N-Methylated Derivatives of Tyramine in Citrus Genus Plants

Mar 17, 2014 - Results indicate that N,N,N-trimethyltyramine is consistently expressed in leaves of clementine, bitter orange, and lemon. Conversely, ...
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N‑Methylated Derivatives of Tyramine in Citrus Genus Plants: Identification of N,N,N‑Trimethyltyramine (Candicine) Luigi Servillo,*,† Alfonso Giovane,† Nunzia D’Onofrio,† Rosario Casale,† Domenico Cautela,‡ Giovanna Ferrari,§ Maria Luisa Balestrieri,† and Domenico Castaldo§,∥ †

Dipartimento di Biochimica, Biofisica e Patologia Generale, Seconda Università degli Studi di Napoli, Via L. De Crecchio 7, 80138 Napoli, Italy ‡ Stazione Sperimentale per le Industrie delle Essenze e dei derivati dagli Agrumi, Azienda Speciale della Camera di Commercio di Reggio Calabria, Via Tommaso Campanella 12, 89127 Reggio Calabria, Italy § Dipartimento di Ingegneria Industriale e ProdAL scarl, Università degli Studi di Salerno, Via Ponte Don Melillo 1, 84084 Fisciano, Salerno, Italy ∥ Ministero dello Sviluppo Economico, Via Molise 2, Roma, Italy ABSTRACT: The distribution of tyramine and its methylated derivatives, N-methyltyramine and N,N-dimethyltyramine, was investigated in tissue parts (leaves and fruits) of several plants of Citrus genus by liquid chromatography−electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). In the course of our study we discovered the occurrence of N,N,Ntrimethyltyramine in all citrus plants examined. This quaternary ammonium compound, known to act in animals as a neurotoxin, was recognized and characterized by mass spectrometric analysis. The substance, never described before in the Citrus genus, is also known as candicine or maltoxin. Results indicate that N,N,N-trimethyltyramine is consistently expressed in leaves of clementine, bitter orange, and lemon. Conversely, low levels were found in the leaves of orange, mandarin, chinotto (Citrus myrtifolia), bergamot, citron, and pomelo. In the edible part of the fruits, N,N,N-trimethyltyramine was found at trace levels. KEYWORDS: N-methylated tyramine derivatives, N,N,N-trimethyltyramine, candicine, Citrus plants, biotic stress

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INTRODUCTION In the course of evolution, vegetal organisms have developed the ability to synthesize a multitude of chemical compounds, some of which are nonessential for their normal growth and development. These compounds are produced with the aim of contributing to the plant’s defense from herbivores and pathogens.1−3 Decarboxylation of aromatic amino acids, catalyzed by specific decarboxylases, is aimed at the biosynthesis of biogenic amines such as tryptamine, tyramine, and phenylethylamine. These compounds are then used as starting substrates for the production of more complex phytochemicals,4,5 which are toxic for the plant aggressors.1,6−8 Despite considerable progress in understanding defense mechanisms against biotic stress, only little research on plant tyrosine decarboxylase (TYDC) has been reported.9−12 TYDC belongs to the family of pyridoxal 5′-phosphate-dependent enzymes that decarboxylate tyrosine or L-dopa to form tyramine or dopamine, respectively.13,14 Except for a study on a TYDC involved in synephrine biosynthesis in Citrus genus,15 further studies on specific TYDC in Citrus genus plants have not yet been reported. Tyramine represents the starting substrate of at least two different and fundamental metabolic routes leading to the formation of protoalkaloids. Specifically, in the first metabolic route, tyrosine decarboxylation catalyzed by TYDC produces tyramine, from which octopamine is then derived through the hydroxylation reaction catalyzed by tyramine-β-hydroxylase. Successively, N-methylation of octopamine forms N-methyloctopamine, commonly known as synephrine. In the second metabolic route, N-methyltyramine © 2014 American Chemical Society

