aboca sansepolcro (ar) june 19...7 scientific program wednesday, june 19th, 2019 14.00-14.30...
TRANSCRIPT
Aboca – Sansepolcro (Ar)
June 19th – 21st, 2019
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Organised by
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COMMITTEES
Scientific Committee
Gianluca Bartolucci Università degli Studi di Firenze
Cecilia Bergamini ARPAE, Bologna
Giuliana Bianco Università degli Studi di della Basilicata, Potenza
Donatella Caruso Università degli Studi di Milano
Roberta Galarini Ist. Zooprofil. Sper. Umbria, Marche, Perugia
Gianluca Giorgi Università degli Studi di Siena
Emiliano Giovagnoni Aboca SpA Società Agricola, Sansepolcro (Ar)
Emanuela Gregori Istituto Superiore di Sanità, Roma
Fulvio Magni Università degli Studi di Milano Bicocca
Luisa Mattoli Aboca SpA Società Agricola, Sansepolcro (Ar)
Valentino Mercati Aboca SpA Società Agricola, Sansepolcro (Ar)
Giorgio Mellerio Università degli Studi di Pavia
Michele Suman Barilla, Parma
Organizing Committee
Luisa Mattoli (Chairperson) Aboca SpA Società Agricola, Sansepolcro (Ar)
Marta Marchesi Università degli Studi di Milano
Chiara Mercati Aboca SpA Scietà Agricola, Sansepolcro (Ar)
Barbara Trenti Aboca SpA Società Agricola, Sansepolcro (Ar)
Conference Secretariat
External Relations Office (Aboca Spa, Sansepolcro)
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The 3rd MS-NatMedDay MASSA2019 conference is kindly
supported and sponsored by:
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SCIENTIFIC PROGRAM
Wednesday, June 19th
, 2019
14.00-14.30 Registration
14.30-15.00
Welcome address
- Valentino Mercati, Aboca SpA founder and president
- Donatella Caruso, DSM president
- Luisa Mattoli, Aboca SpA, Organizing commettee chair
Session 1: MS in biomedical and clinical studies
Chairperson: Luisa Mattoli, Aboca SpA, Sansepolcro
15.00-15.40 PL1 Strategic approaches to improve bioavailability of dietary polyphenols
M. Dell'Agli, E. Sangiovanni, M. Fumagalli, S. Piazza, F. Giavarini, D. Caruso
University of Milano, Milano, Italy
15.40-16.00 OC1
Characterization of bioactive secoiridoids of olive leaves and their enzymatic
hydrolysis by-products by liquid chromatography-electrospray ionization-Fourier
transform mass spectrometry
R. Abbattista, I. Losito, C. De Ceglie, C.D. Calvano, F. Palmisano, T.R.I. Cataldi,
University of Bari “Aldo Moro”, Bari, Italy
16.00-16.20 OC2
Effects of non-banned and non-monitored substances on the parameters of the
urinary steroid profile
M. Iannone, X. de la Torre, V. Di Murro, F. Botrè
Antidoping Laboratory -Italian Medical Sports Federation, Rome, Italy
16.20-16.50 Shotgun Poster presentations: P1, P2, P3, P4, P5
16.50-17.10 OC3
Use of tailored metabolite profiling and identification for drug discovery and
development
A. Vecchi, S. Esposito, A. Lembo, L. Orsatti, E. Monteagudo
IRBM SP, Via Pontina Km 30,600, Pomezia, Italy
17.10 End of section and transfer to Sansepolcro
17.30-19.00 Guided tour of Sansepolcro Museum with paintings by Piero della Francesca and many others
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Thursday, June 20th
, 2019
Session 2: Proteomics, metabolomics, lipidomics and other omics sciences
Chairperson: Giorgio Mellerio, University of Pavia
9.30-10.10 PL2 Using metabolic fingerprints to rationally design combination therapies
A. Campos, M. Zampieri
Institute of Molecular Systems Biology, ETH Zurich, Switzerland
10.10-10.30 OC4
Target metabolomic profiling of natural freeze-dried extracts: the case of
hydrolysable tannins
E. Flamini, M. Burico, G. Fodaroni, G. Proietti, S. Bedont, S. Tamimi, Claudio M.
Quintiero, M. Gianni, L. Mattoli
Department of Phytochemistry Research, Aboca S.p.a. Società Agricola, Sansepolcro,
Italy
10.30-10.50 OC5
Qualitative and quantitative profile of Vaccinium macrocarpon urine metabolites by
HR-MS and evaluation of the urine ex-vivo effect on Candida albicans adhesion and
biofilm formation
G. Baron, A. Altomare, L. Regazzoni, E. Borghi, F. Borgo, E. Ottaviano, P. Allegrini, P.
Morazzoni, A. Riva, L. Arnoldi, M. Carini, G. Aldini
Department of Pharmaceutical Sciences, University of Milano, Milan, Italy
10.50-11.10 OC6
Untargeted profiling of large intestine microbial transformation products of phenolic
compounds in pigmented flours
G. Rocchetti, M. Trevisan, L. Lucini
Department for Sustainable Food Process, Università Cattolica del Sacro Cuore, Piacenza,
Italy
11.10-11.40 Coffee Break
11.40-12.00 Shotgun Poster presentations: P6, P8, P9, P10
12.00-13.30 Meeting of DSM members
13.30-14.30 Lunch at Aboca’s canteen
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Session 3: Omics sciences and biological fluids
Chairperson: Gianluca Giorgi, Siena University
14.30-15.10 PL3
Protein Mass Spectrometry: from complexes to biotherapeutics to biomarkers
J. A. Loo
Department of Chemistry and Biochemistry, Department of Biological Chemistry, David
Geffen School of Medicine, UCLA/DOE Institute for Genomics and Proteomics,
University of California-Los Angeles, Los Angeles, CA, USA
15.10-15.30 OC7
The evolving landscape of diagnostic pathology: a step towards the integration of
MALDI-MSI for the routine diagnosis of thyroid fine needle aspiration biopsies
I. Piga, G. Capitoli, V. Denti, S. Guarnerio, A. Mahajneh, A. Smith, C. Chinello, S.
Galimberti, F. Magni, F. Pagni
Department of Medicine and Surgery, University of Milano Bicocca, Milan, Italy
15.30-15.50 OC8
In-vitro and in-cell cross-linking/mass spectrometry: from 3D-protein structure
investigations to proteome-wide interactome studies
C. Iacobucci, M. Götze, A. Sinz
Department of Pharmaceutical Chemistry and Bioanalytics, Martin-Luther-University
(MLU), Halle-Wittenberg , Germany
15.50-16.10 OC9
Urinary GC-MS untargeted metabolomics for prostate cancer diagnosis.
Preliminary results
E. Amante, A. Biancolillo, R. Bro, F. Porpiglia, A. Salomone, M. Vincenti
Department of Chemistry, University of Turin, Turin, Italy
16.10-16.30 OC10
Tandem mass spectrometry with ion trap in the isomers resolution
M. Menicatti, L. Moracci, L. Braconi, S. Dei, E. Teodori, G. Bartolucci
NEUROFARBA, Department of Neuroscience, Psychology, Drug Area and Child Health
Section of Pharmaceutical and Nutraceutical Sciences, University of Florence, Florence
Italy
16.30-17.00 Coffee Break
17.00-17.10 Poster shotgun presentations: P7, P11
17.10-17.30 OC11 A multiomics approach using metabolomics and lipidomics
C. Ghilardi
Shimadzu Italia srl
17.30-17.50 OC12
Molecular fingerprinting of Pistacia lentiscus hydrosol by ultra-high resolution ESI-
FT-ICR mass spectrometry
A. Onzo, G. Bianco, G. Martelli, R. Pascale, P. Iannece, C. Pifano, C. Gaeta
Department of Sciences, University of Basilicata, Potenza, Italy
17.50 End of section and transfer to Hotels
20.00 Social dinner
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Friday, June 21st, 2019
Special Session: DSM Youth award
Chairperson: Donatella Caruso, University of Milan, DSM president
9.30-9:50 OC13
Isotope evidence of Gravettian and Epigravettian mobility strategies across the Last
Glacial Maximum in Southern Italy
F. Lugli,
University of Modena and Reggio Emilia, Italy
Session 4: Hyphenated Techniques
Chairperson: Roberta Galarini, Ist. Zooprofil. Sper. Umbria, Marche, Perugia
9.50-10.30 PL4 Predictive science and mass spectrometry
G. J. Langley
Chemistry, FNES - University of Southampton, UK
10.30-10.50 OC14
Combination of Pressurized Liquid Extraction with dispersive Liquid Liquid Micro
Extraction for the determination of sixty drugs of abuse in hair
F. Vincenti, C. Montesano, L. Cellucci, A. Gregori, F. Fanti, D. Compagnone, R. Curini,
M. Sergi
“La Sapienza” University of Rome, Department of Chemistry, Rome, Italy
10.50-11.20 Coffee break
11.20-11.40 OC15
Development of Solvent-Assisted Paper Spray Ionization mass spectrometry setup for
the analysis of biomolecules and biofluids
N. Riboni, A. Quaranta, H.V. Motwani, N. Österlund, A. Gräslund, M. Careri, F. Bianchi,
L.L. Ilag
Department of Chemical Sciences, of Life and Environmental Sustainability, University of
Parma, Parma, Italy
11.40-12.00 OC16
Processing effects on the content of bioactive secoiridoids in extra-virgin olive oil
examined by liquid chromatography-electrospray ionization-Fourier transform mass
spectrometry
C. De Ceglie, R. Abbattista, I. Losito, A. Castellaneta, C.D. Calvano, F. Palmisano, T.R.I.
Cataldi,
Department of Chemistry, University of Bari “Aldo Moro”, Bari, Italy
12.00-12.20 OC17
Disclosing the key biological target of Crellastatin A through a combination of
proteomic approaches
E. Morretta, M. Di Mauro, C. Festa, R. Riccio, A, Casapullo, M. C. Monti
Department of Pharmacy, University of Salerno, Salerno, Italy
12.20-12.40 Closing remarks
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SUMMARY
PLENARY AND ORAL COMMUNICATIONS ........................................................................................ 13
PL1 Strategic approaches to improve bioavailability of dietary polyphenols .......................................... 15
OC1 Characterization of bioactive secoiridoids of olive leaves and their enzymatic hydrolysis by-products
by liquid chromatography-electrospray ionization-Fourier transform mass spectrometry .......................... 16
OC2 Effects of non-banned and non-monitored substances on the parameters of the urinary steroid profile
..................................................................................................................................................................... 18
OC3 Use of tailored metabolite profiling and identification for drug discovery and development ............ 20
PL2 Using metabolic fingerprints to rationally design combination therapies ........................................ 21
OC4 Target metabolomic profiling of natural freeze-dried extracts: the case of hydrolysable tannins ...... 22
OC5 Qualitative and quantitative profile of Vaccinium macrocarpon urine metabolites by HR-MS and
evaluation of the urine ex-vivo effect on Candida albicans adhesion and biofilm formation ..................... 24
OC6 Untargeted profiling of large intestine microbial transformation products of phenolic compounds in
pigmented flours .......................................................................................................................................... 26
PL3 Protein Mass Spectrometry: from complexes to biotherapeutics to biomarkers ............................ 27
OC7 The evolving landscape of diagnostic pathology: a step towards the integration of MALDI-MSI for
the routine diagnosis of thyroid fine needle aspiration biopsies .................................................................. 28
OC8 In-Vitro and In-Cell Cross-Linking/Mass Spectrometry: from 3D-Protein Structure Investigations to
Proteome-Wide Interactome Studies ........................................................................................................... 29
OC9 Urinary GC-MS untargeted metabolomics for prostate cancer diagnosis. Preliminary results. ......... 30
OC10 Tandem mass spectrometry with ion trap in the isomers resolution ................................................. 31
OC11 A multiomics approach using metabolomics and lipidomics............................................................ 33
OC12 Molecular fingerprinting of Pistacia lentiscus hydrosol by ultra-high resolution ESI-FT-ICR mass
spectrometry ................................................................................................................................................ 34
OC13 Isotope evidence of Gravettian and Epigravettian mobility strategies across the Last Glacial
Maximum in Southern Italy ......................................................................................................................... 35
PL4 Predictive science and mass spectrometry .......................................................................................... 36
OC14 Combination of Pressurized Liquid Extraction with dispersive Liquid Liquid Micro Extraction for
the determination of sixty drugs of abuse in hair ........................................................................................ 37
OC15 Development of Solvent-Assisted Paper Spray Ionization Mass Spectrometry Setup for the analysis
of biomolecules and biofluids ..................................................................................................................... 38
OC16 Processing effects on the content of bioactive secoiridoids in extra-virgin olive oil examined by
liquid chromatography-electrospray ionization-Fourier transform mass spectrometry............................... 40
OC17 Disclosing the key biological target of Crellastatin A through a combination of proteomic
approaches ................................................................................................................................................... 42
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POSTER COMMUNICATIONS ................................................................................................................. 43
P1 Multiclass screening of banned substances in urine and liver by LC-MS/MS ....................................... 45
P2 Computational approaches for identification and quantification of phytochemicals in different complex
matrices ....................................................................................................................................................... 47
P3 Fast determination of bioactive proresolving lipids in plasma by μSPE-UHPLC-MS/MS ................... 48
P4 UHPLC-HRMS/MS-based rapid identification of bioactive molecules for functional recovery of Greco
grape leaves ................................................................................................................................................. 49
P5 Natural steroidal precursors: a critical issue in anti-doping IRMS analysis. The prednisolone and
prednisone case study .................................................................................................................................. 50
P6 Targeted and non-targeted metabolomics to study developmental neurotoxicity of biocides ............... 52
P7 PASEF for ultra-sensitive shotgun proteomics ...................................................................................... 53
P8 Chemical composition and antioxidant activity of Cannabis sativa L. 'Futura 75' essential oil: effect of
the distillation time ...................................................................................................................................... 54
P9 Allergens in food: how to quantify? ....................................................................................................... 55
P10 Metabolomic approaches to investigate the role of the mitochondrial regulator Zc3h10 in adipocytes
..................................................................................................................................................................... 57
P11 Evaluation of onion skin polyphenols for the production of the biofunctional textiles: a multi-
analytical approach ...................................................................................................................................... 58
Author Index ................................................................................................................................................... 59
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PLENARY AND
ORAL COMMUNICATIONS
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15
PL1
Strategic approaches to improve bioavailability of dietary polyphenols
Mario Dell’Agli, Enrico Sangiovanni, Marco Fumagalli, Stefano Piazza, Flavio Giavarini,
Donatella Caruso
Department of Pharmacological and Biomolecular Sciences, Università degli Studi di Milano
Via Balzaretti 9, 20133 Milan, Italy
Keywords: Polyphenols, bioavailability, glycoprotein-P
The human intestinal epithelium, which is mostly composed by enterocytes, represents an extremely efficient
barrier able to modulate penetration of pathogens and to influence absorption of xenobiotics, including
dietary polyphenols. Intestinal membrane shows a variety of transporters, namely efflux transporters, which
recognize xenobiotics and mediate passage from enterocytes to intestinal lumen. Glicoprotein-P (P-gp),
Breast Cancer Resistance Protein (BCRP), and multidrug resistance-associated protein 2 (MRP2) are well-
known efflux transporters. P-gp is an ATP-dependent transporter protein which is involved in the modulation
of absorption of drugs and bioactive compounds; moreover, P-gp is involved in drug resistance in tumoral
cells. Several dietary compounds are inhibitors of P-gp [1], and recently have been used to improve intestinal
absorption of bioactive compounds. Few studies demonstrated that quercetin, a flavonoid occurring in
several botanicals such as marigold, chamomile and ginkgo, improve digoxin absorption. Other pure
compounds from natural origin, including curcumin and resveratrol, show low bioavailability. Berberine, an
alkaloid occurring in plants traditionally used in Ayurveda medicine (Berberis aristata, Berberis vulgaris) is
very low, and interaction with P-gp reduces by 90% its absorption, leading to reduced ipocolesterolemic
effect. Co-administration of berberine and flavolignans from Silybum marianum is able to improve
bioavailability of berberine showing in vivo a significant reduction of LDL cholesterol in patients with type-
2 diabetes [2].
