Multidimensional Gas Chromatography Coupled to Combustion

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MULTIDIMENSIONAL GC-C-IRMS / QUADRUPOLE MS WITH A LOW-BLEED IONIC LIQUID SECONDARY COLUMN FOR THE AUTHENTICATION OF TRUFFLES AND PRODUCTS CONTAINING TRUFFLE Danilo Sciarrone, Antonino Schepis, Mariosimone Zoccali, Paola Donato, Federico Vita, Donato Creti, Amedeo Alpi, and Luigi Mondello Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b00386 • Publication Date (Web): 07 May 2018 Downloaded from http://pubs.acs.org on May 8, 2018

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Analytical Chemistry

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MULTIDIMENSIONAL GC-C-IRMS / QUADRUPOLE MS WITH A LOW-BLEED IONIC

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LIQUID SECONDARY COLUMN FOR THE AUTHENTICATION OF TRUFFLES AND

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PRODUCTS CONTAINING TRUFFLE

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Danilo Sciarrone1, Antonino Schepis1, Mariosimone Zoccali1, Paola Donato2, Federico Vita3,

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Donato Creti4, Amedeo Alpi5, Luigi Mondello1,6,7*

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1

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Messina, Polo Annunziata, viale Annunziata, 98168 – Messina, Italy

Dipartimento di Scienze Chimiche, Biologiche, Farmaceutiche ed Ambientali, University of

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2

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University of Messina, via Consolare Valeria, 98125 – Messina, Italy

Dipartimento di Scienze Biomediche, Odontoiatriche e delle Immagini Morfologiche e Funzionali,

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3

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– Pisa, Italy

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4

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Scandicci (FI), Italy

Department of Agriculture, Food and Environment, University of Pisa, Via Mariscoglio 34, 56124

Enrico Giotti S.p.A., a subsidiary of McCormick & Company, Inc., Via Pisana 592, 50018 –

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5

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University of Pisa, Via Mariscoglio 34, 56124 – Pisa, Italy

Dipartimento di Scienze Agrarie, Alimentari, Agro-ambientali, Laboratorio di Fisiologia Vegetale,

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6

Chromaleont S.r.l., via Leonardo Sciascia Coop. Fede, Pal. B, 98168 – Messina, Italy

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University Campus Bio-Medico of Rome, via Álvaro del Portillo 21, 00128 – Rome, Italy

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*Corresponding author. Tel. +39 0906766536; fax +39 090358220.

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E-mail address: [email protected] (L. Mondello).

1 Environment ACS Paragon Plus

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Abstract

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Truffles are among the most expensive foods available in the market, usually used as flavouring

35

additives for their distinctive aroma. The most valuable species is Tuber magnatum Pico, better

36

known as “Alba white truffle”, in which bis(methylthio)methane is the key aroma compound. Given

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the high economical value of genuine white truffles, analytical approaches are required, able to

38

discriminate between natural or synthetic truffle aroma. Gas chromatography coupled to

39

combustion-isotope ratio mass spectrometry (GC-C-IRMS), exploiting the 13C/12C ratio abundance

40

of the key flavourings compounds in foods, has been a recognized technique for authenticity and

41

traceability purposes, however a number of issues have greatly limited its widespread use, so far. In

42

the present research, a high-efficiency HS-SPME MDGC-C-IRMS with simultaneous quadrupole

43

MS detection, has been applied for the evaluation of bis(methylthio)methane, resolving the

44

coelution occurring with other components. With the aim to minimize the effect of column bleeding

45

on δ13C measurement, a medium polarity ionic liquid-based stationary phase was preferred to a

46

polyethylene glycol one, as secondary column. Twenty-four genuine white truffles harvested in

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Italy were analysed, attaining δ13C values between -42.6‰ and -33.9‰, with a maximum standard

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deviation lower then 0.7‰. Two commercial intact truffles and 14 commercial samples of pasta,

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sauce, olive oil, cream, honey and fresh cheese flavoured with truffle aroma were analysed, and the

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results from δ13C measurement were evaluated in comparison with those of genuine “white truffle”

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range and commercial synthetic bis(methylthio)methane standard.

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Keywords: Bis(methylthio)methane, Ionic liquids, Isotopic ratio, Low-bleed column, MDGC,

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Tuber magnatum Pico, White truffle aroma.


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1. Introduction

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Truffles (Tuber spp.) belonging to the fungal genus Tuber are ascomycete symbiotic fungi that

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undergo a complex life cycle in association with plant roots. Truffles, in fact, are able to form

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fruiting bodies only in symbiosis with plant roots even if they can be rarely found under the same

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trees. The fruiting bodies produce hundreds of volatile compounds capable of attracting insects and

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mammals, in order to spread their ascospores [1]. Some species of truffle are among the most

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expensive foods available in the market, usually used as flavouring additives for their distinctive

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aroma or consumed raw added to certain dishes [2,3]. The most valuable species are Tuber

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magnatum Pico, better known as “Alba white truffle”, Tuber melanosporum Vittad, the “Périgord

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black truffle” and Tuber aestivum, the

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thousands of euro per kilo. Tuber spp. fungi have been studied for a variety of reasons including

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their beneficial activities (anti-inflammatory, anti-microbial, and anti-oxidant) and their nutritional

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composition, but most studies have been focused on their aroma [4]. Several papers have reported

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the chemical constituents considered to be responsible for the typical truffle aroma. Dimethylsulfide

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has been identified in white, black and summer truffles, whereas bis(methylthio)methane is the key

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aroma compound of white truffle. The most abundant volatiles commonly present in truffle aroma

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include bis(methylthio)methane, dimethylsulfide, hexanal, 2-methylbutanal, and 3-methylbutanal,

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with quantitative and qualitative variations according to the truffle species and the geographical

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origin [5-9]. This explains why the food industry uses bis(methylthio)methane to enhance the aroma

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of commercial food products either containing or claiming to contain truffles. Torregiani et al.

