Food Chemistry 153 (2014) 151–156

Contents lists available at ScienceDirect

Food Chemistry journal homepage: www.elsevier.com/locate/foodchem

Analytical Methods

Determination of melatonin and its isomer in foods by liquid chromatography tandem mass spectrometry Tolgahan Kocadag˘lı, Cemile Yılmaz, Vural Gökmen ⇑ Department of Food Engineering, Hacettepe University, 06800 Beytepe, Ankara, Turkey

a r t i c l e

i n f o

Article history: Received 7 August 2013 Received in revised form 28 November 2013 Accepted 7 December 2013 Available online 12 December 2013 Keywords: Melatonin Melatonin isomer Method development Food analysis Fermented foods

a b s t r a c t This study aimed to develop a reliable analytical method for the determination of melatonin and its isomers in various food products. The method entails ethanol extraction of solid samples (or dilution of liquid samples) prior to liquid chromatography coupled to triple quadruple mass spectrometry (LC–MS/MS) analysis of target analytes. The method was in-house validated and successfully applied to various food matrices. Recovery of melatonin from different matrices were found to be 86.0 ± 3.6%, 76.9 ± 5.4%, 98.6 ± 6.4%, and 67.0 ± 4.5% for beer, walnut, tomato and sour cherry samples, respectively. No melatonin could be detected in black and green tea, sour cherry, sour cherry concentrate, kefir (a fermented milk drink) and red wine while the highest amount of melatonin (341.7 ± 29.3 pg/g) was detected in crumb. The highest amounts of melatonin isomer were detected in yeast-fermented foods such as 170.7 ± 29.9 ng/ml in red wine, 14.3 ± 0.48 ng/ml in beer, and 15.7 ± 1.4 ng/g in bread crumb. Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction Melatonin (N-acetyl-3-(2-aminoethyl)-5-methoxyindole), produced mainly by the pineal gland in vertebrates, is an indoleamine synthesised from L-tryptophan metabolism via serotonin (Reiter, 1991). Melatonin has a significant role in regulation of the circadian rhythm, mitigation of sleeping disorders and jet lag (Reiter, 1993). Moreover, melatonin can directly scavenge free radical species and stimulate the activity of antioxidant enzymes (Reiter, Paredes, Manchester, & Tan, 2009). Besides being neurohormone, melatonin has been detected in edible plants as a phytohormone having physiological functions such as protecting plants against oxidative stress and regulating growth (Paredes, Korkmaz, Manchester, Tan, & Reiter, 2009). Previous studies have shown that consumption of foods containing melatonin increased its level in plasma and urinary 6-sulfatoxymelatonin, a marker of circulating melatonin in human (Johns, Johns, Porasuphatana, Plaimee, & Sae-Teaw, 2013; Reiter, Manchester, & Tan, 2005). The molecular structure of melatonin and the hypothetical structure of the isomer identified in foods are shown in Fig. 1. Although presence of melatonin at varying concentrations in foods including seeds, fruits and beverages has been previously reported (Garcia-Moreno, Calvo, & Maldonado, 2012; Manchester et al., 2000; Sturtz, Cerezo, Cantos-Villar, & Garcia-Parrilla, 2011), there is limited published information on melatonin isomers in foods. To date, melatonin isomers have been detected only in grape ⇑ Corresponding author. Tel.: +90 312 2977108; fax: +90 312 2992123. E-mail address: [email protected] (V. Gökmen). 0308-8146/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2013.12.036

