Subscriber access provided by - Access paid by the | UCSB Libraries
Article
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
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Page 1 of 19
Journal of Agricultural and Food Chemistry
1 2
Ultra High Performance Liquid Chromatography (UHPLC)–Tandem Mass Spectrometry (MS/MS) Quantification of Nine Target Indoles in Sparkling Wines
3 4
Rebeca Tudela†, Albert Ribas-Agustí‡, Susana Buxaderas†, Montserrat Riu-Aumatell† *, Massimo Castellari‡, and Elvira López Tamames†
5 6 7 8
†
9
‡
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
10 11 12
*
13
ABSTRACT
14
An ultra high performance liquid chromatography (UHPLC)–tandem mass spectrometry
15
(MS/MS) method was developed for the simultaneous determination of nine target
16
indoles in sparkling wines. The proposed method requires minimal sample pretreatment,
17
and its performance parameters (accuracy, repeatability, LOD and matrix effect)
18
indicate that it is suitable for routine analysis.
19
Four indoles were found at detectable levels in commercial Cava samples: 5-
20
methoxytryptophol (5MTL), tryptophan (TRP), tryptophan ethyl ester (TEE) and N-
21
acetylserotonin (NSER). Two of them, namely NSER and 5MTL, are reported here for
22
the first time in sparkling wines, with values of 0.3-2 µg/mL and 0.29-29.2 µg/mL,
23
respectively. In the same samples, the contents of melatonin (MEL), serotonin (SER), 5-
24
hydroxytryptophan (5-OHTRP), 5-hydroxyindole-3-acetic acid (5OHIA) and 5-
25
methoxy-3-indoleacetic acid (5MIA) were all below the corresponding limits of
26
detection.
Corresponding author. Tel.: (+34) 934033795; fax: (+34) 934035931 E-mail address:
[email protected] (M. Riu-Aumatell)
27 28
Keywords: UHPLC-MS/MS, melatonin, sparkling wine, indole, tryptophan ethyl ester
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Page 2 of 19
29
INTRODUCTION
30
The presence of the ubiquitous neurohormone melatonin (MEL) in grapes and wines
31
has generated great interest within the wine industry in recent years.1-9 MEL is
32
considered to be a bioactive compound10 with antioxidant activities that have been
33
related to the presence of two side chains in the indole ring.11 Studies from 2006
34
onwards suggest that MEL may be formed in grapes;3,12-18 however, the most recent
35
findings indicate that MEL is only accumulated during the winemaking process. Thus,
36
its presence in wine would be strictly related to the activity of yeast during
37
fermentation.1,2,19 MEL can be formed by Saccharomyces yeasts from precursors of the
38
common tryptophan (TRP) biosynthetic pathway, via 5-hydroxytryptophan (5OHTRP),
39
serotonin (SER) and N-acetylserotonin (NSER). Alternative MEL formation involves a
40
secondary metabolic pathway of S. cerevisiae via the O-methylation of serotonin
41
followed
42
methoxytryptophol (5MTL) is produced by yeasts in association with MEL. 19
43
Several authors have showed that wines may contain only melatonin1,7,8,12,13; or just one
44
tentatively identified MEL isomer3; or, in a few cases, melatonin and up to three
45
possible MEL isomers5,21. More recently, tryptophan ethylester (TEE) has been
46
identified as one of the compounds previously reported to be the most abundant MEL
47
isomers in red wine.22 These recent findings underline the need for advanced analytical
48
techniques to elucidate the indole-derived compounds involved in melatonin
49
metabolism.
50
Determination of MEL and related indoles in wine was initially carried out using HPLC
51
with fluorimetric detection (HPLC-F),4,7 and capillary electro chromatography (CE)
52
with immobilized carboxylic multi-walled carbon nanotubes.12 However, the protocols
by
the
N-acetylation
of
5-methoxytryptamine.20
ACS Paragon Plus Environment
Furthermore,
5-
Page 3 of 19
Journal of Agricultural and Food Chemistry
53
require different sample pretreatments such as microextraction by a packed sorbent
54
(MEPS) or solid-phase extraction (SPE) in order to improve MEL detection; even
55
though they increase the total analysis time.4,5,7,13,23,24 More recently, “hyphenated”
56
techniques such as LC-MS/MS, UHPLC-MS/MS or LC coupled to high resolution mass
57
spectrometry (HRMS) have been used to determine MEL and other related compounds
58
in wines.3,5,21,22 The advantage of these techniques is the simplified sample preparation,
59
e.g. dilution or filtration, and the structural information provided by the MS detectors
60
that supports identification.