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reported that neither the natural nor the artificial truffle oil samples adequately replicated the

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aromas of the species of truffle examined [10]. Since bis(methylthio)methane is the main and the

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most characteristic contributor to the aroma of white truffle, several studies have focused on its

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variation during the storage of Tuber magnatum; the results indicated a high relative percentage of

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bis(methylthio)methane in fresh truffles, diminishing with time [11].

“summer truffle”; the first one is routinely sold for



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Bis(methylthio)methane can be easily added to commercial products, due to its solubility in oil and

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stability. Its use as a flavouring agent is permitted by the law, as reported by the Joint FAO/WHO

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Expert Committee on Food Additives (JECFA) [12]. However, there are major differences in the

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market price, between the synthetic (100$/Kg) and the biotechnologic (5000$/Kg) compound.

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Since aroma is made out of volatile organic compounds, the most used analytical techniques for its

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determination in foods aroma have obviously consisted of headspace (HS) extraction coupled to gas

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chromatography mass spectrometry (GC-MS) [13]. Several studies reported the use of HS solid

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phase micro extraction (HS-SPME) as a sensitive technique for the evaluation of truffle aroma and

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composition, also with regard to different storage conditions [3,13-15]. The headspace analysis of

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white truffles fresh and after storage showed significant modifications of the aroma profile, with the

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formation of several compounds including alcohols and acids, or changes in their quantity [16];

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such variations would hamper the assessment of authenticity based on the relative abundance of the

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key aroma compounds. As a consequence of the increasing consumers’ demand for genuine food

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and ingredients, in the last years there was a need for analytical approaches able to determine

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natural and synthetic food ingredients, in order to highlight fraudulent or unsafe practices. From the

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earliest stages of its development, gas chromatography coupled to combustion-isotope ratio mass

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spectrometry (GC-C-IRMS) has been a recognized technique for authenticity and traceability

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purposes, in that it allows to determine the

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compounds used in food products [17]. GC-C-IRMS may be useful for the detection of fraudulent

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actions across a wide range of foods, beverages, and ingredients [18]. With regard to the

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determination of the carbon isotopic ratio, during a GC-C-IRMS analysis compounds eluting from

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the GC column are oxidized to CO2 and transferred to Faraday cups for m/z 44, 45 and 46 by using

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a magnetic sector. Unlike what happens when using a GC hyphenated to a quadrupole mass

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spectrometer (GC–qMS), where co-eluting compounds can still be identified and quantified in the

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selected ion monitoring (SIM) mode, excellent chromatographic resolution is mandatory for GC-C-

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IRMS analysis, since the isotopic ratio of the CO2 generated from coeluted peaks cannot be treated

13

C/12C ratio abundance of the key flavourings


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in the same way. Baseline-to-baseline integration over an entire peak is in fact required for accurate

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measurement of its isotopic composition, since column fractionation would retain in a slightly

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different way the heavier isotopes, eluted in the first part of the peak, and the lighter ones, eluted in

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the tail [19]. In order to separate compounds of interest from non-target compounds,

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multidimensional gas chromatography (MDGC) in the heart-cut mode may be successfully applied

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in different analytical fields [20], including isotope ratio measurements [21-29]. The MDGC-C-

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IRMS approach has been used in the last decade to analyse flavor components [21,22], steroids in

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urine [23,24], wax compounds in tobacco leaf and smoke samples [25], selected congeners in

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polychlorobiphenyls (PCB) and polychloronaphthalene (PCN) [26], C2 to C5 hydrocarbons

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produced during biomass burning experiments [27], benzene, toluene, ethylbenzene, and xylenes

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(BTEX) in environmental samples [28] and monoaromatic hydrocarbons in complex groundwater

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and gas-phase samples [29]. In the present research, MDGC-C-IRMS with simultaneous quadrupole

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MS detection after the second dimension (2D), was exploited for the δ13C evaluation of

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bis(methylthio)methane in different natural truffles and food-aromatized samples, after extraction

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by HS-SPME.

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2. Experimental Section

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2.1. Sampling of fruiting bodies

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Twenty-four samples of fruiting bodies of Tuber magnatum Pico were collected from different

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areas of Italy during the autumn 2016. For each sample, different naturally grown carpophores were

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harvested in their specific regional areas, and namely: 6 from Tuscany (San Miniato), 3 from

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Molise, 1 from Calabria (Catanzaro), 1 from Basilicata, 2 from Abruzzo (Teramo), 6 from Piedmont

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(Alba, Mombercelli), 5 from Umbria (Assisi). Detailed information on the geographical origin,

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weight, and species of the samples are reported in Table 1, together with the name of the symbiotic

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plant species, if known. Three standards of bis(methylthio)methane (two synthetic and one natural

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flavouring substance) were kindly provided by MilliporeSigma/Supelco (Bellefonte, PA, USA) and



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Axxence Aromatic GmbH (Emmerich, Germany). Indiana standard (Indiana University,

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Bloomington, IN, USA), calibrated against an international standard [Vienna-Pee Dee Belemnite

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(V-PDB)] with defined δ13C content, was used for multipoint calibration of the CO2 reference gas

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δ13C value. All samples were stored at 4 °C.

