactucopicrin Lactucopicrin
17.3 20.6 27.7 27.8
C21H28O9 C25H24O10 C23H24O7 C23H22O7
423.16606 483.12967 411.14493 409.12928
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Table 3 Concentration (μg/g) of sesquiterpene lactones in four different chicory varieties determined by high resolution mass spectrometry. Values are means (± standard deviations) of three samples; different letters indicate statistically different levels of sesquiterpene lactones between samples according to the Duncan test (P = 0.05). Compound
Cicoria Catalogna
Rosso di Chioggia
Treviso Precoce
Treviso Tardivo
11β,13-Dihydrolactucin Lactucin Lactuside C (jaquinellin glucoside) Dihydro-lactucopicrin oxalate Deoxy-lactucin Jaquinellin Dihydro-lactucopicrin Lactucopicrin Total
4.035 13.160 1.193 0.217 6.484 1.464 6.421 54.937 87.910
2.988 5.367 3.124 0.360 6.661 2.298 4.393 29.720 54.911
13.794 5.876 36.296 1.377 11.057 10.208 11.747 99.358 189.713
4.348 1.052 9.021 0.995 4.152 5.149 7.566 13.502 45.785
± ± ± ± ± ± ± ± ±
0.167b 0.071b 0.045a 0.012a 0.058b 0.030a 0.235b 2.518c 3.137c
have been determined. The m/z accuracy ranged between −2.57 ppm and +4.33 ppm, confirming the identity of the investigated compounds. The concentrations of the SLs in chicory cultivars change depending on several factors such as production location, temperature, harvest date, production year and nitrogen fertilization (Foster et al., 2006). Arakawa, Minami, Nakamura, Matsushima, and Nemoto (2008) compared the SL content of various Lactuca species cultivated in Japan and reported that the major SL is lactucopicrin, however they did not mention the presence of lactucopicrin oxalate as found in our samples. As mentioned in other studies (Sessa et al., 2000), SL oxalate derivatives are unstable in methanol and therefore the absence in our extracts of lactucin, deoxylactucin and lactucopicrin oxalates could be due to their decomposition during the extraction steps. Alternatively, it cannot be ruled out that the natural variability among chicory types is the simple explanation for the absence of these compounds as well as of the matricarin derivatives previously reported for chicory leaves (Shah et al., 2011). Comparing the performance of HRMS technique with those adopted thus far it is possible to highlight that the use of the Orbitrap mass spectrometer increased the information obtained from any single sample compared to other LC–MS platforms currently used. In Table 3 the concentrations of different sesquiterpene lactones in four different varieties of chicory were reported. The levels of SLs varied considerably between samples analyzed; in accordance with the findings of Wulfkuehler et al. (2013), lactucopicrin was the predominant SL in chicory representing on average 50% of the total sesquiterpenes. The type “Treviso Precoce”, the less famous and the most bitter radicchio variety on the market, actually exhibited significantly higher total SL concentrations compared to the other samples. The type “Treviso Tardivo” presented the lowest content of SL in four different varieties of chicory. This result was mainly determined by the peculiar agricultural practices (forcing process) adopted by producers to obtain good quality attributes in terms of crispness, antioxidants, phenol compounds and reduced bitter taste (Nicoletto & Pimpini, 2009, 2010). All in all, data confirmed that the most valuable type within the radicchio variety is also the one with lower SL concentration. As regards the other compounds, results obtained showed that jaquinellin-glucoside is the second most abundant compound (on average 19% of total compounds) for “Treviso Precoce” and “Treviso Tardivo” varieties while lactucin and 8-deoxylactucin are more representative compounds (on average 15% and 12%, respectively) in the case of “Cicoria Catalogna” and “Rosso di Chioggia” varieties. Between these varieties “Rosso di Chioggia”, has a sweeter taste than the Cicoria Catalogna in agreement with the low amounts of SLs found. In all analyzed varieties, dihydro-lactucopicrin oxalate is the
± ± ± ± ± ± ± ± ±
0.027a 0.016b 0.106b 0.009b 0.164b 0.023b 0.058a 0.468b 0.839b
± ± ± ± ± ± ± ± ±
0.181c 0.519b 2.179d 0.240d 0.281c 0.017d 0.387d 5.216d 7.620d
± ± ± ± ± ± ± ± ±
0.225b 0.040a 0.585c 0.030c 0.087a 0.254c 0.202c 0.675a 2.098a
less abundant compound, reaching an average concentration of about 1% compared to the total amount of sesquiterpene lactones. Chicory and especially radicchio is appreciated as a vegetable for its fine, slightly bitter taste, determined by SLs. His profile should be carefully controlled as an excessive amount or the unbalanced composition can compromise the typical taste and flavor. The procedure above-described represented a reliable tool to monitor SL profile in radicchio and coupled to a sensory study might allow one to predict its bitterness. This tool would be of great importance to breeders and at markets to valorize and properly address the product to the final market destination. Linear regression parameters are reported in Table 4. A good linearity was verified, with all correlation coefficients exceeding 0.971. Slopes were very similar for curves representing a different sample, suggesting a comparable matrix effect. LODs and LOQs are also shown in Table 4. The noise level depended on the matrix and, therefore, santonin was characterized by different LODs and LOQs in the four analyzed samples. The LODs varied between 0.08 and 0.11 μg/g while the LOQs ranged between 0.25 and 0.29 μg/g. The sensitivity of this method represents a significant improvement for SL analysis when compared to published LC–MS methods (Ferioli & D'Antuono, 2012). Considering that it has been observed a comparable matrix effect, only Cicoria Catalogna sample was used for precision method trials. The precision of the method, expressed as % RSD, met acceptance criteria, since % RSD was lower than 10% at each tested concentration level for intra- and interday precision (Table 5). The accuracy of SL analysis ranged between 85.90% and 113% and was acceptable for all concentrations tested. 