products (Gomez, Raba, Cerutti, & Silva, 2012; Tan et al., 2012; Vitalini, Gardana, Simonetti, Fico, & Iriti, 2013). Biosynthesis of melatonin isomers is still not clear, however it was reported by Gomez et al. (2012) that the level of melatonin isomers increased during fermentation process in wine making. From the viewpoint of biological functions, melatonin isomers show antioxidant and cytoprotective activity depending on changing the position of two side chains in the indole ring (Spadoni et al., 2006). The analysis of melatonin is a difficult task as it occurs in low ppb levels in foods. Various analytical methods have been used to analyse melatonin in foods. These include liquid chromatography coupled to fluorescence detector (Hattori et al., 1995; Mills, Finlay, & Haddad, 1991) or mass spectrometer (Cao, Murch, O’Brien, & Saxena, 2006; Dubbels et al., 1995; Lewy & Markey, 1978), gas chromatography–mass spectrometry (Lewy & Markey, 1978), radioimmunoassay (Kennaway, Frith, Phillipou, Matthews, & Seamark, 1977), enzyme-linked immunosorbent assay (Iriti, Rossoni, & Faoro, 2006; Maldonado, Moreno, & Calvo, 2009) and immunoprecipitation (Harumi & Matsushima, 2000; Pape & Luning, 2006). In melatonin assay, a chromatographic separation prior to detection is a must to prevent any interference of complex food matrices. This study aimed to develop an analytical method for the determination of melatonin and its isomers in various food matrices. The method is based on ethanol extraction of melatonin and its isomers from foods, reversed phase liquid chromatographic separation, and specific detection by tandem mass spectrometry. The method was validated in-house by analysing number of native and spiked food samples.

T. Kocadag˘lı et al. / Food Chemistry 153 (2014) 151–156

152

(a) H C 3

2.3. LC–MS/MS analysis of melatonin O

H N

H 3C

O

N H

(b)

CH 3

O N O N H

CH3

Fig. 1. Molecular structures. (a) Melatonin, and (b) melatonin isomer identified in foods.

2. Materials and methods 2.1. Chemicals and consumables Acetonitrile (HPLC grade), ethanol (HPLC grade) and melatonin (N-acetyl-5-methoxytryptamine) (>98%) were obtained from Sigma–Aldrich (Steinheim, Germany). Formic acid (98%) was purchased from Merck Co. (Darmstadt, Germany). Ultra-pure water was used throughout the experiments (Milli Q-System, Millipore, Milford, MA, USA). Syringe filters (nylon, 0.45 lm) were supplied from Waters (Millford, MA) and ZORBAX Rapid Resolution SBC18 column (4.6  50 mm, 3.5 lm) was supplied from Agilent Technologies (Waldbronn, Germany). 2.2. Sample preparation Black and green tea, green coffee, walnut, cacao powder, black olive, tomato, frozen sour cherry, probiotic yogurt, kefir, bread, red wine and beer were purchased from a local market. Low moisture samples (tea, walnut, cacao powder, green coffee, bread crumb and crust) were grinded using a home type grinder prior to extraction. Higher moisture samples (sour cherry, tomato, kefir, probiotic yogurt, black olive) were homogenised at 20 000 rpm for 3 min (Heidolph, Germany) and freeze-dried to obtain fine powder prior to extraction. Removal of water prevented the increase in the polarity of extraction solvent during extraction. Liquid samples (red wine, beer, sour cherry concentrate) were diluted up to fivefolds with the mixture of ethanol–water (50:50, v/v). Beer was degassed in an ultrasonic bath prior to analysis. One milliliter was filtered through a 0.45 lm syringe filter into an autosampler vial prior to LC–MS/MS analysis. 2.2.1. Extraction One gram of ground sample was extracted with ethanol in three stages (5.0, 2.5, and 2.5 ml). In each stage, vortexing (3 min) and centrifugation (7500g for 5 min) were performed. Supernatants obtained from oily samples were stored at 80 °C for 30 min and filtered through a filter paper (125-mm circle No.:6, Advantec Toyo Kaisha, Ltd., Tokyo, Japan) to remove oil without using a non-polar solvent. Extracts were combined and evaporated to dryness under a gentle stream of nitrogen. The final residue was re-dissolved in 1 ml of the mixture of ethanol–water (50:50, v/v). After cold centrifugation (13 201g for 15 min at 10 °C), supernatant was passed through a 0.45 lm syringe filter into an autosampler vial prior to LC–MS/MS analysis.