and N,N-dimethyltyramine (hordenine) are formed from tyramine by two consecutive methylation steps. Moreover, synephrine can be also produced through hydroxylation of Nmethyltyramine. Octopamine and synephrine, both present in Citrus genus plants, are of pharmacological relevance.16−21 Another substance, N,N,N-trimethyltyramine (Figure 1), known as candicine or maltoxin, was first discovered in barley malt rootlets (Hordeum distichon L.)22,23 and acts in animals as a neurotoxin.24,25 In this investigation we studied the distribution of Nmethylated tyramine derivatives in several plants of Citrus genus and quantitated their levels in leaf tissue.

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MATERIALS AND METHODS

Reagents. Tyramine, N-methyltyramine, N,N-dimethyltyramine, phenylalanine, methyl iodide, and 0.1% solution of formic acid in water were from Sigma−Aldrich (Milan, Italy). SPE-C18 columns for flash chromatography were obtained from Phenomenex (Anzola Emilia, Italy). All other solvents and reagents used were of analytical grade. Synthesis and Purification of N,N,N-Trimethyltyramine. For the conversion of tyramine into its quaternary ammonium compound N,N,N-trimethyltyramine, we used a modified heterogeneous-phase reaction employing methyl iodide as the methylation agent in the presence of KHCO3.26,27 Tyramine (200 mg) was dissolved in 20 mL of methanol, and then 1 g of KHCO3 and, subsequently, 10 mL of Received: Revised: Accepted: Published: 2679

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methyltyramine, 0.020 mg/L for N,N-dimethyltyramine, and 0.009 mg/L for N,N,N-trimethyltyramine. Flow Injection Analysis and HPLC-ESI-MS/MS Measurements. HPLC-ESI-MS analyses were performed with an HPLC Agilent 1100 series coupled online with an Agilent LC-MSD SL quadrupole ion trap. MS acquisition was performed by using electrospray inonization (ESI) in positive-ion mode. Nitrogen was used at a flow rate of 7 L/min and a pressure of 30 psi as both a drying and a nebulizing gas. The nebulizer temperature was set at 350 °C. The ion charge control (ICC) was applied with a target set at 30 000 and maximum accumulation time at 20 ms. Measurements were performed from the peak area of the extracted ion chromatogram (EIC). Quantitation was achieved by comparison with calibration curves obtained with standard solutions. Optimization of instrumental parameters for analyses of tyramine and its methylated derivatives was performed by continuous infusion [flow injection analysis (FIA)-ESIMS/MS measurements] of 5 μM standard solution in 0.1% formic acid. Mass cutoff and amplitude were optimized to obtain the most efficient MS/MS transitions from the positively charged precursor ion [M + H+] to the fragment ions. Multiple reaction monitoring (MRM) was used for quantitation of analytes. The transitions utilized for MS/ MS quantitation were 138.1 → 121 for tyramine, 152.1 → 121 for Nmethyltyramine, 166.1 → 121 for N,N-dimethyltyramine, 180.1 → 121 for N,N,N-trimethyltyramine, and 166.1 → 120 for phenylalanine. LCESI-MS/MS analyses were performed with a 150 mm × 3.0 mm i.d., 5 μm Discovery-C8 analytical column (Supelco). The mobile phase was composed of 0.1% formic acid in water and pumped at 0.1 mL/min under isocratic conditions at room temperature. Volumes of 10−20 μL of standard solutions or samples were injected. Statistical Analysis. Data are expressed as mean ± std of n = 4 determinations, each in duplicate. Differences were assessed by t-test, and a p value less than 0.05 was considered to be significant.