In vitro study carried on by our group demonstrated that lignans occurring in flaxseed (Linum usitatissimum)
and their metabolites are able to improve intestinal absorption of berberine. Caco-2 cells were differentiated
to enterocytes in transwell plates and subjected to different concentrations of dietary polyphenols in the
presence of berberine. Apical and basolateral compartments were collected, extracted with nBuOH, and
analysed by LC-MS/MS (triple quadrupole API-4000, AB-SCIEX). Papaverine was used as reference
standard. Results showed that metabolites from flaxseed lignans improve berberine absorption up to 5-fold
with respect to berberine alone. LC-MS can be efficiently used to identify dietary compounds able to
improve bioavailability of bioactive compounds.
References
1. Abdallah, HM et al., Journal of Advanced Research, 2015. 6 (1): p. 45-62.
2. Di Pierro, F et al., Clinical Pharmacology, 2013. 5: p. 167-74.
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OC1
Characterization of bioactive secoiridoids of olive leaves and their enzymatic hydrolysis by-
products by liquid chromatography-electrospray ionization-Fourier transform mass
spectrometry
R. Abbattista1, I. Losito
1,2, C. De Ceglie
1, C.D. Calvano, F. Palmisano
1,2, T.R.I. Cataldi
1,2
1Department of Chemistry and
2SMART Inter-department Research Center, University of Bari
“Aldo Moro” Via E. Orabona 4, 70126 Bari, Italy
Keywords: bioactive secoiridoids, olive leaves, liquid chromatography-electrospray ionization-Fourier
transform mass spectrometry
Plant phenolic compounds are secondary metabolites mainly arising from the shikimate- phenylpropanoid-
flavonoid pathways, playing a relevant role in plant metabolome and representing one of the largest bioactive
metabolomic heritages present in nature. Indeed, 15-20% of a plant genome, which has been estimated to
contain from 20,000 to 60,000 genes, is usually designed specifically to encode enzymes for secondary
metabolism. Just to make a comparison, the entire genome of Drosophila melanogaster is made of ca.
13,000 genes [1]. Such a difference in genetic complexity is related to the different ways available to plants
and animals to protect themselves against predators, pests, diseases, and abiotic stress. Differently from
animals, that may escape from their predators using movement, plants are forced to synthesize an array of
secondary compounds and use them as chemical weapons [1]. Phenolic compounds are among this type of
metabolites, due to their relevant antibiotic, antinutritional or unpalatable properties. Moreover, they play an
important role for plant pigmentation, growth and reproduction as well [1]. From this point of view, olive
tree (Olea europaea) is not an exception; several bioactive phenolic compounds are distributed in the olive
drupe mesocarp, pulp and seed and in olive leaves and occur in several parts of the plant cells, either in
simple form or bound to sugar molecules. Among them, seco-biophenols, to which secoiridoids belong, are
both terpenic and hydroxy-aromatic secondary metabolites, arising from both shikimate-phenylpropanoid
and from mevalonic metabolism, representing the major constituents of defence mechanisms against olive-
trees infesting pathogens [2]. The most abundant secoiridoids of O. europea drupes and leaves are glycosidic
derivatives named oleuropein and ligstroside. Their involvement in the plant resistance to infestation implies
the β-glucosidase-catalyzed production of the corresponding aglycone isoforms, which act as harmful
glutaraldehyde-like structure phytoalexins, with strong protein denaturant ability: the protein-crosslinking
and lysine-decreasing activities induce the cell death of host pathogens [3]. After microbial/pathogen
infection, or when the leaf tissue is destroyed/damaged by herbivores or by an insect stinger, a significant
increment of the β-glucosidase activity occurs, resulting in content increase of oleuropein and ligstroside
aglycones, rapidly converted into highly reactive aldehydic compounds, accompanied by their enol-
aldheydic and dienolic counterparts, also considered as active forms [4]. In situ studies of the enzymatic
activity in olive fruit tissue after Bactrocera oleae punctures showed the strongest β-glucosidase activity
after 20 min at the infection site, with subsequent inactivation extending to all adjacent tissue in ca. 3 h. A
concurrent increase in the level of peroxidase activity was detected as well [5].
Starting from this background, the characterization of the main isoforms of oleuropein and ligstroside,
together with glycosidic iridoids, like loganine and secologanoside, present in olive leaves and of their
enzymatic by-products, has been recently undertaken in our laboratory using reversed-phase liquid
chromatography coupled to electrospray ionization Fourier-transform high resolution/accuracy mass
spectrometry (RPLC-ESI-FTMS). Firstly, the enzymatic hydrolysis was investigated in vitro by introducing
almond -glucosidase into an aqueous extract of olive leaves and incubating the mixture at 30 °C for up to
40 h. The resulting aglycone derivatives were examined by tandem MS and the presence of several isomers
was ascertained. In plot (A) of Figure 1 the eXtracted Ion Current (XIC) chromatogram of a sample extract
of olive tree leaves (Coratina cultivar), showing the most intense peak of oleuropein at 10.28 min, is
displayed. Upon -glucosidase addition, the same solution exhibited the occurrence of oleuropein aglycone
(OA) isoforms (see plot (B) in the same figure). In the present communication the formation of such OA
isomers, with molecular structures named as open and closed forms I/II (see insets of plot B), will be
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discussed. Notably, many of these OA isomers were identified also in olive oil extracts (see plot (C) in
Figure 1) since -glucosidases are typically present in olive drupes and released during their crushing in the
first stage of oil production.
Fig. 1. XIC chromatograms obtained by RPLC-ESI-FTMS of an aqueous extract of olive leaves (Coratina cultivar)
before (A) and after (B) enzymatic hydrolysis with β-glucosidase. The peak of oleuropein is the most intense in plot A
(10.28 min). In panel C the XIC chromatogram obtained for the extract of an olive oil, whereby OA isomers are
generated through the involvement of endogenous -glucosidase, is displayed. Structures inferred for oleuropein
aglycone isoforms are reported in insets of plot B.
Secondly, the same analytical approach was applied to assess the occurrence of secoiridoid aglycones and
their by-products, including elenolic acid and hydroxytyrosol, in leaves of olive trees affected by the Xylella
fastidiosa bacterium. The content increase of aglyconic isoforms in infested leaves, likely related to the
increased expression and action of endogenous -glucosidase as a response to bacterial infection, will be
discussed in the present communication.
References
1. V. Lattanzio, V.M.T. Lattanzio, A. Cardinali, Phytochemistry: Advances in Research, 2006, pp 23-67.
2. N. Uccella, Trends in Food Science & Technology 11 (2001), pp 315–327.
3. K. Konno, C. Hirayama, H. Yasui, M. Nakamura, Proc. Natl. Acad. Sci. USA, 96 (2009) pp. 9159–9164.
4. I. Kubo, A. Matsumoto, I. Takase, Journal of Chemical Ecology, 11 (1985) pp. 251-263.
5. A. Spadafora, S. Mazzuca, F. Chiappetta, A. Parise, E. Perri and A. M. Innocenti, Agricultural Sciences in China, 7
(2008) pp. 703-712.
6 8 10 12 14 16 18 20 22 24 26 28 30 32 340
50
100
0
50
100
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ve
Ab
un
dan
ce
10.28
13.4412.23
24.53
8.657.31 20.43 25.9611.476.64 17.4815.38 19.78 22.1913.499.44
NL: 9.74E7
m/z= 539.17500-539.17900
NL: 4.09E6
m/z= 377.12219-377.12619
Rela
tive
Ab
un
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Open Forms I Open Forms II
Closed Forms I
Closed Forms II
0
50
100 NL: 8.07E8
m/z= 377.12219-377.12619
Re
lati
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Ab
un
dan
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A
C
B
6 8 10 12 14 16 18 20 22 24 26 28 30 32 34
6 8 10 12 14 16 18 20 22 24 26 28 30 32 34
22.3220.70
24.81
21.6924.48
13.4917.7311.51 26.9211.85 19.43 26.1515.80
Time/ min
Time/ min
Time/ min
18
OC2
Effects of non-banned and non-monitored substances on the parameters of the urinary steroid
profile
Michele Iannone1, Xavier de la Torre
1, Veronica Di Murro
1, Francesco Botrè
1,2.
1 Laboratorio Antidoping, Federazione Medico Sportiva Italiana (FMSI)
Largo Giulio Onesti 1, Rome, Italy
2 Dipartimento di Medicina Sperimentale, “Sapienza” Università di Roma
Viale Regina Elena 324, Rome, Italy
Keywords: urinary steroid profile, Athlete Biological Passport (ABP), confounding factors.
The detection of doping by pseudo-endogenous steroids (endogenous steroids when administered
exogenously) by gas-chromatography coupled to mass spectrometry or tandem mass spectrometry (GC-MS
or GC-MS/MS) represents a complex analytical challenge for the World Anti-Doping Agency (WADA)
accredited laboratories, mostly for the need of discriminating between their endogenous and exogenous
origin. The analytical procedure is based on the longitudinal monitoring of six steroidal urinary markers
(testosterone (T), epitestosterone (E), androsterone (A), etiocholanolone (Etio), 5α-androstane-3α,17α-diol
(5αAdiol), 5β-androstane-3α,17α-diol (5βAdiol)) and their relative ratios (T/E, A/T, A/Etio,
5αAdiol/5βAdiol and 5αAdiol/E) by the application of a Bayesian adaptive model, that is able to predict the
maximum variability for each marker based on the previous data to outline atypical results [1-3]. Although
the introduction of the longitudinal steroid profile clearly improved the detection of pseudo-endogenous
steroid doping, it does not yet allow to gather any information on the occurrence of atypical profiles due to
the presence of endogenous (i. e. enzyme induction or inhibition, genetic polymorphisms) or exogenous (i. e.
banned drugs, masking agents, ethanol, bacterial contamination) confounding factors, that could influence
the urinary concentration of the described markers [4-5].
The aim of the present work is to verify whether the administration of specific substances, neither prohibited
nor considered in the WADA Technical Document TD2018EAAS [1] as possible confounding factors,
present in dietary supplements and/or other similar over the counter products, can interfere with the correct
evaluation of the markers of the steroidal module of the Athlete Biological Passport (ABP) [2], previously
described. Particularly, this work considers the potential confounding effects of two synthetic isoflavones,
methoxyisoflavone and ipriflavone, previously described as in vitro aromatase inhibitors [6], and of the
stilbenoid resveratrol and its biological precursor polydatin, known in vitro inhibitors of different CYP450
isoforms involved in androgen biosynthesis [7].
Five healthy caucasican volunteers were selected for the study that was carried out by the analysis of urinary
samples collected before, during and after the administration of (i): methoxyisoflavone (Methoxyisoflavone
– MyProtein), (ii): ipriflavone (Osteofix ® - Chiesi Farmaceutici), (iii): resveratrol (Resveratrol – Solgar)
and (iv): polydatin (Polidal – Ghimas). After enzymatic hydrolysis and liquid-liquid extraction, all urinary
samples were analyzed by gas-chromatography coupled to tandem mass spectrometry (GC-MS/MS),
according to the validated and routinely applied analytical procedure performed to determine the steroid
profile of each athlete [8].
Our results indicate that the administration of the selected substances cause an alteration of the urinary
concentrations of the investigated steroids and an increase in data dispersion, that makes more difficult the
interpretation of the longitudinal steroid profile based on the definition of individual excretion ranges for
each athlete. Our data are also consistent with previous evidence reported in the literature regarding the in
vitro effects of the selected substances [6-7], suggesting their monitoring in doping routine analysis.
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References
1. WADA Technical Document TD2018EAAS “Endogenous Anabolic Androgenic Steroids. Measurement and
Reporting”. (2018).
2. Athlete Biological Passport (ABP). Operating Guidelines (2018).
3. PE. Sottas, N. Baume, C. Saudan, C. Schweizer, M. Kamber, M. Saugy; Biostatistics, 8 (2007), pp 285-296.
4. U. Mareck, H. Geyer, G. Opfermann, M. Thevis, W. Schanzer; Journal of Mass Spectrometry, 43 (2008), pp 877-
891.
5. T. Kuuranne, M. Saugy, N. Baume; British Journal of Sport Medicine, 48 (2014), pp 848-855.
6. M. Iannone, F. Botrè, N. Cardillo, X. de la Torre; Drug Testing and Analysis, 11 (2019), pp 208-214.
7. V. Mohos, T. Bencsik, G. Boda, E. Fliszar-Nyul, B. Lemli, S. Kunsagi-Matè, M. Poor; Biomedicine &
Pharmacotherapy, 107 (2018), pp 777-784.
8. X. de la Torre, C. Colamonici, D. Curcio, D. Jardines, F. Molaioni, MK. Parr, F. Botrè; Drug Testing and Analysis,
6 (2014), pp 1133-1140.
20
OC3
Use of tailored metabolite profiling and identification for drug discovery and development
Andrea Vecchi, Simone Esposito, Angelo Lembo, Laura Orsatti, Edith Monteagudo
IRBM SP, Via Pontina Km 30,600, Pomezia, Italy
Keywords: Met ID, HRMS, radiometabolite profiling
Understanding the metabolic fate of a compound is crucial to support the development of a drug candidate at
each stage of a drug discovery program. Metabolic transformations are frequently associated with adverse
effects or unfavorable pharmacokinetic and pharmacodynamics profiles. Here we present a metabolite
profiling approach tailored to support the various steps of a drug discovery and development program. Three
levels of investigations based on LC-HRMS and software-assisted metabolite identification or LC-fraction
collection and micro-scintillation counting will be discussed:
1. A Tier I Met ID workflow, designed as a screening/targeted approach using incubations with liver
microsomes from rodents, non-human primates and human, to identify the main metabolic soft spots
and assess the interspecies differences of newly synthesized compounds. This approach provides the
medicinal chemists with information on potential metabolism-related liabilities at an early stage and
with a higher throughput than classical Met ID studies. The early identification of sites of
metabolism (SoM) and the structural elucidation of the corresponding metabolites can be decisive
for the design of compounds with appropriate metabolic stability.
2. A Tier II Met ID workflow, consisting in in vitro (hepatocyte and microsomal incubations) as well as
in vivo Met ID studies to identify metabolic routes, inter-species differences, in vitro/in vivo
correlation and relevance of possible troublesome metabolites. This approach should be used to
characterize selected leads showing good efficacy and favorable pharmacokinetic profile in the
appropriate animal models.
3. A Tier III Met ID workflow, consisting in the quantification of radio-metabolites in plasma and
tissues of interest in preclinical species. Metabolites radioprofiling is particularly useful during the
late Lead Optimization stage.
Case studies from each level of investigation will be presented in detail.