Melatonin and its isomers were determined by using an Agilent 1200 series HPLC system coupled to Agilent 6460 triple quadruple mass spectrometer (Waldbronn, Germany). The chromatographic separations were performed on a ZORBAX Rapid Resolution SB-C18 column (50  4.6 mm i.d., 3.5 lm) using the mixture of 0.1% formic acid in water and 0.1% formic acid in acetonitrile (65:35, v/v) at a flow rate of 0.5 ml/min at 40 °C. The injection volume was 5 ll. The electrospray source had the following settings: drying gas (N2) flow of 10 l/min at 325 °C, nebulizer pressure of 30 psi, sheath gas (N2) flow of 10 l/min at 375 °C, nozzle voltage of 1000 V, and positive capillary voltage of 4000 V. MS data were acquired in the positive mode and melatonin was identified by multiple reaction monitoring (MRM). Fragmentor voltage of precursor ion was 80 V. The transitions for melatonin were determined as 233.2 ? 174.2 (collision energy of 10 V) and 233.2 ? 216.2 (collision energy of 4 V). A dwell time was set at 250 ms for each transition. 2.4. Validation of the method The 233.2/174.2 MRM transition was selected to quantify melatonin. Concentration of melatonin was calculated by means of a external calibration curve built in the range between 0.05 and 20 ng/ml. All melatonin standard solutions were prepared in ethanol–water mixture (50:50). Determination of the melatonin isomer was semi-quantitative and its content was calculated from total ion chromatogram (sum of the area of the MRM transitions (233.2 ? 174.2 and 233.2 ? 216.2 of melatonin). The linearity was evaluated by plotting the peak area against the concentrations of melatonin standards. Limit of detection (LOD) and limit of quantification (LOQ) were determined at a signal to noise ratio of 3 and 10, respectively. Reproducibility of the method was determined by analysing five replicates in three consecutive days. Solid and liquid food samples spiked with different levels of melatonin (1, 5 and 10 ng/ml) were analysed to determine percentage recovery. 3. Results and discussion The chromatographic separation and ion transitions of melatonin and its isomer in different food matrices analysed were illustrated in Fig. 2. Melatonin was identified with its retention time, specific MRMs (233.2 ? 174.2 and 233.2 ? 216.2) and ion ratios acquired from the standard compound. Fig. 2a illustrates the chromatogram of 1 ng/ml melatonin standard for the MRM transitions of 233.2 ? 174.2 (upper panel) and 233.2 ? 216.2 (bottom panel). Relative abundance of the fragment ion of 174.2 was higher than that of 216.2 for melatonin. Therefore, the fragment ion of 174.2 was used for the quantification of melatonin. The ratio of 174.2/ 216.2 for melatonin was found to be 14.0 ± 2.0 (Table 1). This ratio was used to confirm the presence of melatonin in foods. Fig. 2b–d illustrate the chromatograms of walnut, beer, and red wine, respectively. As seen in these chromatograms, another compound with the same fragment ions of melatonin was detected. This compound having relatively shorter retention time was identified as melatonin isomer. Although melatonin and its identified isomer had the same fragment ions, the ratios of these ions were different for melatonin and melatonin isomer. Relative abundance of the fragment ion of 216.2 was higher than that of 174.2 for melatonin isomer. The ratio of 216.2/174.2, which was used to confirm the presence of melatonin isomer in foods, was found to be 3.0 ± 0.3 (Table 1). The fragment ion 174.2 is originated by the cleavage of amide substituent (–NH2COCH3) from main ion. Diamantini, Tarzia,

T. Kocadag˘lı et al. / Food Chemistry 153 (2014) 151–156

153

(a) x10 2

(b)

+ESI MRM Frag=80.0V [email protected] (233.2 -> 174.2) 29_03_13_003.d

1.25 1 0.75 0.5 2

x10 1

2.5

3

3.5 4 4.5 5 5.5 6 Counts vs. Acquisition Time (min)