4. Conclusion Numerous attempts have been made to correlate the guaianolide content of C. intybus plants with the bitter taste determined by sensorial testing and revealed a high correlation of various sesquiterpene lactones with the bitterness perceived by a sensory panel (Dirinck, Van Poucke, Van Acker, & Schamp, 1985; Mazijk-Bokslag et al., 1991; Price et al., 1990). In the literature studies in which chicory SL profile was performed with LC/MS/MS and HRMS spectrometry are not described. In LC/MS/MS analysis the measured compounds are limited to those targeted by MRM events programmed in the method. The Exactive Orbitrap HRMS detected all ions and a full scan method can be used, looking for each ion in the appropriate scan range. The number of detectable compounds is virtually unlimited and the same analysis can be used to study a complete metabolic pattern of different classes of metabolites. Therefore HRMS could be proposed as an analytical technique to study the complete metabolic profile of both SLs and other bioactive
Table 4 Method validation parameters: slopes (m), correlation coefficients (R2) and limits of detection (LOD) and quantification (LOQ). Cicoria Catalogna
Rosso di Chioggia
Treviso Precoce
Treviso Tardivo
m
R2
LOD (μg/g)
LOQ (μg/g)
m
R2
LOD (μg/g)
LOQ (μg/g)
m
R2
LOD (μg/g)
LOQ (μg/g)
m
R2
LOD (μg/g)
LOQ (μg/g)
5E + 06
0.998
0.09
0.27
4.6E + 06
0.981
0.08
0.25
4.8E + 06
0.971
0.11
0.29
5E + 06
0.991
0.09
0.27
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Table 5 Accuracy and intraday and interday precision: 6 replicates of a freeze dried Cicoria Catalogna spiked at 3 different concentration levels (1, 10 and 50 ppm); interday precision: data were obtained in 3 different days. Santonin added (ppm)
Accuracy (%)
1 10 50
92.50 ± 2.51 113.00 ± 7.11 85.90 ± 6.16
Intraday (RSDr) 1
2
3
4.20 9.00 2.20
2.80 4.90 1.00
4.40 4.80 5.30
Interday (RSDR)
7.82 8.43 4.94
compounds in a fast and reproducible way also by eliminating the purification steps used in the extraction. References Adegawa, S., Miyase, T., Ueno, A., Noro, T., Kuroyanagi, M., & Fukushima, S. (1985). Sesquiterpene glycosides from Crepidiastrum keiskeanum Nakai. Chemical and Pharmaceutical Bulletin, 33, 4906. Ames, B.N., Profet, M., & Gold, L.S. (1990). Dietary pesticides (99.99% all natural). Proceedings of the National Academy of Sciences of the United States of America, 87, 7777–7781. Arakawa, K., Minami, M., Nakamura, K., Matsushima, K., & Nemoto, K. (2008). Variation of sesquiterpene lactones content in Lactuca and Chichorium species. Horticultural Research, 7, 499–504. Beharav, A., Ben-David, R., Malarz, J., Stojakowska, A., Michalska, K., Doležalová, I., et al. (2010). Variation of sesquiterpene lactones in Lactuca aculeata natural populations from Israel, Jordan and Turkey. Biochemical Systematics and Ecology, 38, 602–611. Craig, W.J. (1997). Phytochemicals: guardians of our health. Journal of the American Dietetic Association, 97, 199–204. Dirinck, P., Van Poucke, P., Van Acker, M., & Schamp, N. (1985). Objective measurement of bitterness in chicory heads (Cichorium intybus L.). In W. Baltes, P.B. Czedik-Eysenberg, H. Deeistra, P. Dirinck, W. Ooghe, & W. Pfannhauser (Eds.), Strategies in food quality assurance: Analytical, industrial and legal aspects. Vol. 3. (pp. 62–68). Antwerpen, Belgium: De Sikkel. Drewnowski, A., & Gomez-Carneros, C. (2000). Bitter taste, phytonutrients, and the consumer: A review. The American Journal of Clinical Nutrition, 72, 1424–1435. Ferioli, F., & D'Antuono, L.F. (2012). An update procedure for an effective and simultaneous extraction of sesquiterpene lactones and phenolics from chicory. Food Chemistry, 135, 243–250. Foster, J.G., Clapham, W.M., Belesky, D.P., Labreveux, M., Hall, M.H., & Sanderson, M.A. (2006). Influence of cultivation site on sesquiterpene lactone composition of forage chicory (Cichorium intybus L.). Journal of Agricultural Food Chemistry, 54, 1772–1778. Higdon, J.V., Delage, B., Williams, D.E., & Dashwood, R.H. (2007). Cruciferous vegetables and human cancer risk: Epidemiologic evidence and mechanistic basis. Pharmacological Research, 55, 224. Kisiel, W., & Barszcz, B. (1997). Minor sesquiterpene lactone lactones from Lactuca virosa. Phytochemistry, 46, 1241–1243. Kisiel, W., & Zielińska, K. (2001). Guaianolides from Cichorium intybus and structure revision of Cichorium sesquiterpene lactones. Phytochemistry, 57, 523–527. Lesschaeve, I., & Noble, A.C. (2005). Polyphenols: factors influencing their sensory properties and their effects on food and beverage preferences. The American Journal of Clinical Nutrition, 81, 330–335.