21
PL2
Using metabolic fingerprints to rationally design combination therapies
Adrian Campos, Mattia Zampieri
Institute of Molecular Systems Biology, ETH Zurich
Keywords: Metabolomics, Antibiotics, Drug Mode of Action
Despite rapid technological progress, the discovery of novel antibiotics has been stalled for the past 50 years.
To combat the growing burden of antibiotic resistance, innovative drug discovery paradigms are required to
improve and expedite the antibiotic discovery process. A crucial bottleneck in drug discovery is the
identification of compounds’ Mode of Action (MoA). To address this problem we developed a rapid and
systematic metabolome profiling strategy to classify the MoA of bioactive compounds. In contrast to existing
methods based on phenotypic drug profiling, mostly on the basis of growth assays, we exploit here the
intracellular response of about 1000 metabolites as a truly multiparametric readout of the cellular response.
The specific advance over existing metabolic platforms is a faster throughput of 1-2 orders of magnitude,
allowing our combined MS-based metabolomics and computational workflow to scale with the size of
typical compound libraries. I will present our recent results obtained from analyzing an open access set of
~200 novel anti-tuberculosis compounds with unknown MoAs and discuss how our metabolome-based
screening approach is directly applicable to extract multiple quantitative signatures indicative of functional
properties of MoAs in large compound libraries. I will illustrate how this technology enables monitoring the
metabolic response of Escherichia coli to 1279 human targeted drugs and revealed an unexpectedly large
spectrum of metabolic effects in E. coli. Combining metabolic profiling with chemogenomic data, we
predicted epistatic drug interactions and showed how to expand the search for new antimicrobial treatments
to compounds with no growth-inhibitory activity.
22
OC4
Target metabolomic profiling of natural freeze-dried extracts: the case of hydrolysable
tannins
Enrico Flamini, Michela Burico, Giada Fodaroni, Giacomo Proietti, Stella Bedont, Sara
Tamimi, Claudio M. Quintiero, Mattia Gianni, Luisa Mattoli
Aboca S.p.A. Società̀ Agricola, Località̀ Aboca, 52037 San Sepolcro, Arezzo, Italy
Tannins are a class of polyphenolic compounds produced by plants characterized by polymeric structures.
These compounds include two important classes: hydrolysable tannins (HTs) and condensed tannins (CTs).
In contrast to CTs, HTs are of limited distribution in nature. HTs, which are studied in this project, include
gallo-tannins (GTs) and ellagi-tannins (ETs) and are abundant in leaves, fruits, pods and galls (in some cases
also wood and bark) of dicots such as oak (Quercus spp), chestnut (Castanea spp) and sumac (Rhus coriaria
L.), but have not been detected in monocots.
Castanea spp Quercus spp Rhus coriaria L.
GTs have a polyol core with different esterified gallic acid items and the most frequently found member is
pentagalloyl-β-D-glucose. The name of ETs, instead, is due to ellagic acid, a dilactone form of
hexahydroxydiphenic acid (HHDPA) [1].
Pentagalloyl-β-D-glucose Hexahydroxydiphenic acid
(HHDPA)
Here is described a metabolomic LC-HRMS method using All-Ions Fragmentation acquisition (AIF)[2] [3]
to increase natural hydrolysable tannins (HT) identification in Green tea (Camellia sinensis L.) leaves
extracts (GTE) and Witch-hazels (Hamamelis virginiana L.) leaves extracts (WHE). Here, also, the
chromatographic method optimized on WHE is presented.
Many times, it is not possible to find on the market reference standards for all the possible HTs so it is
necessary follow another approach to better understanding the molecular structure of HTs present in natural
complex extracts. The analysis in AIF shows its potential in these cases. The technique analyze the fragment
ion of UHPLC eluted compounds and correlate it to its precursor (precursor ion approach). Combining the
23
MS/MS information with retention time, high-resolution accurate mass and product/precursor ion intensity
ratios, it has been possible the identification and annotation of various HTs in WHE and GTE. Mass
spectrometric investigations in GTE and WHE were carried out on a UHPLC-qToF system, in high
resolution (2GHz) and using AIF. In negative ion mode, AIF has been acquired at different energies. The
EICs of each tannin subclass typical-fragments has been used to get tannins semi-quantitative data. A
specific tannins database is required, of course. Studying the fragmentation behavior of the few tannins
available on the market, the following fragments common to each HTs subclasses have been identified: ions
at m/z 125.0236 and m/z169.0140 for GTs, ions at m/z 300.9980 for ETs. In GTE and WHE some HTs have
been identified; for many other compounds putative compound annotation and putative class annotation have
been done. The annotated GTs in GTE and WHE have been semi-quantified by external regression curves of
Gallic acid (ions at m/z169.0140). This compromise is necessary, otherwise any quantitative information
cannot be obtained on annotated compounds. By this approach also Pseudo-tannins in GTE can be analyzed.
Semi-quantitative results have a rational correlation with the targeted determination on the same compounds.
Anyone ET has been identified or annotated in GTE and WHE, at the moment. The UHPLC-qToF method
first developed for GTE applied to WHE revealed the need to be improved. So an enhanced chromatographic
separation have been worked out.
In conclusion, to get validated quantitative data it is mandatory to have an effective chromatographic peak
separation! If reference standards are available, tannins can be analyzed by comparison with the
corresponding compounds. Incase this it is not possible, metabolomic screening by AIF-precursor ion
approach is an effective alternative. Semi-quantitative data from putatively annotated compounds can also be
obtained and in complex natural extracts can be very useful for screening purposes.
References
1. Richard W., Hemingway and Joseph J. Karchesy, Chemistry and Significant of Condensed Tannins p.28-32, (1989)
2. Application note 5991-2465EN, Agilent Technologies Inc., (2013).
3. Naz S., Gallart-Ayala H., Reinke S. N., Mathon C., Blankley R., Chaleckis R., Wheelock C.E., Analytical
Chemistry, 89, 7933-7942, (2017).
24
OC5
Qualitative and quantitative profile of Vaccinium macrocarpon urine metabolites by HR-MS
and evaluation of the urine ex-vivo effect on Candida albicans adhesion and biofilm formation
Giovanna Baron1, Alessandra Altomare
1, Luca Regazzoni
1, Elisa Borghi
2, Francesca Borgo
2,
Emerenziana Ottaviano2, Pietro Allegrini
3, Paolo Morazzoni
3, Antonella Riva
3, Lolita
Arnoldi3, Marina Carini
1, Giancarlo Aldini
1
1 Department of Pharmaceutical Sciences, Università degli Studi di Milano, Via Luigi Mangiagalli 25, 20133, Milano,
Italy; 2 Department of Health Sciences, Università degli Studi di Milano, Via Di Rudinì 8, 20142, Milano, Italy;
3 Indena S.p.A., Viale Ortles 12, 20139, Milan, Italy.
Keywords: mass spectrometry, Vaccinium macrocarpon, urine metabolites.
Cranberry (Vaccinium macrocarpon) is a rich source of polyphenols, which possess beneficial properties
towards pathogenic infections. In fact, cranberry based products are mainly used in the prevention of urinary
tract infections (UTIs) [1]. In vitro studies showed proanthocyanidins (PACs) as the most active compounds
[2], but many studies reported their abscence in human urine after cranberry intake [3,4]. The use of different
dosages and non-standardized extract could be the explanation of such dissimilar results. The aim of this
work was to profile cranberry components and metabolites in human urine after the oral intake of a highly
standardized cranberry extract (AnthocranTM
), which has been found effective in human studies [5], and to
test the urine ex-vivo effect on Candida albicans adhesion and biofilm formation. Eight young healthy
female volunteers took 2 capsules Anthocran™/day for 7 days. Urine samples were collected before starting
supplementation and at the following time-points: 1, 2, 4, 6, 10, 12, 24 hours. For the analyses, an HPLC-
MS/MS method was set up using an LTQ-XL-Orbitrap mass spectrometer, working in data-dependent scan
mode.
A targeted and an untargeted approaches were adopted to obtain the qualitative profile of the cranberry
components and metabolites: this approach lead to the identification of 33 compounds (Table 1), confirmed
by the use of pure standards, where commercially available. Standards were also used for the quantitative
analysis and ethylgallate was used as internal standard. Some known PACs metabolites were identified, but
not intact PACs were detected as reported in previous studies. Collected urine were then tested on the
reference strain C. albicans SC5314, a biofilm-forming strain. Urine fractions collected after 1 and 12 hours
were found effective, significantly reducing the adhesion compared to the control (p < 0.001). Among the
cranberry components/metabolites peaking at 1 and/or 12 hours, and hence most likely responsible for the
bioactivity, we found four valerolactones, known as PACs metabolites. The effect on C. albicans adhesion
and biofilm formation of each metabolite as well as a mixture of the metabolites that reach their maximum
concentration in the active urine fractions was tested. In conclusion, several cranberry components and
metabolites, including four valerolactones as PACs metabolites, were identified and quantified in human
urine by targeted and an untargeted HPLC-MS/MS approaches. Ex vivo analyses demonstrated that urine
fractions collected at certain time points, when peaking some cranberry metabolites, reduce C. albicans
adhesion and biofilm formation.
25
Table 1 – Cranberry components and metabolites identified in human urine
Targeted Untargeted
Sinapinic acid
3,4-Dihydroxyhydrocinnamic acid
p-Hydroxyhippuric acid
m-Hydroxyhippuric acid
o-Hydroxyhippuric acid
Protocatchuic acid Quinic acid
p-Coumaric acid 2-Methylhippuric acid
Gallic acid 5-(3',4'-Dihydroxyphenyl)-gamma-valerolactone
2-Hydroxybenzoic acid 5-(4'-Hydroxyphenyl)-gamma-valerolactone-3'-O-sulphate
4-Hydroxybenzoic acid 5-(3'-Hydroxyphenyl)-gamma-valerolactone-4'-O-sulphate
2,3-Dihydroxybenzoic acid 5-(3',4',5'-Trihydroxyphenyl)-gamma-valerolactone-3'-O-
sulphate
2,5-Dihydroxybenzoic acid 4-Hydroxy-5-(dihydroxyphenyl)-valeric acid-O-sulphate
2,4-Dihydroxybenzoic acid Salicyluric glucuronide
3-(4-Hydroxyphenyl)-propionic acid 3-O-Methylcatechin-sulphate
Vanillic acid
3,4-Dihydroxyphenylacetic acid
Hippuric acid
Kaempferol
Quercetin
Syringetin
Quercetin-3-O-arabinofuranoside
Quercetin-3-O-rhamnoside
Quercetin-3-O-galactoside
Isorhamnetin-3-O-arabinopyranoside
References
1. Vasileiou, A. Katsargyris, S. Theocharis, C. Giaginis; Nutrition Research, 33 (2013), pp 595-607.
2. A.B. Howell, J.D. Reed, C.G. Krueger, R. Winterbottom, D.G. Cunningham, M.Leahy; Phytochemistry, 66 (2005),
pp 2281-2291.
3. R.P. Feliciano, A. Boeres, L. Massacessi, G. Istas, M.R. Ventura, C. Nunes dos Santos, C. Heiss, A. Rodriguez-
Mateos; Archives of Biochemistry and Biophysics, 599 (2016), pp 31-41.
4. I.Iswaldi, D. Arráez-Román, A.M. Gómez-Caravaca, M. del Mar Contreras, J. Uberos, A. Segura-Carretero, A.
Fernández-Gutiérrez; Food and Chemical Toxicology, 55 (2013), pp 484-492.
5. A. Ledda, G. Belcaro, M. Dugall, A. Riva, S. Togni, R. Eggenhoffner, L. Giacomelli; European Review for
Medical and Pharmacological Sciences, 21 (2017), pp 389-393
26
OC6
Untargeted profiling of large intestine microbial transformation products of phenolic
compounds in pigmented flours
Gabriele Rocchetti, Marco Trevisan, Luigi Lucini
Università Cattolica del Sacro Cuore, Department for sustainable food process, Piacenza, Italy
Keywords: phenolic profiling; in vitro fermentation; catabolism
In this work, 18 flours prepared from pigmented cereals, pseudocereals and legumes differing in
pigmentation, were screened for their phenolic profiles, cooked, and then subjected to in vitro digestion and
colonic fermentation. An untargeted metabolomic approach was used to elucidate the microbial
biotransformation processes of polyphenols following digestion. Our results demonstrated an increase in 3,5-
dihydroxybenzoic acid (on average from 0.67 up to 1.30 mol/g dry matter) throughout the fermentation of
pseudocereals (esp. quinoa), due to their high alkylresorcinol contents. Isoflavones were converted into
equol- or O-desmethylangolensin- derivatives, whereas anthocyanins were degraded into lower-molecular-
weight phenolics (i.e., protocatechuic aldehyde and 4-hydroxybenzoic acid, with the latter exhibiting the
highest increase over time). These findings highlight that pigmented flours are able to deliver bioaccessible
polyphenols to the colon.
Fig. 2. Proposed catabolic pathways for microbial transformation of phenolic compounds in pigmented flours
27
PL3
Protein Mass Spectrometry: from complexes to biotherapeutics to biomarkers
Joseph A. Loo
Department of Chemistry and Biochemistry, Department of Biological Chemistry, David Geffen
School of Medicine
UCLA/DOE Institute for Genomics and Proteomics, University of California-Los Angeles, Los
Angeles, CA USA
Mass spectrometry (MS) has advanced over the past decades to address the sensitive measurement of large
biomolecules such as proteins. “Native” MS of proteins and protein assemblies reveals size and binding
stoichiometry. Elucidating their structures using MS to understand their function is more challenging, but
native top-down MS, i.e., fragmentation of the gas-phase protein, can be effective for deriving structural
information for soluble and membrane protein complexes and biotherapeutics.
Native top-down MS generates information on the surface topology, ligand binding sites, and post-
translational modifications (PTMs) of protein complexes. We use native MS/MS to investigate the molecular
action of compounds that prevent amyloid fibril formation in neurodegenerative diseases such as
Alzheimer’s and Parkinson’s disease.
Protein MS is extended to discovery of novel biomarkers for improving human health. Our lab is engaged in
efforts to discover new protein biomarkers for head trauma, i.e., traumatic brain injury (TBI). TBI is an
expanding public health epidemic with pathophysiology that is difficult to diagnose and thus treat. Our
primary objective has been to select candidate neurotrauma biomarkers that are robustly released by trauma
and that are brain specific.
28
OC7
The evolving landscape of diagnostic pathology: a step towards the integration of MALDI-
MSI for the routine diagnosis of thyroid fine needle aspiration biopsies
Isabella Piga1, Giulia Capitoli
2, Vanna Denti
1, Sonia Guarnerio
1, Allia Mahajneh
1, Andrew
Smith1, Clizia Chinello
1, Stefania Galimberti
2, Fulvio Magni
1, Fabio Pagni
3
1Dipartimento di Medicina e Chirurgia, Unità di Proteomica e Metabolomica Clinica,
2Dipartimento di Medicina e Chirurgia, Centro di Biostatistica per Epidemiologia Clinica,
3Dipartimento di Medicina e Chirurgia, Sezione di Patologia, Università degli Studi di Milano-
Bicocca Via Cadore 49, 20900, Monza, Italy
Keywords: Thyroid, MALDI-MSI Proteomics, Fine needle aspiration biopsies
Fine Needle Aspiration biopsy (FNAB) is the gold standard procedure to determine the malignant nature of
thyroid nodules. However, approximately 20% of FNABs are diagnosed as “indeterminate for malignancy”,
and these patients undergo diagnostic thyroidectomy, often unnecessary. MALDI-MSI represents an ideal
tool to explore the spatial distribution of proteins directly in-situ, integrating molecular and
cytomorphological information. This enables the discovery of potential diagnostic markers in thyroid
cytopathology. The first aim of the study was to standardise the sample preparation of ex-vivo and in-vivo
thyroid FNABs for proteomic MALDI-MSI analysis, in order to minimise haemoglobin interference, which
suppresses other proteins signals in the sample. Then, we investigated the proteomic stability of the samples.