6.5

7

7.5

7

7.5

+ESI MRM Frag=80.0V [email protected] (233.2 -> 216.2) 29_03_13_003.d

4.6 4.4 4.2 4 2

2.5

3

3.5 4 4.5 5 5.5 6 Counts vs. Acquisition Time (min)

6.5

(c)

(d)

Fig. 2. LC–MS/MS chromatograms for (a) 1.0 ng/ml melatonin standard, (b) walnut, (c) beer, and (d) wine. Upper panel illustrates the MRM of 233.2 ? 174.2, and lower panel illustrates the MRM of 233.2 ? 216.2.

Table 1 Ion ratios of the fragments 216.2 and 174.2 for melatonin and its isomer in different food matrices (n = 3 for all food samples and n = 20 for melatonin standard solutions). Sample Pure melatonin standard Walnut Green coffee Tomato Sour cherry Bread (crumb) Bread (crust) Beer Red wine

Melatonin 174.2/216.2

Melatonin isomer 216.2/174.2

14.0 ± 2.0 15.1 ± 0.3 15.5 ± 0.3 14.2 ± 0.1 nd 14.0 ± 0.6 15.3 ± 0.8 14.9 ± 0.4 nd

– 3.1 ± 0.003 2.9 ± 0.1 2.9 ± 0.2 2.9 ± 0.1 3.0 ± 0.1 3.1 ± 0.1 2.9 ± 0.2 2.9 ± 0.03

nd: Not detected.

Spadoni, D’Alpaos, and Traldi (1998) indicated that 174.2 is the base peak for all isomers except where the substituent is bound to nitrogen of the pyrrole ring as shown in Fig. 1b. However, the position of the methoxy group on indole ring could not be determined by tandem mass spectrometry. The purification of the isomer is needed for the identification of exact molecular structure by using either NMR spectroscopy or X-ray diffraction spectroscopy. Other investigators have also observed similar fragmentation

Table 2 LOD and LOQ values of melatonin in different food matrices (pg/ml or pg/g). Food

LOD

LOQ

Walnut Green coffee Cacao powder Tomato Green tea Black tea Sour cherry Sour cherry concentrate Probiotic yogurt Kefir (fermented milk drink) Black olive (naturally fermented) Bread (crumb) Bread (crust) Beer Red wine

33.7 9.6 40.9 12.0 24.1 52.9 14.4 17.2 14.4 16.8 28.9 52.9 26.5 16.8 33.7

112.3 32.1 136.4 40.1 80.2 176.5 48.1 56.3 48.1 56.2 96.3 176.5 88.2 56.2 112.3

patterns (Gomez et al., 2012; Rodriguez-Naranjo, Gil-Izquierdo, Troncoso, Cantos, & Garcia-Parrilla, 2011). Vitalini et al. (2013) used MS/MS and orbitrap MS with a collision cell for the detection of melatonin and its isomers, but they did not mention the 233.2 ? 216.2 transition for both.

154

T. Kocadag˘lı et al. / Food Chemistry 153 (2014) 151–156

Since there is no standard compound commercially available for melatonin isomer, its semi-quantification was performed by means of the calibration curve of melatonin. Although the abundant ion of 216.2 is suitable for the quantification of the isomer its abundance in melatonin itself was very low, i.e. the transition ratio of 233.2/ 216.2 is quite different as mentioned. Thus, sum of the ions of 174.2 and 216.2 were used for calibration and quantification of the isomer in food samples. Rodriguez-Naranjo et al. (2011) used 233 > 216 transition for quantification of both melatonin and the isomer. Vitalini et al. (2013) and Gomez et al. (2012) did not mention the quantifier ion for both. An external calibration was built between 0.05 and 20 ng/ml with melatonin standard and a good linearity was obtained for the fragment ion 174.2 and for the sum of both fragments. Linearity equations of y = 619.5x + 44.2 and y = 658.5x + 58.6 and good determination coefficients (R2 = 0.9994 and 0.9995, respectively) were obtained in both quantifications. LOD and LOQ values were given in Table 2 for melatonin and relatively low levels were obtained. LOD was ranged between 9.6–52.9 pg/g and LOQ was 32.1–176.5 pg/g for the foods analysed. Percent recovery of melatonin was determined by analysing the samples spiked with three levels (1, 5 and 10 ng/ml). Fig. 3 shows the chromatograms of native and spiked sour cherry (1 ng/ml). Mean percentage recoveries of melatonin from beer, green coffee,