Martínez-Ballesta, M.C., López-Pérez, L., Hernández, M., López-Berenguer, C., FernándezGarcía, N., & Carvajal, M. (2008). Agricultural practices for enhanced human health. Phytochemistry Reviews, 7, 251–260. Mazijk-Bokslag, D.M., Cramwinckel, A.B., Essers, M.L., & Hollman, P.C.H. (1991). The relationship between the taste and a HPLC profile of chicory. Report 91.50. Wageningen, The Netherlands: DLO-State Institute for Quality Control of Agricultural Products. Merfort, I. (2002). Review of the analytical techniques for sesquiterpenes and sesquiterpene lactones. Journal of Chromatography A, 967, 115–130. Nagy, K., Redeuil, K., Bertholet, R., Steiling, H., & Kussmann, M. (2009). Quantification of anthocyanins and flavonols in milk-based food products by ultra performance liquid chromatography–tandem mass spectrometry. Analytical Chemistry, 81(15), 6347–6356. Nicoletto, C., & Pimpini, F. (2009). Influence of the forcing process on some qualitative aspects in radicchio “Rosso di Treviso tardivo” (Cichorium intybus L., group rubifolium). 1. Nitrate, nitrite and organic nitrose. Italian Journal of Agronomy, 4, 137–145. Nicoletto, C., & Pimpini, F. (2010). Influence of the forcing process on some qualitative aspects in Radicchio “Rosso di Treviso Tardivo” (Cichorium Intybus L., Group Rubifolium). 2. Antioxidant capacity, phenols and ascorbic acid. Italian Journal of Agronomy, 5, 43–52. Nishimura, K., Miyase, T., Ueno, A., Noro, T., Kuroyanagi, M., & Fukushima, S. (1986). Sesquiterpene lactones from Lactuca laciniata. Phytochemistry, 25, 2375–2379. Ou, B., Huang, D., Hampsch-Woodill, M., Flanagan, J.A., & Deemer, E.K. (2002). Analysis of antioxidant activities of common vegetables employing oxygen radical absorbance capacity (ORAC) and ferric reducing antioxidant power (FRAP) assays: A comparative study. Journal of Agricultural Food Chemistry, 50, 3122–3128. Poli, F., Sacchetti, G., Tosi, B., Fogagnolo, M., Chillemi, G., Lazzarin, R., et al. (2002). Variation in the content of the main guaianolides and sugars in Cichorium intybus var. “Rosso di Chioggia” selections during cultivation. Food Chemistry, 76, 139–147. Price, K.R., Dupont, M.S., Shepherd, R., Chan, H.W.S., & Fenwick, G.R. (1990). Relationship between the chemical and sensory properties of exotic salad crops-colored lettuce (Lactuca sativa) and chicory (Cichorium intybus). Journal of the Science of Food and Agriculture, 53, 185–192. Rousseff, R.L. (1990). Bitterness in food products: An overview. In R.L. Rousseff (Ed.), Bitterness in foods and beverages (pp. 1–14). Elsevier. Roy, G.M. (1990). The applications and future implications of bitterness reduction and inhibition in food products. Critical Reviews in Food Science and Nutrition, 29, 59–71. Sanchez-Rabaneda, F., Jauregui, O., Casals, I., Andres-Lacueva, C., Izquierdo-Pulido, M., & Lamuela-Ravento, R.M. (2003). Liquid chromatographic/electrospray ionization tandem mass spectrometric study of the phenolic composition of cocoa (Theobroma cacao). Journal of Mass Spectrometry, 38, 35–42. Scigelova, M., & Makarov, A. (2006). Orbitrap mass analyzer—Overview and applications in proteomics. Proteomics, 16–21. Sessa, R.A., Bennett, M.H., Lewisi, M.J., Mansfield, J.W., & Bealei, M.H. (2000). Metabolite profiling of sesquiterpene lactones from Lactuca species. The Journal of Biological Chemistry, 275, 26877–26884. Shah, S., Ali, Z., Malik, A., Khan, I.A., & Saieda, S. (2011). Sesquiterpene lactones from Cichorium intybus. Journal for Nature Research, 66b, 729–732. Vinson, J.A., Hao, Y., Su, X., & Zubik, L. (1998). Phenol antioxidant quantity and quality in foods: Vegetables. Journal of Agricultural Food Chemistry, 46, 3630–3634. Wulfkuehler, S., Gras, C., & Carle, R. (2013). Sesquiterpene lactone content and overall quality of fresh-cut witloof chicory (Cichorium intybus L. var. foliosum Hegi) as affected by different washing procedures. Journal of Agricultural Food Chemistry, 61, 7705–7714. Xie, J., Zhang, Y., Kong, D., & Rexit, M. (2011). Rapid identification and determination of 11 polyphenols in Herba lycopi by HPLC–MS/MS with multiple reactions monitoring mode (MRM). Journal of Food Composition and Analysis, 24, 1069–1072. Yan‐Hwa, C., Chang, C., & Hsu, H. (2000). Flavonoid content of several vegetables and their antioxidant activity. Journal of the Science of Food and Agriculture, 80, 561–566.