In-vivo thyroid FNABs were collected from 14 patients (San Gerardo Hospital, Monza, Italy) and transferred
into CytoLyt solution, centrifuged and re-suspended in PreservCyt solution. Cytospin spots have been
positioned onto ITO-conductive slides and MALDI-MSI intact proteins analysis was performed using an
ultrafleXtreme MALDI-TOF/TOF. Each FNAB was split into several samples in order to evaluate the
experimental repeatability (intra-day and inter-day) of the proteomics analysis and the cytological samples
stability after 7, 14 days and 2 months in preservative solution at 4°C. Results showed that liquid-based
preparation efficiently remove hemoglobin, allow to preserve the proteomic integrity of the sample, and to
store sample at 4°C for 14 days in the preservative solution before deposing it onto the conductive slide.
This study represents a step forward towards the implementation of MALDI-MSI, combined with a
trustworthy and robust sample preparation methodology, into the cytopathology routine, integrating the
morphology with the proteomics data to improve the diagnosis.
FUNDING: This work was funded thanks to AIRC (Associazione Italiana per la Ricerca sul Cancro) MFAG
GRANT 2016 - Id. 18445.
29
OC8
In-Vitro and In-Cell Cross-Linking/Mass Spectrometry: from 3D-Protein Structure
Investigations to Proteome-Wide Interactome Studies
Claudio Iacobucci1, Michael Götze
1,2, Andrea Sinz
1
1 Department of Pharmaceutical Chemistry and Bioanalytics, Martin-Luther University Halle-
Wittenberg, Germany 2 Present address: Institute of Molecular Systems Biology, ETH Zürich, Switzerland
Keywords: Cross-Linking Mass Spectrometry, 3D Protein Structure, Protein-Protein Interactions
Chemical cross-linking/mass spectrometry (XLMS) has emerged as a powerful tool for the 3D-structure
analysis of proteins and protein complexes and is becoming increasingly popular in structural biology.[1]
We have developed and successfully applied advanced cross-linkers[2-7] and integrated workflows[8,9] to
perform XLMS at different levels, ranging from isolated protein and protein assemblies to highly complex
protein mixtures, such as cell lysates and intact cells. Our protocols can be conducted within one week and
are based on the commercially available MS-cleavable cross-linker disuccinimidyl dibutyric urea (DSBU).
The workflows can be employed by every lab having access to a mass spectrometer with tandem MS
capabilities. We developed a novel version 2.0 of the freeware software tool MeroX (www.StavroX.com)
that allows a fully automated analysis to deliver insights into protein interaction networks and protein
conformations on a proteome-wide scale.[9] We demonstrate the successful application of our workflow for
Drosophila embryo extracts as well as intact E.coli cells and human embryonic kidney cells.[9] Principles of
modern cross-linkers and our recent applications of XLMS will be discussed.
References 1. C. Iacobucci, et al., submitted (2019), preprint doi: https://doi.org/10.1101/424697.
2. C. Iacobucci, C. Piotrowski, A. Rehkamp, C. H. Ihling and A. Sinz, J. Am. Soc. Mass Spectrom., 30 (2019), pp 139-
148.
3. C. Iacobucci, M. Schäfer, A. Sinz, Mass Spectrom. Rev., 38 (2018), pp 187-201.
4. C. Iacobucci, M. Götze, C. Piotrowski, C. Arlt, A. Rehkamp, C. Ihling, C. Hage and A. Sinz, Anal. Chem., 90
(2018), pp 2805-2809.
5. C. Hage, C. Iacobucci, A. Rehkamp, C. Arlt and A. Sinz, Angew. Chem. Int. Ed., 56 (2017), pp 14551-14555.
6. C. Iacobucci and A. Sinz, Anal. Chem., 89 (2017), pp 7832-7835.
7. C. Iacobucci, C. Hage, M. Schäfer and A. Sinz, J. Am. Soc. Mass Spectrom., 28(2017), pp 2039-2053.
8. C. Iacobucci, M. Götze, C. Hage, C. Arlt, C. H. Ihling, R. Schmidt, C. Piotrowski and A. Sinz, Nat. Protoc., 13
(2018), pp 2864-2889.
9. M. Götze, C. Iacobucci, C. Ihling and A. Sinz, submitted (2019), preprint doi: https://doi.org/10.1101/524314.
30
OC9
Urinary GC-MS untargeted metabolomics for prostate cancer diagnosis. Preliminary results.
Eleonora Amante 1,2
, Alessandra Biancolillo3, Rasmus Bro
4, Francesco Porpiglia
5, Alberto
Salomone1,2
, Marco Vincenti1,2
1 Department of Chemistry, University of Torino, Via P. Giuria 7, 10125 Torino, Italy,
2 Anti-
Doping Centre “A. Bertinaria”, Regione Gonzole 10/1, 10043 Orbassano, Torino, Italy, 3
Department of Chemistry – Sapienza, University of Roma, P.le Aldo Moro 5, 00185 Rome, Italy, 4
Department of food science, Faculty of Science, University of Copenhagen, Rolighedsvej 30 - 1958
Frederiksberg, Denmark, 5 Division of Urology, San Luigi Gonzaga Hospital and University of
Torino, Orbassano, Italy
Keywords: gas chromatography – mass spectrometry (GC-MS), untargeted metabolomics, cancer
diagnosis
Prostate carcinoma is the principal cause of cancer-related death in men [1]. The prostate specific antigen
(PSA) dosage is, at the present, the unique clinical test available to screen for this pathology. PSA is highly
sensitive but not enough specific for malignancy, providing a huge amount of misleading results. Thus, new
biomarkers which could improve the diagnostic power of prostate screenings are needed.
The present work is a preliminary study aimed to discover new urinary biomarkers suitable for this purpose.
Ninety subjects, divided into controls and prostate carcinoma-affected, were recruited. Two aliquots of each
sample were treated as follows: proteins were precipitated, two subsequent extractions (at acid and basic
conditions) were performed for each aliquot and the dried extracts were derivatized using trifluoroacetic
anhydride (aliquot A) and trimethylsilyl derivatizing mixture (aliquot B). The full-SCAN GC-MS spectra
(two for each sample) were then processed with the following chemometric approach:
(i) The alignment was performed by double warping algorithm [2];
(ii) The spectral deconvolution was carried out using the PARADISe software, based on the
PARAFAC2 algorithm [3];
(iii) The semiquantitative report provided by the software was then subjected to two different and
independent variable selection procedures:
a. Variable Importance Projection followed by genetic algorithms [4];
b. Covariance Selection algorithm [5];
(iv) The reduced datasets were used to build two classification models, which performances were
compared.
The validation of the models was performed using a repeated double cross validation approach [6], and the
obtained preliminary results are promising, resulting near to 90% for both specificity and sensitivity.
References 1. V.M. Velonas, H.H. Woo, C.G. dos Remedios, S.J. Assinder; International Journal of Molecular Science, 14(2013)
pp 11034-11060
2. G. Tomasi, F. Van Den Berg, C. Andersson; Journal of Chemometrics, 18(2004) pp 231-241
3. L.G. Johnsen, P.B. Skou, B. Khakimov, R. Bro; Journal of Chromatography A, 1503(2017) pp 57-64
4. R. Leardi; Journal of Chemometrics, 15(2001) pp 559-569
5. J.M. Roger, B. Palagos, D. Bertrand, E. Fernandez-Ahumada; Journal of Chemometrics; 23(2009) pp 160-171
31
OC10
Tandem mass spectrometry with ion trap in the isomers resolution
M. Menicatti, L. Moracci, L. Braconi, S. Dei, E. Teodori, G. Bartolucci
NEUROFARBA - Dipartimento di Neuroscienze, Psicologia, Area del Farmaco e Salute del
Bambino Sezione Scienze Farmaceutiche e Nutraceutiche, Università di Firenze
Via U. Schiff, 6 50019 Sesto Fiorentino (FI), Italy
Keywords: MS/MS, Linear Equations Deconvolution Analysis, ERMS
The analysis of isobaric molecules by tandem mass spectrometry, especially if they are isomers, is often
complicated by the similarity of their fragmentation patterns. In fact, it is common that the same MS/MS
product ions are present in the spectra of all isomers. In this case an adequate chromatographic separation
between compounds should be developed in order to eliminate mutual interferences. The resolution of
mixtures of isomers by liquid chromatography requires peculiar stationary phases and specific elution
programs, which leads to lack of productivity, in terms of number of samples per time unit, and rise of
analysis costs. The use of MS/MS experiments and the application of LEDA (Linear Equations
Deconvolution Analysis) algorithm to the obtained data allows a remarkable reduction of these issues.
Considering the energetic fragmentation profiles of pure isomers, the LEDA tool permits the deconvolution
of isomers mixtures, assigning to each component the correct relative percentage. [1-3]
The aim of this work is the validation of a method to analyze five positionally isomers of multi drug
resistance inhibitors (MDR) using an ion trap (IT) mass spectrometer. In fact, some issues were occur in the
triple quadrupole (QqQ) LEDA application, that showed in some cases lack of precision on the obtained
results. These problems were mainly due to the difficult evaluation of the abundance of the precursor ion
before its decomposition and/or to the extensive number of formed product ions, which involves the
subdivision of the ionic signal. To limit these occurrences, the MS/MS experiments in IT were carried out.
The IT fragmentation mechanism allows a better management of the ions (precursor/products) ensuring their
correct abundance rating. Indeed, the different energy transfer during collision process, lead to the formation
of product ions directly by precursor ion (Figure 1).
Figure 1: General MS/MS fragmentation pathway of studied compounds in QqQ and (X) limited in IT
32
However, this characteristic often produce a poor number of product ions, which contrasts with necessary
information for the LEDA application.
The LEDA algorithm consists in the application of a matrix of linear regression equations to different
experimental data (equation 1).
n
x
xxm Ri
PiRi
Pi
1
%
Where: (Pi/Ri)m is the ratio between the abundances of product (Pi) and reference (Ri) ions measured
(m) in the sample;
(Pi/Ri)x are the characteristic abundance ratios between the product and reference ions of pure
isomers
[%]x: is the concentration (%) of isomers in the sample
The LEDA tool was proposed to establish the relative proportions of individual isomers in the sample.
This work is part of a wider project comprehending drug plasma stability and activity assays of these
compounds. [4]
Considering the pharmaceutical interest of the compounds under investigation, the LC-MS/MS method
developed was tested to be effective at the pharmacological active concentration levels of studied
compounds, corresponding to a range between nM to µM (corresponding to ng mL-1
of processed sample).
The performance evaluation of the proposed algorithm (LEDA) in IT application confirmed its effectiveness
allowing an accurate and precise quantitative analysis of complex mixtures of isomers.
Furthermore the product ion scan acquisition allows to measure the isotopic cluster of fragment ions and to
determine the number of carbon atoms of these fragments.
It is worth to observe that the LEDA tool is able not only to give the relative quantities of the mixture
components but, overall, to distinguish immediately their combination (e.g. binary, ternary, quaternary, ....
mixtures) or if the sample is represented by a pure compound.
The LEDA approach has the advantage that isomers can be quantified without the need of LC separation or
additional specialized ion mobility instrumentation.
References
1. M. Menicatti, L. Guandalini, S. Dei, E. Floriddia, E. Teodori, P. Traldi, G. Bartolucci; Rapid Commun Mass
Spectrom., 30, 423-432 (2016).
2. M. Menicatti, L. Guandalini, S. Dei, E. Floriddia, E. Teodori, P. Traldi, G. Bartolucci; Eur. J. Mass Spectrom., 22
(5), 235-243 (2016).
3. M. Menicatti, M. Pallecchi, S. Bua, D. Vullo, L. Di Cesare Mannelli, C. Ghelardini, F. Carta, C T. Supuran, G
Bartolucci, J Enzyme Inhib Med Chem. 2018; 33(1): 671–679.
4. S. Dei, M. Coronnello, E. Floriddia, G. Bartolucci, C. Bellucci, L. Guandalini, D. Manetti, M. N. Romanelli, M.
Salerno, I. Bello, E. Mini, E. Teodori; Eur J Med Chem. 30, 398-412 (2014).
Eq. 1
33
OC11
A multiomics approach using metabolomics and lipidomics
Claudio Ghilardi
Shimadzu Italia srl
Keywords: LCMS, triple quadrupole, Q-TOF
Microorganisms including Escherichia coli and yeast are used to mass produce useful substances in a variety
of industrial sectors such as food, biotechnology, and energy. In the food sector, for example, fermentation
processes that utilize microorganisms are widely used for alcoholic beverages and fermented foods, and
microbial breeding is performed for the purpose of more efficient fermentative production and the
production of high value-added metabolites. In order to improve the production efficiency of useful
substances and increase the production capacity of such high value-added compounds, it is necessary to
monitor metabolic changes using metabolomics. Since monitoring of metabolic changes not only requires an
understanding of the target substance but also an understanding of the metabolic changes of the precursor
and intermediates, the metabolomics approach is expected to be very effective because of the ability to
simultaneously analyze a large number of compounds. This research attempts to understand metabolic
changes from a multiomics approach by evaluating metabolic changes using metabolomics and combining
the lipidomics results of phospholipids. The sample used was E. coli, which efficiently produces the sulfur-
containing metabolite ergothioneine. Thiosulfate or sulfate was added as a sulfur source for the synthesis of
cysteine which acts as the substrate for ergothioneine. By applying the approaches of metabolomics and
lipidomics, this article introduces an example of evaluating how related sulfur-containing metabolites change
depending on cultivation progression
References
T. Nakanishi, Internal review, Shimadzu Corporation
34
OC12
Molecular fingerprinting of Pistacia lentiscus hydrosol by ultra-high resolution ESI-FT-ICR
mass spectrometry
A. Onzoa, G. Bianco
a, G. Martelli
a, R. Pascale
a, P. Iannece
b, C. Pifano
c, C. Gaeta
b
a)
Università degli Studi della Basilicata, Dipartimento di Scienze, Via dell’Ateneo Lucano 10 -
85100, Potenza (Italy), b)
Università degli Studi di Salerno, Dipartimento di Chimica e Biologia, Via
Giovanni Paolo II 132 – 84084 Fisciano (Italy), c)
BioInnova s.r.l., via Ponte Nove Luci 22 - 85100
Potenza (Italy)
Keywords: Metabolomics, Pisticia lentiscus, hydrosol
Pistacia lentiscus is part of a large family (Anacardiaceae) widely distributed in the Mediterranean basin
ecosystems where it grows wild. Previous studies led to the quantification and/or identification of many
constituents such as flavonoids and anthocyanins, responsible for its health-promoting properties [1]. Indeed,
it’s widely used in Algeria for the treatment of inflammation, burns and gastrointestinal complaints and
related essential oils are commercially available all over the world and they are mostly used as decongestant
and analgesic [2]. However, less has been done on related hydrosol, mostly considered as a by-product of
essential oil production. The aim of this work was to perform the molecular fingerprinting of a sample of
Pisticia lentiscus hydrosol by using Ultra-High Resolution ESI-FT-ICR Mass Spectrometry, in order to
obtain information about its metabolic content. Obtained data were used to perform a rapid analysis of
metabolome by converting accurate m/z values in putative elemental formulas in order to better understand
the chemical composition of the sample. Molecular formula maps were obtained by making 2D Van
Krevelen plots, that lead to a direct identification of different classes of metabolites [3]. The presence of
several metabolite classes, i.e. fatty acid derivatives, tannins, amino acids and peptides, carbohydrates and
polyphenolic derivatives, was assessed. Moreover, in this work, an evaluation of biological activity of
Pistacia lentiscus hydrosol was done by performing a series of in vitro tests, during which the sample
showed marked anti-inflammation and inducing-cell regeneration activities. Results shed some light on
metabolic profile of Pistacia lentiscus hydrosol, thus supporting the idea of it as a rich source of important
metabolites and its utilization for therapeutic purposes.