61
Production of Spanish “Cava” sparkling wines follows the classical or traditional
62
method, which includes a second fermentation of the base wine to obtain the
63
characteristic foam, and aroma. At the end of the second fermentation, yeast cells die
64
and remain in contact with the wine for a long time (up to several months). At this
65
stage, the intracellular compounds, including TRP, are released into the wine,3,24-27 and
66
contribute to the development of the particular flavor and aroma of sparkling wines.
67
To the best of our knowledge, no study of MEL and its metabolic pathway has been
68
performed before now in sparkling wine. Since traditional sparkling wines undergo two
69
fermentations, it might be expected that the assessment of the metabolic pathways of
70
indole-derived compounds could be important to clarify the role of yeast and lees
71
autolysis in the development of the characteristic qualities of sparkling wine.25-27
72
Therefore, the aim of this study was to develop a suitable UHPLC-MS/MS method for
73
the simultaneous identification and quantification of MEL and other indole compounds
74
(Figure 1) involved in the melatonin metabolism pathway in sparkling wines. The levels
75
of L-tryptophan (TRP), 5-hydroxy-L-tryptophan (5-OHTRP), serotonin (SER), N-
76
acetylserotonin (NSER), melatonin (MEL), 5-hydroxyindole-3-acetic acid (5OHIA), 5-
77
methoxytryptophol (5MTL), 5-methoxy-3-indoleacetic acid (5MIA) and tryptophan
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
78
ethyl ester (TEE) in 18 commercial Spanish sparkling wines (Cava) were evaluated; as
79
were the performance parameters of the proposed methods.
80
MATERIALS AND METHODS
81
Chemicals. L-tryptophan, 5-hydroxy-L-tryptophan, serotonin hydrochloride, n-acetyl-
82
5-hydroxytryptamine, melatonin, 5-methoxytryptophol, 5-hydroxyindole-3-acetic acid,
83
5-methoxy-3-indoleacetic acid, L-tryptophan ethyl ester hydrochloride, L-tryptophan-d5
84
with an isotopic purity of more than 97% (indole-d5) (internal standard; I.S.),
85
hydrochloric acid and ammonium hydroxide ACS were supplied by Sigma-Aldrich (St.
86
Louis, USA).
87
Acetonitrile LC–MS grade was from Riedel-de Haën (Seelze, Germany), methanol,
88
hexane and acetone HPLC grade were purchased from Merck (Darmstadt, Germany).
89
Ultra-pure water was obtained using a Milli-Q system (Millipore, Milford, MA).
90
The single standard stock solutions and I.S. solution were prepared weekly by
91
dissolving known amounts of pure standards in methanol (1,000 mg/L) and then stored
92
in the dark at 4ºC until analysis. Spiking standard solutions (20 mg/L) were prepared
93
weekly by diluting standard stock solutions with methanol.
94
Samples. Several brands of commercial Cava sparkling wine (n=18) were purchased in
95
local supermarkets, produced at different wineries in the Cava region (Catalonia, Spain).
96
Cava is produced following the traditional method in which a second fermentation is
97
followed by aging in contact with the lees (minimum 9 months) in which the sparkling
98
wine develops the characteristic foam and aroma.26-28
99
Once the bottles were opened, samples were degassed by magnetic stirring for 5 min,
100
and then stored in 250 mL amber flasks at -20ºC until analysis. The entire process was
101
carried out under reduced light conditions to prevent degradation of the melatonin. Just
ACS Paragon Plus Environment
Page 4 of 19
Page 5 of 19
Journal of Agricultural and Food Chemistry
102
before analysis, the samples were filtered with a nylon membrane filter (0.2 µm)
103
(Waters, Milford, MA).