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Food Research International journal homepage: www.elsevier.com/locate/foodres
Profiling chicory sesquiterpene lactones by high resolution mass spectrometry Giulia Graziani a,⁎, Rosalia Ferracane a, Paolo Sambo b, Silvia Santagata b, Carlo Nicoletto b, Vincenzo Fogliano c a b c
Department of Agricultural and Food Science, University of Napoli “Federico II”, Parco Gussone, 80055 Portici, Napoli, Italy Department of Agronomy, Food, Natural resources, Animals and Environment (DAFNAE), Agripolis — University of Padova, Viale dell'Università 16, 35020 Legnaro, PD, Italy Food Quality & Design Group, Wageningen University, PO Box 8129, 6700 EV Wageningen, The Netherlands
a r t i c l e
i n f o
Article history: Received 31 July 2014 Accepted 13 November 2014 Available online 20 November 2014 Keywords: Chicory Radicchio Bitterness Sesquiterpene lactones High resolution mass spectrometry Tandem mass spectrometry
a b s t r a c t Sesquiterpene lactones (SLs) are the main determinants of radicchio bitterness and the control of their concentrations is a key point for the market value of this product. An innovative analytical approach based on two complementary mass spectrometers, Orbitrap-HRMS and MS/MS, was used for quantitative analysis of SLs in aerial part of four different varieties of chicory. Data highlighted the presence of eight SLs: 11β,13-dihydrolactucin, lactucin, 8-deoxy-lactucin, dihydro-8-deoxylactucin, dihydrolactucopicrin, lactucopicrin, lactuside C (jaquinellin glucoside) and dihydro-lactucopicrin oxalate. Significant varietal differences were found. The highest amount of SLs was found in the radicchio “Treviso Precoce” variety (189.71 μg/g), the lowest amount in “Treviso Tardivo” variety (45.78 μg/g). Lactucopicrin was the most abundant compound with concentration ranged between 99.36 in “Treviso Precoce” and 13.50 μg/g in “Treviso Tardivo” while dihydro-lactucopicrin oxalate was the less abundant in all analyzed varieties with an average concentration of about 1% on the total amount of SLs. Published by Elsevier Ltd.
1. Introduction Diet rich in vegetables and fruit has been linked with lower rates of cancer and coronary disease (Drewnowski & Gomez-Carneros, 2000; Ou, Huang, Hampsch-Woodill, Flanagan, & Deemer, 2002; Vinson, Hao, Su, & Zubik, 1998; Yan‐Hwa, Chang, & Hsu, 2000). Plant-based phenols, flavonoids, isoflavones, glucosinolates, terpenes and other compounds that are present in the everyday diet have antioxidant and anticarcinogenic properties and a wide spectrum of tumor-blocking activities (Drewnowski & Gomez-Carneros, 2000; Higdon, Delage, Williams, & Dashwood, 2007). These compounds are known as phytochemicals or phytonutrients and were often bitter, acrid or astringent (Craig, 1997; Drewnowski & Gomez-Carneros, 2000; Lesschaeve & Noble, 2005). Unfortunately, bitter taste lowered consumer acceptability despite the fact that bitter compounds were often potentially helpful to human health (Ames, Profet, & Gold, 1990). Bitter taste also posed considerable problems at industrial level because de-bittering processes are complex and can reduce the overall quality, especially for vegetable food (Rousseff, 1990; Roy, 1990). Therefore, the control and modulation of the bitter taste in food played a pivotal role in order to match health quality and marketing needs. Genetic improvement techniques or innovative farming practices can be used to handle the bitter taste (Martínez-Ballesta et al., 2008). However, rapid and precise analytical methods to identify and quantify the chemical components responsible ⁎ Corresponding author. Fax: +39 81 7762580. E-mail address: [email protected] (G. Graziani).
http://dx.doi.org/10.1016/j.foodres.2014.11.021 0963-9969/Published by Elsevier Ltd.
for the bitter taste are needed. In this framework, chicory species and in particular radicchio (Cichorium intybus L., group rubifolium) represented a peculiar case: in fact, they are well-known for their bitter taste which is differently desired for consumers. Radicchio is one of the most popular and expensive type of chicory also due to the smart red colored leaves, which makes it very eye-catching among leafy vegetables. The bitter taste of chicory originates from sesquiterpene lactones (SLs) mainly present in the latex (Poli et al., 2002). SLs are C-15 terpenoids which occur as hydrocarbons or in oxygenated forms such as alcohols, ketones, aldehydes, acids or lactones in nature. The principal SLs found in species of Lactuca and chicory were lactucin, lactucopicrin, 8-deoxylactucin and derivatives such as 11,13 dihydro-analogs. The SLs lactucin, 8-deoxylactucin, and lactucopicrin were also intensely bitter, so their presence within salad lettuce and chicory should be carefully controlled to avoid product depreciation (Poli et al., 2002; Price, Dupont, Shepherd, Chan, & Fenwick, 1990). Previous studies also reported the presence of SL glycosides such as 15-glycosyl conjugate of 11,13-dihydrolactucopicrin that has been identified in roots of Lactuca tatarica, Lactuca aculeata and C. intybus (Beharav et al., 2010; Kisiel & Zielińska, 2001; Kisiel & Barszcz, 1997). This glycoside and others such as picriside A (lactucin 15-glycoside) and crepidiaside A (8-deoxylactucin-15-glycoside), have also been identified in other members of the Lactuceae species (Adegawa et al., 1985; Nishimura et al., 1986). The presence of 15-oxalates and 8sulfates as additional major conjugates of lactucin, 8-deoxylactucin, and lactucopicrin in lettuce and chicory has also been reported by Sessa, Bennett, Lewisi, Mansfield, and Bealei (2000). The most frequently used