References
1. A. Dellai, H. Souissi, W. Borgi, A. Bouraoui, N. Chouchane, Ind. Crop. Prod. 2013 49, 879–882.
2. E. H. Benyoussef, S. Charchari , N. Nacer-Bey , N. Yahiaoui , A. Chakou, M. Bellatreche, Journal of Essential Oil
Research 2005, 17(6), 642-644.
3. R. Pascale, G. Bianco, T.R.I. Cataldi, P. Schmitt-Kopplin, F. Bosco, L. Vignola, J. Uhl, M. Lucio, L. Milella, Food
Chemistry 2018, 242, 497–504.
35
OC13
Isotope evidence of Gravettian and Epigravettian mobility strategies across the Last Glacial
Maximum in Southern Italy
Federico Lugli
University of Modena and Reggio Emilia. Italy
Understanding the reason(s) behind changes of human mobility strategies through space and time is a major
challenge in paleoanthropology. Most of the time this is due to the lack of suitable temporal sequences of
human skeletal specimens during critical climatic or cultural shifts. Here, we present temporal variations in
the Sr isotope composition of 14 human deciduous teeth and the N and C stable isotope ratios of 4 human
remains from the Grotta Paglicci site (Apulia, Southern Italy). The specimens were recovered from the
Gravettian and Epigravettian layers, across the Last Glacial Maximum, and dated between 31210-33103 and
18334-19860 cal BP (2σ). The two groups of individuals exhibit different 87
Sr/86
Sr ratios and while the
Gravettians are similar to the local macrofauna in terms of Sr isotopic signal, the Epigravettians are shifted
towards higher radiogenic Sr ratios. These data, together with stable isotopes, can be explained by the
adoption of different mobility strategies between the two groups with the Gravettians exploiting logistical
mobility strategies and the Epigravettian applying residential mobility.
36
PL4
Predictive science and mass spectrometry
G. John Langley
Chemistry, FNES - University of Southampton, Southampton SO17 1BJ (U.K.)
Today we are in an era where data are readily produces. Large data repositories, data lakes are becoming
commonplace and simply curating these is a challenge. Extracting information from large datasets is a
significant challenge and needs and data-mining tools to access this information. The expectation that we
will find ‘something important’ if we collect a large data set is not unlike the combinatorial chemistry
approach of the early part of this century, where the ethos was ‘if we make big enough haystacks we will
find a needle’. Given our experience of the combichem revolution there is a need to reconsider this approach
or tailor it with some front end knowledge and improved automated data analysis tools.
To deal with these large data, and more importantly to aid confidence in data assignment there needs to be an
understanding many factors, including ionisation technique(s), mass spectrometer type, experiments used
and how these factors can influence the mass spectral data. It is also critical that we capture the correct
metadata associated with these data.
The importance of the initial question and thought experiment cannot be underestimated. From that point
prediction can begin, and may even start before the question is fully formed.
Different aspects of predictive science will be discussed, in relation to where this plays a role across different
areas of analysis, focusing on predictive approaches to tandem mass spectrometry. Interpretation of MS/MS
data is often a laborious, time-consuming manual process, requiring highly skilled analysts. We already
have some tools to aid accelerate this process.
Ultimately the goal is to understand how we take ‘data to knowledge’ and ‘knowledge to information’ and
improve confidence in any measurement or interpretation.
37
OC14
Combination of Pressurized Liquid Extraction with dispersive Liquid Liquid Micro
Extraction for the determination of sixty drugs of abuse in hair
Flaminia Vincentia,b
, Camilla Montesanoa, Luana Cellucci
a, Adolfo Gregori
c, Federico Fanti
d,
Dario Compagnoned, Roberta Curini
a, Manuel Sergi
d
aSapienza University of Rome, Department of Chemistry,
bDepartment of Public Health and
Infectious Disease, 00185 Rome, Italy, cCarabinieri, Department of Scientific Investigation (RIS),
00191 Rome, Italy, dUniversity of Teramo, Faculty of Bioscience and Technology for Food,
Agriculture and Environment, 64100 Teramo, Italy
Keywords: Illicit drugs; Multiclass analysis; HPLC-HRMS/MS
The increasing phenomenon of drug addiction and the introduction of New Psychoactive Substances (NPS)
has led to a progressive growth of research in the field of forensic analytical toxicology, with the need to
develop modern and faster analytical procedures. Hair testing has gained increasing attention and recognition
as a complement to blood and urine analysis, since it is a unique material for the retrospective detection of
drugs, due to its large detection window [1].
In this work, a multiclass method for the simultaneous extraction, identification and quantitation of sixty
drugs of abuse belonging to different chemical classes in hair is proposed. This method can provide a valid,
fast, simple and low-cost alternative to common tests [2]; at the same time it provides quantitative results,
which can confirm concurrently the assumption of one or more illicit substances. Both the decontamination
step and the extraction of the analytes from the inner core of the hair were carried out by means of
pressurized liquid extraction (PLE) while the clean-up by dispersive liquid/liquid microextraction (dLLME),
giving the great advantage of a very high enrichment factor. The optimized chromatographic conditions
allowed a satisfying separation of the 60 analytes in 14 min, while the detection was conducted with a high-
resolution mass spectrometer with Orbitrap technology.
In order to evaluate the method performances, four different types of samples were analysed: a matrix
obtained simply by spiking the standard solution on the hair; the second one was performed by soaking; and
finally, CRM and real samples obtained from volunteers. This multiclass method has demonstrated to be
suitable for analytes with different chemical characteristics allowing to reduce time and cost of analysis,
organic solvent volume and the amount of matrix needed. The whole method was fully validated as
confirmatory method following SWGTOX guidelines [3].
References 1. A. Verstraete, Therapeutic Drug Monitoring (2005) pp. 200-205.
2. C. Montesano, M.C. Simeoni, G. Vannutelli, A. Gregori, L. Ripani, M. Sergi, D. Compagnone, R. Curini, Journal of
Chromatography A (2015) pp. 192–200.
3. G.A.A. Cooper, R. Kronstrand, P. Kintz, Society of Hair Testing guidelines for drug testing in hair, Forensic
Science International 218 (2012) pp.20-24.
38
OC15
Development of Solvent-Assisted Paper Spray Ionization Mass Spectrometry Setup for the
analysis of biomolecules and biofluids
N. Riboni1, A. Quaranta
2, H.V. Motwani
2, N. Österlund
3, A. Gräslund
3, M. Careri
1,
F. Bianchi1, L.L. Ilag
2
1
Dipartimento di Scienze Chimiche, della Vita e della Sostenibilità Ambientale, Università di
Parma, Parma, Italy 2
Department of Environmental Science and Analytical Chemistry and 3
Department of Biochemistry and Biophysics, Stockholm University, Stockholm, Sveden
Keywords: Ambient Mass Spectrometry, Paper Spray Mass Spectrometry, Biomolecules Analysis
Ambient Mass Spectrometry (AMS) allows the analysis of samples in their native state without or with a
reduced sample preparation [1-3]. In Paper Spray Ionization (PSI) the sample solution is spotted onto a
triangular paper tip, held in front of the MS inlet by a conductive stainless steel clip. After drying the sample,
few microliters of solvent are deposited on the paper probe and a potential in the kV range is applied to the
clip, resulting in the formation of a Taylor cone [4]. PSI, due to its simplicity, versatility and low costs, is
applied for the detection of small molecules, e.g. pesticides, drugs and metabolites, in complex matrices
[5,6]. However, some drawbacks are related both to the short data acquisition time and to the not constant
ionization conditions.
In order to overcome these limitations and expand the applicability of PSI toward the detection of large
biomolecules, a new setup called Solvent Assisted Paper Spray Ionization (SAPSI) was developed. This
setup is an integrated solution able to provide ionization potential and constant solvent flow to the paper tip:
a commercially available Waters Synapt nanoESI source was modified to host a support for the PSI clip. The
NanoLockSpray reference probe capillary holder was also removed to introduce a support for peek tubes and
the ion source was directly connected to the instrument fluidics (Figure 1).
Fig. 1. Schematic representation of SAPSI ion source connected to the Waters Synapt fluidic system
The constant ionization conditions resulted in enhanced sensitivity, repeatability and possibility to perform
real-time monitoring. SAPSI was tested toward the analysis of three classes of biomolecules: amyloid-β (1-
40) (Aβ) peptide and its oligomeric forms, intact proteins, and glycans. Finally, diluted untreated human
serum and cerebrospinal fluid (CSF) were directly analysed by SAPSI. Aggregation and disaggregation of
Aβ (1-40) were investigated by monitoring the presence and the evolution of its oligomeric states in real
39
time. Their differentiation was obtained by coupling SAPSI with ion mobility spectrometry (SAPSI-IMS-
MS). Oligomeric species up to octamer were identified in incubated Aβ samples. Real time monitoring
demonstrated the sequential disaggregation of the oligomeric states during the analysis due to the use of
isopropanol in the ionization solvent mixture.
Four standard proteins, namely human serum albumin (HSA), human hemoglobin, human transferrin (TFN),
and bovine superoxide dismutase were analysed in the unfolded form. The constant ionization conditions
resulted in very stable, resolved and sharp peaks characterized by highly charge states. By performing peaks
deconvolution, it was possible to identify protein forms having different post-translational modifications
(PTMs), with a resolution of 15 Da. Cysteinylated, glycated and oxidized forms of HSA were detected,
whereas different glycoforms of TFN were identified. Glycans were analysed by MS/MS analysis of a
standard H5N4S2 glycan and of enzymatically released N-glycans from TFN and α-1-antitrypsin, with and
without sialic acids respectively.
Finally, untreated human serum and CSF were analysed by SAPSI-MS. Three different zones (Figure 2)
could be identified: the first, below m/z 700, characterized by signals attributable to compounds having a low
molecular weight and to background noise; the second, around m/z 750-850, presenting intense singly
charged ions related to phosphatidylcholine lipids (identified by MS-MS analysis); the third, between m/z
850 and 2600, showing two different envelopes. After peaks deconvolution, forms of HSA and
apolipoprotein A1 (ApoA1) presenting different PTMs were identified.
Fig. 2. MS spectrum of diluted human serum and related MaxEnt™1 deconvolution.
Protein alkylated species were also identified, both in aqueous solution and in serum, after incubation with
acrylamide at concentration levels compatible with in vivo toxicological experiments.
It can be concluded that the developed setup, due to the constant ionization conditions and the prolonged
data acquisition time, is suitable for the real-time monitoring of chemical and physical processes taking place
on the paper tip. In addition, presenting enhanced peak resolution compared to PSI, SAPSI could be used to
monitor alterations in the PTMs of serum and CSF proteins related to inflammation and diseases and/or as a
tool to evaluate the exposure to alkylating compounds.
References 1. F. Bianchi, N. Riboni, V. Termopoli, L. Mendez, I. Medina, L. Ilag, A. Cappiello, M. Careri, Journal of Analytical
Methods in Chemistry, 2018 (2018), pp. 1–24.
2. R. Chen, J. Deng, L. Fang, Y. Yao, B. Chen, X. Wang, T. Luan, Trends in Environmental Analytical Chemistry, 15
(2017), pp. 1–11.
3. P.M. Peacock, W.-J. Zhang, S. Trimpin, Analytical Chemistry, 89 (2017), pp. 372–388.
4. H. Wang, J. Liu, R.G. Cooks, Z. Ouyang, Angewandte Chemie International Edition in English, 49 (2010), pp. 877–
80.
5. W.R. de Araujo, T.M.G. Cardoso, R.G. da Rocha, M.H.P. Santana, R.A.A. Muñoz, E.M. Richter, T.R.L.C. Paixão,
W.K.T. Coltro, Analytica Chimica Acta, 1034 (2018), pp. 1–21.
6. C. Black, O.P. Chevallier, C.T. Elliott, TrAC Trends in Analytical Chemistry, 82 (2016), pp. 268–278.
40
OC16
Processing effects on the content of bioactive secoiridoids in extra-virgin olive oil examined by
liquid chromatography-electrospray ionization-Fourier transform mass spectrometry
C. De Ceglie1, R. Abbattista
1, I. Losito
1,2, A. Castellaneta
1, C.D. Calvano, F. Palmisano
1,2,
T.R.I. Cataldi1,2
University of Bari “Aldo Moro”, 1Department of Chemistry and
2SMART Inter-department
Research Center, Via E. Orabona 4, 70126 Bari, Italy
Keywords: bioactive secoiridoids, olive oil production technology, liquid chromatography-electrospray
ionization-Fourier transform mass spectrometry
Extra-virgin olive oil (EVOO) is one of the main components of the Mediterranean diet, recognized as an
important source of phenolic bioactive compounds [1]. A relevant role among the latter is played by
secoiridoids, a class of phenolic compounds present mainly in O. europaea leaves and drupes, which have
raised a significant interest in recent years due to their beneficial effects in human health [1]. The main
secoiridoids present in olive drupes, i.e., oleuropein, ligstroside and demethyl-oleuropein, in order of
abundance, are glycosides. Enzymatic reactions occurring during olive oil production, catalyzed by β-
glucosidase, methylesterase and decarboxylase enzymes, convert these glycosides in the corresponding
aglycones and then in the corresponding decarboxymethylated forms, known as oleocanthal and oleacin [2].
Therefore, the abundance of secoiridoids in EVOOs depends not only on olive cultivar, ripening stage,
geographical origin and agronomic practices, but also on the employed technological process; each stage of
olive oil production (i.e. crushing, malaxation, extraction, final centrifugation) may affect the secoiridoid
content and nutraceutical properties along with sensory traits and nutritional quality [2]. Oil extraction from
the olive paste upon malaxation is based either on three- or two-phase horizontal centrifugation. In the
former processing system, water is normally added to the olive paste during centrifugation, thus, along with
humid olive pomace, olive mill wastewater, partly dissolving hydrophilic phenolic compounds, is obtained
as a by-product. A higher content of phenolic compounds, including secoiridoids, is expected in EVOO
obtained by two-phase centrifugation; information on the total phenolic content of two- and three-phase
EVOOs has usually been reported in the literature, confirming this suggestion [2]. Currently, only a few
studies have been focused on the content of secoiridoids, evidencing a distinction between three- and two-
phase horizontal centrifugation [3].
As part of a more general investigation on secoiridoids in EVOOs based on reversed-phase liquid
chromatography (RPLC) and high-resolution Fourier-transform mass spectrometry with electrospray
ionization (ESI-FTMS), this issue has been recently investigated in our laboratory. Twenty-two EVOO
samples, mostly produced during the 2018/2019 campaign, using olives of Coratina cultivar, and either a
three- (14) or a two-phase (8) horizontal centrifugation, were collected from Apulian olive oil producers.
