Quality control and determination of melatonin in food supplements

Depósito de investigación de la Universidad de Sevilla https://idus.us.es/ “This is an Accepted Manuscript of an article published by Elsevier in: Journal of Food Composition and Analysis on 2016, available at: https://doi.org/10.1016/j.jfca.2015.09.013” 1 Original Research Article Quality Control and Determination of Melatonin in Food Supplements Ana B. Cerezo, Ángela Leal, M. Antonia Álvarez-Fernández, Ruth Hornedo-Ortega, Ana M. Troncoso, M. Carmen García-Parrilla* Nutrition and Food Science, Faculty of Pharmacy, University of Seville. C/ P. García González nº 2. Sevilla 41012, Spain. * Corresponding author: e-mail: [email protected]; fax +34954233765; phone +34954556759 2 Abstract 1 Melatonin supplements are used widely in Europe and the United States (US) to 2 alleviate jet-lag and other sleep-delay disorders. Recently, the European Commission 3 authorized two health claims for food containing melatonin, with a minimum dose per 4 quantified portion. This study aims to determine the dose of melatonin in food 5 supplements marketed in Europe (Spain) and the US by validating a Liquid- 6 Chromatography couple to Diode-Array-Detector (LC-DAD) method. Additionally, 7 contaminants present in melatonin supplements were analyzed using an Ultra-High- 8 Performance-Liquid-Chromatography coupled to an ion trap-orbitrap mass 9 spectrometer. 10 We tentatively identified 8 tryptophan-related contaminants in melatonin supplements, 11 suggesting tighter impurity control in melatonin supplements is necessary. The LC- 12 DAD validated method achieved reliable results regarding recovery, precision, 13 repeatability, limit of detection, limit of quantitation and linearity parameters. Label 14 melatonin dose varied from 1–1.95 mg/unit and 0.3–5 mg/unit for supplements 15 marketed in Europe (Spain) and the US, respectively. Four out of seventeen 16 supplements showed significant deviations from melatonin content declared on the label 17 (from -60% to -20%). Regarding health claims, three out of the ten supplements 18 purchased in Spain should not include the claim “reduction of time taken to fall asleep” 19 due to the fact that their melatonin content is lower than 1 mg. 20 Key words: melatonin, food/dietary supplements, OrbiTrap, LC-DAD, health claim, 21 dose, food analysis, food composition. 22 23 Chemical compounds studied in this article 24 3 Melatonin (PubChem CID: 896); Tryptophan (PubChem CID: 6305); 3- 25 (phenylamino)alanine (PubChem CID: 22062824); 1,1’-ethylidenebis(tryptophan) 26 (PubChem CID: 3905118). 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 4 1. Introduction 50 The food supplements markets has undergone exponential growth in recent years 51 (Nutrition Business Journal, 2010; Vargas-Murga et al., 2011). Currently, supplements 52 are consumed by a broad sector of the population, having become very attractive 53 because of relatively low prices, their non-prescription status, huge promotion, and the 54 perception that natural products are safe (Zakaryan & Martin, 2012). 55 Despite their formulation, food supplements are not considered medicinal products but 56 food. The European Union (EU) consider them food as regard European food law 57 (Regulation (EC) 178/2002), and Directive 2002/46/EC regulates them. However, the 58 United States (US) uses the term dietary supplements and has a special regulation 59 exclusively for them (Food and Drug Administration 1994). Both Europe and the US 60 define food/dietary supplements as food whose purpose is to supplement the diet, with 61 concentrated sources of nutrients or other substances with a nutritional or physiological 62 effect, and marketed in dose forms such as capsules, tablets, pills, liquids, soft gels, and 63 powders (Food and Drug Administration 1994; Directive 2002/46/EC). The same 64 substance (ie. melatonin, caffeine, lovastatin, etc.) can have an ambivalent status as food 65 supplement and medicine in the EU depending on its dose (Lachenmeier et al., 2012). 