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analytical techniques for the separation and analysis of SLs are reversed-phase HPLC (RP-HPLC) with UV detection, supercritical fluid chromatography, micellar electrokinetic chromatography (MEKC), gas chromatography and thin-layer chromatography (Merfort, 2002). Among these, HPLC-UV, mainly on reversed phase HPLC was the most applied analytical method using mostly acetonitrile–water or methanol–water gradients, less numerous were data reporting SL analysis performed by mass spectrometry. Sessa et al. (2000) studied chicory SL profile by HPLC-UV and electrospray mass spectral data for each HPLC peak were obtained from analysis of components of individual peaks collected from preparative HPLC. Ferioli and D'Antuono (2012), reported the identification of chicory SLs by HPLC-mass spectrometry using a single quadrupole mass spectrometer and Selected Ion Monitoring (SIM) acquisition. The use of two complementary mass spectrometry techniques such as high resolution mass spectrometry (HRMS) and ion spray triple quadrupole could be very useful for qualitative analysis of target compounds and can be adopted for the identification and quantification of the maximum number of SLs. LC/MS/MS in MRM (Multiple Reaction Monitoring) acquisition allows one to obtain high specificity for each selected analyte, even in complex matrices. The measured compounds are limited to those targeted by MRM events programmed in the method. In addition, quantitative performance decreases with an increasing number of MRM scan events (Nagy, Redeuil, Bertholet, Steiling, & Kussmann, 2009; Sanchez-Rabaneda et al., 2003; Xie, Zhang, Kong, & Rexit, 2011). Moreover, with Orbitrap a generic full scan method can be used, looking for every compound in the selected scan range. Ion-specific chromatograms are generated for all observed ion signals, using a 5 ppm m/z window. The number of compounds that can be detected is virtually unlimited (Scigelova & Makarov, 2006). In this work, we adopted an innovative analytical technique based on combination of mass spectrometry (MS) techniques, liquid chromatography/electrospray ionization HRMS (LC/ESI-HRMS) and liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ ESI-MS/MS) on a triple quadrupole, for the identification and quantification of SLs in four different chicory varieties. 2. Materials and methods 2.1. Plant material and chemicals The marketable aerial part of four different varieties of chicory “Cicoria Catalogna” (C. intybus L. var dentatum), radicchio “Rosso di Chioggia”, radicchio “Rosso di Treviso Precoce” and radicchio “Rosso di Treviso Tardivo” (C. intybus L. Rubifolium Group) was considered. Plants were cultivated in Northern-East of Italy (Veneto Region), transplanted on loamy soil in the second half of July following normal agronomical practices. For each chicory type three plots 25 m2 (5 × 5 m) were singled out and 35 representative plants were uprooted from each of these at harvest time. The harvest was carried out at marketable maturity in November for “Cicoria Catalogna” and in December for radicchio cultivars. After harvest radicchio “Rosso di Treviso Tardivo” was subjected to the usual forcing process before commercialization as reported by Nicoletto and Pimpini (2009, 2010). For all varieties, the marketable material of each plot was cut in pieces, immediately frozen at − 80 °C overnight, freeze-dried and ground before extraction. All reagents and solvents of HPLC grade were purchased from Merck (Darmstadt, Germany). Santonin used to quantify SLs was obtained from Sigma (Milano, Italy). 2.2. Extraction of sesquiterpene lactones (SLs) 0.5 g of freeze-dried sample was exactly weighed in a screw-cap plastic centrifuge tube to which was added 100 μg of santonin (internal standard, methanolic solution) and 15 mL of 2% (v/v) formic acid in methanol/water 4/1 (v/v). The sample was then shaken on a vortex
stirrer for 1 min, sonicated at room temperature for 10 min and then centrifuged at 4000 g for 10 min. After collection of the supernatant fraction, the extraction procedure was repeated. The extracts were pooled, dried under reduced pressure at 35 °C, subjected to nitrogen flux for 5 min, and recovered with 2 mL of methanol. Before SPE, the SL fraction was centrifuged at 4000 g for 10 min to remove solid particles. SLs were purified from phenols and other interfering compounds by SPE, employing silica cartridges (3 mL reservoir, 500 mg sorbent mass). The cartridges were conditioned with 6 mL of dichloromethane/ isopropanol 1/1 (v/v), equilibrated with 6 mL of dichloromethane and, after sample loading, eluted with 6 mL of dichloromethane/ethyl acetate 3/2 (v/v). Both the loading and elution fractions were collected, dried under reduced pressure at 35 °C, recovered with 1 mL of methanol/water 1/1 (v/v), filtered in a HPLC glass vial through a nylon syringe filter (diameter: 13 mm; pore dimension: 0.45 μm) and stored at −18 °C until mass spectrometry analyses.
2.3. LC/MS/MS analysis Chromatographic separation was performed using an HPLC apparatus equipped with two micropumps Series 200 (Perkin Elmer, CanadaShelton, USA) and Luna C18, 250 × 4.6 mm, 5 μm (Phenomenex, Torrance, CA, USA) (Phenomenex, CA, USA). The solvent system consisted of (A) water and (B) acetonitrile. The gradient program was as follows: 10–42% B (30 min), 10% B (5 min), constant to B (5 min), at a constant flow of 1 mL/min. Injection volume was 20 μL. These conditions were also used for preparative HPLC. MS/MS analyses of SLs were performed on an API 3000 triple quadrupole mass spectrometer (Applied Biosystems, Canada) equipped with a Turbo Ion Spray source. The LC flow was split and 0.2 mL/min was sent to the mass spectrometer. The declustering potential (DP) and the collision energy (CE) were optimized for each compound infusing directly into the mass spectrometer standard solutions (10 μg/mL) at a constant flow rate of 10 μL/min using a model 11 syringe pump (Harvard Apparatus, Holliston, MA, USA). Analysis was performed in the positive and negative ion mode in MRM (Multiple Reaction Monitoring). Drying gas (air) was heated to 350 °C and the capillary voltage (IS) was set to + 5000 V in positive ion mode and −4000 V in negative ion mode. Data acquisition and processing were performed using Analyst software version 1.4.2.