According to the protocol of Vichi et al. [4], with some modifications, secoiridoids were extracted in
duplicate from EVOO samples using a CH3OH/H2O 60:40 (v/v) mixture. Each extract was spiked with 100
mg/L of oleuropein as an internal standard (IS) and examined by RPLC-ESI-FTMS analysis, using a C18
core-shell column and a binary elution gradient based on water and acetonitrile. MS detection was performed
in negative ion polarity using a hybrid quadrupole-Orbitrap mass spectrometer, operated under high mass
resolution (resolving power > 100,000) and accuracy (< 1 ppm).
The ratios between peak areas obtained from eXtracted Ion Current (XIC) chromatograms referred to the
four secoiridoids of interest, i.e. the aglycones of oleuropein and ligstroside, oleacin and oleocanthal, and the
area obtained for the IS peak were correlated to the processing system. In the present communication the
results of such comparison will be discussed, emphasizing the outcomes of cluster analysis (CA) and
principal component analysis (PCA) performed on data. As a general trend, PCA confirmed the higher
content in secoiridoids of two-phase processing EVOOs, although two notable exceptions were found. As
displayed in Figure 1, the bidimensional scatterplot referred to the first two principal components (total
41
variance explained, 88.49%), two-phase EVOO samples were displaced towards higher values of PC1, due
to their generally higher content of secoiridoids (especially oleuropein aglycone).
Fig. 3. Scatterplot referred to the first two components (PC1 and PC2) obtained after PCA on normalized XIC peak
areas of oleuropein aglycone, ligstroside aglycone, oleacin and oleocanthal, resulting from the LC-ESI-FTMS analysis
of 22 Apulian olive oils produced using three- (3F) or two-phase (2F) decanters.
We wish to mention that two EVOO samples obtained by three-phases (3F) processing were in the same
region as the two-phases (2F) ones, i.e., were characterized by a higher content in secoiridoids with respect
to typical three-phases oils. The following explanations were attempted for these samples: one of them was
subjected to a prolonged oil/water decantation, instead of vertical centrifugation, which is known to decrease
the olive oil secoiridoid content [5]. Moreover, the same oil was finally stored in a nitrogen-saturated
container, a procedure able to minimize the secoiridoid loss due to oxidation. The second exception in the
scatterplot of Fig. 1 was represented by the only olive oil, in the analyzed set, produced from a blend of
Coratina and Leccino cultivars. This might have influenced positively the original content of oleuropein and
ligstroside, i.e., the precursors of the four secoiridoids subsequently analyzed in olive oil.
References
1. E. Tripoli, M. Giammanco, G. Tabacchi, D. Di Majo, S. Giammanco, M. La Guardia, Nutr. Res. Rev., 18 (2005) 98-
112.
2. D. Boskou (Ed.), Olive and Olive Oil Bioactive Constituents, AOCS Press, 2015.
3. M. Servili, A. Taticchi, S. Esposto, B. Sordini, S. Urbani, Technological Aspects of Olive Oil Production in I.
Muzzalupo (Ed.), Olive Germoplasm, IntechOpen (2012) DOI: 10.5772/3314.
4. S. Vichi, N. Cortes-Francisco, J. Caixach; J. Chromatog. A, 1301 (2013), pp 48-59.
5. R. Pascale, G. Bianco, R.I. Cataldi, A. Buchicchio, I. Losito, G. Altieri, F. Genovese, A. Tauriello, G.C. Di Renzo,
M.C. Lafiosca, J. Am. Oil Chem. Soc., 95 (2018), pp 665-671.
42
OC17
Disclosing the key biological target of Crellastatin A through a combination of proteomic
approaches
E. Morretta1, M. Di Mauro
1, C. Festa,
2 R. Riccio,
1 A. Casapullo,
1 M. C. Monti
1
1Department of Pharmacy, University of Salerno, via Giovanni Paolo II, 132 84084, Fisciano, Italy
2Department of Pharmacy, University of Naples “Federico II”, Naples, Italy
Keywords: Proteomics, limited proteolysis, multiple reaction monitoring.
The identification of target proteins of natural products is crucial to understand their mechanism of action in
order to develop new molecular probes and/or therapeutic drugs.
In this scenario, we applied the Drug Affinity Responsive Target Stability (DARTS) approach to characterize
the interactome of Crellastatin A (CreA), a marine sulphated bis-steroid from the sponge Crella sp. [1].
This strategy relies on the evidence that a protein might become less susceptible to proteolysis when it is
bound to a molecule, as a result in the shift of its thermodynamic landscape to favor the more stable ligand-
bound state [2]. The conformational changes between the protein and its ligand-bound form can be detected
by SDS-PAGE and mass spectrometry, through the altered proteolytic pattern reached by the protein when
exposed to an unspecific protease, such as subtilisin. DARTS led to the identification of Poly [ADP-ribose]
polymerase 1 (PARP-1) as CreA most reliable partner, as also validated by immunoblotting.
In order to identify PARP1 regions involved in the interaction with CreA, we also performed LiP-MRM
(Limited Proteolysis-Multiple Reaction Monitoring) experiments [3].
Because of the natural compound exerted protection, PARP1 region(s) interacting with CreA are less prone
to subtilisin digestion and thus generate more abundant fully tryptic peptides compared to a control sample,
which is in turn more prone to subtilisin and will produce more half-tryptic peptides. Tryptic peptides
intensities can be easily measured by MRM-MS directly in a complex proteome matrix, without sample
enrichment or protein labeling. Finally, LiP MRM obtained data were validated through conventional limited
proteolysis, carried out on the human recombinant PARP1. References 1. D'Auria, M. V.; Giannini, C.; Zampella, A.; Minale, L.; Debitus, C. and Roussakis, C. J. Org. Chem., 63 (1998), pp
7382-7388.
2. Lomenick, B.; Hao, R.; Jonai, N.; Chin, R. M.; Aghajan, M.; Warburton, S.; Wang, J.; Wu, R. P.; Gomez, F.; Loo, J.
A.; Wohlschlegel, J. A.; Vondriska, T. M.; Pelletiere, J.; Herschman, H. R.; Clardy, J.; Clarke, C. F.; Huang, J. PNAS,
106 (2009), pp 21984–21989.
3. Feng, Y.; De Franceschi, G.; Kahraman, A.; Soste, M.; Melnik, A.; Boersema, P. J.; Polverino de Laureto, P.;
Nikolaev, Y.; Oliveira, A. P.; Picotti, P. Nat. Biotecnology, 32 (2014), pp 1036-1044.
43
POSTER COMMUNICATIONS
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45
P1
Multiclass screening of banned substances in urine and liver by LC-MS/MS
S. Moretti1, C. Barola
1, D. Giusepponi
1, G. Scortichini
2, M. Pallecchi
2, G. Bartolucci
2,
R. Galarini1
1 Istituto Zooprofilattico Sperimentale dell’Umbria e delle Marche “Togo Rosati”, Via G.
Salvemini, 1-06126 – Perugia, Italy; 2Department of Neurosciences, Psychology, Drug Research
and Child Health, University of Florence, Via U. Schiff, 6 -50019 Sesto F.no (FI), Florence, Italy Keywords: urine, liver, banned substances
Treatments of food producing animals with growth-promoting agents are prohibited in European Union from
about 30 years. The traditional single-class analytical methods are more and more criticized because of the
low and, probably, unrealistic percentages of non-compliant results found against the high cost of official
controls annually performed all along the European territory. According to the last available EFSA Report
(2016), of the 185179 samples analysed in all animal species and product categories for banned substances
(groups from A1 to A6), only 133 samples were not compliant (0.07%) [1]. Starting from a previous method
developed in our laboratory in bile and urine using liquid-chromatography coupled to high resolution mass
spectrometry (LC-HRMS) [2], in this study we implemented a multiclass screening procedure in urine and
liver applying liquidchromatography coupled to triple quadrupole mass spectrometry (LC-MS/MS, 6500
QTRAP, AB Sciex). The sample preparation scheme is shown in Figure 1.
Fig. 1. Sample treatment scheme for urine and liver
In order to purify and recover the wide range of the tested analytes (57), two separate clean-up were carried
out in urine applying three types of SPE sorbents. In liver only two classes of anabolic agents (beta-agonists
and corticosteroids) were included in the official plans, allowing a simpler sample treatment.
46
After its development, the procedure was validated following the European guidelines analysing forty
samples of different origin per matrix without (blank) and with fortification [3]. In urine, for 53 analytes out
of 57, the percentage of false compliant results was lower than 5%, as required by the EU criteria. The
spiking level was 0.2 µg/L for beta-agonists and chloramphenicol and 1 µg/L for all the other drugs. In liver,
14 out of the sixteen tested analytes were detectable at 1 µg/kg (false negative rate < 5%). In Figure 2, the
LC-MS/MS chromatograms of a blank and spiked liver sample are reported. The peaks of two beta-agonists
(salbutamol and terbutaline) and three corticosteroids (betamethasone, dexamethasone and flumethasone) are
shown in the bottom of the Figure.
Fig. 2. Blank bovine liver (top) and blank bovine liver fortified at 1 µg/kg (bottom)
The method was able to detect more than 50 molecules in urine (beta-agonists, corticosteroids, resorcylic
acid lactones, steroids, stilbenes, tranquillizers and nitroimidazoles) and 14 in liver (betaagonists and
corticosteroids) encompassing the majority of group A substances included in the National Residue Plans.
Although laborious, the procedure can replace several of the current singleclass tests, dramatically
decreasing the number of procedures, which have to be accredited and managed within the official
laboratories. Last but not least, multiclass approaches can improve the food safety standard in EU.
Acknowledgements This work was supported by the Italian Health Ministry (Ricerca Corrente IZS UM
011/2015 RC “Development and validation of new screening methods to determine residues of banned
substances in urine and liver”).
References
1 EFSA “Report for 2016 on the results from the monitoring of veterinary medicinal product residues and other
substances in live animals and animal products” www.efsa.europa.eu/en/supporting/pub/en-1358
2 S. Moretti et al. “Multiclass screening method to detect more than fifty banned substances in bovine bile and
urine” Analytica Chimica Acta, 32 (2015), pp 25-34.
3 Community Reference Laboratories “Guidelines for the Validation of Screening Methods for Residues of
Veterinary Medicines” 20/1/2010, https://ec.europa.eu/food/sites/food/files/safety/docs/cs_vet-med-
residues_guideline_validation_screening_en.pdf
47
P2
Computational approaches for identification and quantification of phytochemicals in different
complex matrices
Stefano Di Bona1, Laura Goracci
1, Gabriele Cruciani
1
1 Department of Chemistry, Biology and Biotechnology, Università degli Studi di Perugia, Via Elce
di Sotto 8, 06123 Perugia
Keywords: Tandem mass spectrometry, untargeted, omics sciences
The metabolome fingerprints identification in plants extracts is a bottleneck for the untargeted studies in
different research fields. These fingerprints are constituted by a large number of compounds with different
structures and in different concentration [1]. Consequently, the development of fast identification methods
for different compounds classes in plant extracts results very interesting. In this study, we want to obtain a
software where it is possible to identify different phytochemicals into a complex matrix and to do a statistical
analysis of different samples similar at Lipostar [2], but with a database of phytochemicals classes based on
their fragmentation rules in tandem mass spectrometry.
Fig. 4. An example of identification of phytochemical costituents in a sample of wine
References 1. V. Bhatt, S. Sharma, N. Kumar et al, J. Pharm. and Biomed. Analysis, (2017), 132, pp 46–55.
2. L. Goracci, S. Tortorella, P.Tiberi, R. M. Pellegrino, A. Di Veroli, A. Valeri, and G. Cruciani, Anal. Chem., (2017),
89 (11), pp 6257-6264.
48
P3
Fast determination of bioactive proresolving lipids in plasma by μSPE-UHPLC-MS/MS
F. Fanti1, D. Tortolani
1, F. Vincenti
2, C. Rapino
1, D. Compagnone
1, M. Maccarone
3, M. Sergi
1
1University of Teramo, Faculty of Bioscience and Technology for Food, Agriculture and
Environment, Teramo, Italy 2Sapienza University of Rome, Department of Chemistry, 00185 Rome, Italy,
3Campus Biomedico
University of Rome, Faculty of Medicine and Surgery, 00128 Roma, Italy
Keywords: Bioactive Lipids, μSPE, UHPLC-MS/MS
In the event of tissue damages or infections, innate immune cells, such as granulocytes and
monocytes/macrophages, are recruited to the damaged site and rapidly generate classical eicosanoids; this
class of lipid mediators is responsible for acute inflammation characterized by the so-called “cardinal signs”
of inflammation: redness, heat, swelling, pain, and loss of function (1). But, in other hand, some lipids can
actively terminate inflammation and drive the restoration of full tissue homeostasis by activating the signs of
resolution: removal, relief, restoration, regeneration, and remission (2).
In this context, the outcome of inflammation depends also on the other two families of bioactive lipids,and
endocannabinoidss; these compounds regulate numerous important cellular processes triggering the
mechanisms that underlie cell and tissue adaption to inflammatory events. Indeed, chronic inflammation
represents often the causative agent of the damage associated to many pathologies, such as cancer,
autoimmune, metabolic, cardiovascular, and neurodegenerative diseases.
Resolvins, where D series derived from the n-3 fatty acid docosahexaenoic acid (DHA) and E-series
resolvins derived from the n-3 fatty acid eicosapentaenoic acid (EPA), acting via G-coupled protein
receptors, have potent anti-inflammatory and proresolving actions and reduce airway inflammation, dermal
inflammation, colitis, arthritis, and postoperative pain. Studies have shown that these mediators increase with
time during the inflammatory process (3), in other hand there is increasing and exciting evidence showing
that endocannabinoids regulate the immune response at both the innate and adaptive immune response.
Immune cells are not only able to be influenced but are also able to generate and secrete endocannabinoids
that lead to changes in immune cell behavior as well as the production of other inflammatory factors that
subsequently influence tissue inflammation (4).
Because of their clinical interest, the development of high-throughput analytical methods for targeted
determination of eicosanoid panels is demanded. Quantitative analysis of pro-resolving lipids in biological
samples calls for highly sensitive and selective detection techniques. Therefore, to better understand the
processes underlying the resolution of inflammation a simultaneous knowledge of these bioactive lipids is
necessary.
The aim of this work is the development of an analytical method that allows the screening of two classes of
bioactive lipids that take part in the pro-resolving phase through a unique LC-MS/MS method preceded by a
μSPE extraction from human plasma, by which it is possible to extract both classes of analytes from the
same sample, not only to obtain more precise information regarding the relationships between the two
different classes of bioactive lipids, but also with the aim of minimizing the use of biological fluids human in
analysis.