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2.2. Commercial samples

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Fourteen commercial food products aromatized with truffles were purchased from local stores

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(Messina, Italy), and namely: 3 olive oils, 3 sauces, 3 fresh cheeses, 1 honey, 1 cream, 1 pasta, 1

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summer truffle and 1 white truffle (Table 2). Isotope ratios of the samples of interest were obtained

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by the following formula:

145 δ13CV-PDB = 1000 (Rsample - Rstandard)/Rstandard

146 147 148

where R represents the abundance ratio of the heavier isotope of the element against the lighter

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isotope (13C/12C).

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2.3. HS-SPME conditions

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Truffles were finely chopped and 0.3 g of each were put into a 20 mL crimped vial for SPME, and

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the extractions were carried out in the headspace mode. A 1-cm long fused silica fiber coated with a

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50/30 µm layer of divinylbenzene/carboxen/polydimethylsiloxane (MilliporeSigma/Supelco,

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Bellefonte, PA, USA) was chosen to extract the volatile components from the truffles. The fiber

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was conditioned following the manufacturer’s instructions previous to its use (270 °C for 0.5

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hours). Samples were conditioned for 5 min at 50 °C, under agitation (clockwise rotation at 500

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rpm), and then underwent the extraction step for 15 min at 50 °C, still under agitation as previously

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indicated. Analytes were then desorbed into the GC injection port for 1 min. Blanks were run

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between consecutive analyses. The same procedure was used for the analyses of the commercial

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samples (using 1 g of sample) and standard solutions (1000 ppm). Each sample was analysed in

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triplicate.


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2.4. Instrumentation and operational conditions

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The MDGC-C-IRMS/qMS prototype system consisted of two GC-2010 Plus gas chromatographs

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(defined as GC1 and GC2), connected by means of a Deans-switch (DS) transfer device, an MS-

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QP2010 Ultra quadrupole mass spectrometer, and an AOC-20i autosampler (Shimadzu Corporation,

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Kyoto, Japan). The instrument was coupled to a VisION IRMS system (Elementar

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Analysensysteme GmbH, Langenselbold, Germany) by means of a GC V furnace system

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(Elementar Analysensysteme GmbH, Langenselbold, Germany) maintained at 850 °C (See Figure

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S-1 in Supporting Information for more details). GC1 was equipped with a split/splitless injector

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and a flame ionization detector (FID), while GC2 was connected to a rapid scanning qMS. The

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MDGC transfer device, located in GC1, was connected to an advanced pressure control unit (APC),

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which supplied the carrier gas (He). GC1: an SLB-5ms 30 m × 0.25 mm ID × 0.25 µm df

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(MilliporeSigma/Supelco, Bellefonte, PA, USA) was used as column 1 at a constant flow rate of 1

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mL/min; the following inlet pressure program was applied: 185 kPa for 1 min, then to 330 kPa at

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1.81 kPa/min. Injection mode (260 °C): splitless (1 min), then a 10:1 split ratio was applied.

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Temperature program: 40 oC (1 min) to 280 °C at 3 °C/min. FID (300 °C; H2 flow: 40.0 mL/min; air

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flow rate: 400.0 mL/min; sampling rate: 80 msec) was connected to the DS switch via a 0.25 m ×

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0.18 mm ID stainless steel uncoated column. GC2: an SLB-IL59 30 m × 0.25 mm ID × 0.2 µm df

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(MilliporeSigma/Supelco, Bellefonte, PA, USA) was used as column 2 with the same temperature

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program as for column 1. The following pressure program was applied by means of the APC: 140

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kPa for 1 min, then to 265 kPa at 1.56 kPa/min. The effluent from the 2D column was splitted

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through a zero dead-volume tee-union (Valco) to the GC V furnace and to the IRMS system via a

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0.85 m × 0.25 mm ID uncoated column and to the qMS via a 1.5 m × 0.1 mm ID uncoated column.

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The following settings were applied to the VisION system: acceleration voltage, 3789.907 V; trap

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current, 600 µA; magnet current, 3685.990 mA. MS settings were: ion source, 200 °C; interface

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temp., 200 °C; mass range, 40-400 m/z; scan speed, 1250 amu/sec. The MDGCsolution control

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software package (Shimadzu, Kyoto, Japan) allowed to set the analytical conditions for both the



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GC1 (FID) and GC2 (MS) together. The analysed compounds were identified by searching their

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qMS spectra against the FFNSC 3.0 mass spectral library database (Shimadzu, Kyoto, Japan). The

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IRMS used in the study was a bench top 5kV system with integrated monitoring gas delivery

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system with a high performance silicon carbide tube furnace for providing combustion/pyrolisis

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temperatures for the quantitative, fractionation-free conversion of organic compounds to pure gases

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(CO2 and H2O). The CO2 produced by combustion of each component was transferred to the MS,

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while the resulting water was removed through a Nafion tube. The system was designed to maintain

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the chromatographic integrity of the separated compounds, preserving the chromatographic

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resolution at the IRMS. An auxiliary He line (sample line He), automatically controlled through the

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second channel of the APC, was used in the furnace to allow a proper control over the open split

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conditions for the IRMS (see Figure S-1 in Supporting Information). Since in the micro-combustion

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interface the open split was placed inside the furnace tube, therefore demanding that the open split

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is in a steady state, the APC was operated in constant flow mode. An electron-impact ionization

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(EI) gas source, a variable field, stigmatically focused electromagnet for beam separation and multi-

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channel Faraday collectors for beam detection were used. IRMS data were collected by IonOS

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stable isotope data processing software ver. 3.0.0.5196 (Elementar Analysensysteme GmbH,

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Langenselbold, Germany); the apex track integration method was exploited to automatically find

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the correct starting and finishing point of the peaks. A multipoint linear drift calibration was used

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for the correction of the IRMS data during the runs since, as discussed by Skrzypek et al. [30], such

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treatment is crucial to obtain the most accurate and precise results.