Figure 1. Chemical structures of analyzed compounds: tyramine (1), N-methyltyramine (2), N,N-dimethyltyramine (hordenine) (3), 2-(4hydroxyphenyl)ethyl-trimethylazanium (candicine or N,N,N-trimethyltyramine) (4), and phenylalanine (5).

methyl iodide were added. The mixture was stirred at room temperature and the formation of the reaction products N-methyltyramine, N,N-dimethyltyramine, and N,N,N-trimethyltyramine was monitored by liquid chromatography−electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). The addition of methyl iodide (10 mL) and KHCO3 (1 g) was repeated twice more. Finally, after centrifugation of the mixture, the supernatant was collected and evaporated to dryness at 40 °C in a rotary evaporator. The residue containing N,N,N-trimethyltyramine was dissolved in 10 mL of Milli Q-grade water and purified by flash chromatography on SepPac C18 cartridge (Phenomenex, Anzola Emilia, Italy). The sample-loaded column was washed with 100 mL of Milli Q water, and N,N,Ntrimethyltyramine was eluted from the column by applying 50 mL of a solution of H2O/acetonitrile (80:20). The eluted product was evaporated to dryness under a stream of air and dried overnight in vacuum over P2O5. The yield was 88%. Preparation of Citrus Leaf Extracts. Citrus leaf samples were obtained from the Research Centre for Citrus Crops and the Mediterranean (CRA-ACM), Arboretum of Reggio Calabria (Italy). Four lots of 100 g were taken from different branches from each citrus plant species. Leaves, in the late development stage, were harvested in January and February. The leaves were washed with distilled water and dried with filter paper. Then 50 g of product, finely chopped, was homogenized in a blender with 100 mL of 0.2% formic acid in Milli Qgrade water and the mixture was stirred for 1 h. In these conditions the extraction yield was higher than 96% for all the analyzed compounds. Finally, the homogenate was centrifuged at 18000g for 30 min and the supernatant was stored in 20 mL vials at −20 °C. Citrus varieties included in this study were mandarin, sweet orange, bitter orange, chinotto (Citrus myrtifolia), pomelo, citron, grapefruit, bergamot, and clementine. Preparation of Standards. Standard stock solutions of tyramine, N-methyltyramine, N,N-dimethyltyramine, and N,N,N-trimethyltyramine were prepared at concentration of 2 mg/L and stored at 4 °C. Prior to injection, stock solutions were appropriately diluted with water containing 0.1% formic acid before being used as working solutions. Quantitation was achieved by comparison with calibration curves obtained with the standard solutions. Additional calibration levels (2, 1, 0.5, 0.1, and 0.05 mg/L) were prepared by serial dilution with water containing 0.1% formic acid. The calibration curve was built by use of these standard solutions. The response of the MS/MS detection followed a linear calibration curve between 0.05 and 2 mg/L with a correlation coefficient of 0.99. Detection limits, determined as the compound concentration that gave a peak height 3 times that of background noise, were 0.021 mg/L for tyramine, 0.016 mg/L for N-

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RESULTS AND DISCUSSION

Identification of N,N,N-Trimethyltyramine in Citrus Genus Plants. Recently, we reported the occurrence of secondary metabolites derived from tryptamine in Citrus genus plants.28 Specifically, we described the presence of Nmethyltryptamine and N,N-dimethyltryptamine in all parts of the fruit (peel and edible part) with higher levels in leaves and seeds. Interestingly, we also revealed the occurrence of N,N,Ntrimethyltryptamine, a metabolite never described before in plants.28 Subsequently, in several Citrus genus plants, we quantitated 5-hydroxytryptamine (serotonin) and its Nmethylated forms such as 5-hydroxy-N-methyltryptamine, 5hydroxy-N,N-dimethyltryptamine (bufotenine), and 5-hydroxyN,N,N-trimethyltryptamine. In particular, we reported the occurrence of 5-hydroxy-N,N,N-trimethyltryptamine, also named bufotenidine or cinobufotenine.29 It is worth noting that the occurrence of tyramine, N-methyltyramine, and N,Ndimethyltyramine in Citrus genus plants has been known for a long time, whereas the occurrence of N,N,N-trimethyltyramine has never been reported so far. Therefore, we sought to verify the occurrence of N,N,N-trimethyltyramine in the most common and economically important plants of the Citrus genus. Moreover, we also aimed to quantitatively compare, among them, the distribution of tyramine and its N-methylated derivatives. The analysis, performed on a lemon leaf extract and conducted with the same HPLC-ESI-MS/MS methodology previously described,30,31 showed complete chromatographic separation of all examined metabolites in less than 30 min (Figure 2A). In particular, tyramine eluted at a retention time (TR) of 12.7 min, followed by N-methyltyramine at TR 15.5 min and N,N-dimethyltyramine (hordenine) at TR 19.5 min. Interestingly, an unknown peak emerged at TR 23.8 min. Moreover, this peak occurred at m/z 180 and showed a very 2680

