Analysis of tomato glycoalkaloids by liquid chromatography coupled with electrospray ionization tandem mass spectrometry.

Steroidal glycoalkaloids (SGAs) extracted from tomato leaves and berries (Lycopersicon esculentum Mill.) were separated and identified using optimized reversed-phase liquid chromatography with electrospray ionization (ESI) and ion trap mass spectrometry (ITMS). The ESI source polarity and chromatographic conditions were evaluated. The ESI spectra contain valuable information, which includes the mass of SGAs, the mass of the aglycones, and several characteristic fragment ions. Cleavage at the interglycosidic bonds proximal to the aglycones is the most prominent process in the ESI process. A protonated molecule, [M+H]+, accompanied by a mixed adduct ion, [M+H+Na]2+, was observed for a-tomatine (i.e., m/z 1034.7 and 528.9) and dehydrotomatine (i.e., m/z 1032.6 and 527.9) in positive ion mode spectra. The structures of these tomato glycoalkaloids were confirmed using tandem mass spectrometry. The identification of a new a-tomatine isomer glycoalkaloid, named filotomatine (MW 1033), which shares a common tetrasaccharide structure (i.e., lycotretraose) with a-tomatine and dehydrotomatine, and soladulcidine as an aglycone, is described for the first time. It occurs in significant amounts in the extracts of wild tomato foliage. Multistage mass spectrometry both of the protonated molecules and of the doubly charged ions was used for detailed structural elucidation of SGAs. Key fragmentations and regularities in fragmentation pathways are described and the fragmentation mechanisms involved are propose

Assay of riboflavin in sample wines by capillary zone electrophoresis and laser-induced fluorescence detection

To routinely assay the concentration of riboflavin (RF) in wines, a rapid and sensitive method was developed and evaluated. The method is based on a simple sample preparation, capillary zone electrophoretic separation and laser-induced fluorescence detection (CZE-LIF). Sample-preparation required only dilution and filtration. Under optimized conditions, the limit of detection of riboflavin was 0.5 mug/L, using a hydrodynamic sample introduction of 10 s at 54 mbar. The method was fully validated: the recovery of RF in wines was >95%. The concentrations of RF within the three sample types of Italian wines investigated here ranged from 69 to 151 mug/L with a mean value(+/-SD) of 112 +/- 25 mug/L, from 74 to 193 mug/L with a mean value of 115 +/- 45,ug/L, and from 156 to 292 mug/L with a mean value of 226 +/- 40 mug/L, for white, rose and red wines, respectively. Such an accurate and highly sensitive CZE-LIF method represents a powerful improvement over previous methods in terms of sensitivity, simplicity, and efficiency. It is well suited to satisfy the demands for accurate and sensitive detection with minimal sample preparation and cleanup

Establishing the occurrence of major and minorglucosinolates in Brassicaceae by LC-ESI-hybrid linearion-trap and Fourier-transform ion cyclotron resonancemass spectrometry

Glucosinolates (GLSs) are sulfur-rich plant secondary metabolites which occur in a variety of cruciferous vegetables and among various classes of them, genus Brassica exhibits a rich family of these phytochemicals at high, medium and low abundances. Liquid chromatography (LC) with electrospray ionization in negative ion mode (ESI-) coupled to a hybrid quadrupole lineariontrap (LTQ) and Fouriertransformioncyclotronresonancemass spectrometer (FTICRMS) was employed for the selective and sensitive determination of intact GLSs in crude sample extracts of broccoli (Brassica oleracea L. Var. italica), cauliflower (B. oleracea L. Var. Botrytis) and rocket salad (Eruca sativa L.) with a wide range of contents. When LTQ and FTICR mass analyzers are compared, the magnitude of the limit of detection was ca. 5/6-fold lower with the FTICR MS. In addition, the separation and detection by LC–ESI-FTICR MS provides a highly selective assay platform for unambiguous identification of GLSs, which can be extended to lower abundance (minor) GLSs without significant interferences of other compounds in the sample extracts. The analysis of Brassicaceae species emphasized the presence of eight minor GLSs, viz. 1-methylpropyl-GLS, 2-methylpropyl-GLS, 2-methylbutyl-GLS, 3-methylbutyl-GLS, n-pentyl-GLS, 3-methylpentyl-GLS, 4-methylpentyl-GLS and n-hexyl-GLS. The occurrence of these GLSs belonging to the saturated aliphatic side chain families C4, C5 and C6, presumably formed by chain elongation of leucine, homoleucine and dihomoleucine as primary amino acid precursors, is described. Based on their retention behavior and tandem MS spectra, all these minor compounds occurring in plant extracts of B. oleracea L. Var. italica, B. oleracea L. Var. Botrytis and E. sativa L. were tentatively identified