(a)

Table 3 Percentage recoveries of melatonin from different food matrices for the spiking levels of 1, 5 and 10 ng/g or ng/ml (n = 3 for each spiking level). Food matrices

Beer Green coffee Walnut Tomato Sour cherry

Recovery ± SD% 1 ng/g

5 ng/g

10 ng/g

81.5 ± 5.1 48.2 ± 2.2 79.2 ± 9.5 101.4 ± 3.3 64.5 ± 4.5

85.4 ± 2.8 54.5 ± 0.2 83.1 ± 3.8 – –

91.0 ± 3.7 45.8 ± 3.6 68.8 ± 6.4 95.8 ± 6.4 69.4 ± 4.9

Table 4 Levels of melatonin and melatonin isomers determined in various food samples. Food sample

Melatonin (pg/g or pg/ml)

Melatonin isomer (ng/g or ng/ml)

Walnut Green coffee Cacao powder Tomato Green tea Black tea Sour cherry Sour cherry concentrate Probiotic yogurt Kefir (fermented milk drink) Black olive (naturally fermented) Bread (crumb) Bread (crust) Beer Red wine

137.9 ± 27.40 39.0 ± 6.50 7.2 ± 0.50 28.9 ± 4.50 nd nd nd nd 126.7 ± 9.00 nd 5.3 ± 0.10 341.7 ± 29.30 138.1 ± 23.20 94.5 ± 6.70 nd

0.3 ± 0.02 1.2 ± 0.14 0.4 ± 0.03 1.6 ± 0.14 0.3 ± 0.02 0.3 ± 0.10 4.6 ± 0.14 5.2 ± 0.36 0.9 ± 0.06 0.6 ± 0.04 0.1 ± 0.01 15.7 ± 1.40 0.4 ± 0.12 14.3 ± 0.48 170.7 ± 29.90

nd: Not detected.

(b)

Fig. 3. LC–MS/MS chromatograms for (a) native sour cherry, and (b) sour cherry spiked with 1.0 ng/ml melatonin. Upper panel illustrates the MRM of 233.2 ? 174.2, and lower panel illustrates the MRM of 233.2 ? 216.2.

walnut, tomato and sour cherry are given in Table 3. As melatonin is a lipophilic compound, lower recoveries were observed in oily foods as seen in walnut and coffee. The analytical method had very high day-to-day reproducibility test under stated chromatographic conditions. Percentage standard deviation of the retention time of melatonin was found to be 1.4%. The mass spectrometric detection reproducibility was also found very high with a percentage deviation of 4.1% for the peak area of melatonin. Table 4 illustrates melatonin and the isomer contents of various foods. According to these results, it was found that sour cherry and its concentrate did not contain melatonin. In aprevious study, although similar result was observed for sour cherry concentrate, frozen Montmorency and frozen Balaton cherries contained 12.3 and 2.9 ng/g dry weight melatonin, respectively (Kirakosyan, Seymour, Llanes, Kaufman, & Bolling, 2009). Burkhardt, Tan, Manchester, Hardeland, and Reiter (2001) also reported similar results for same varieties of frozen cherries. Melatonin was also not detected in black tea, green tea, kefir and red wine samples. Gomez et al. (2012) reported that melatonin was not detected in finished wines made from grapes containing melatonin within the range 120–160 ng/g in dry weight basis. However, RodriguezNaranjo, Gil-Izquierdo, Troncoso, Cantos-Villar, and Garcia-Parrilla (2011) reported melatonin in wine within the range of 74.13– 423.01 ng/ml depending on different stages of the wine making process. Melatonin was also detected up to 129.5 ng/ml in wines produced from common grape varieties by HPLC/MS–MS (Rodriguez-Naranjo et al., 2011). As given in Table 4, melatonin was detected in crumb (341.7 pg/g), crust (138.1 pg/g), probiotic yogurt (126.7 pg/g), cacao powder (7.2 pg/g) and black olive (5.3 pg/g). To the best of authors’ knowledge, presence of melatonin in these foods has not been previously reported in literature. de la Puerta et al. (2007) reported melatonin in olive oil within the range of 70–120 pg/ml. Melatonin content appeared to be low in tomato (28.88 pg/g), walnut