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Figure 2. (A) LC-ESI-MS/MS chromatogram of lemon leaf extract. (B) MS2 fragmentation pattern of the unknown peak (peak X) at retention time of 24 min.

simple MS/MS fragmentation pattern (Figure 2B) with only two fragments at m/z 60 and 121, which strongly suggested the possibility that the it might be N,N,N-trimethyltyramine. Synthesis and ESI-MSn Characterization of N,N,NTrimethyltyramine. With the aim to confirm the presence of N,N,N-trimethyltyramine in Citrus genus plants, we synthesized the substance according to the method of Chen and Benoiton,26,27 as it was not commercially available. Under the reaction conditions adopted, the methylation process of tyramine, which was used as starting substrate for the synthesis, was essentially complete in about 6 h at room temperature. Formation of N,N,N-trimethyltyramine and the less methylated derivatives (N-methyltyramine and N,N-dimethyltyramine) was monitored by withdrawing 50 μL of reaction mixture at preset times of 2, 6, 12, and 24 h. Each aliquot was then diluted to a final volume of 1 mL with 0.1% formic acid. Then 20 μL portions of the diluted samples were analyzed by LC-ESI-MS/ MS. After 2 h, the reaction mixture contained tyramine and noticeable amounts of N-methyltyramine and N,N-dimethyltyramine compared to N,N,N-trimethyltyramine (Figure 3A). High levels of the desired compound were found in the reaction mixture in 6 h (Figure 3B). Conversely, other Nmethylated tyramine derivatives occurred at lower levels. The synthesis was complete in 12 h of reaction, as demonstrated by the presence in the reaction mixture of only N,N,Ntrimethyltyramine and the disappearance of other components (Figure 3C). In order to find the optimal instrumental conditions for detection and quantitation, the synthesized N,N,N-trimethyltyramine was purified and subjected to mass spectrometric analysis. In particular, amplitude and cutoff, the main parameters for an ion-trap mass spectrometer to achieve the most effective collision-induced dissociation (CID) of the parent ion toward its ion fragments, were optimized. This study was performed in positive-ion mode by direct infusion of 5 μM N,N,N-trimethyltyramine solution in 0.1% formic acid. N,N,NTrimethyltyramine showed the highest retention time relative to the less methylated tyramine derivatives and eluted at about 24 min (Figure 4A). It is interesting to note that the MS2 fragmentation pattern of the synthesized N,N,N-trimethyltyramine is essentially constituted by only two ions (Figure 4B). The one at m/z 60 corresponds to trimethylammonium ion,

Figure 3. Time course of tyramine methylation performed according to the method of Chen and Benoiton:26,27products of tyramine methylation after (A) 2 h, (B) 6 h, and (C) 12 h of reaction. The MS2 transitions used to monitor tyramine (1), N-methyltyramine (2), N,Ndimethyltyramine (3), and N,N,N-trimethyltyramine (4) are also reported.