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4. Results and discussion

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4.1. System configuration and optimization

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In order to evaluate the quality of commercial available food products labelled with a valuable

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aroma, as in the case of truffle, the isotopic ratio measurement of a key component was exploited as

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a

criterion

to

discriminate

between

genuine,

fortified,


or

synthetic

samples.

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Bis(methylthio)methane is known to be one of the main components of truffle flavor and for such a

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reason it is commonly added as an additive in ”truffle flavoured” products. This organosulfur

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compound is in fact a convenient, low cost characterizing aroma for food truffle preparation, able to

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provide or to enhance the flavor of truffle where it is present in low amount or even absent. In this

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concern, the present investigation was focused on the evaluation of the δ13C value of this key

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compound in natural fruiting bodies collected during autumn 2016 in different regions of Italy, and

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on commercial products bought in different local supermarkets. Twenty-four fruiting bodies were

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collected from different geographical origins and analyzed as soon as shipped to our lab, in order to

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prevent spoiling of the aroma profile. After extraction, if available in sufficient amount, the samples

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were stored at 4 °C. A first step consisted in the conventional approach used for this analysis, based

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on monodimensional GC after SPME extraction. In order to evaluate the occurrence of carbon

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isotope fractionation during the HS-SPME extraction, different experimental conditions were tested

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(see 2.3. HS-SPME conditions section) on a reference bis(methylthio)methane standard solution in

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hexane, and compared with the results obtained after SPME. After proper optimization of the

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extraction conditions, the δ13C values obtained from triplicate GC analyses of the reference sample

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after HS-SPME were practically superimposable with those obtained on the same sample directly

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injected into GC, with only a slight variation in precision (namely, 0.1σ for the reference standard

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solution directly injected into GC, and 0.2σ for the reference standard solution after SPME). With

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the aim to find the most suitable stationary phase for the complete separation of

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bis(methylthio)methane, which is mandatory to avoid isotopic ratio errors. The two most

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widespread phases for the analysis of the volatile fraction were investigated, namely apolar (A:

235

equivalent to poly(5% diphenyl/95% dimethylsiloxane)) and mid polar (B: polyethylenglycole), in

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order to highlight possible criticisms in terms of coelution of the target compound with other

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sample components. Figure 1 reports as an example, coelutions occurring on both columns for two

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different

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bis(methylthio)methane: on the 5% column (A) bis(methylthio)methane, with a linear retention

samples.

The

chromatograms

showed

a

certain


degree

of

coelutions

of


240

index (LRI) value of 893, in fact coeluted with heptan-2-one; the latter peak was assigned by its EI

241

spectrum (96% similarity) and LRI value of 898. On the wax phase (B), bis(methylthio)methane

242

peak (LRI 634) coeluted with hexan-2-ol (LRI 630); however for the latter peak identification was

243

only tentative, for two main reasons. First, LRI values on such stationary phase are not reproducible

244

as they are on the apolar column, and furthermore hexan-2-ol and its (methyl) substituted isomers

245

show very similar EI fragmentation patterns, preventing their unambiguous identification.

246

Furthermore, the amount of bis(methylthio)methane decreases proportionally with the storage

247

period, probably related to its very high volatility, therefore coelutions are more likely to occur, e.g.

248

on a wax stationary phase, with samples that are just harvested (Figure 2A); on the other hand the

249

separation from neighbour peaks would be easier when the concentration of bis(methylthio)methane

250

in sample is reduced after a certain storage period (Figure 2B). It is evident from Figures 1 and 2

251

that in monodimensional GC analysis, different issues can greatly affect the separation degree,

252

possibly leading to a partial coelution of the bis(methylthio)methane peak, for which evaluation of

253

the isotopic ratio value is likely to be undermined due to isotopic column fractionation along the

254

chromatographic band [21]. It must be emphasised that all the organic matter is combusted to CO2

255

before reaching the IRMS detector, and spectroscopic resolution of the CO2 obtained from a

256

chromatographic coelution is not feasible, unlike what is practical in MS, both during data

257

monitoring (i.e. in selected ion monitoring or multiple reaction monitoring mode) and in post-run

258

data processing (i.e. extracted ion mode). That being said, the complete resolution of a peak and its

259

conservation avoiding extra-column band broadening due to dead volumes along the IRMS path is

260

crucial for getting accurate results. Considering the increased complexity of commercial samples in

261

which truffle aroma is mixed with matrix components, an MDGC was deemed as necessary to solve

262

coelutions before the IRMS detection. The use of two-dimensional GC in heart-cut mode coupled to

263

IRMS is not a novel concept [21], however the technology exploited in this research presents a

264

serious step forward in its applicability. Recently, 2D systems based on a microfluidic chip have

265

been coupled to IRMS [23,25,28,29]. These systems have a common drawback, related to the


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266

nature of the transfer system employed, such that each cut causes a slight change in the

267

backpressure of the first dimension (1D), in this way changing the relative retention times of the

268

next eluting components. On the contrary, the DS used in the present work was not affected by this

269

drawback: the main feature of such a transfer device is in fact the capability to generate the same 1D

270

backpressure in both stand-by and cut conditions, allowing the transfer of substantially unlimited

271

heart-cut fractions. Furthermore, the risk of isotopic fractionation resulting from the not quantitative

272

transfer of peaks due to retention time shifts during consecutive heart-cuts is completely avoided

273

(See Figure S-2 in Supporting Information for more details). From the IRMS side, the interface

274

(furnace) with the MDGC system has been optimized in terms of dead volumes, to accommodate

275

the demand that a high-efficiency separation, namely a two-dimensional analysis, is realized.

