Ultrahigh-Performance Liquid Chromatography (UHPLC)–Tandem

May 5, 2016 - Departament de Nutrició i Bromatologia, Xarxa de Referència en Tecnologia dels Aliments (XaRTA), Institut de recerca en Nutrició i Se...
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Ultra high performance liquid chromatography (UHPLC)–tandem mass spectrometry (MS/MS) quantification of nine target indoles in sparkling wines Rebeca Tudela, Albert Ribas-Agustí, Susana Buxaderas, Montserrat Riu-Aumatell, Massimo Castellari, and Elvira Lopez-Tamames J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b01254 • Publication Date (Web): 05 May 2016 Downloaded from http://pubs.acs.org on May 7, 2016

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Ultra High Performance Liquid Chromatography (UHPLC)–Tandem Mass Spectrometry (MS/MS) Quantification of Nine Target Indoles in Sparkling Wines

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Rebeca Tudela†, Albert Ribas-Agustí‡, Susana Buxaderas†, Montserrat Riu-Aumatell† *, Massimo Castellari‡, and Elvira López Tamames†

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Departament de Nutrició i Bromatologia, Xarxa de Referència en Tecnologia dels Aliments (XaRTA), Institut de recerca en Nutrició i Seguretat Alimentària (INSA), Universitat de Barcelona, Campus de l’Alimentació de Torribera, Av. Prat de la Riba 171, 08921 Santa Coloma de Gramenet, Spain. IRTA-Monells, Finca Camps i Armet s/n, 17121 Monells, Spain

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*

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ABSTRACT

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An ultra high performance liquid chromatography (UHPLC)–tandem mass spectrometry

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(MS/MS) method was developed for the simultaneous determination of nine target

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indoles in sparkling wines. The proposed method requires minimal sample pretreatment,

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and its performance parameters (accuracy, repeatability, LOD and matrix effect)

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indicate that it is suitable for routine analysis.

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Four indoles were found at detectable levels in commercial Cava samples: 5-

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methoxytryptophol (5MTL), tryptophan (TRP), tryptophan ethyl ester (TEE) and N-

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acetylserotonin (NSER). Two of them, namely NSER and 5MTL, are reported here for

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the first time in sparkling wines, with values of 0.3-2 µg/mL and 0.29-29.2 µg/mL,

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respectively. In the same samples, the contents of melatonin (MEL), serotonin (SER), 5-

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hydroxytryptophan (5-OHTRP), 5-hydroxyindole-3-acetic acid (5OHIA) and 5-

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methoxy-3-indoleacetic acid (5MIA) were all below the corresponding limits of

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detection.

Corresponding author. Tel.: (+34) 934033795; fax: (+34) 934035931 E-mail address: [email protected] (M. Riu-Aumatell)

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Keywords: UHPLC-MS/MS, melatonin, sparkling wine, indole, tryptophan ethyl ester

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INTRODUCTION

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The presence of the ubiquitous neurohormone melatonin (MEL) in grapes and wines

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has generated great interest within the wine industry in recent years.1-9 MEL is

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considered to be a bioactive compound10 with antioxidant activities that have been

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related to the presence of two side chains in the indole ring.11 Studies from 2006

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onwards suggest that MEL may be formed in grapes;3,12-18 however, the most recent

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findings indicate that MEL is only accumulated during the winemaking process. Thus,

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its presence in wine would be strictly related to the activity of yeast during

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fermentation.1,2,19 MEL can be formed by Saccharomyces yeasts from precursors of the

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common tryptophan (TRP) biosynthetic pathway, via 5-hydroxytryptophan (5OHTRP),

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serotonin (SER) and N-acetylserotonin (NSER). Alternative MEL formation involves a

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secondary metabolic pathway of S. cerevisiae via the O-methylation of serotonin

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followed

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methoxytryptophol (5MTL) is produced by yeasts in association with MEL. 19

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Several authors have showed that wines may contain only melatonin1,7,8,12,13; or just one

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tentatively identified MEL isomer3; or, in a few cases, melatonin and up to three

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possible MEL isomers5,21. More recently, tryptophan ethylester (TEE) has been

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identified as one of the compounds previously reported to be the most abundant MEL

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isomers in red wine.22 These recent findings underline the need for advanced analytical

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techniques to elucidate the indole-derived compounds involved in melatonin

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metabolism.