which is peculiar for most betaines,30,32 and the other, at m/z 121, is formed as a consequence of neutral loss of the trimethylamine moiety33 from the parent ion. An intense fragment at m/z 121 is present also in the MS2 spectra of tyramine, N-methyltyramine, and N,N-dimethyltyramine. This fragment was observed for the first time by Cram and Thompson33 in the solvolysis of 2-arylethyl derivatives and is supposed to have the very stable bicyclo structure shown in the inset of Figure 4B. The fragment is produced from tyramine by neutral loss of ammonia, whereas N-methyltyramine and N,Ndimethyltyramine produce it by neutral loss of methylamine and dimethylamine, respectively. The MS3 fragmentation pattern was obtained by isolating the MS2 fragment at m/z 121 of the synthesized N,N,N-trimethyltyramine (Figure 4C). The MS3 fragment at m/z 93 likely originated from C2H4 neutral loss from the m/z 121 parent ion.33 The identity of the fragment at m/z 121 in the MS2 fragmentation pattern of the synthesized N,N,N-trimethyltyramine was further supported by the observation that its MS3 fragmentation pattern was the same as those of the MS2 fragments, isolated at m/z 121, generated by tyramine, N-methyltyramine, and N,N-dimethyltyramine. It is important, from an analytical point of view, to highlight that the stability of the N-methylated derivatives of tyramine toward in-source CID is strongly dependent on the degree of 2681

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point of view, N,N,N-trimethyltyramine was most abundant in the leaves of clementine (0.88 ± 0.64 mg/kg), followed by bitter orange and lemon. In contrast, substantially lower levels were found in orange, bergamot, citron, pomelo, and mandarin leaves (Table 1). In order to unambiguously confirm the presence of N,N,Ntrimethyltyramine in these matrices, chromatographic and mass spectrometric behaviors of the putative N,N,N-trimethyltyramine peak in leaf extracts were compared with those of the synthesized standard. In each case, both peaks showed the same chromatographic retention time. Moreover, under the same instrumental settings of the ion trap, the peaks showed the same fragmentation patterns and the same intensity ratios of main fragments in both MS2 and MS3 measurements. On the whole, these results leave no doubt that the compound eluting at about 24 min in the extracts of citrus leaves is N,N,Ntrimethyltyramine. Among the other analyzed metabolites, tyramine is wellrepresented in leaves of citrus plants. Particularly, 49.75 ± 12.16 and 21.63 ± 7.80 mg/kg tyramine were found in leaves of chinotto and clementine, respectively (Table 1), while values of 0.55 ± 0.38 and 0.41 ± 0.33 mg/kg were found in pomelo and bergamot leaves. N-Methyltyramine is the tyramine derivative most abundant in citrus leaves. Particularly, leaves of chinotto and clementine contained mean levels above 20 mg/kg of this metabolite, whereas leaves of bergamot, pomelo, citron, mandarin, lemon, bitter orange, and sweet orange contained amounts from a minimum of 0.45 ± 0.31 mg/kg up to a maximum of 6.91 ± 2.04 mg/kg (Table 1). N,N-Dimethyltyramine was contained in discrete amounts only in the leaves of clementine (0.30 ± 0.19 mg/kg). In all other citrus matrices N,N-dimethyltyramine was at levels equal or below 0.07 ± 0.04 mg/kg (Table 1). It is important to point out that phenylalanine, an amino acid well represented in the citrus matrices,34,35 has the same mass as N,N-dimethyltyramine. Fortunately, complete chromatographic separation of all examined tyramine derivatives and phenylalanine was obtained under the chromatographic conditions employed (Figure 5). In particular, phenylalanine, which in all samples examined was always present in much higher amounts than N,N-dimethyltyramine, was well resolved from it (Figure 6A). Therefore, phenylalanine did not interfere in the quantitation of N,Ndimethyltyramine in the samples examined. It is important to achieve good chromatographic resolution between these two compounds, especially when phenylalanine is much more abundant then N,N-dimethyltyramine, despite the fact that phenylalanine and N,N-dimethyltyramine seem resolvable by mass spectrometric measurements. In fact, the analytical MS2 transition employed for N,N-dimethyltyramine determination was at m/z 166 → 121, while for phenylalanine it was at m/z 166 → 120. However, besides the intense peak corresponding