276

Another implementation of the system consists in the absence of the valve used to vent the solvent

277

between GC and IRMS. In fact, in a standard GC-C-IRMS configuration, a heart-cut valve provides

278

a way of removing the solvent peak from the system before it enters the micro-combustion oven;

279

this in order to prevent exhausting the copper oxide catalyst, and provide a long-living furnace

280

setup. Since in an MD system only the fractions of interest are transferred to a 2D column, and

281

hence to the C-IRMS, such a device useless, and therefore it was removed, to limit post-column

282

peak broadening. An additional advantage of the MDGC-C-IRMS coupling is related to a longer

283

duration of the copper oxide catalyst, since only a limited part of the sample is transferred to the 2D

284

column and then combustion involves a reduced amount of organic matter. The two GC columns

285

were selected in order to guarantee the highest possible purification of the band prior to the IRMS

286

analysis. In addition, special attention was paid in selecting the column to be used in 2D,

287

considering the foreseeable influence of column bleeding on the δ13C measurement. From the GC-

288

MS standpoint, it is well known that a reduced bleeding will positively affect identification,

289

resulting in a better spectral matching against a database of reference spectra; on the other hand,

290

further investigation is needed, concerning GC-C-IRMS analysis, since the influence of column

291

bleeding on δ13C measurement needs to be evaluated. As well known, the amount of stationary



292

phase released from the column depends on the column phase, and furthermore will be also affected

293

by the operating temperature. Being most of the GC applications accomplished in the temperature

294

program mode, the bleeding effect is most likely to affect the δ13C measurement of those

295

components eluted at high oven temperatures, while it may be negligible for early-eluting

296

components, with higher volatility. Two different mid-polarity stationary phases were evaluated,

297

and namely a polyethylene glycol column (PEG) and an ionic liquid-based SLB-IL59, which are

298

characterized by similar polarity but exhibit different selectivity [31-36]. At a glance, a substantial

299

advantage of the IL phase with respect to the PEG one is represented by the higher maximum

300

operational temperature (300 °C vs. 280 °C). Ragonese et al. [37] reported a comparison of the two

301

stationary phases, in terms of bleeding, revealing that at the column max temperature, the noise

302

level was over one order of magnitude higher for the PEG column (1.8 108 pA s), with respect to

303

the ionic liquid one (4.9 106 pA s). Aiming to assess the influence of the bleeding effect on the

304

accuracy and precision of the δ13C measurement, in this work some experiments were accomplished

305

on the two stationary phases, using a standard solution. Figure 3 shows a comparison of a vanillin

306

ex-lignin standard solution, previously determined by elemental analysis (EA) IRMS (-27.1‰),

307

analysed with the MDGC system in triplicate with an oven temperature ramp of 5 °C/min from 50

308

°C in both the dimensions, using a 5% column as 1D and: (A) the PEG or (B) the IL-based SLB-

309

IL59 column as 2D. As can be appreciated, the vanillin peak eluted approximately in the same zone,

310

even if the ionic liquid phase presented a slightly higher retention with respect to the PEG one (∆rt:

311

1.5 min). For the IL59i column at the elution temperature of vanillin (about 220 °C), the bleeding

312

was negligible, with an average δ13C value of -27.1‰, thus in accordance with the EA-IRMS

313

measured value and a good precision (0.2 σ). On the other hand, as predicted, a higher noise was

314

registered for the PEG phase at the elution temperature of vanillin (about 210 °C), which affected

315

the δ13C result both in terms of accuracy (-25,7‰) and precision (1.1σ). Based on these preliminary

316

investigations, the SLB-IL59 phase was selected as 2D column in the MDGC system, coupled to a


Page 12 of 27

Page 13 of 27 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


317

5% column (SLB-5ms) in 1D, useful for allowing the use of LRIs, to provide complementary

318

selectivity with the lowest bleeding interference [31-36].

319 320

4.2 Analysis of genuine white truffle

321

The results obtained from δ13C measurements (three replicates) of the 24 genuine white truffle

322

samples are reported in Table 1. The lowest value was obtained for sample 2 from Molise (-42.6‰),

323

while the less negative was obtained for sample 11 from Piedmont (-33.9‰). The data collected

324

were in accordance with those reported by Werning et al [38]. A good precision was in general

325

achieved employing the SPME fiber to extract the truffle flavour, with standard deviation (SD)

326

values always lower than 0.5‰, except for 6 samples, available in low amounts, for which higher

327

SD values were obtained.