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Determination of MEL and related indoles in wine was initially carried out using HPLC

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with fluorimetric detection (HPLC-F),4,7 and capillary electro chromatography (CE)

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with immobilized carboxylic multi-walled carbon nanotubes.12 However, the protocols

by

the

N-acetylation

of

5-methoxytryptamine.20

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5-

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require different sample pretreatments such as microextraction by a packed sorbent

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(MEPS) or solid-phase extraction (SPE) in order to improve MEL detection; even

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though they increase the total analysis time.4,5,7,13,23,24 More recently, “hyphenated”

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techniques such as LC-MS/MS, UHPLC-MS/MS or LC coupled to high resolution mass

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spectrometry (HRMS) have been used to determine MEL and other related compounds

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in wines.3,5,21,22 The advantage of these techniques is the simplified sample preparation,

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e.g. dilution or filtration, and the structural information provided by the MS detectors

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that supports identification.

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Production of Spanish “Cava” sparkling wines follows the classical or traditional

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method, which includes a second fermentation of the base wine to obtain the

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characteristic foam, and aroma. At the end of the second fermentation, yeast cells die

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and remain in contact with the wine for a long time (up to several months). At this

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stage, the intracellular compounds, including TRP, are released into the wine,3,24-27 and

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contribute to the development of the particular flavor and aroma of sparkling wines.

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To the best of our knowledge, no study of MEL and its metabolic pathway has been

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performed before now in sparkling wine. Since traditional sparkling wines undergo two

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fermentations, it might be expected that the assessment of the metabolic pathways of

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indole-derived compounds could be important to clarify the role of yeast and lees

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autolysis in the development of the characteristic qualities of sparkling wine.25-27

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Therefore, the aim of this study was to develop a suitable UHPLC-MS/MS method for

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the simultaneous identification and quantification of MEL and other indole compounds

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(Figure 1) involved in the melatonin metabolism pathway in sparkling wines. The levels

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of L-tryptophan (TRP), 5-hydroxy-L-tryptophan (5-OHTRP), serotonin (SER), N-

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acetylserotonin (NSER), melatonin (MEL), 5-hydroxyindole-3-acetic acid (5OHIA), 5-

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methoxytryptophol (5MTL), 5-methoxy-3-indoleacetic acid (5MIA) and tryptophan

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ethyl ester (TEE) in 18 commercial Spanish sparkling wines (Cava) were evaluated; as

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were the performance parameters of the proposed methods.

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MATERIALS AND METHODS

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Chemicals. L-tryptophan, 5-hydroxy-L-tryptophan, serotonin hydrochloride, n-acetyl-

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5-hydroxytryptamine, melatonin, 5-methoxytryptophol, 5-hydroxyindole-3-acetic acid,

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5-methoxy-3-indoleacetic acid, L-tryptophan ethyl ester hydrochloride, L-tryptophan-d5

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with an isotopic purity of more than 97% (indole-d5) (internal standard; I.S.),

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hydrochloric acid and ammonium hydroxide ACS were supplied by Sigma-Aldrich (St.

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Louis, USA).

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Acetonitrile LC–MS grade was from Riedel-de Haën (Seelze, Germany), methanol,

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hexane and acetone HPLC grade were purchased from Merck (Darmstadt, Germany).

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Ultra-pure water was obtained using a Milli-Q system (Millipore, Milford, MA).

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The single standard stock solutions and I.S. solution were prepared weekly by

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dissolving known amounts of pure standards in methanol (1,000 mg/L) and then stored

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in the dark at 4ºC until analysis. Spiking standard solutions (20 mg/L) were prepared

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weekly by diluting standard stock solutions with methanol.

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Samples. Several brands of commercial Cava sparkling wine (n=18) were purchased in

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local supermarkets, produced at different wineries in the Cava region (Catalonia, Spain).

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Cava is produced following the traditional method in which a second fermentation is

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followed by aging in contact with the lees (minimum 9 months) in which the sparkling

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wine develops the characteristic foam and aroma.26-28

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Once the bottles were opened, samples were degassed by magnetic stirring for 5 min,

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and then stored in 250 mL amber flasks at -20ºC until analysis. The entire process was

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carried out under reduced light conditions to prevent degradation of the melatonin. Just

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before analysis, the samples were filtered with a nylon membrane filter (0.2 µm)

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(Waters, Milford, MA).