Figure 4. Mass spectrometric characterization of N,N,N-trimethyltyramine (4) standard solution. (A) Extracted-ion chromatogram at m/z 180.1 from LC-ESI-MS analysis of the synthesized N,N,N-trimethyltyramine (4) standard solution. (B) MS2 fragmentation pattern of the peak in panel A. Arrows indicate the structure of the generated MS2 fragments. (C) MS3 fragmentation pattern of the peak in panel A, obtained by isolating the MS2 fragment at m/z 121.1. Arrows indicate the structure of the MS3 fragment at m/z 93.1, likely generated by C2H4 neutral loss.

methylation. In fact, we observed that about 85% of tyramine, 70% of N-methyltyramine, and 10% of N,N-dimethyltyramine was fragmented by in-source CID. Instead, no in-source fragmentation of N,N,N-trimethyltyramine was observed. Therefore, especially for tyramine and N-methyltyramine, if needed, more analytical sensitivity can be achieved by selecting the fragment at m/z 121 produced in the ion source than that at the m/z corresponding to the protonated parent molecules. Distribution of N-Methylated Tyramine Derivatives in Citrus Genus Plant Leaves. Analysis of the content of tyramine and its N-methylated derivative in citrus leaf extracts was conducted by HPLC-ESI-MS/MS. From a quantitative

Table 1. Distribution of N-Methylated Tyramine Derivatives in Citrus Leaves contenta (mg/kg) compd tyramine N- methyltyramine N,Ndimethyltyramine N,N,Ntrimethyltyramine a

citron

mandarin

pomelo

bitter orange

sweet orange

chinotto

clementine

lemon

bergamot

1.42 ± 0.45 0.63 ± 0.43 0.07 ± 0.04

2.47 ± 0.47 0.66 ± 0.52 0.06 ± 0.04

0.55 ± 0.38 0.58 ± 0.41 0.03 ± 0.02

0.70 ± 0.56 2.22 ± 0.82 0.04 ± 0.02

5.74 ± 1.39 6.91 ± 2.04 0.03 ± 0.02

49.75 ± 12.16 22.63 ± 8.62 0.05 ± 0.03

21.63 ± 7.80 23.25 ± 6.88 0.30 ± 0.19

1.92 ± 1.01 1.88 ± 0.97 0.05 ± 0.04

0.41 ± 0.33 0.45 ± 0.31 0.03 ± 0.02

0.01 ± 0.01

0.01 ± 0.01

0.01 ± 0.01

0.64 ± 0.50

0.01 ± 0.01

0.07 ± 0.05

0.88 ± 0.64

0.31 ± 0.19

0.01 ± 0.01

Content is expressed as mean ± std of n = 4 determinations, each in duplicate. 2682