66 Therefore, some authors consider the European legislator should rethink the definition 67 of medicinal products, as the current situation based on complicated case-by-case 68 decision on pharmacological action leads to an unregulated market flooded with 69 potentially illegal food supplements (Lachenmeier et al., 2012). 70 Melatonin (N-acetyl-5-methoxytryptamine) is an indoleamine that synchronizes 71 circadian rhythms (Reiter et al., 2007). Originally, it was considered a neurohormone 72 exclusive to the pineal gland in vertebrates. Recently, it has been identified in a wide 73 range of invertebrates, plants, fruit, wine, bacteria and fungi, being considered an 74 5 ubiquitous molecule (De la Puerta et al., 2007; Paredes et al., 2009; Rodriguez–Naranjo 75 et al., 2011; Stürtz et al., 2011; Aguilera et al., 2014; Reiter et al., 2015). An array of 76 methodologies, such as liquid chromatography (LC) coupled to different detectors 77 (fluorescence, electrochemical detector or MS/MS), gas chromatography/mass 78 spectrometry, immunoprecipitation and ELISA, have been used to analyze melatonin in 79 plants, seeds, fruits, olive oil and wine (Manchester et al., 2000; De la Puerta et al., 80 2007; Murch et al., 2010; Rodriguez–Naranjo et al., 2011; Stürtz et al., 2011; Oladi et 81 al., 2014). The main limitation of melatonin determination in foods and plants is its low 82 concentration (pg - µg per g or ml), which make necessary the use of sophisticated and 83 highly sensitive detectors. However, as far as we know, only few studies have 84 developed and validated simple analytical methods (capillary electrophoresis system, 85 thin layer chromatography, LC with UV detector and LC amperometric detector) for the 86 determination of melatonin in food supplements at higher concentrations (3-20 87 mg/tablet or capsule) (Costantini & Paoli, 1998; Saracino et al., 2008). 88 It has been demonstrated, although in vitro and in animal studies and at high dosages, 89 that melatonin has antioxidant properties (Fischer et al., 2013; Reiter et al., 2014; Gurer- 90 Orhan & Suzen, 2015), anticancer activity (Xin et al., 2015; Cos et al., 2014), 91 immunomodulatory properties (Carrillo-Vico et al., 2013; Medrano-Campillo et al., 92 2015) and neuroprotective activity among others (Singh & Jadhav, 2014). On the other 93 hand, melatonin or its contaminants has also been reported to induce eosinophilia in 94 patients with cancer (Lissoni et al., 1993a; 1993b) and to affect the reproductive 95 systems of rodents, sheep and primates (Kennaway, 2015). Despite this, melatonin has 96 been widely used as food supplement for the alleviation of jet-lag and other disorders 97 resulting from sleep delay (Brzezinski et al., 2005; Herxheimer & Petrie, 2002). 98 Recently, the European Commission has authorized two health claims for food 99 6 containing melatonin (Regulation (EU) 432/2012) regarding alleviation of jet-lag (0.5 100 mg/day) and to fall asleep (1mg/day) based on the positive scientific opinion of the 101 European Food Safety Authority (European Food Safety Authority 2010; 2011). 102 However, the Food and Drug Administration (FDA) does not evaluate the claims for 103 melatonin supplements (Food and Drug Administration 2002; 2006; 2008). Instead, a 104 disclaimer on the label declaring “these statements have not been evaluated by the 105 FDA” is mandatory. The dose is not controlled either. 106 Despite the European Commission clearly stablishes a minimum melatonin content per 107 quantified portion to fulfill the authorized health claims (Regulation (EU) 432/2012), 108 the literature focused on determination of melatonin content in food supplements for 109 quality control is highly scarce and dated before this regulation entered into force 110 (Costantini & Paoli, 1998; Saracino et al., 2008). 