2.4. High resolution mass spectrometry (HRMS) analysis LC–MS data were acquired on an Accela U-HPLC system coupled to an Exactive mass spectrometer (Thermo Fisher Scientific, San Jose, CA). The Accela system consisted of a quaternary pump, an autosampler and a column oven. The analysis was performed using the same column and chromatographic condition reported in LC/MS/MS section. The injection volume was 10 μL and the partial loop was used as injection technique. The Exactive Orbitrap MS equipped with a heated electrospray interface (HESI) was operated in the full spectral acquisition mode, in the positive and negative ionization mode in the mass range of m/z 150–900. The resolving power was set to 25,000 full width at half-maximum resulting in a scan time of 0.5 s. An automatic gain control target was set into high dynamic range, and the maximum injection time was 250 ms. The interface parameters were as follows: the spray voltage was + 3500 V and − 3000 V, respectively in positive and negative ion mode, the tube lens was at 20 V, the capillary voltage was 30 V, the capillary temperature was 275 °C, and a sheath and auxiliary gas flow of 45 and 15 arbitrary units were used. Chromatographic data acquisition, peak integration, and quantification were performed using Xcalibur software (Thermo Fisher Scientific, San Jose, USA). Xcalibur software (Thermo Fisher Scientific, San Jose, USA), with a specifically modified Excel macro was used. Quantitative results were obtained using santonin as internal standard.
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Table 1 MS/MS conditions used for the quantification of sesquiterpene lactones present in the chicory extracts: precursor ion, product ion, collision energy (CE), declustering potential (DP). Compound
Peak ID nr.
Precursor ion [M + H]+ (m/z)
Product ions (m/z)
DP
CE
11-β,13-Dihydro-lactucin Lactucin 8-Deoxy-lactucin Dihydro-8-deoxy-lactucin (jaquinellin) Santonin
1 2 3 5 8
279 277 261 263 247
215–243–187 213–241-185 243–215–187 245–217–189 201
40 30 45 40 60
20–16–26 19–17–23 18–20–24 17–21–25 21
151–215–259 151–213–257
35 37
18–23–14 18–24–16
Precursor ion [M − H]− (m/z) Dihydro-lactucopicrin Lactucopicrin
6 7
411 409
2.5. Method validation The method was validated for linearity, accuracy, precision (repeatability and reproducibility) and sensitivity. Linearity was evaluated using the calibration curve of santonin standard at five concentration levels from 0.3 to 50 ppm. The accuracy of the extraction method was evaluated with the recovery test as the ratio of the mean observed concentration and the known spiked concentration in the matrix and was expressed as [(mean observed concentration) / (added concentration)] × 100. In particular, the performance of the extraction was evaluated spiking six replicates of freeze dried Cicoria Catalogna with known quantities of santonin standard (1, 10 and 50 ppm). The spiked samples were then extracted and analyzed as described above. Intraday precision (repeatability) was assessed by calculating the relative standard deviation (RSDr), calculated from results generated under repeatability conditions of three determinations for concentration in a single day. Interday precision (reproducibility) was calculated by the relative standard deviation (RSDR) calculated from results generated under reproducibility conditions by one determination per concentration on three days. Sensitivity was evaluated by limit of detection (LOD) and limit of quantification (LOQ) values. The LOD was calculated as the quantity of analyte able to produce a chromatographic peak three times higher than the noise of the baseline in a chromatogram (S/N = 3) of a nonspiked sample, after having estimated the endogenous amount. The LOQ was set at ten times higher than the noise of the baseline in a chromatogram (S/N = 10). Five replicates were carried out for LOD and LOQ determination. 2.6. Statistical analysis We performed analysis of variance (ANOVA) in order to test the significance of the observed differences. When the effects of variety were
significant (P ≤ 0.05), the mean values for each value were compared by a multiple comparison Duncan test to look for grouping (at P = 0.05). 3. Results and discussion The first part of the work was focused at developing a new analytical method to study SLs in chicory applying an innovative approach based on a combined use of tandem mass spectrometry (LC/MS/MS) and highresolution mass spectrometry (Orbitrap MS). The SL extracts were initially analyzed by preparative HPLC, afterwards each peak on the chromatogram was collected, freeze-dried and used for the optimization of the analytical parameters to the mass spectrometer. The optimum LC/MS/ MS conditions were set up using different SL solutions of 10 μg/mL by infusion experiments, in particular declustering potential (DP) and collision energy (CE) were optimized for each compound. In Table 1 the LC/MS/MS data for each chicory SL were summarized, while in Fig. 1 the total ion chromatogram (TIC) of chicory SL extract was reported. LC/MS/MS highlighted the presence of 11β,13-dihydrolactucin, lactucin, 8-deoxylactucin, dihydro-8-deoxylactucin (jaquinellin), dihydrolactucopicrin, and lactucopicrin. LC/MS/MS data showed that positive ESI was more sensitive for the determination of the different SLs except for dihydrolactucopicrin and lactucopicrin which were detected at higher sensitivity in the negative ion mode. Dihydro-lactucin and lactucin showed [M + H]+ ions at m/z 279 and 277, respectively. The ion fragments were in accord to those reported in literature (Wulfkuehler, Gras, & Carle, 2013). The most abundant fragments (m/z 215 and 243 for dihydro-lactucin, m/z 213 and 241 for lactucin), corresponded to the loss of formic acid and water (64 amu) and two molecules of water, respectively. Deoxy-lactucin and dihydro-deoxy-lactucin showed [M + H]+ ions at 261 and 263, respectively. The main ion fragments were formed by
Fig. 1. Total ion chromatogram (TIC) of chicory SL extracts obtained by MS/MS working in the positive and negative ion mode in MRM (Multiple Reaction Monitoring).