References
1. C. Nathan, Nature (2002), 420(6917), 846–852;
2. C. N. Serhan, Nature (2014), 510(7503), 92–101. doi:10.1038/nature13479
3. N. Barrie, and N. Manolios, European Journal of Rheumatology (2017), 4(3), 210–218.
4. E. Mas, K.D. Croft, P. Zahra, A. Barden, and T.A. Mori, Clinical Chemistry (2012), 58(10), 1476–1484.
49
P4
UHPLC-HRMS/MS-based rapid identification of bioactive molecules for functional recovery
of Greco grape leaves
Marialuisa Formato, Giuseppina Crescente, Maria Tommasina Pecoraro,
Alessandro Bianco, Simona Piccolella, Severina Pacifico
Department of Environmental Biological and Pharmaceutical Sciences, University of Campania
Luigi Vanvitelli, Caserta, Italy
Keywords: food waste recovery, grape leaves, UHPLC-HR MS/MS analysis, flavonol glycuronides
recovery
Grape cultivation is one of the main agro-economic activities worldwide, with over 60 million tons produced
globally every year. The production is intended for fresh consumption (e.g. table fruit, juice), and
winemaking process.[1]
Indeed, wine production involves the genesis of large amounts of by-products and
waste residues, causing considerable concern for environmental sustainability, waste of resources and human
health. In this context, the actual and growing demand for green materials and renewable resources of
bioactive compounds (for pharmaceutical, cosmetic and food sectors) represents a valuable response to
recycle, reuse and recover wine waste residues. The valorisation of wine by-products and wastes is prompt to
realize a virtuous system, able to enhance every single phase of the working process and thus reduce its
environmental impact. Leaves are an example of waste in the wine supply chain, but they, unlike pomace
and seeds, are not included as by-products in the Ministerial Decree 27/11/2008. Indeed, their production is
massive in the early stages of winemaking, in de-stemming and peeling processes. In this consciousness,
leaves of Vitis vinifera cv. Greco di Tufo, collected in the Benevento province (October 2017), underwent,
after reduction, lyophilization and pulverization, ultrasound assisted maceration in ethanol. The alcoholic
extract, as evidenced by a preliminary UHPLC HRMS analysis, showed a high metabolic complexity being
rich in (poly)phenol, alkylphenol, glycerolipid and glycerophospholipid components. Thus, the extract was
further fractionated, obtaining, among the others, a fraction enriched in flavonol glycosides and
glycuronides. In fact, several derivatives of myricetin, quercetin, kaempferol, and isorhamnetin were
tentatively identified based on their relative retention time and TOF-MS2 data. As the localization of
saccharidic moiety in glycuronide derivatives proved to be difficult due to the lack of well-established
fragmentation pattern and/or the absence of characteristic key fragments,[2]
to obtain useful MS information
and to eliminate matrix effect redundancies, the isolation of the most abundant extract’s compound was
achieved, and its structure was elucidated by means of UV-Vis, and NMR spectroscopy. HR MS/MS spectra
of the compound, quercetin-3-O-glucuronide, allowed us to deeply rationalize its fragmentation pattern, and
to unravel the main differences between MS/MS behavior of flavonol glycosides and glycuronides.
Furthermore, cytotoxicity assessment on alcoholic extracts, its (poly)phenol rich fraction and the pure
isolated compound was carried out towards central nervous system cell lines. Based on data acquired, which
showed the chemoprotective effect of both the (poly)phenol fraction and quercetin-3-O-glucuronide, their
anti-acetylcholinesterase activity was also evaluated. New investigations at cell level will aim at ascertaining
the neuroprotective potential for the functional recovery of this precious waste made in Campania.
References
1. Zhang, L., Zhu, M., Shi, T., Guo, C., Huang, Y., Chen, Y., Xie, M. (2017). Recovery of dietary fiber and polyphenol
from grape juice pomace and evaluation of their functional properties and polyphenol compositions. Food Funct.
8(1):341-351.
2. Dueñas, M., Mingo-Chornet, H., Pérez-Alonso, J.J., Di Paola-Naranjo, R., González-Paramás, A.M., Santos-Buelga,
C. (2008). Preparation of quercetin glucuronides and characterization by HPLC–DAD–ESI/MS. Eur Food Res
Technol 227: 1069-1076.
50
P5
Natural steroidal precursors: a critical issue in anti-doping IRMS analysis. The prednisolone
and prednisone case study
Loredana Iannella1,2
, Cristiana Colamonici1, Davide Curcio
1, Francesco Botrè
1,3,
Xavier de la Torre1
1 Laboratorio Antidoping, Federazione Medico Sportiva Italiana, Rome, Italy,
2 Dipartimento di
Chimica e Tecnologie del Farmaco and 3 Dipartimento di Medicina Sperimentale, “Sapienza”
Università di Roma, Rome, Italy
Keywords: anabolic androgenic steroids, pseudoendogenous steroids, 13
C signature
The anabolic androgenic steroids (AAS) are a class of pharmaceutical drugs widely used for a broad range of
therapeutic applications and often abused by athletes to increase the muscle mass and enhance the sportive
performance. As ergogenic doping agents, they are banned by the World Anti-Doping Agency (WADA) and
included in the Prohibited List [1]. The steroidal hormones are physiologically produced in the body and
then, doping violation cannot be based on their mere presence. Prohibited steroids (such as 19-norsteroids,
boldenone and its metabolite and prednisone and prednisolone) could be also enzymatically produced ex-vivo
from endogenous steroids naturally excreted in urine as a result of urinary microbial flora activity. The
discrimination between the exogenous drugs and the endogenously or in situ produced compounds is
performed through their 13
C composition study in a gas chromatography coupled to isotope ratio mass
spectrometry (GC-C-IRMS) analysis. The 13
C/12
C ratio (expressed as delta values, δ13
C ‰) of synthetic
steroids depends on the natural sources selected for their pharmaceutical synthesis. They are mainly
produced by a combination of microbial and chemical processes: phytosterols and sapogenins have been
classically used as the basic natural precursors in their industrial manufacturing [2]. Phytosterols are
collected as discarded products during the soybean-oil production: stigmasterol, β-sitosterol, campesterol and
brassicasterol are the most typical ones [3-4]. Sapogenins, such as hecogenin, tiogenin and diosgenin, are
primarily extracted from roots of various Dioscorea species of Mexico yams [5]. Both phytosterols and
sapogenins derive from plants that assimilate CO2 through the C3 photosynthetic pathway. Compared to C4
plants (δ13
C from –6 to –19 ‰), the soybeans and yams are characterized by a low 13
C/12
C ratio and δ13
C
from –22 to –34 ‰ [6]. The isotopic endogenous profile is affected by the dietary habits: differences
between people living in different geographic areas are extensively known reflecting their C3 or C4 plant
enrich diet [7]. The endogenous reference delta values range from –16 ‰ to –25 ‰ in the worldwide
population, and then are normally less negative than the exogenous formulations synthetized from C3 plants
[8]. A critical issue in the anti-doping IRMS analyses is the commercial availability of pharmaceutical
preparations with delta values overlapping the endogenous δ13
C.
Ten prednisolone and prednisone pharmaceutical preparations were analyzed: eight were purchased in Italy
and two were provided by a Belgian pharmacy. Prednisolone and prednisone are two synthetic
glucocorticoids banned “in competition” when administered by systemic routes [9]. The anti-doping IRMS
analysis of prednisolone and prednisone is a mandatory requirement to disclose if their urinary presence can
be attributed to an exogenous administration or to an in situ microbial formation from endogenous cortisone
and cortisol respectively. The analysis was performed through a specific GC method developed and validated
for each compound. The exogenous average δ13
C (‰) value of the Italian pharmaceutical products was -
28.96 ± 0.39 ‰ which is more negative than endogenous reference delta values.
The two Belgian prednisolone drugs displayed one a delta value of -30.29 ± 0.23 ‰, and the other a δ13
C less
negative (-16.49 ± 0.40 ‰) than the previous preparations. This exogenous prednisolone δ13
C is still
distinguishable from the European reference delta values, but within the endogenous steroids values range
measured in the Americas. The unexpected δ13
C value can be reasonably explained by presuming the
selection of a starting natural material not derived from C3 plants.
51
References
1. WADA. International standard - The Prohibited List,https://www.wada-ama.org/en/what-we-do/the-prohibited-list
2. Y. Al Jasem, M. Khan, A. Taha, and T. Thiemann; Mediterranean Journal of Chemistry 3 (2014), pp. 796–830.
3. F.Q. Wang, K. Yao, D.Z. Wei and H. El-Shemy editor, Soybean and health, InTech—Open Access Publisher,
Rijeka, 2011, pp 232–252.
4. L. Fernández‐ Cabezón, B. Galán, and J.L. García; Frontiers in Microbiology 9: 958 (2018), pp 1-15.
5. I. Herráiz; Methods in Molecular Biology 1645 (2017), pp 15-27.
6. D. Cornet, J. Sierra, R. Bonhomme; Photosynthetica 45 (2007), pp 303–305.
7. A.T. Cawley, G.J. Trout, R. Kazlauskas, C.J. Howe, A.V. George; Steroids 74 (2009), pp 379–392.
8. X. de La Torre, D. Jardines, D. Curcio, C. Colamonici, F. Botrè; Rapid Communication in Mass Spectrometry 33
(2019), pp 579–586.
9. WADA. Technical Letter. TL03/2017- In situ Formation of Exogenous Compounds in urine Samples.
52
P6
Targeted and non-targeted metabolomics to study developmental neurotoxicity of biocides
Pim Leonards1, Arnd Ingendoh
2, Magdalene Reinkensmeier
2, Aiko Barsch
2, Heiko Neuweger
2
1VU University Amsterdam, Netherlands, ²Bruker Daltonics, Bremen, Germany
Keywords: metabolomics, biocides, neurotoxicity
Serious concern has arisen worldwide regarding the dramatic increase in incidences of learning and
developmental disorders in children. Recently, various epidemiological studies have indicated that during
childhood, exposure to low doses of biologically active contaminants in the environment can have
deleterious effects on cognitive and behavioral development. The aim of the current study was to investigate
the behavioral and cognitive effects of pesticide exposure in mice and to study the underlying molecular
mechanisms of the observed associations using metabolomics. This study was part of the European
DENAMIC project.
Mice were exposed to a single dose at PND10 of the pesticides chlorpyrifos, carbaryl, cypermethrin, PFHxS
or endosuldfan (0.5 to 20 mg/kg bw). Metabolomic analysis was carried out using methanolic:chloroform
extracts of mouse brain tissues (cerebral cortex and hippocampus). Data for targeted and non-targeted
metabolite analysis was acquired by HILIC-QTOF-MS/MS (Compact, Bruker Daltonics). MetaboScape 3.0
(Bruker Daltonics) was used for data processing.
The applied software provides a fully integrated workflow for discovery metabolomics and enabled a higher
throughput for profiling of complex tissue extracts. Data analysis time was reduced due to the simultaneous
and confident assignment of known targets and tentative annotation of unknown peaks. Early-life exposure
of pesticides can result in persistent effects on behavior and cognition later in life.
53
P7
PASEF for ultra-sensitive shotgun proteomics
Romano Hebeler1, Heiner Koch
1, Markus Lubeck
1, Martina Stella
2, Scarlet Koch
1
1Bruker Daltonik GmbH, Bremen, Germany
2 Bruker Daltonics, Macerata, Italy
Keywords: shotgun proteomics, trapped ion mobility spectrometry, proteome coverage
Time and space focusing of ion clusters focused by their collisional cross section in (TIMS) boosts the
sensitivity of QTOF MS. In addition, TIMS enables parallel accumulation–serial fragmentation (PASEF),
which couples high sequencing speed (>120 Hz) at 100% duty cycle without sacrificing spectral quality. We
show the combined increase in sensitivity and speed can be used for deep proteome coverage using very low
sample amounts (< 10 ng).
A dilution series of a tryptic digest from a human cancer cell line (HeLa) was used. LC was performed on a
nanoElute (Bruker Daltonics) using nano-flow HPLC and a 25 cm column with integrated emitter
(IonOpticks, Australia) and a 60 min gradient. Data was generated using the PASEF acquisition mode with a
cycle time of 1.1 s. Data analysis was performed using PEAKS studio (Bioinformatics Solution Inc.) and
MaxQuant (Jürgen Cox, Max Planck Institute of Biochemistry).
Sensitivity of the timsTOF Pro instrument with PASEF was evaluated on sample concentrations of ~ 3 ng
and up to 100 ng on column. Using the lowest amount on column, which corresponds to 10 HeLa cells (~ 3
ng), more than 1,650 protein families from 7,000 unique peptide sequences were identified. A linear increase
in ID numbers was observed up to the identification of 5,091 and a sample amount of 100 ng. This linear
increase is a testament to the 100% duty cycle coupled with fast sequencing speed. We tested four different
admixtures of HeLa and E.coli where the final protein mix was 100 ng and the ratios varied from 3/2, 3/1,
7/1 and 16/1, respectively. Proteome admixtures showed good reproducibility and sensitivity. Applying
PASEF on the timsTOF Pro mass spectrometer provides deep proteome coverage at low sample
concentrations.
54
P8
Chemical composition and antioxidant activity of Cannabis sativa L. 'Futura 75' essential oil:
effect of the distillation time
Sara Palmieri, Antonella Ricci, Claudio Lo Sterzo
Faculty of Bioscience and Technology for Food, Agriculture and Environment, University of
Teramo, Via Balzarini 1, 64100 Teramo, Italy
Keywords: Cannabis sativa L., GC-MS, antioxidant activity
In the last decades, there is an increasing interest in the cultivation of industrial hemp, Cannabis sativa L.
with low content of delta9-tetrahydrocannabinol (THC), the psychotropic agent, due to its use in different
field, among them food, cosmetic and pharmaceutical industry [1]. In particular, have been investigated C.
sativa extracts with high content of cannabidiol (CBD), that is a molecule showing different pharmacological
activities, including an antitumoral action [2]. At the same time, characterization studies of the hemp
essential oil (EO) were conducted and its antioxidant and antimicrobial activity was shown [3,4,5].
The aim of this work was to explore the effect of the distillation time on the chemical composition and the
antioxidant properties of the EOs obtained by steam distillation from C. sativa cv. Futura 75, cultivated in the
Abruzzo Region.
Dried inflorescence, provided by a local farm and preserved in vacuum bags until distillation, were subjected
to steam distillation at two, four and six hours, affording EOs with 0,13%, 0,16% and 0,26% yield,
respectively. The chemical composition of the obtained EOs was determined by GC-MS analysis, and was
compared with the volatile fraction composition of dried inflorescences matrix, carried out by solid phase
microextraction (SPME) coupled to GC-MS technique. EOs at different time contain the same main
compounds, but with different relative abundance.
The EOs were evaluated for the total phenolic content (TPC) by Folin-Ciocâlteu method, and for the
antioxidant activity (AOC) by TEAC/ABTS, FRAP and DPPH assays.
EOs distilled at more time showed higher yield, TPC and AOC, with four and six hours more similar to each
other, and more performing than two hours one.
References
1. O. Ranalli, G. Venturi; Euphytica, 140 (2004), pp 1-6
2. S. Burstein; Biorganic &Medicinal Chemistry, 23 (2015), pp 1377-85
3. J. Novak, K. Zitterl-Eglseer, S.G Deans, C.M Franz; Flavour and fragrance journal, 16 (2001), pp 259-262
4. L. Nissen, A. Zatta, I. Stefanini, S. Grandi, B. Sgorbati, B. Biavati, A. Monti; Fitoterapia, 81 (2010), pp 413-419.
5. A. Nafis, A. Kasrati, C.A. Jamali, N. Mezriouj, W. Setzer, A. Abbad, L. Hassani; Industrial Crops and Products,
137 (2019) pp 396-400.
55
P9
Allergens in food: how to quantify?