328 329

Table 1. Genuine Tuber Magnatum white truffle samples analysed. The symbiotic plant,

330

geographical origin, and δ13C average values of three consecutive replicates together with the

331

measured standard deviation (SD) are reported. avg δ13C ‰ (n=3) SD

ID

Symbiotic plant

Origin

1

Unknown

Molise

-36.8

0.1

2

Unknown

Molise

-42.6

0.8

3

Unknown

Molise

-40.0

0.4

4

Unknown

Tuscany

-41.2

0.3

5

Populus alba

Tuscany

-37.6

0.2

6

Populus alba

Tuscany

-40.1

0.6

7

Populus alba

Tuscany

-38.5

0.2

8

Populus alba

Tuscany

-37.6

0.2

9

Populus alba

Tuscany

-41.9

0.6

10

Populus alba

Piedmont

-38.3

0.2

11

Populus alba

Piedmont

-33.9

0.1

12

Unknown

Piedmont

-35.4

0.3

13

Unknown

Piedmont

-37.7

0.1

14

Quercus Petraea

Piedmont

-37.9

0.2

15

Quercus Petraea

Piedmont

-39.0

0.2

16

Unknown

Abruzzo

-40.5

0.1

17

Unknown

Abruzzo

-42.0

0.2

18

Populus alba

Umbria

-38.7

0.1



19

Quercus cerris

Umbria

-40.7

0.3

20

Unknown

Umbria

-39.1

0.1

21

Quercus cerris

Umbria

-38.4

0.1

22 Juniperus communis

Umbria

-37.8

0.6

23

Unknown

Basilicata

-41.5

0.6

24

Unknown

Calabria

-38.5

0.7

Page 14 of 27

332 333

By evaluating these results in relation to the geographical origin of the samples, a slightly different

334

variability of the δ13C values can be evidenced within the different places, as reported in Figure 4.

335

The most numerous samples were harvested in Tuscany (6) and Piedmont (6), followed by Umbria

336

(5), Molise (3), Abruzzo (2), Basilicata (1) and Calabria (1). The samples from Molise showed the

337

wider range (around 6) with an SD (σ) of 2.9 and an average δ13C value of -39.8‰. Truffle

338

samples from Tuscany and Umbria showed similar ranges: the 6 samples from Tuscany showed a

339

range of around 4, with an average δ13C value of -39.5‰ and 1.8σ, while the 5 samples from

340

Umbria showed a range of around 3, with an average δ13C value of -38.9‰ and 1.1σ. The two

341

samples from Abruzzo showed an average value of -41.2‰ with 1.1σ, while samples from

342

Piedmont showed the less negative range, with a range of around 5, an average value of -37.0‰

343

and 2.0σ. The other 2 samples were from Basilicata (-41.5‰) and Calabria (-38.5‰), respectively.

344

It has to be underlined that such evaluation cannot be regarded as a statistical reference, since a

345

limited number of samples were available; on the other hand, given the difficulty and costs related

346

to the collection of a large number of samples for a deeper statistical evaluation, the present data

347

represent the widest study in the field of δ13C measurement of bis(methylthio)methane, together

348

with a recent published data on the same topic [38].

349 350

4.3 Analysis of commercial white truffle samples

351

Before

352

bis(methylthio)methane were analysed. Two of them were of petrochemical synthetic origin,

353

characterized by very low δ13C values (-56.4‰, 0.1σ and -77.1‰, 0.3σ). The third standard

the

analysis

of

the

commercial

white

truffle


samples,

three

standards

of

Page 15 of 27 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


354

presented the most positive value, viz. -28.5‰, 0.1σ, being a natural flavouring substance obtained

355

by physical, enzymatic or microbiological processes from material of vegetable, animal or

356

microbiological origin [39]. Subsequently, fourteen samples purchased from local stores (Messina)

357

were analysed using the same MDGC approach (Table 2).

358 359

Table 2. Description of the commercial truffle samples analysed. The average values of five

360

consecutive replicates together with the measured standard deviation (SD) are reported.

361

Abbreviation: n.d. = not detected ID

Description

Flavour

Avg δ13C ‰ (n=5)

SD

A

Olive oil 1

White truffle

-39.6

0.2

B

Olive oil 2

White truffle

-37.4

0.3

C

Olive oil 3

Black truffle, aroma

-40.3

0.1

D Fresh cheese 1

Summer truffle, aroma

-57.6

0.1

E Fresh Cheese 2

Black truffle, aroma Black and summer truffle, aroma White truffle

-66.5

0.8

-53.9

0.2

-42.1

0.3

F Fresh Cheese 3 G

Sauce 1

H

Sauce 2

I

Sauce 3

L

Cream Dehydrated truffle Honey

M N O P

Pasta Summer truffle

Summer truffle, aroma Black and summer truffle, aroma White truffle, aroma

-43.7

0.2

-63.7

0.1

-55.0

0.1

White truffle

-38.0

0.1

White truffle

-39.1

0.5

White truffle

-36.7

0.8

-

n.d.