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UHPLC-MS/MS Conditions. Analysis was carried out with an Acquity Ultra-

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PerformanceTM liquid chromatography system (UPLC©), equipped with a binary pump

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system (Waters, Milford, MA). Detection was carried out using a TQD triple

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quadrupole MS detector (Waters, Milford, MA). Chromatographic separation was

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carried out with a BEH C18 Shield column (150 x 1.0 mm i.d.) with a 1.7 µm particle

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size (Waters, Milford, MA), kept at 30ºC. Linear gradient elution was carried out from

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95% mobile phase A (5:94.9:0.1 v/v/v ACN:water:formic acid) and 5% B (95:4.9:0.1

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v/v/v ACN:water:formic acid) to 25% A in 8 minutes, with a flow rate of 0.125

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mL/min. After each run, the system was stabilized with the initial conditions for one

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minute before the next injection. Samples were maintained at 10ºC and the injected

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volume was 8 µL. A standard washing procedure (with weak and strong eluents, i.e. the

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mobile phases A & B) was included in the chromatographic method after each injection.

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Carry over was checked for at the beginning of each sample batch by injecting a blank

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sample (water) after a spiked sample.

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The electrospray interface (ESI) was operated in positive mode; the source temperature

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was fixed at 130ºC, the capillary voltage was set at +3.0 kV and the desolvation

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temperature was set at 350ºC. The cone gas (nitrogen) flow rate was 350 L/h. The gas

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used in the collision cell was argon at a flow rate of 0.1 mL/min. Multiple reaction

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monitoring (MRM) mode was used to quantify the compounds. MS/MS experiments

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were also performed in product ion scan mode29 to study the fragmentation patterns of

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specific molecular ions. The MRM conditions were optimized using Autotune Wizard

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Software by infusing standard solutions (10 mg/L) of each compound. Two specific

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MRM transitions of the deprotonated molecular ions were monitored for each

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compound; for quantification purposes, the product ion with the higher signal was used.

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Data acquisition and integration were controlled by MassLynxTM software (version 4.1).

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Analytic Performance. Standard addition calibration was created by spiking a cava

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sample with known amounts of standards at five levels (ranging from 1 to 50 µg/L for

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5MTL, NSER, MEL, 5OHTRP and TEE; from 5 to 50 µg/L for 5MIA and 5OHIA;

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from 5 to 1,000 µg/L for TRP; and from 100 to 500 µg/L for SER), and by using

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tryptophan-d5 as the I.S. The stock solution of the I.S. was prepared at 1 mg/mL in

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mobile phase A. Then the working solution was prepared by diluting to a final

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concentration of 20 mg /L. Calibration curves were created by plotting the ratio between

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the area of the more intense transition and the area of the I.S. in the MRM mode, vs. the

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corresponding concentrations of each target compound.

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Identification of the target compounds was carried out following the criteria of the

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Commission Decision 2002/657/CE, by comparing their retention time and MRM

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product ion ratios with those obtained from pure standards.

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The performance parameters of the method were established following Long and

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Winefordner,31 and IUPAC.32 Signal-to-noise ratios of 3:1 and 10:1 were considered for

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LOD and LOQ, respectively.

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Repeatability was assessed by injection in triplicate of three Cava sparkling wines.

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Three Cava samples spiked with standard solutions were used in the case of compounds

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that fell below the LOD in the commercial samples.

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The accuracy of the analytical assay was determined by analyzing three Cava sparkling

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wines spiked at two levels with standard solutions (20 µg/L and 50 µg/L for 5MTL,

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NSER, MEL, 5OHTRP, TEE, 5MIA and 5OHIA; and 100 µg/L and 500 µg/L for TRP

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and SER respectively), and calculating the ratio between expected and calculated

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concentrations for each compound.

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Matrix effects were estimated for each compound by comparing the response in the

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presence of the matrix (three samples spiked at two levels (50 and 100 µg/L) as for the

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accuracy assays) with the response of pure compounds at the same concentrations

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dissolved in a neat solution (mobile phase A).

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RESULTS AND DISCUSSION

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Analytic Performance

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Table 1 shows the MRM conditions and calibration parameters for the target

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compounds. Under our conditions, the best responses were obtained by working in

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positive mode for all the nine compounds that were the objects of this study.

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The MRM profiles for a mixture of pure standards of the nine target compounds (plus

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the I.S.), and the profile of a cava sample are shown in the Supporting Information.

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Separation of all the target compounds was completed within 5.2 minutes which, jointly

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with the minimal sample preparation, allows a high sample throughput.