Synthesis and matrix properties of α-cyano-5-phenyl-2,4-pentadienic acid (CPPA) for intact proteins analysis by matrix-assisted laser desorption/ionization mass spectrometry

The effectiveness of a synthesized matrix, α-cyano-5-phenyl-2,4-pentadienic acid (CPPA), for protein analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) in complex samples such as foodstuff and bacterial extracts, is demonstrated. Ultraviolet (UV) absorption along with laser desorption/ionization mass spectrometry (LDI-MS) experiments were systematically conducted in positive ion mode under standard Nd:YLF laser excitation with the aim of characterizing the matrix in terms of wavelength absorption and proton affinity. Besides, the results for standard proteins revealed that CPPA significantly enhanced the protein signals, reduced the spot-to-spot variability and increased the spot homogeneity. The CPPA matrix was successful employed to investigate intact microorganisms, milk and seed extracts for protein profiling. Compared to conventional matrices such as sinapinic acid (SA), α-cyano-4-hydroxycinnamic acid (CHCA) and 4-chloro-α-cyanocinnamic acid (CClCA), CPPA exhibited better signal-to-noise (S/N) ratios and a uniform response for most examined proteins occurring in milk, hazelnut and in intact bacterial cells of E. coli. These findings not only provide a reactive proton transfer MALDI matrix with excellent reproducibility and sensitivity, but also contribute to extending the battery of useful matrices for intact protein analysis

Bioactive Secoiridoids in Italian Extra-Virgin Olive Oils: Impact of Olive Plant Cultivars, Cultivation Regions and Processing

In the last two decades, phenolic compounds occurring in olive oils known as secoiridoids have attracted a great interest for their bioactivity. Four major olive oil secoiridoids, i.e., oleuropein and ligstroside aglycones, oleacin and oleocanthal, were previously characterized in our laboratory using reversed-phase liquid chromatography with electrospray ionization-Fourier transform-mass spectrometry (RPLC-ESI-FTMS). The same analytical approach, followed by multivariate statistical analysis (i.e., Principal Component Analysis), was applied here to a set of 60 Italian extra-virgin olive oils (EVOO). The aim was to assess the secoiridoid contents as a function of olive cultivars, place of cultivation (i.e., different Italian regions) and olive oil processing, in particular two- vs. three-phase horizontal centrifugation. As expected, higher secoiridoid contents were generally found in olive oils produced by two-phase horizontal centrifugation. Moreover, some region/cultivar-related trends were evidenced, as oleuropein and ligstroside aglycones prevailed in olive oils produced in Apulia (Southern Italy), whereas the contents of oleacin and oleocanthal were relatively higher in EVOO produced in Central Italy (Tuscany, Lazio and Umbria). A lower content of all the four secoiridoids was generally found in EVOO produced in Sicily (Southern Italy) due to the intrinsic low abundance of these bioactive compounds in cultivars typical of that region

Mass spectrometric evidence for collisionally induced removal of H2 from monoanions of 10B nido-carborane derivatives investigated by electrospray ionization quadrupole linear ion trap and Fourier transform ion cyclotron resonance mass spectrometry