-

362 363

Eight products, namely three olive oils (A-C), two sauces (G,H), one dehydrated truffle (M), one

364

honey (N) and one pasta (O) presented δ13C values compatible with the natural white truffle range

365

measured. Within these samples some of them, i.e. olive oil 1 (A: -39.6‰, 0.2σ) and olive oil 2 (B:

366

-37.4‰, 0.3σ), were characterized by the presence of the so called “witness” of declared white

367

truffle, arising from a small piece of genuine truffle added to attract the customer and to

368

demonstrate the originality of the product. Dehydrated truffle was declared as white truffle from



369

Alba: also in this case, the results obtained from δ13C measurement (M: -38.0‰, 0.1σ) were

370

compatible with the presence of an original white truffle, especially those within the range typical

371

of the samples from Piedmont region (-33.9‰/-39.0‰). Sauce 1 (G: -42.1‰, 0.3σ), honey (N: -

372

39.1‰, 0.5σ) and pasta (O: -36.7‰, 0.8σ) samples were declared to be flavoured with white truffle:

373

the analysis confirmed what labelled on the products. A different situation was found for olive oil 3

374

(C: -40.3‰, 0.1σ) and sauce 2 (H: -43.7‰, 0.2σ). Both samples were declared to be added with

375

black and summer truffle, respectively: however, both these species are characterized by the

376

absence of bis(methylthio)methane [9], as hereby confirmed by the analysis of the summer truffle

377

sample (P) that did not contain any bis(methylthio)methane. Addiction of an aroma was declared

378

for samples C and H and, most probably, it was a mixture of a synthetic and a biotechnological

379

molecule, combined to obtain a δ13C value compatible with the natural white truffle flavour. The

380

situation would be similar for the remaining samples, where no white truffle was declared among

381

the ingredients. In detail, δ13C values ranging from -53‰ to -57‰, were registered for fresh cheese

382

1 (D: -57.67‰, 0.1σ), fresh cheese 3 (F: -53.9‰, 0.2σ) and cream (L: -55.0‰, 0.1σ), all of them

383

declared as flavoured with aroma and summer truffle (sample D), aroma, black and summer truffles

384

(sample F), and aroma and white truffle (sample L). Regarding sample L, being the isotopic ratio

385

outside the range of natural bis(methylthio)methane, it is the authors’ opinion that, a low quality

386

(probably not fresh) white truffle was used, fortified with a synthetic flavour (consistently with a

387

reduced residue amount of bis(methylthio)methane). In this concern, in order to verify the influence

388

of the sample aging on the δ13C value, sample 15 was re-analysed after 2 months of storage. As

389

expected, the amount of bis(methylthio)methane was reduced to about 25% (n=3), but no

390

considerable variations of the δ13C value was observed (-40.5‰ vs. -40.7‰), as reported in Figure

391

5. Finally, values greatly outside the natural white truffle range were measured for fresh cheese 2

392

(E: -66.5‰, 0.8σ) and sauce 3 (I: -63.7‰, 0.1σ), clearly flavoured with a synthetic aroma having a

393

more negative δ13C value.


Page 16 of 27

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394 395

5. Conclusions

396

The present work demonstrates the capability of an MDGC-C-IRMS system to overcome some of

397

the historical problems of IRMS, associated with the combustion and measurement of impure

398

peaks. Extra-column band broadening has been greatly reduced thanks to the optimization of the

399

micro-combustion furnace and to the elimination of the heart-split valve, not necessary in a

400

multidimensional configuration. The high-efficiency 2D system has been applied for evaluation of

401

the key aroma compound of white truffle, namely bis(methylthio)methane, resolving the coelution

402

occurring with other components present in the different commercial products investigated.

403 404 405 406 407 408

Supporting information.

409

Figure S-1: scheme of the instrumentation is shown.

410

Figure S-2(A-B): operation of the transfer device employed for multi heart-cut.

411

Detailed description of the transfer device employed

412

Explanation of the operation in “stand-by” and “cut” position of the Deans switch.

413



414

References

415

[1] Splivallo, R.; Ottonello, S.; Mello, A.; Karlovsky, P. New Phytol. 2011, 189, 688-699.

416

[2] Pacioni, G.; Cerretani, L.; Procida, G.; Cichelli, A. Food Chem. 2014, 146, 30-35.

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[3] Costa, R.; Fanali, C.; Pennazza, G.; Tedone, L.; Dugo, L.; Santonico, M.; Sciarrone, D.;

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Cacciola, F.; Cucchiarini, L.; Dachà, M.; Mondello, L. LWT-Food Sci. Technol. 2015, 60, 905-913.

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[4] Wang, S.; Marcone, M.F. Food Res. Int. 2011, 44, 2567-2581.

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[5] Fiecchi, A.; Kienle, M.G.; Scala, A.; Cabella, P. Tetrahedron Lett. 1967, 8, 1681-1682.

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[6] Gioacchini, A.M.; Menotta, M.; Guescini, M.; Saltarelli, R.; Ceccaroli, P.; Amicucci, A.;

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Barbieri, E.; Giomaro, G.; Stocchi, V. Rapid Commun. Mass Spectrom. 2008, 22, 3147-3153.

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[7] Mauriello, G.; Marino, R.; D'Auria, M.; Cerone, G.; Rana, G.L. J. Chromatogr. Sci. 2004, 42,

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299-305.

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[8] Pelusio, F.; Nilsson, T.; Montanarella, L.; Tilio, R.; Larsen, B.; Facchetti, S.; Madsen, J.Ø. J.

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Agric. Food Chem. 1995, 43, 2138-2143.

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[9] Culleré, L.; Ferreira, V.; Chevret, B.; Venturini, M.E.; Sánchez-Gimeno, A.C.; Blanco, D. Food

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Chem. 2010, 122, 300–306.

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[10] Torregiani, E.; Lorier, S.; Sagratini, G.; Maggi, F.; Vittori, S.; Caprioli, G. Food Anal. Methods

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[11] Pennazza, G.; Fanali, C.; Santonico, M.; Dugo, L.; Cucchiarini, L.; Dachà, M.; D’Amico, A.;

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Costa, R.; Dugo, P.; Mondello, L. Food Chem. 2013, 136, 668-674.