T. Kocadag˘lı et al. / Food Chemistry 153 (2014) 151–156

(137.90 pg/g) and beer (94.50 pg/ml) as compared to previously reported results by Garcia-Moreno et al. (2012), Reiter et al. (2005), Sturtz et al. (2011). The differences in the literature might be due to varietal differences of the foods. Additionally, agronomical factors might have an effect on melatonin concentrations. Among the foods analysed, the highest content of melatonin isomer was detected in red wine (170.7 ng/ml). Gomez et al. (2012) reported that the isomer concentration ranges within 18–24 ng/ml in laboratory made wine. Rodriguez-Naranjo et al. (2011) reported up to 22 ng/ml of melatonin isomer in several commercial wines. Higher concentrations in wines up to 72 ng/ml were also reported by Vitalini et al. (2013). Although fermentation process and yeasts might yield different concentrations of melatonin isomer, much higher concentration was detected in this study. As discussed above, transition ratios for the fragments are quite different for melatonin and the isomer hence total ions (approximately equals to the amount of main ion which they fragmented) must be count in order not to get misleading results. More samples need to be analysed to conclude about the variations in melatonin concentrations of different wine types. Apart from that, red wine contained two additional melatonin isomers having both fragment ions. The concentrations of these additional isomers eluting at 3.9 and 4.3 min were calculated to be 0.75 and 0.30 ng/ml, respectively, using the external calibration curve of melatonin. Presence of three melatonin isomers in wine was previously reported by Vitalini et al. (2013). Bread and beer as yeast-fermented samples were also found to contain relatively higher amounts of melatonin isomer comparing to other food samples analysed. Concentration of melatonin isomer in crumb and crust was found to be 15.7 and 0.4 ng/g, respectively. It is thought that temperatures exceeding 100 °C caused a remarkable degree of degradation in melatonin isomers in bread during baking. Beer was found to contain 14.3 ng/ml of melatonin isomer. However, lower concentrations of melatonin isomer were observed in probiotic yogurt (0.9 ng/g), kefir (0.6 ng/g) and black olives (0.1 ng/g) that known to be fermented by bacteria rather than yeasts. Moreover, relatively high amounts of melatonin isomer were found in sour cherry (4.6 ng/g) and sour cherry concentrate (5.2 ng/g). It is well known that the yeasts are one of the causative agents for the spoilage of acid foods like fruits and fruit juices during storage. Non-fermented foods were also found to contain remarkable amounts of melatonin isomer. Whether the isomer is produced by the microflora of the non-fermented foods or it is originated from the food itself is unknown. However, it was reported that the concentration of melatonin isomer is increased by the activity of yeasts (Tan et al., 2012). It was also reported by Gomez et al. (2012) that plays an important role in the production of melatonin isomer during wine fermentation process.