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In all the Cava wine samples, a relevant peak was found at 2.92 minutes with an

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[M+H]+ of m/z = 232. This peak (MRM profiles of pure standards and cava samples are

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provided in the Supporting Information) fits the retention time and the product ion

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spectra of the TEE pure standard (profile of product ions from collision-induced

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dissociation at 15 V, for the TEE standard is provided in the Supporting Information),

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while the ratio between ions at m/z 216 and m/z 174 is in agreement with the results of

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the recent work of Gardana et al.22

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The linearity of the calibration curves could be considered satisfactory in the ranges of

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concentration included in the protocol of analysis (Table 1).

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The LOD of our method in the case of MEL was similar to the values reported by other

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authors operating with both HPLC-F and LC-MS/MS setups.1,3,4,7,12,13,19,21 The LOD for

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TRP was better than those previously obtained by other authors working with HPLC-

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F.13,23,24 The LOD of SER was slightly higher than that reported by Mercolini et al.13

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with an HPLC-F method; but it was comparable to that previously reported in fruit

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analysis by HPLC–MS/MS, where the detection limit was 50 µg/L.9

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The LODs for the other target compounds were always lower than 1 µg/L (Table 1) and

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only in the case of 5OHIA was the LOD approximately 3 times higher than in the study

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by Mercolini et al.13 mentioned above using HPLC-F. The LODs for TEE were

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comparable to that obtained by Vigentini et al.19

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Both repeatability (with values always lower than 10%) and accuracy (Table 1) can be

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considered adequate for all the target compounds, taking into account that their levels in

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real samples are mainly in the range of a few µg/L. Matrix effects were in general lower

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than 20%; a good result considering that the samples were injected without any pre-

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treatment to reduce the interfering compounds. Only in the case of 5OHTRP was the

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ionization suppression higher than 30% (Olajnik et al., 2013)36.

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Sparkling Wine Results

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The performance parameters of the method confirm that it is fit for routine analysis

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(Table 1). To check the suitability of the proposed protocol, a total of 18 samples of

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commercial sparkling wines were analyzed with the proposed method (Table 2). TRP

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was the compound detected with the highest concentrations in all the samples. The

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concentrations of TRP found in Cava sparkling wines were in accordance with the

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results of Mattivi et al.23 obtained by an HPLC–F method in Chardonnay wines (62-417

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µg/L); but were somewhat lower than those cited by Hoenicke et al.24 in different white

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wines.

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TEE was found in all the Cava samples (Table 2); however, the amounts found in the

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current study, ranging from 0.1 to 2.7 µg/L, were lower than those found by Gardana et

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al.22 in Syrah wine (84 µg/L).

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To the best of our knowledge, no data have previously been reported in the literature for

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the content of NSER and 5MTL in wines. The indole NSER, which is the immediate

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precursor of MEL according to its biosynthetic pathway,20 was detected in all the Cava

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samples. The amount of NSER varied between 0.3 and 2.0 µg/L. Moreover, 5MTL,

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another MEL precursor,20 was also detected in variable amounts but in only three of the

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18 samples analyzed (LOD: 29.2 µg/L) (Table 2).

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MEL, SER, 5MIA, 5OHIA and 5OHTRP were all under the LOD in the samples

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analyzed in this study (Table 2 and MRM profiles of pure standards and cava samples

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provided in the Supporting Information). MEL has been found in different kinds of

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wine at levels ranging from 0.5 to 423 µg/L,1,4,7,12,13,19; with 390.82 µg/L being the

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higher concentration found in white wine.1 The absence of detectable levels of MEL in

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Cava wines could be due to the specific technology used for their preparation, which

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includes a second fermentation and a long aging process (always over a year) which

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could contribute to the transformation and fading of the MEL present in the base wines.

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It should also be emphasized that MEL was only found at detectable levels in wines

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produced at the laboratory scale or analyzed by HPLC-F.1,4,7,13,19 In contrast, the

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samples included in our study are commercially available Cava sparkling wines with a

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long ageing (over 12 months) which could have influenced the final levels of MEL.

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SER was previously found in Albana wine at concentrations below the LOD of our

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method; while 5OHIA, a SER metabolite, was previously found by Mercolini et al.13 at

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105 µg/L in white wine. The undetectable levels of these compounds in our samples

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could be related to the different conditions of elaboration of the wine and/or the

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differences in the LOD values between the methods.