G. Saluti1, F. Paoletti
1, M.S. Altissimi
1, M.N. Haouet
1, T. Bladek
2, R. Galarini
1
1 Istituto Zooprofilattico Sperimentale dell’Umbria e delle Marche “Togo Rosati”, Via Salvemini,
1- 06126 – Perugia, Italy; 2Department of Pharmacology and Toxicology, National Veterinary
Research Institute, Al. Partyzantow 57, 24-100 - Pulawy, Poland
Keywords: allergens, food, LC-HR-MS/MS
Food allergy is a rising global health problem. In order to protect consumers, the presence of 14 ingredients
causing potential allergenic reactions is required to be stated in food labelling by European legislation
(Regulation UE 1169/2011). An accidental occurrence of allergens in food is also possible due to cross-
contamination phenomenon. In this work, the issue of quantification of egg and milk proteins in cookies has
been investigated. HQGLPQEVLNENLLR (α-S1-casein, milk), GGLEPINFQTAADQAR (ovalbumin,
white egg) and NIPFAEYPTYK (vitellogenin-2, yolk egg) were selected for quantification purposes together
with the relevant labelled peptides (IS1). In addition, also the so-called “long isotope-labelled peptides” (IS2)
were synthetized as analogues of the target peptides, but with slight modification on the amino acid sequence
to correct variations during the tryptic digestion [1]. The instrumental analysis was performed by liquid-
chromatography coupled to a quadrupole-orbitrap detector (LC-Q-Exactive Plus, Thermo Scientific, San
Jose, CA, USA) acquiring three product ions for each peptide in Parallel Reaction Monitoring (PRM) mode.
The sample preparation was that proposed by Planque et al. (2017) with some modifications [2]. Figure 1
shows the LC-MS/MS chromatograms of an allergen-free cookie sample (left) and the same sample spiked
with egg and milk proteins (right).
Fig. 5. No-spiked and spiked cookies at 2 mg/kg and 5 mg/kg with milk and egg proteins, respectively.
Peptides with asterisks(*)
are isotopically labelled with 13
C and 15
N
56
The results obtained using internal calibration are listed in Table 1. This strategy consisted in the addition of
the relevant IS2s at the beginning of the sample preparation and then quantifying the peptides through
matrix-matched curves using the ratio between the peak area of the native peptide and of its IS1. Three
different types of cookies (A, B and C) were tested with matrix-matched curves prepared using the same
sample (AA, B
B and C
C) or different ones (A
B, A
C, B
A etc). Unsatisfactory data were obtained being
recoveries out of the accepted range (60-120%) [3]. These results were due to the concomitant influence of
the relative matrix effect in MS detection and the inefficiency of IS2 to correct digestion-step-related effects.
On the other hand, the standard addition method consisting in the addition of known amounts of the allergen
standard solutions directly in the sample to be analysed, allowed better recoveries (86, 92 and 96 % for
NIPFAEYPTYK, HQGLPQEVLNENLLR and GGLEPINFQTAADQAR, respectively). Further
experiments are in progress.
Table 1 – Apparent recoveries (%) and SD (%) obtained in spiked cookies using internal calibration with
long chain labelled peptides as IS
Peptide HQGLPQEVLNENLLR GGLEPINFQTAADQAR NIPFAEYPTYK
ID sample Trueness
(n=3)
Trueness
(n=3)
Trueness
(n=3)
AA 89 ± 2 65 ± 2 116 ± 11
BA 120 ± 16 41 ± 8 77 ± 12
CA 106 ± 9 53 ± 2 152 ± 12
AB 81 ± 1 62 ± 1 113 ± 11
BB 108 ± 14 39 ± 8 75 ± 11
CB 96 ± 8 51 ± 2 148 ± 11
AC 100 ± 2 61 ± 1 90 ± 9
BC 134 ± 18 38 ± 8 60 ± 9
CC 119 ± 10 50 ± 2 117 ± 9
Data in bold are out of the required interval (60-120%)[3]
Although laborious, at present the standard addition method seems to be the more suitable strategy for
allergen quantification in complex matrices. On the other hand, the “gold standard” approach, i.e. the
application of the isotopic dilution methodology using labelled proteins is not feasible for its cost. Our data
confirm the observations recently published by Planque et al. (2019) who studied other foodstuffs, selecting
different signature peptides and IS2 [4].
Acknowledgements
This work was supported by the Italian Health Ministry (Ricerca Corrente IZS UM 007/2017 RC
“Relationship between food allergy and territory: epidemiological evolution, variation of the food allergic
power and analytical tools”). T.B. was funded by KNOW (Leading National Research Centre) Scientific
Consortium “Healthy Animal - Safe Food”, decision of Polish Ministry of Science and Higher Education No.
05 1/KNOW2/2015”
References 1. Q. Chen et al. “Quantification of bovine β-casein allergen in baked foodstuffs based on ultra-performance liquid
chromatography with tandem mass spectrometry” Food Additives & Contaminants: Part A, 32 (2015), pp 25-34.
2. M. Planque at al. “Liquid chromatography coupled to tandem mass spectrometry for detecting ten allergens in
complex and incurred foodstuffs” Journal of Chromatography A, 1530 (2017), pp 138-151.
3. AOAC SMPR 2016.002. Standard Method Performance Requirements (SMPRs®) for Detection and Quantitation of
Selected Food Allergens.
4. M. Planque et al. “Development of a strategy for the quantification of food allergens in several food products by
mass spectrometry in a routine laboratory” Food Chemistry, 274 (2019), pp. 35-45.
57
P10
Metabolomic approaches to investigate the role of the mitochondrial regulator Zc3h10
in adipocytes
Silvia Pedretti, Matteo Audano, Emma De Fabiani, Donatella Caruso, Nico Mitro
Dipartimento di Scienze Farmacologiche e Biomolecolari (DiSFeB), Università degli Studi di
Milano, Milano, Italy
Keywords: Zc3h10, metabolism; adipocytes;
Mitochondria play a crucial role in many cellular processes and they are essentials organelles for the health
of the cell. Beyond their contribution to energy production, they are key regulators of tissue development and
cell differentiation. We recently isolated the new mitochondrial regulator zinc finger CCCH-type containing
10 (Zc3h10) [1] and here, we validated its role during adipocytes differentiation.
C3H/10T1/2 cell line (mesenchymal stem cells) can be differentiated into white adipocytes using a specific
adipogenic cocktail. Quantification of different metabolites was performed with a liquid
chromatography/tandem mass spectrometry (LC-MS/MS) on an API-4000 triple quadrupole mass
spectrometer coupled with a HPLC system using a C18 column for amino acids and cyano-phase LUNA
column for metabolites.
Zc3h10 protein levels increases during C3H/10T1/2 differentiation. Zc3h10 silencing significantly affects
adipocyte differentiation and mitochondrial activity. To demonstrate that Zc3h10 plays a role in energy
metabolism we evaluated the intracellular levels of by-products belonging to the main metabolic pathways
(i.e. glycolysis, tricarboxylic acid (TCA) cycle and pentose phosphate pathway (PPP), amino acids) using
LC-MS/MS. Steady-state metabolomics indicated that lack of Zc3h10 led to decreased AMP, ADP, ATP and
NADH levels. We also observed reduced levels of acetyl-CoA, α-ketoglutarate (α-KG), citrate, and succinyl-
CoA and increased levels of glutamate. We also used metabolic tracers ([U-13
C6]-glucose, [U-13
C16]-
palmitate or [U-13
C5]-glutamine) to confirm that the flow of energy substrates into the TCA is affected by
Zc3h10 silencing.
Our results indicate that Zc3h10 expression increases during murine white adipocyte differentiation. Further,
Zc3h10 silencing in white preadipocytes and adipocytes deeply impaired mitochondrial function, decreased
adipogenic potential and altered metabolic profile.
These data annotate Zc3h10 as a new regulator of mitochondrial function and cell differentiation in
adipocytes.
References
1. Audano M., Pedretti S., Cermenati G., Brioschi E., Diaferia G.R., Ghisletti S., Cuomo A., Bonaldi T., Salerno F.,
Mora M., Grigore L., Garlaschelli K., Baragetti A., Bonacina F., Catapano A.L., Norata G.D., Crestani M., Caruso
D., Saez E., De Fabiani E., Mitro N., EMBO Reports, (2018) Mar 5, pii: e45531. doi: 10.15252/embr.201745531.
58
P11
Evaluation of onion skin polyphenols for the production of the biofunctional textiles:
a multi-analytical approach
Lucia Pucciarini1, Federica Ianni
1, Valentina Petesse
2, Federica Pellati
3, Virginia Brighenti
3,
Claudia Volpi4, Marco Gargaro
4, Benedetto Natalini
1, Catia Clementi
2, Roccaldo Sardella
1
1Department of Pharmaceutical Sciences, University of Perugia, Via Fabretti 48, 06123 Perugia,
Italy; 2Department of Chemistry Biology and Biotechnology, University of Perugia, Via Elce di
Sotto 8, 06123 Perugia, Italy, 3Department of Life Sciences, University of Modena and Reggio
Emilia, Via G. Campi 103, 41125 Modena, Italy; 4Department of Experimental Medicine,
University of Perugia, P.le Severi, 06132 Perugia, Italy
Keywords: onion skin; antioxidant activity; biofunctional textiles
The aqueous extract of dry onion skin waste from the ‘Dorata di Parma’ cultivar was evaluated as a new
potential source of biomolecules for the environmentally friendly production of colored biofunctional wool
yarns, obtained through dyeing procedures. The attention was especially addressed to their antioxidant and
UV protection properties. Based on the results of spectrophotometric and mass spectrometry analyses, the
obtained deep red-brown color was ascribed to quercetin and its glycoside derivatives. The application of the
Folin–Ciocalteu assay revealed good phenol uptakes on the wool fiber (higher than 27% for the textile after
the first dyeing cycle). The manufactured materials showed remarkable antioxidant activity along with a
pronounced ability to protect human skin against lipid peroxidation following UV radiation: 7.65 ± 1.43
(FRAP assay) and 13.60 (ORAC assay) mg equivalent trolox/g textile; lipid peroxidation inhibition up to
89.37%. The measured photoprotective and antioxidant activities were therefore attributed to the polyphenol
pool contained in the outer dried gold skins of onion. It is worth highlighting that citofluorimetric analysis
demonstrated that the aqueous extract does not have a significative influence on cell viability, neither is
capable of inducing a proapoptotic effect.
59
Author Index
A
Abbattista, R. OC1, OC16 16, 40
Aldini, G. OC5 24
Allegrini, P. OC5 24 Altissimi, M.S. P9 55
Altomare, A. OC5 24
Amante, E. OC9 30 Arnoldi, L. OC5 24
Audano, M. P10 57
B Barola, C. P1 45
Baron, G. OC5 24
Barsch, A. P6 52 Bartolucci, G. OC10, P1 31, 46
Bedont, S. OC4 22
Bianchi, F. OC15 38 Bianco, A. P4 49
Bianco, G. OC12 34
Biancolillo, A. OC9 30 Bladek, T. P9 55
Borghi, E. OC5 24
Borgo, F. OC5 24 Botrè, F. OC2, P5 18, 50
Braconi, L. OC10 31
Brighenti, V. P11 58 Bro, R. OC9 30
Burico, M. OC4 22
C Calvano, C.D. OC1, OC16 16, 40
Campos, A. PL2 22 Capitoli, G. OC7 28
Careri, M. OC15 38
Carini, M. OC5 24 Caruso, D. PL1, P10 15, 57
Casapullo, A OC17 42
Castellaneta, A. OC16 40 Cataldi, T.R.I. OC1, OC16 16, 40
Cellucci, L. OC14 37
Chinello, C. OC7 28 Clementi, C. P11 58
Colamonici, C. P5 50
Compagnone, D.lo P3 48 Compagnone, D.io OC14 37
Crescente, G. P4 49
Cruciani, G. P2 47 Curcio, D. P5 50
Curini, R. OC14 37
D De Ceglie, C. OC1, OC16 16, 40
De Fabiani, E. P10 57
de la Torre, X. OC2, P5 18, 50 Dei, S. OC10 31
Dell’Agli, M. PL1 16
Denti, V. OC7 28 Di Bona, S. P2 47
Di Mauro, M. OC17 42
Di Murro, V. OC2 18
E Esposito, S. OC3 20
F Fanti, F. OC14, P3 37, 48
Festa, C. OC17 42
Flamini, E. OC4 22 Fodaroni, G. OC4 22
Formato, M.L. P4 49
Fumagalli, M. PL1 16
G Gaeta, C. OC12 34
Galarini, R. P1, P9 45, 55 Galimberti, S. OC7 28
Gargaro, M. P11 58
Ghilardi, C. OC11 33 Gianni, M. OC4 22
Giusepponi, D. P1 45
Goracci, L. P2 47 Götze, M. OC8 29
Gräslund, A. OC15 38
Gregori, A. OC14 37 Guarnerio, S. OC7 28
H Haouet, M.N. P9 55
Hebeler, R. P7 53
I Iacobucci, C. OC8 29
Iannece, P. OC12 34
Iannella, L. P5 50
Ianni, F. P11 58 Iannone, M OC2 18
Ilag, L.L. OC15 38
Ingendoh, A. P6 52
K Koch, H. P7 53
Koch, S. P7 53
L Langley, G.J. PL4 37
Lembo, A. OC3 20 Leonards, P. P6 52
Lo Sterzo, C. P8 54
Loo, J.A. PL3 28 Losito, I. OC1, OC16 16, 40
Lubeck, M. P7 53 Lucini, L. OC6 26
Lugli, F. OC13 35
M Maccarone, M. P3 48
Magni, F. OC7 28
Mahajneh, A. OC7 28 Martelli, G. OC12 34
Mattoli, L. OC4 22
Menicatti, M. OC10 31 Mitro, N. P10 57
Monteagudo, E. OC3 20
Montesano, C. OC14 37 Monti, M. C. OC17 42
Moracci, L. OC10 31
Morazzoni, P. OC5 24 Moretti, S. P1 45
Morretta, E OC17 42
Motwani, H.V. OC15 38
N Natalini, B. P11 58
Neuweger, H. P6 52
O Onzo, A. OC12 34
Orsatti, L. OC3 20 Österlund, N. OC15 38
Ottaviano, E. OC5 24
P Pacifico, S. P4 49
Pagni, F. OC7 28
Pallecchi, M. P1 45 Palmieri, S. P8 54
Palmisano, F. OC1, OC16 16, 40
Paoletti, F. P9 55 Pascale, R. OC12 34
Pecoraro, M.T. P4 49
Pedretti, S. P10 57 Pellati, F. P11 58
Petesse, V. P11 58
Piazza, S. PL1 16 Piccolella, S. P4 49
Pifano, C. OC12 34
Piga, I. OC7 28 Porpiglia, F. OC9 30
Proietti, G. OC4 22
Pucciarini, L. P11 58
Q Quaranta, A. OC15 38
Quintiero, C.M. OC4 22
R Rapino, C. P3 48
Regazzoni, L. OC5 24 Reinkensmeier, M. P6 52
Riboni, N. OC15 38
Ricci, A. P8 54 Riccio, R. OC17 42
Riva, A. OC5 24
Rocchetti, G. OC6 26
S Salomone, A. OC9 30
Saluti, G. P9 55 Sangiovanni, E. PL1 16
Sardella, R. P11 58
Scortichini, G. P1 45 Sergi, M. OC14, P3 37, 48
Sinz, A. OC8 29
Smith, A. OC7 28 Stella, M. P7 53
T Tamimi, S. OC4 22
Teodori, E. OC10 31
Tortolani, D. P3 48 Trevisan, M. OC6 26
V Vecchi, A. OC3 20
Vincenti, F. OC14, P3 37, 48
Vincenti, M. OC9 30
Volpi, C. P11 58
Z Zampieri, M. PL2 22