4. Conclusion Validated analytical method described here was rapid and useful to analyse melatonin and melatonin isomers in food products. The results revealed that concentration of melatonin isomer was higher than that of melatonin in all foods analysed. Although several researchers have indicated that melatonin isomer is produced during yeast fermentation, whether it is found naturally in nonfermented foods or produced by the microflora is still unknown. However, it is obvious that yeast fermented foods contain several folds higher melatonin isomer than those fermented by bacteria. Lower amounts of melatonin and the isomer in crust than crumb indicated their degradation during thermal processing of foods. In-depth studies are necessary to understand the effects of food processing conditions (i.e. fermentation by and thermal treatments) on formation and degradation of melatonin and its isomers.

155

Since melatonin isomer is found in several tenfold higher amount than melatonin in yeast-fermented foods, its bioavailability and biological consequence needs to be clarified. The degradation products should be also investigated in thermally processed foods.

References Burkhardt, S., Tan, D. X., Manchester, L. C., Hardeland, R., & Reiter, R. J. (2001). Detection and quantification of the antioxidant melatonin in montmorency and balaton tart cherries (Prunus cerasus). Journal of Agricultural and Food Chemistry, 49(10), 4898–4902. Cao, J., Murch, S. J., O’Brien, R., & Saxena, P. K. (2006). Rapid method for accurate analysis of melatonin, serotonin and auxin in plant samples using liquid chromatography–tandem mass spectrometry. Journal of Chromatography A, 1134(1–2), 333–337. de la Puerta, C., Carrascosa-Salmoral, M. P., Garcia-Luna, P. P., Lardone, P. J., Herrera, J. L., Fernandez-Montesinos, R., et al. (2007). Melatonin is a phytochemical in olive oil. Food Chemistry, 104(2), 609–612. Diamantini, G., Tarzia, G., Spadoni, G., D’Alpaos, M., & Traldi, P. (1998). Metastable ion studies in the characterization of melatonin isomers. Rapid Communications in Mass Spectrometry, 12(20), 1538–1542. Dubbels, R., Reiter, R. J., Klenke, E., Goebel, A., Schnakenberg, E., Ehlers, C., et al. (1995). Melatonin in edible plants identified by radioimmunoassay and by high-performance liquid chromatography–mass spectrometry. Journal of Pineal Research, 18(1), 28–31. Garcia-Moreno, H., Calvo, J. R., & Maldonado, M. D. (2012). High levels of melatonin generated during the brewing process. Journal of Pineal Research, 5. http:// dx.doi.org/10.1111/JPI.12005. Gomez, F. J. V., Raba, J., Cerutti, S., & Silva, M. F. (2012). Monitoring melatonin and its isomer in cv. Malbec by UHPLC–MS/MS from grape to bottle. Journal of Pineal Research, 52(3), 349–355. Harumi, T., & Matsushima, S. (2000). Separation and assay methods for melatonin and its precursors. Journal of Chromatography B, 747(1–2), 95–110. Hattori, A., Migitaka, H., Iigo, M., Itoh, M., Yamamoto, K., Ohtanikaneko, R., et al. (1995). Identification of melatonin in plants and its effects on plasma melatonin levels and binding to melatonin receptors in vertebrates. Biochemistry and Molecular Biology International, 35(3), 627–634. Iriti, M., Rossoni, M., & Faoro, F. (2006). Melatonin content in grape: Myth or panacea? Journal of the Science of Food and Agriculture, 86(10), 1432–1438. Johns, N. P., Johns, J., Porasuphatana, S., Plaimee, P., & Sae-Teaw, M. (2013). Dietary intake of melatonin from tropical fruit altered urinary excretion of 6sulfatoxymelatonin in healthy volunteers. Journal of Agricultural and Food Chemistry, 61(4), 913–919. Kennaway, D. J., Frith, R. G., Phillipou, G., Matthews, C. D., & Seamark, R. F. (1977). Specific