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The results show that also N,N,N-trimethyltyramine is present at very different levels in the various plant species examined. Clementine, lemon, and bitter orange leaves contained the most significant amounts. The highest level was found in clementine leaves (0.88 mg/kg) and the lowest in citron, mandarin, pomelo, and bergamot leaves (0.01 mg/kg) (p < 0.05) (Table 1). From a physiological point of view, the simultaneous presence of tyramine and all other N-methylated derivatives in the leaf, a part of the plant highly specialized in biosynthesis, secretion, and accumulation of toxic substances, suggests that these substances are aimed at plant defense. It is well-known that plants under pathogenic attacks respond with the activation of defense mechanisms regulated by several phytohormones. These rapid defensive responses occur through metabolic pathways involving tryptophan, tyrosine, and phenylalanine, leading to the production and accumulation of specific metabolites aiming to prevent or alleviate the action of the pathogens.36,37 Among the products of these enzymatic activities, an important role is played by tyramine and tryptamine, produced by the actions of tyrosine decarboxylase and tryptophan decarboxylase. These biogenic amines are precursors of many specialized defensive secondary metabolites.38,39 In this context, the formation of N,N,N-trimethyltyramine may be the result of a series of reactions starting from tyrosine decarboxylation and continuing with successive methylation catalyzed by the same or a different Nmethyltransferase.29 This process is likely aimed to increase the plant’s defense through formation of a final product with powerful physiological activity. Such a possibility is supported by pharmacological studies reporting that N,N,N-trimethyltyramine is a neuromuscular blocker of depolarizing type that possesses muscarine-like and sympathomimetic effects.22−25 It was demonstrated that in some animals N,N,N-trimethyltyramine induces cardiovascular and respiratory disturbances. Furthermore, it has been shown that N,N,N-trimethyltyramine is a stimulant of autonomic ganglia and can liberate catecholamines from the adrenal medulla. Finally, the hypothesized defensive role of N,N,N-trimethyltyramine could occur in synergy with other biogenic amine derivatives also well-represented in citrus leaves, such as those arising from tryptophan.29

Figure 5. Chromatographic separation of standard solutions of phenylalanine and methylated tyramine derivatives by HPLC-ESIMS/MS. For each compound, the extracted ion chromatogram (EIC) at the indicated MS2 transition is reported. (1) tyramine (TR 12.7 min); (2) N-methyltyramine (TR 15.5 min); (3) N,N-dimethyltyramine (TR 19.5 min); (5) phenylalanine (TR 21.3 min); (4) N,N,Ntrimethyltyramine (TR 23.8 min).

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Figure 6. Representative HPLC-ESI-MS/MS chromatogram of lemon leaf extract. (A) Peaks of components present at higher levels: Nmethyltyramine (2) and phenylalanine (5) (intensity × 10−7). (B) Peaks of components present at lower levels: tyramine (1), N,Ndimethyltyramine (3), and N,N,N-trimethyltyramine (4) (intensity × 10−5). The M + 1 peak originating from phenylalanine 13C content, obtained by monitoring the MS2 transition at 166−121, is also shown in panel B.

AUTHOR INFORMATION

Corresponding Author

*Phone +39-08-15665865; fax+39-08-15665863; e-mail luigi. [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are grateful to Dr. Giuseppe Cicciarello, peripheral operational section of Reggio Calabria Research Centre for Citrus Crops and the Mediterranean (CRA-ACM), Acireale (Italy), for providing citrus plant materials.

to the transition at m/z 166 → 120, phenylalanine, being much more abundant than N,N-dimethyltyramine in all the samples examined, also showed a peak at m/z 166 → 121, arising from a not-negligible 13C contribution, with intensity similar to the peak at m/z 166 → 121 of N,N-dimethyltyramine. Therefore, without chromatographic resolution, the two compounds would strongly interfere (Figure 6B). The data reported in Table 1 show that distribution of the biogenic amine tyramine and its other two methylation products, N-methyltyramine and N,N-dimethyltyramine, is somewhat uneven among the various Citrus species. The novelty of this work has been the identification and quantitation of N,N,N-trimethyltyramine in Citrus plant leaves.

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REFERENCES

(1) Kutchan, T. M. A role for intra-and intercellular translocation in natural product biosynthesis. Curr. Opin. Plant Biol. 2005, 8, 292−300. (2) Murata, J.; Roepke, J.; Gordon, H.; De Luca, V. The leaf epidermome of Catharanthus roseus reveals its biochemical specialization. Plant Cell 2008, 20, 524−542. (3) Ballhorn, D. J.; Kautz, S.; Heil, M.; Hegeman, A. D. Analyzing plant defences in nature. Plant Signal Behav. 2009, 8, 743−745.

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Journal of Agricultural and Food Chemistry

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dx.doi.org/10.1021/jf5001698 | J. Agric. Food Chem. 2014, 62, 2679−2684