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[12] Evaluations of the Joint FAO/WHO Expert Committee on Food Additives (JECFA)

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http://apps.who.int/food-additives-contaminants-jecfa-database/chemical.aspx?chemID=2952

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[13] Biniecka, M.; Caroli, S. TRAC-Trend. Anal. Chem. 2011, 30, 1756-1770.

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[14] Costa, R.; Tedone, L.; De Grazia, S.; Dugo, P.; Mondello, L. Anal. Chim. Acta 2013, 770, 1-6.

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[15] Bellesia, F.; Pinetti, A.; Bianchi, A.; Tirillini, B. Flavour Frag. J. 1996, 11, 239–243.

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[16] Piloni, M.; Tat, L.; Tonizzo, A.; Battistutta, F. Ital. J Food Sci. 2005, 17(4), 463-468.


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[18] Muccio, Z.; Jackson, G.P. Analyst 2009, 134, 213-222.

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[19] Ricci, M.P.; Merritt, D.A.; Freeman, K.H.; Hayes, J.M. Org. Geochem. 1994, 21, 561–571.

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[20] Tranchida, P.Q.; Sciarrone, D.; Dugo, P.; Mondello, L. Anal. Chim. Acta 2012, 716, 66– 75.

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[21] Juchelka, D.; Beck, T.; Hener, U.; Dettmar, F.; Mosandl, A. J. High Resolut. Chromatogr.

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1998, 21, 145–151.

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[22] Sewenig, S.; Bullinger, D.; Hener, U.; Mosandl, A. J. Agric. Food Chem. 2005, 53, 838–844.

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[23] Brailsford, A.D.; Gavrilovìc, I.; Ansell, R.J.; Cowan, D.A.; Kicman, A.T. Drug Test Anal.

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2012, 4, 962–969.

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[24] Casilli, A.; Piper, T.; Azamor de Oliveira, F.; Costa Padilha, M.; Pereira, H.M.; Thevis, M.; de

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Aquino Neto, F.R. Drug Test Anal. 2016, 8, 1204–1211.

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[25] Brailsford, A.D.; Gavrilovìc, I.; Ansell, R.J.; Cowan, D.A.; Kicman, A.T. Drug Test Anal.

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[26] Dumont, E.; Tienpont, B.; Higashi, N.; Mitsui, K.; Ochiai, N.; Kanda, H.; David, F.; Sandra, P.

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[30] Skrzypek, G.; Sadler, R. Rapid Commun. Mass Spectrom. 2011, 25, 1625–1630.

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[31] Ragonese, C.; Sciarrone, D.; Grasso, E.; Dugo, P.; Mondello, L. J. Sep. Sci. 2016, 39, 537-544.

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[35] Pantò, S.; Sciarrone, D.; Maimone, M.; Ragonese, C.; Giofrè, S.; Donato, P.; Farnetti, S.;

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Mondello, L. J. Chromatogr. A 2015, 1417, 96–103.

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[36] Sciarrone, D.; Pantò, S.; Donato, P.; Mondello, L. J. Chromatogr. A 2016, 1475, 80-85.

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2011, 83, 7947-7954.

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[38] Wernig, F.; Buegger, F.; Pritsch, K.; Splivallo, R. Food Control 2018, 87, 9-16.

473

[39] Regulation (EC) no 1334/2008 of the european parliament and of the council of 16 December

474

2008 on flavourings and certain food ingredients with flavouring properties for use in and on foods

475

and amending Council Regulation (EEC) No 1601/91, Regulations (EC) No 2232/96 and (EC) No

476

110/2008 and Directive 2000/13/EC.


Page 20 of 27

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477

Figure legends

478

Figure 1. Coelutions of bis(methylthio)methane after monodimensional GC analysis on apolar (A)

479

and mid polar (B) stationary phase.

480

Figure 2. Comparison of the bis(methylthio)methane peak obtained on a wax column after analysis

481

of a white truffle sample: just harvested (A) and after two months storage (B).

482

Figure 3. Analysis of standard vanillin ex-lignin for bleeding evaluation on the polyethylene glycol

483

(A) and ionic liquid-based SLB-IL59 (B) stationary phase.

484

Figure 4. δ13C ranges on the basis of the geographical origin for the genuine white truffle samples.

485

Figure 5. D2 IRMS chromatograms of bis(methylthio)methane in: just harvested A) and two-month

486

aged truffle sample B).

487



488 489

For TOC only

490


Page 22 of 27

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Figure 1. Coelutions of bis(methylthio)methane after monodimensional GC analysis on apolar (A) and mid polar (B) stationary phase. 83x85mm (300 x 300 DPI)

ACS Paragon Plus Environment


Figure 2. Comparison of the bis(methylthio)methane peak obtained on a wax column after analysis of a white truffle sample: just harvested (A) and after two months storage (B). 83x48mm (300 x 300 DPI)


Page 24 of 27

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Figure 3. Analysis of standard vanillin ex-lignin for bleeding evaluation on the polyethylene glycol (A) and ionic liquid-based SLB-IL59i (B). 83x116mm (300 x 300 DPI)



Figure 4. δ13C ranges on the basis of the geographical origin for the genuine white truffle samples. 83x86mm (300 x 300 DPI)


Page 26 of 27

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Figure 5. D2 IRMS chromatograms of bis(methylthio)methane in: just harvested A) and two- month aged truffle sample B). 83x38mm (300 x 300 DPI)


Multidimensional Gas Chromatography Coupled to Combustion

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