radioimmunoassay for melatonin in biological tissue and fluids and its validation by gas chromatography mass spectrometry. Endocrinology, 101(1), 119–127. Kirakosyan, A., Seymour, E. M., Llanes, D. E. U., Kaufman, P. B., & Bolling, S. F. (2009). Chemical profile and antioxidant capacities of tart cherry products. Food Chemistry, 115(1), 20–25. Lewy, A. J., & Markey, S. P. (1978). Analysis of melatonin in human-plasma by gaschromatography negative chemical ionization mass-spectrometry. Science, 201(4357), 741–743. Maldonado, M. D., Moreno, H., & Calvo, J. R. (2009). Melatonin present in beer contributes to increase the levels of melatonin and antioxidant capacity of the human serum. Clinical Nutrition, 28(2), 188–191. Manchester, L. C., Tan, D. X., Reiter, R. J., Park, W., Monis, K., & Qi, W. B. (2000). High levels of melatonin in the seeds of edible plants – Possible function in germ tissue protection. Life Sciences, 67(25), 3023–3029. Mills, M. H., Finlay, D. C., & Haddad, P. R. (1991). Determination of melatonin and monoamines in rat pineal using reversed-phase ion-interaction chromatography with fluorescence detection. Journal of Chromatography Biomedical Applications, 564(1), 93–102. Pape, C., & Luning, K. (2006). Quantification of melatonin in phototrophic organisms. Journal of Pineal Research, 41(2), 157–165. Paredes, S. D., Korkmaz, A., Manchester, L. C., Tan, D. X., & Reiter, R. J. (2009). Phytomelatonin: A review. Journal of Experimental Botany, 60(1), 57–69. Reiter, R. J. (1991). Pineal melatonin – Cell biology of its synthesis and of its physiological interactions. Endocrine Reviews, 12(2), 151–180. Reiter, R. J. (1993). The melatonin rhythm – Both a clock and a calendar. Experientia, 49(8), 654–664. Reiter, R. J., Manchester, L. C., & Tan, D. X. (2005). Melatonin in walnuts: Influence on levels of melatonin and total antioxidant capacity of blood. Nutrition, 21(9), 920–924. Reiter, R. J., Paredes, S. D., Manchester, L. C., & Tan, D. X. (2009). Reducing oxidative/ nitrosative stress: A newly-discovered genre for melatonin. Critical Reviews in Biochemistry and Molecular Biology, 44(4), 175–200. Rodriguez-Naranjo, M. I., Gil-Izquierdo, A., Troncoso, A. M., Cantos, E., & GarciaParrilla, M. C. (2011a). Melatonin: A new bioactive compound in wine. Journal of Food Composition and Analysis, 24(4–5), 603–608. Rodriguez-Naranjo, M. I., Gil-Izquierdo, A., Troncoso, A. M., Cantos-Villar, E., & Garcia-Parrilla, M. C. (2011b). Melatonin is synthesised by yeast during alcoholic fermentation in wines. Food Chemistry, 126(4), 1608–1613.

156

T. Kocadag˘lı et al. / Food Chemistry 153 (2014) 151–156

Spadoni, G., Diamantini, G., Bedini, A., Tarzia, G., Vacondio, F., Silva, C., et al. (2006). Synthesis, antioxidant activity and structure-activity relationships for a new series of 2-(-acylaminoethyl)indoles with melatonin-like cytoprotective activity. Journal of Pineal Research, 40(3), 259–269. Sturtz, M., Cerezo, A. B., Cantos-Villar, E., & Garcia-Parrilla, M. C. (2011). Determination of the melatonin content of different varieties of tomatoes (Lycopersicon esculentum) and strawberries (Fragaria ananassa). Food Chemistry, 127(3), 1329–1334.

Tan, D. X., Hardeland, R., Manchester, L. C., Rosales-Corral, S., Coto-Montes, A., Boga, J. A., et al. (2012). Emergence of naturally occurring melatonin isomers and their proposed nomenclature. Journal of Pineal Research, 53(2), 113–121. Vitalini, S., Gardana, C., Simonetti, P., Fico, G., & Iriti, M. (2013). Melatonin, melatonin isomers and stilbenes in Italian traditional grape products and their antiradical capacity. Journal of Pineal Research, 54(3), 322–333.