104
UHPLC-MS/MS Conditions. Analysis was carried out with an Acquity Ultra-
105
PerformanceTM liquid chromatography system (UPLC©), equipped with a binary pump
106
system (Waters, Milford, MA). Detection was carried out using a TQD triple
107
quadrupole MS detector (Waters, Milford, MA). Chromatographic separation was
108
carried out with a BEH C18 Shield column (150 x 1.0 mm i.d.) with a 1.7 µm particle
109
size (Waters, Milford, MA), kept at 30ºC. Linear gradient elution was carried out from
110
95% mobile phase A (5:94.9:0.1 v/v/v ACN:water:formic acid) and 5% B (95:4.9:0.1
111
v/v/v ACN:water:formic acid) to 25% A in 8 minutes, with a flow rate of 0.125
112
mL/min. After each run, the system was stabilized with the initial conditions for one
113
minute before the next injection. Samples were maintained at 10ºC and the injected
114
volume was 8 µL. A standard washing procedure (with weak and strong eluents, i.e. the
115
mobile phases A & B) was included in the chromatographic method after each injection.
116
Carry over was checked for at the beginning of each sample batch by injecting a blank
117
sample (water) after a spiked sample.
118
The electrospray interface (ESI) was operated in positive mode; the source temperature
119
was fixed at 130ºC, the capillary voltage was set at +3.0 kV and the desolvation
120
temperature was set at 350ºC. The cone gas (nitrogen) flow rate was 350 L/h. The gas
121
used in the collision cell was argon at a flow rate of 0.1 mL/min. Multiple reaction
122
monitoring (MRM) mode was used to quantify the compounds. MS/MS experiments
123
were also performed in product ion scan mode29 to study the fragmentation patterns of
124
specific molecular ions. The MRM conditions were optimized using Autotune Wizard
125
Software by infusing standard solutions (10 mg/L) of each compound. Two specific
126
MRM transitions of the deprotonated molecular ions were monitored for each
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
127
compound; for quantification purposes, the product ion with the higher signal was used.
128
Data acquisition and integration were controlled by MassLynxTM software (version 4.1).
129
Analytic Performance. Standard addition calibration was created by spiking a cava
130
sample with known amounts of standards at five levels (ranging from 1 to 50 µg/L for
131
5MTL, NSER, MEL, 5OHTRP and TEE; from 5 to 50 µg/L for 5MIA and 5OHIA;
132
from 5 to 1,000 µg/L for TRP; and from 100 to 500 µg/L for SER), and by using
133
tryptophan-d5 as the I.S. The stock solution of the I.S. was prepared at 1 mg/mL in
134
mobile phase A. Then the working solution was prepared by diluting to a final
135
concentration of 20 mg /L. Calibration curves were created by plotting the ratio between
136
the area of the more intense transition and the area of the I.S. in the MRM mode, vs. the
137
corresponding concentrations of each target compound.
138
Identification of the target compounds was carried out following the criteria of the
139
Commission Decision 2002/657/CE, by comparing their retention time and MRM
140
product ion ratios with those obtained from pure standards.
141
The performance parameters of the method were established following Long and
142
Winefordner,31 and IUPAC.32 Signal-to-noise ratios of 3:1 and 10:1 were considered for
143
LOD and LOQ, respectively.
144
Repeatability was assessed by injection in triplicate of three Cava sparkling wines.
145
Three Cava samples spiked with standard solutions were used in the case of compounds
146
that fell below the LOD in the commercial samples.
147
The accuracy of the analytical assay was determined by analyzing three Cava sparkling
148
wines spiked at two levels with standard solutions (20 µg/L and 50 µg/L for 5MTL,
149
NSER, MEL, 5OHTRP, TEE, 5MIA and 5OHIA; and 100 µg/L and 500 µg/L for TRP
150
and SER respectively), and calculating the ratio between expected and calculated
151
concentrations for each compound.
ACS Paragon Plus Environment
Page 6 of 19
Page 7 of 19
Journal of Agricultural and Food Chemistry
152
Matrix effects were estimated for each compound by comparing the response in the
153
presence of the matrix (three samples spiked at two levels (50 and 100 µg/L) as for the
154
accuracy assays) with the response of pure compounds at the same concentrations
155
dissolved in a neat solution (mobile phase A).
156 157
RESULTS AND DISCUSSION
158
Analytic Performance
159
Table 1 shows the MRM conditions and calibration parameters for the target
160
compounds. Under our conditions, the best responses were obtained by working in
161
positive mode for all the nine compounds that were the objects of this study.
162
The MRM profiles for a mixture of pure standards of the nine target compounds (plus
163
the I.S.), and the profile of a cava sample are shown in the Supporting Information.
164
Separation of all the target compounds was completed within 5.2 minutes which, jointly
165
with the minimal sample preparation, allows a high sample throughput.