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In summary, a UHPLC MS/MS method for the simultaneous assessment of nine indole

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compounds involved in MEL metabolism in sparkling wines was developed and its

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main performance parameters estimated. The proposed protocol requires limited sample

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pre-treatment, requires about 6 minutes for the complete separation of the nine target

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compounds and is suitable for routine analysis to give a quick overview of the levels of

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some of the main indole-derived compounds involved in the MEL metabolism pathway.

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Among the nine indoles studied, only four were detected in our Cava samples:

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tryptophan (TRP), tryptophan ethyl ester (TEE), N-acetylserotonin (NSER), and 5-

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methoxytryptophol (5MTL). The presence of NSER and 5MTL in sparkling wine is

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reported here for the first time. The proposed method is a promising starting point for

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further studies to clarify the effect of sparkling wine technology on the MEL

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biosynthetic pathway.

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Supporting information

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MRM profiles for a mixture of pure standards of the nine target compounds (plus the

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I.S.), and the profile of a cava sample (Figure S1); Profiles of product ions from

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collision-induced dissociation at 15 V, for the MEL standard (a); the TEE standard (b);

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and the peak in Cava sparkling wines at 2.92 min (c) (Figure S2) (PDF).

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This material is available free of charge via the Internet at http://pubs.acs.org.

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ACKNOWLEDGMENTS

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This study was made possible thanks to the financial assistance provided by the

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Comisión Interministerial de Ciencia y Tecnología (CICYT) (Spain), via award

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AGL2011-23872, and the Generalitat de Catalunya (Spain), via project 2014SGR1438.

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REFERENCES

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(10) Mas, A.; Guillamon, J.M.; Torija, M.J.; Beltran, G.; Cerezo, A.B.; Troncoso, A.M.; Garcia-Parrilla, M.C. Bioactive Compounds Derived From the Yeast Metabolism of Aromatic Amino Acids During Alcoholic Fermentation. Biomed. Res. Int. 2014, 2014, 7 pp.

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(11) Spadoni, G.; Diamantini, G.; Bedini, A.; Tarzia, G.; Vacondio, F.; Silva, C.; Rivara, M.; Mor, M.; Plazzi, P. V.; Zusso, M.; Franceschini, D.; Giusti, P. Synthesis, Antioxidant Activity and Structure-Activity Relationships for a New Series of 2-(Acylaminoethyl) Indoles with Melatonin-Like Cytoprotective Activity. J. Pineal Res. 2006, 40, 259-269.

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(20) Sprenger, J.; Hardeland, R.; B. Fuhrberg, Han, S.-Z. Melatonin and Other 5Methoxylated Indoles in Yeast: Presence in High Concentrations and Dependence on Tryptophan Availability. Cytologia. 1999, 64, 209-213.

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(21) Kocadağlı, T.; Yılmaz, C.; Gökmen, V. Determination of Melatonin and its Isomer in Foods by Liquid Chromatography Tandem Mass Spectrometry. Food Chem. 2014, 153, 151-156.

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(22) Gardana, C.; Iriti, M.; Stuknyte, M.; De Noni, I.; Simonetti, P. ‘Melatonin Isomer’ in Wine Is Not an Isomer of the Melatonin but Tryptophan-Ethylester. J. Pineal Res. 2014, 57, 435-441.

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(23) Mattivia, F.; Vrhovsek, U.; Versini, G. Determination of Indole-3-Acetic Acid, Tryptophan and Other Indoles in Must and Wine by High-Performance Liquid Chromatography with Fluorescence Detection. J. Chromatogr. A. 1999, 855, 227-235.

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(24) Hoenicke, K.; Simat, T. J.; Steinhart, H.; Köhler, H. J.; Schwab, A. Determination of Free and Conjugated Indole-3-Acetic Acid, Tryptophan, and Tryptophan Metabolites in Grape Must and Wine. J. Agric. Food Chem. 2001, 49, 5494-5501.

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(25) Henschke, P.A.; Jiranek, V. Yeasts - Metabolism of Nitrogen Compounds. In: Wine Microbiology and Biotechnology; G.H. Fleet (Ed.), Harwood Academic Publishers: Chur, Switzerland, 1993; pp.77-164.

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(26) Alexandre, H.; Guilloux-Benatier, M. Yeast Autolysis in Sparkling Wine A Review. Aust. J. Grape Wine Res. 2006, 12, 112-127.

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(27) Buxaderas, S.; López-Tamames, E. Sparkling Wines: Features and Trends From Tradition. Adv. Food Nutr. Res. 2012, 66, 1-45.