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Fig. 2. Extracted ion chromatograms (XICs) of sesquiterpene lactones obtained using the high-resolution mass Orbitrap spectrometry in positive and negative ions.
the loss of water and formic acid (243 and 215 for deoxy-lactucin, 245 and 217 for dihydro-deoxy-lactucin). Santonin showed [M + H]+ ions at m/z 247 and a fragment ion at m/z 201 due to the loss of two molecules of water. Dihydro-lactucopicrin and lactucopicrin were analyzed in negative ion mode and showed [M − H]− ions at m/z 411 and 409. The main fragments, according to literature data (Ferioli & D'Antuono, 2012) derived from the loss of p-hydroxyphenylacetic moiety. Up to now, the SL concentration was measured mainly by HPLC and after treatment with enzymes or acidified methanol by which the glucosides, oxalates or sulfates were converted to their free forms (Mazijk-Bokslag, Cramwinckel, Essers, & Hollman, 1991; Price et al., 1990). In a recent study (Shah, Ali, Malik, Khan, & Saieda, 2011) two new compounds were identified in the aerial parts of C. intybus, 15hydroxytaraxacin and 6,8,11-epi-desacetylmatricarin along with three known compounds such as desacetylmatricarin, 11β,13-dihydrolactucin and 11β,13-dihydrolactucopicrin. Only few studies dealt with the SL analysis by MS: Ferioli and co-workers (Ferioli & D'Antuono, 2012) reported that methanol extract of chicory leaves contains lactucin, 8deoxylactucin, lactucopicrin and the corresponding 11,13-dihydro
derivatives. They performed single quadrupole MS analysis both in positive and negative atmospheric pressure ionization-electrospray source (API-ES). In this paper SL chicory extracts were analyzed using a triple quadrupole mass spectrometry in MRM acquisition (Multiple Reaction Monitoring) so results represented a significant improvement about specificity, sensitivity and quantitation. Sessa et al. (2000), reported in chicory the presence of lactucin-15-oxalate, lactucin, 8-deoxylactucin15-oxalate, lactucopicrin-15-oxalate and lactucopicrin using HPLC with UV absorption at 264 nm and diode array detection and confirming the peak identity by coupled HPLC-MS or by 1H and 13C NMR. In this paper for the first time the high-resolution mass spectrometry (HRMS) was combined with the MS/MS detection to study qualitative and quantitative profile of SLs in chicory. In Fig. 2 the chromatographic detection of SLs obtained using the HRMS is shown. The first result was that two further compounds namely lactuside C (jaquinellin-glucoside) and dihydro-lactucopicrin oxalate were found with respect to those found by MS/MS. As summarized in Table 2, a total of nine SLs could be detected with a mass accuracy b 5.0 ppm. Mass differences (delta mass error) between the theoretical and experimental m/z values
Table 2 High resolution mass spectral identification of sesquiterpene lactones (SLs) in chicory extracts: theoretical (exact) and experimental masses of SL ions with calculated absolute mass accuracies. Compound
RT (min)
Molecular formula
Theoretical
Experimental
Mass accuracy (ppm)
m/z+ 11-β,13-Dihydro-lactucin Lactucin 8-Deoxy-lactucin Dihydro-8-deoxy-lactucin (jaquinellin) Santonin
10.5 11.7 20.8 21.5 29.8
C15H18O5 C15H16O5 C15H16O4 C15H18O4 C15H18O3
279.12270 277.10705 261.11214 263.12779 247.13287
279.12390 277.10825 261.11301 263.12888 247.13364
4.30 4.33 3.33 4.14 3.12
Compound
RT (min)
Molecular formula
Theoretical
Experimental
Mass accuracy (ppm)
423.16577 483.12949 411.14490 409.12823
−0.69 −0.37 −0.07 −2.57
−
m/z Lactuside C (jaquinellin glucoside) Dihydro-lactucopicrin oxalate Dihydro-lactucopicrin Lactucopicrin
17.3 20.6 27.7 27.8
C21H28O9 C25H24O10 C23H24O7 C23H22O7
423.16606 483.12967 411.14493 409.12928
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Table 3 Concentration (μg/g) of sesquiterpene lactones in four different chicory varieties determined by high resolution mass spectrometry. Values are means (± standard deviations) of three samples; different letters indicate statistically different levels of sesquiterpene lactones between samples according to the Duncan test (P = 0.05). Compound
Cicoria Catalogna
Rosso di Chioggia
Treviso Precoce
Treviso Tardivo
11β,13-Dihydrolactucin Lactucin Lactuside C (jaquinellin glucoside) Dihydro-lactucopicrin oxalate Deoxy-lactucin Jaquinellin Dihydro-lactucopicrin Lactucopicrin Total
4.035 13.160 1.193 0.217 6.484 1.464 6.421 54.937 87.910
2.988 5.367 3.124 0.360 6.661 2.298 4.393 29.720 54.911
13.794 5.876 36.296 1.377 11.057 10.208 11.747 99.358 189.713
4.348 1.052 9.021 0.995 4.152 5.149 7.566 13.502 45.785
± ± ± ± ± ± ± ± ±
0.167b 0.071b 0.045a 0.012a 0.058b 0.030a 0.235b 2.518c 3.137c
have been determined. The m/z accuracy ranged between −2.57 ppm and +4.33 ppm, confirming the identity of the investigated compounds. The concentrations of the SLs in chicory cultivars change depending on several factors such as production location, temperature, harvest date, production year and nitrogen fertilization (Foster et al., 2006). Arakawa, Minami, Nakamura, Matsushima, and Nemoto (2008) compared the SL content of various Lactuca species cultivated in Japan and reported that the major SL is lactucopicrin, however they did not mention the presence of lactucopicrin oxalate as found in our samples. As mentioned in other studies (Sessa et al., 2000), SL oxalate derivatives are unstable in methanol and therefore the absence in our extracts of lactucin, deoxylactucin and lactucopicrin oxalates could be due to their decomposition during the extraction steps. Alternatively, it cannot be ruled out that the natural variability among chicory types is the simple explanation for the absence of these compounds as well as of the matricarin derivatives previously reported for chicory leaves (Shah et al., 2011). Comparing the performance of HRMS technique with