166
In all the Cava wine samples, a relevant peak was found at 2.92 minutes with an
167
[M+H]+ of m/z = 232. This peak (MRM profiles of pure standards and cava samples are
168
provided in the Supporting Information) fits the retention time and the product ion
169
spectra of the TEE pure standard (profile of product ions from collision-induced
170
dissociation at 15 V, for the TEE standard is provided in the Supporting Information),
171
while the ratio between ions at m/z 216 and m/z 174 is in agreement with the results of
172
the recent work of Gardana et al.22
173
The linearity of the calibration curves could be considered satisfactory in the ranges of
174
concentration included in the protocol of analysis (Table 1).
175
The LOD of our method in the case of MEL was similar to the values reported by other
176
authors operating with both HPLC-F and LC-MS/MS setups.1,3,4,7,12,13,19,21 The LOD for
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
177
TRP was better than those previously obtained by other authors working with HPLC-
178
F.13,23,24 The LOD of SER was slightly higher than that reported by Mercolini et al.13
179
with an HPLC-F method; but it was comparable to that previously reported in fruit
180
analysis by HPLC–MS/MS, where the detection limit was 50 µg/L.9
181
The LODs for the other target compounds were always lower than 1 µg/L (Table 1) and
182
only in the case of 5OHIA was the LOD approximately 3 times higher than in the study
183
by Mercolini et al.13 mentioned above using HPLC-F. The LODs for TEE were
184
comparable to that obtained by Vigentini et al.19
185
Both repeatability (with values always lower than 10%) and accuracy (Table 1) can be
186
considered adequate for all the target compounds, taking into account that their levels in
187
real samples are mainly in the range of a few µg/L. Matrix effects were in general lower
188
than 20%; a good result considering that the samples were injected without any pre-
189
treatment to reduce the interfering compounds. Only in the case of 5OHTRP was the
190
ionization suppression higher than 30% (Olajnik et al., 2013)36.
191
Sparkling Wine Results
192
The performance parameters of the method confirm that it is fit for routine analysis
193
(Table 1). To check the suitability of the proposed protocol, a total of 18 samples of
194
commercial sparkling wines were analyzed with the proposed method (Table 2). TRP
195
was the compound detected with the highest concentrations in all the samples. The
196
concentrations of TRP found in Cava sparkling wines were in accordance with the
197
results of Mattivi et al.23 obtained by an HPLC–F method in Chardonnay wines (62-417
198
µg/L); but were somewhat lower than those cited by Hoenicke et al.24 in different white
199
wines.
ACS Paragon Plus Environment
Page 8 of 19
Page 9 of 19
Journal of Agricultural and Food Chemistry
200
TEE was found in all the Cava samples (Table 2); however, the amounts found in the
201
current study, ranging from 0.1 to 2.7 µg/L, were lower than those found by Gardana et
202
al.22 in Syrah wine (84 µg/L).
203
To the best of our knowledge, no data have previously been reported in the literature for
204
the content of NSER and 5MTL in wines. The indole NSER, which is the immediate
205
precursor of MEL according to its biosynthetic pathway,20 was detected in all the Cava
206
samples. The amount of NSER varied between 0.3 and 2.0 µg/L. Moreover, 5MTL,
207
another MEL precursor,20 was also detected in variable amounts but in only three of the
208
18 samples analyzed (LOD: 29.2 µg/L) (Table 2).
209
MEL, SER, 5MIA, 5OHIA and 5OHTRP were all under the LOD in the samples
210
analyzed in this study (Table 2 and MRM profiles of pure standards and cava samples
211
provided in the Supporting Information). MEL has been found in different kinds of
212
wine at levels ranging from 0.5 to 423 µg/L,1,4,7,12,13,19; with 390.82 µg/L being the
213
higher concentration found in white wine.1 The absence of detectable levels of MEL in
214
Cava wines could be due to the specific technology used for their preparation, which
215
includes a second fermentation and a long aging process (always over a year) which
216
could contribute to the transformation and fading of the MEL present in the base wines.
217
It should also be emphasized that MEL was only found at detectable levels in wines
218
produced at the laboratory scale or analyzed by HPLC-F.1,4,7,13,19 In contrast, the
219
samples included in our study are commercially available Cava sparkling wines with a
220
long ageing (over 12 months) which could have influenced the final levels of MEL.