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(28) Commission Regulation (EC) No 607/2009 of 14 July 2009 Laying Down Certain Detailed Rules for the Implementation of Council Regulation (EC) No 479/2008 as Regards Protected Designations of Origin and Geographical Indications, Traditional Terms, Labelling and Presentation of Certain Wine Sector Products.

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(29) Murray, K.K.; Boyd, R.K.; Eberlin, M.N.; Langley, G.J.; Li, L.; Naito, Y. Definition of Terms Relating to Mass Spectrometry (IUPAC Recommendations 2013). Pure Appl. Chem. 2013, 85, 1515-1609.

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(30) European Commission, Decision 2002/657/EC of 12 August 2002, Off. J. European Union L221. 2002, 8-36.

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(31) Long, G.L.; Winefordner, J.D. Limit of Detection A Closer Look at the IUPAC Definition. Anal. Chem. 1983, 55, 712-724.

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(33) Olajnik, M.; Jedziniak, P.; Szprengier-Juszkiewicz, T.; Żmudzki, J. Influence of Matrix Effect on the Performance of the Method for the Official Residue Control of Non-Steroidal Anti-Inflammatory Drugs in Animal Muscle. Rapid Commun. Mass Spectrom. 2013, 27, 437-442.

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Table 1: MRM conditions andlinearity parameters for the target indoles analyzed Compound

[M+H]+

Cone voltage

5MIA

5-Methoxyindole-3-acetic acid

206.2

24

5MTL

5-Methoxytryptophol

192.1

28

NSER

N-acetylserotonin (N-Acetyl-5-hydroxytryptamine)

219.2

22

5OHIA

5-Hydroxyindole-3-acetic acid

192.3

24

TRP

Tryptophan

205.1

20

TRPd5

Deuterated tryptophan

210.3

20

MEL

Melatonin

233.1

24

5OHTRP

5-Hydroxy-L-tryptophan

221.3

20

SER

Serotonin (5-Hydroxytryptamine)

177.3

16

TEE

Tryptophan ethyl ester

233.1

20

a

(IS)

Collision energy

MRM transitiona

30 42 26 34 20 38 38 48 22 30 22 30 18 12 20 28 24 28 18 20

206.2 > 117.7 206.2 > 145.6 192.1 > 159.6 192.1 > 131.5 219.2 > 160.5 219.2 > 115.7 192.3 > 117.9 192.3 > 91.7 205.1 > 146.6 205.1 > 118.7 210.3 > 151.6 210.3 > 123.4 233.1 > 174.2 233.1 > 216.1 221.3 > 162.5 221.3 > 134.6 177.3 > 160.5 177.3 > 115.7 233.1 > 174.0 233.1 > 159.0

R2

Repeatability

b

Matrix c effects (n=6)

Accuracyd

LOD

LOQ

(µg/L)

(µg/L)

(n=3)

0.9988

0.61

2.01

7.5

15.6 %

97.9

0.9973

0.29

0.96

8.0

12.9 %

97.1

0.998

0.05

0.17

0.5 %

97.0

0.9982

1.46

4.82

-26.8 %

99.2

0.9999

0.13

0.43

- 10.1%

98.5

-

-

-

-

-

-

0.9935

0.02

0.17

4.8

19.7 %

98.3

0.9997

0.11

0.36

3.6

- 38.8 %

99.0

0.9987

19.8

65.34

8.2

- 14.2 %

98.2

0.9953

0.01

0.03

-4.6 %

101.0

) First transition as ben used for Quantification

b

) n=3 samples in triplicate. Cava samples spiked with pure standards of the compounds below detectable levels in real samples (5 ppb for 5MIA, 5MTL, 5OHIA, MEL and 5OHTRP; 100 ppb for SER) c) Mean variation of the signals in spiked sample compared to those of pure standards dissolved in mobile phase A d ) Calculated as: (expected value/ observed value) * 100

358 359

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(n=3)

8.2 9.3 5.9

6.4

(n=6)

Journal of Agricultural and Food Chemistry

Table 2. Indole levels in sparkling wine samples (expressed as µg/L sample) Compound (Abbr)

Category

% vol

Cava type

TRP

TEE

5MTL

NSER

Cava Cava Cava Cava Cava Cava Cava Cava

35.7 5.9 8.0 18.8 35.6 178.9 574.9 22.7

0.4 0.1 0.1 0.4 0.3 1.9 2.7 0.1