those adopted thus far it is possible to highlight that the use of the Orbitrap mass spectrometer increased the information obtained from any single sample compared to other LC–MS platforms currently used. In Table 3 the concentrations of different sesquiterpene lactones in four different varieties of chicory were reported. The levels of SLs varied considerably between samples analyzed; in accordance with the findings of Wulfkuehler et al. (2013), lactucopicrin was the predominant SL in chicory representing on average 50% of the total sesquiterpenes. The type “Treviso Precoce”, the less famous and the most bitter radicchio variety on the market, actually exhibited significantly higher total SL concentrations compared to the other samples. The type “Treviso Tardivo” presented the lowest content of SL in four different varieties of chicory. This result was mainly determined by the peculiar agricultural practices (forcing process) adopted by producers to obtain good quality attributes in terms of crispness, antioxidants, phenol compounds and reduced bitter taste (Nicoletto & Pimpini, 2009, 2010). All in all, data confirmed that the most valuable type within the radicchio variety is also the one with lower SL concentration. As regards the other compounds, results obtained showed that jaquinellin-glucoside is the second most abundant compound (on average 19% of total compounds) for “Treviso Precoce” and “Treviso Tardivo” varieties while lactucin and 8-deoxylactucin are more representative compounds (on average 15% and 12%, respectively) in the case of “Cicoria Catalogna” and “Rosso di Chioggia” varieties. Between these varieties “Rosso di Chioggia”, has a sweeter taste than the Cicoria Catalogna in agreement with the low amounts of SLs found. In all analyzed varieties, dihydro-lactucopicrin oxalate is the
± ± ± ± ± ± ± ± ±
0.027a 0.016b 0.106b 0.009b 0.164b 0.023b 0.058a 0.468b 0.839b
± ± ± ± ± ± ± ± ±
0.181c 0.519b 2.179d 0.240d 0.281c 0.017d 0.387d 5.216d 7.620d
± ± ± ± ± ± ± ± ±
0.225b 0.040a 0.585c 0.030c 0.087a 0.254c 0.202c 0.675a 2.098a
less abundant compound, reaching an average concentration of about 1% compared to the total amount of sesquiterpene lactones. Chicory and especially radicchio is appreciated as a vegetable for its fine, slightly bitter taste, determined by SLs. His profile should be carefully controlled as an excessive amount or the unbalanced composition can compromise the typical taste and flavor. The procedure above-described represented a reliable tool to monitor SL profile in radicchio and coupled to a sensory study might allow one to predict its bitterness. This tool would be of great importance to breeders and at markets to valorize and properly address the product to the final market destination. Linear regression parameters are reported in Table 4. A good linearity was verified, with all correlation coefficients exceeding 0.971. Slopes were very similar for curves representing a different sample, suggesting a comparable matrix effect. LODs and LOQs are also shown in Table 4. The noise level depended on the matrix and, therefore, santonin was characterized by different LODs and LOQs in the four analyzed samples. The LODs varied between 0.08 and 0.11 μg/g while the LOQs ranged between 0.25 and 0.29 μg/g. The sensitivity of this method represents a significant improvement for SL analysis when compared to published LC–MS methods (Ferioli & D'Antuono, 2012). Considering that it has been observed a comparable matrix effect, only Cicoria Catalogna sample was used for precision method trials. The precision of the method, expressed as % RSD, met acceptance criteria, since % RSD was lower than 10% at each tested concentration level for intra- and interday precision (Table 5). The accuracy of SL analysis ranged between 85.90% and 113% and was acceptable for all concentrations tested. 4. Conclusion Numerous attempts have been made to correlate the guaianolide content of C. intybus plants with the bitter taste determined by sensorial testing and revealed a high correlation of various sesquiterpene lactones with the bitterness perceived by a sensory panel (Dirinck, Van Poucke, Van Acker, & Schamp, 1985; Mazijk-Bokslag et al., 1991; Price et al., 1990). In the literature studies in which chicory SL profile was performed with LC/MS/MS and HRMS spectrometry are not described. In LC/MS/MS analysis the measured compounds are limited to those targeted by MRM events programmed in the method. The Exactive Orbitrap HRMS detected all ions and a full scan method can be used, looking for each ion in the appropriate scan range. The number of detectable compounds is virtually unlimited and the same analysis can be used to study a complete metabolic pattern of different classes of metabolites. Therefore HRMS could be proposed as an analytical technique to study the complete metabolic profile of both SLs and other bioactive
Table 4 Method validation parameters: slopes (m), correlation coefficients (R2) and limits of detection (LOD) and quantification (LOQ). Cicoria Catalogna
Rosso di Chioggia
Treviso Precoce
Treviso Tardivo
m
R2
LOD (μg/g)
LOQ (μg/g)
m
R2
LOD (μg/g)
LOQ (μg/g)
m
R2
LOD (μg/g)
LOQ (μg/g)
m
R2
LOD (μg/g)
LOQ (μg/g)
5E + 06
0.998
0.09
0.27
4.6E + 06
0.981
0.08
0.25
4.8E + 06
0.971
0.11
0.29
5E + 06
0.991
0.09
0.27
198
G. Graziani et al. / Food Research International 67 (2015) 193–198
Table 5 Accuracy and intraday and interday precision: 6 replicates of a freeze dried Cicoria Catalogna spiked at 3 different concentration levels (1, 10 and 50 ppm); interday precision: data were obtained in 3 different days. Santonin added (ppm)
Accuracy (%)
1 10 50
92.50 ± 2.51 113.00 ± 7.11 85.90 ± 6.16
Intraday (RSDr) 1
2
3
4.20 9.00 2.20
2.80 4.90 1.00
4.40 4.80 5.30
Interday (RSDR)
7.82 8.43 4.94
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