221
SER was previously found in Albana wine at concentrations below the LOD of our
222
method; while 5OHIA, a SER metabolite, was previously found by Mercolini et al.13 at
223
105 µg/L in white wine. The undetectable levels of these compounds in our samples
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
224
could be related to the different conditions of elaboration of the wine and/or the
225
differences in the LOD values between the methods.
226 227
In summary, a UHPLC MS/MS method for the simultaneous assessment of nine indole
228
compounds involved in MEL metabolism in sparkling wines was developed and its
229
main performance parameters estimated. The proposed protocol requires limited sample
230
pre-treatment, requires about 6 minutes for the complete separation of the nine target
231
compounds and is suitable for routine analysis to give a quick overview of the levels of
232
some of the main indole-derived compounds involved in the MEL metabolism pathway.
233
Among the nine indoles studied, only four were detected in our Cava samples:
234
tryptophan (TRP), tryptophan ethyl ester (TEE), N-acetylserotonin (NSER), and 5-
235
methoxytryptophol (5MTL). The presence of NSER and 5MTL in sparkling wine is
236
reported here for the first time. The proposed method is a promising starting point for
237
further studies to clarify the effect of sparkling wine technology on the MEL
238
biosynthetic pathway.
239
Supporting information
240
MRM profiles for a mixture of pure standards of the nine target compounds (plus the
241
I.S.), and the profile of a cava sample (Figure S1); Profiles of product ions from
242
collision-induced dissociation at 15 V, for the MEL standard (a); the TEE standard (b);
243
and the peak in Cava sparkling wines at 2.92 min (c) (Figure S2) (PDF).
244
This material is available free of charge via the Internet at http://pubs.acs.org.
245
246
ACS Paragon Plus Environment
Page 10 of 19
Page 11 of 19
Journal of Agricultural and Food Chemistry
247
ACKNOWLEDGMENTS
248
This study was made possible thanks to the financial assistance provided by the
249
Comisión Interministerial de Ciencia y Tecnología (CICYT) (Spain), via award
250
AGL2011-23872, and the Generalitat de Catalunya (Spain), via project 2014SGR1438.
251 252
REFERENCES
253 254 255
(1) Rodriguez-Naranjo, M.I.; Gil-Izquierdo, A.; Troncoso, A.M. Melatonin is Synthesised by Yeast During Alcoholic Fermentation in Wines. Food Chem. 2011a, 126, 1608-1613.
256 257 258
(2) Rodriguez-Naranjo, M.I.; Torija, M.J.; Mas, A.; Cantos-Villar, E.; Garcia-Parrilla, M del C. Production of Melatonin by Saccharomyces Strains Under Growth and Fermentation Conditions. J. Pineal Res. 2012, 53, 219-224.
259 260 261
(3) Gomez, F.J.; Raba, J.; Cerutti, S.; Silva, M.F. Monitoring Melatonin and its Isomer in Vitis Vinifera cv. Malbec by UHPLC-MS/MS from Grape to Bottle. J. Pineal Res. 2012, 52, 349-355.
262 263 264
(4) Rodriguez-Naranjo, M. I.; Gil-Izquierdo, A.; Troncoso, A. M.; Cantos-Villar, E.; Garcia-Parrilla, M. C. Melatonin: A New Bioactive Compound in Wine. J. Food Compost. Anal. 2011b, 24, 603-608.
265 266 267
(5) Vitalini, S.; Gardana, C.; Simonetti, P.; Fico, G.; Iriti, M. Melatonin, Melatonin Isomers and Stilbenes in Italian Traditional Grape Products and their Antiradical Capacity. J. Pineal Res. 2013, 54, 322-333.
268 269 270
(6) Arevalo-Villena, M.; Bartowsky, E.J.; Capone, D.; Sefton, M.A. Production of Indole by Wine-Associated Microorganisms Under Oenological Conditions. Food Microbiol. 2010, 27, 685-690.
271 272 273
(7) Mercolini, L.; Addolorata Saracino, M.; Bugamelli, F.; Ferranti, A.; Malaguti, M.; Hrelia, S.; Raggi, M.A. HPLC-F Analysis of Melatonin and Resveratrol Isomers in Wine Using an SPE Procedure. J. Sep. Sci. 2008, 31, 1007-1014.
274 275 276
(8) Vitalini, S.; Gardana, C.; Zanzotto, A.; Fico, G.; Faoro, F.; Simonetti, P.; Iriti, M. From Vineyard to Glass: Agrochemicals Enhance the Melatonin and Total Polyphenol Contents and Antiradical Activity of Red Wines. J. Pineal Res. 2011, 51, 278-285.