111 The aim of this study is to evaluate the purity and dose of melatonin supplements in a 112 representative sample of food/dietary supplements available on the EU (Spain) and US 113 markets. To this end, we detected the presence of contaminants by ultra hight 114 performance liquid chromatography-orbitrap-mass spectrometry (UHPLC-Orbitrap- 115 MS). Additionally, we set up and validated a sensitive, specific and reproducible LC 116 couple to a diode-array detector (LC-DAD) method for routine determination of 117 melatonin in food supplements. 118 119 2. Materials and methods 120 121 2.1. Samples 122 The study was conducted with a total of 17 melatonin supplements (S1-S17) marketed 123 in the EU (Spain) and the US (Table 1). Seven supplements were purchased in San 124 7 Francisco, California (US) supermarkets and the remaining ten in pharmacies in Spain. 125 There were three types of dose forms: tablets (13 samples), capsules (4 samples) and 126 sachets (1 sample). 127 128 2.2. Reagents 129 Reagents were purchased from Sigma (St. Louis, MO, US) [melatonin (≥ 98%)], Merck 130 (Darmstadt, Germany) [methanol] and Panreac (Barcelona, Spain) [formic acid]. All 131 reagents used have HPLC (High Performance Liquid Chromatography) quality. 132 133 2.3. Sample extractions 134 Melatonin was extracted from supplements following the method described by Saracino 135 et al (2008) with slight modifications. A total of 3 tablets, 3 capsules or 2 sachets of 136 each supplement were used for each analysis. In the case of tablets, a prior grinding step 137 was necessary. Subsequently, samples were diluted in 20 mL of a methanol/water 138 (50:50, v/v) mixture and shaken for 1 hour in an orbital shaker. The mixture was 139 centrifuged at 1500 g for 3 minutes. The supernatant was collected and filtered prior to 140 injection. Each extraction was performed twice. 141 142 2.4. UHPLC-MS Orbitrap analysis 143 All analyses were performed using a Thermo Scientific liquid chromatography system 144 consisting of a binary Rs Pump Dionex Ultimate 3000 and Rs autosampler Dionex 145 Ultimate 3000, connected to a hybrid ion trap-orbitrap mass spectrometer (Orbitrap 146 8 Elite) with a heated-electrospray ionization probe (HESI-II) (Thermo Fisher Scientific, 147 Bremen, Germany). Xcalibur software (version 3.0.63) (Thermo Fisher Scientific, 148 Bremen, Germany) was used for instrument control, data acquisition and data analysis. 149 Separation was performed on an SB-C18 column (2.1 x 100mm, 1.8 µm) (Agilent 150 Technology, Santa Clara, United States). The MS spectra were acquired by full MS. 151 Injection volume was 5 µL and flow rate was 0.5 mL/min. Two different solvents were 152 used as a mobile phase: solvent A (water/0.1% formic acid) and solvent B 153 (methanol/0.1% formic acid), in the following gradient: 0–2 min 5% B, 2–10 min 5- 154 100% B, 10–12 min 100% B, 12–15 min 100-5% B. The mass spectrometer was 155 operated in positive mode. HESI source parameters were as follows: source voltage 3.5 156 Kv; tube lens voltage 50 V; capillary temperature 230 ºC; source heater temperature 440 157 ºC; sheath, auxiliary and sweep gas flow rate (N2) 53, 14 and 3, respectively (arbitrary 158 units). 159 Compounds were tentatively identified according to their mass spectra, exact mass, 160 molecular formula and Δmass (ppm) compared with the literature data available (Hill et 161 al., 1993; Williamson et al., 1997a; Williamson et al., 1997b; Williamson et al., 1998a; 162 Williamson et al., 1998b; Naylor et al., 1999; Simat et al., 2003; Darwish & Attia, 163 2012). 164 165 2.5. 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LC-DAD Chromatogram of melatonin standard (top) and melatonin in 499 supplement S4 (bottom). 500 501 502 503 23 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 Figure 1. 520 24 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 Figure 2. 538