277 278 279
(9) Huang, X.; Mazza, G. Simultaneous Analysis of Serotonin, Melatonin, Piceid and Resveratrol in Fruits Using Liquid Chromatography Tandem Mass Spectrometry. J. Chromatogr. A. 2011, 1218, 3890-3899.
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
280 281 282 283
(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.
284 285 286 287 288
(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.
289 290 291 292
(12) Stege, P.W.; Sombra, L.L.; Messina, G.; Martinez, L.D.; Silva, M.F. Determination of Melatonin in Wine and Plant Extracts by Capillary Electrochromatography with Immobilized Carboxylic Multi-Walled Carbon Nanotubes as Stationary Phase. Electrophoresis. 2010, 31, 2242-2248.
293 294 295
(13) Mercolini, L.; Mandrioli, R.; Raggi, M.A. Content of Melatonin and Other Antioxidants in Grape-Related Foodstuffs: Measurement Using a MEPS-HPLC-F Method. J. Pineal Res. 2012, 53, 21-28.
296 297
(14) Iriti, M.; Rossoni, M.; Faoro, F. Melatonin Content in Grape: Myth or Panacea?. J. Sci. Food Agric. 2006, 86, 1432–1438.
298 299 300
(15) Murch, S.J.; Hall, B.A.; Le, C.H.; Saxena, P.K. Changes in the Levels of Indoleamine Phytochemicals During Véraison and Ripening of Wine Grapes. J. Pineal Res. 2010, 49, 95-100.
301 302 303
(16) Vitalini, S.; Gardana, C.; Zanzotto, A.; Simonetti, P.; Faoro, F.; Fico, G.; Iriti, M. The Presence of Melatonin in Grapevine (Vitis Vinifera L.) Berry Tissues. J. Pineal Res. 2011, 51, 331-337.
304 305 306 307
(17) Boccalandro, H.E.; Gonzáles, C.V.; Wunderlin, D.A.; Silva, M.F. Melatonin Levels, Determined by LC-ESI-MS/MS, Deeply Fluctuate During the Day in Vitis Vinifera cv Malbec. Evidences for its Antioxidant Role in Fruits. J. Pineal Res. 2011, 51, 226-232.
308 309 310
(18) Gomez, F.J.; Hernández, I.G.; Martinez, L.D.; Silva, M.F.; Cerutti, S. Analytical Tools for Elucidating the Biological Role of Melatonin in Plants by LC-MS/MS. Electrophoresis. 2013, 34, 1749-1756
311 312 313 314
(19) Vigentini, I.; Gardana, C.; Fracassetti, D.; Gabrielli, M.; Foschino, R.; Simonetti, P.; Tirelli, A.; Iriti, M. Yeast Contribution to Melatonin, Melatonin Isomers and Tryptophan-Ethylester During Alcoholic Fermentation of Grape Musts. J. Pineal Res. 2015, 58, 388-396.
ACS Paragon Plus Environment
Page 12 of 19
Page 13 of 19
Journal of Agricultural and Food Chemistry
315 316 317
(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.
318 319 320
(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.
321 322 323
(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.
324 325 326
(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.
327 328 329
(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.
330 331 332
(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.
333 334
(26) Alexandre, H.; Guilloux-Benatier, M. Yeast Autolysis in Sparkling Wine A Review. Aust. J. Grape Wine Res. 2006, 12, 112-127.
335 336
(27) Buxaderas, S.; López-Tamames, E. Sparkling Wines: Features and Trends From Tradition. Adv. Food Nutr. Res. 2012, 66, 1-45.
337 338 339 340
(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.
341 342 343
(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.
344 345
(30) European Commission, Decision 2002/657/EC of 12 August 2002, Off. J. European Union L221. 2002, 8-36.
346 347
(31) Long, G.L.; Winefordner, J.D. Limit of Detection A Closer Look at the IUPAC Definition. Anal. Chem. 1983, 55, 712-724.
348 349
(32) IUPAC Compendium of Chemical Terminology, second ed., 1997, see http://iupac.org/publications/analytical compendium/Cha18sec437.pdf.
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
350 351 352 353
(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.
354 355 356 357
ACS Paragon Plus Environment
Page 14 of 19
Page 15 of 19
Journal of Agricultural and Food Chemistry
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
ACS Paragon Plus Environment
(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