Some newly synthesized 10B nido-carborane derivatives, i.e., 7,8-dicarba-nido-undecaborane monoanions ([7-Me-8-R-C2B9H10]-K+, R = H, butyl, hexyl, octyl and decyl), have been fully characterised and examined by electrospray ionization and Fourier transform ion cyclotron resonance mass spectrometry with liquid chromatographic separation (LC/ESI-FTICR-MS). These boron-containing compounds exhibit abundant molecular ions ([M]−) at m/z 140.22631 [CB9H14]−, m/z 196.28883 [CB9H22]−, m/z 224.32032 [CB9H26]−, m/z 252.35133 [CB9H30]− and m/z 280.38354 [CB9H34]− at the normal tube lens voltage setting of −90 V, which was an instrumental parameter value selected in the tuning operation. Additional [M–nH2]− (n = 1−4) ions were observed in the mass spectra when higher tube lens voltages were applied, i.e., −140 V. High-resolution FTICR-MS data revealed the accurate masses of fragment ions, bearing either an even or an odd number of electrons. Collision-induced dissociation of the [M–nH2]− ions (n = 0–4) in the quadrupole linear ion trap (LTQ) analyzer confirmed the loss of hydrogen molecules from the molecular ions. It is suggested that the loss of H2 molecules from the alkyl chain is a consequence of the stabilization effect of the nido-carborane charged polyhedral skeleton

Lipidomics of the edible brown alga wakame (Undaria pinnatifida) by liquid chromatography coupled to electrospray ionization and tandem mass spectrometry

The lipidome of a brown seaweed commonly known as wakame (Undaria pinnatifida), which is grown and consumed around the world, including Western countries, as a healthy nutraceutical food or supplement, was here extensively examined. The study was focused on the characterization of phospholipids (PL) and glycolipids (GL) by liquid chromatography (LC), either hydrophilic interaction LC (HILIC) or reversed-phase LC (RPLC), coupled to electrospray ionization (ESI) and mass spectrometry (MS), operated both in high and in low-resolution mode. Through the acquisition of single (MS) and tandem (MS/MS) mass spectra more than 200 PL and GL of U. pinnatifida extracts were characterized in terms of lipid class, fatty acyl (FA) chain composition (length and number of unsaturations), and regiochemistry, namely 16 SQDG, 6 SQMG, 12 DGDG, 5 DGMG, 29 PG, 8 LPG, 19 PI, 14 PA, 19 PE, 8 PE, 38 PC, and 27 LPC. The FA (C16:0) was the most abundant saturated acyl chain, whereas the monounsaturated C18:1 and the polyunsaturated C18:2 and C20:4 chains were the prevailing ones. Odd-numbered acyl chains, i.e., C15:0, C17:0, C19:0, and C19:1, were also recognized. While SQDG exhibited the longest and most unsaturated acyl chains, C18:1, C18:2, and C18:3, in the sn-1 position of glycerol, they were preferentially located in the sn-2 position in the case of PL. The developed analytical approach might pave the way to extend lipidomic investigations also for other edible marine algae, thus emphasizing their potential role as a source of bioactive lipids

Biosensore amperometrico per la L-lisina basato su co-crosslinking di lisina-ossidasi su elettrodi di Pt modificati con polipirrolo overossidato

La determinazione dell’aminoacido essenziale L-lisina è di particolare interesse in campo biochimico, biotecnologico ed alimentare poichè, ad es., i suoi livelli sono in genere associabili, rispettivamente, a talune disfunzioni patologiche ed alle qualità nutrizionali di un prodotto alimentare. In questo contesto, l’impiego di metodi enzimatici specifici, basati su elettrodi ad enzima immobilizzato, costituisce certamente una valida alternativa alle metodiche analitiche convenzionali. Diversi biosensori per la determinazione della lisina sono descritti in letteratura. White e Guilbault [1] hanno esplorato la determinazione potenziometrica della lisina accoppiando elettrodi a CO2 con la lisina-decarbossilasi mentre Dempsey et al. [2] hanno sviluppato un biosensore amperometrico immobilizzando la lisina-deidrogenasi su elettrodi di Pt. Sfortunatamente, il metodo potenziometrico è limitato dall’interferenza dovuta alla CO2 atmosferica che ne riduce notevolmente la sensibilità mentre il secondo necessita la presenza dell’NAD+ come cofattore e di un mediatore in soluzione. Conseguentemente, l’approccio più efficiente risulta essere quello amperometrico basato sull’impiego della L-lisina-α-ossidasi che catalizza l’ossidazione dell’aminoacido ad α-cheto-ε-aminocaproato, ione ammonio ed acqua ossigenata. Nella realizzazione di un biosensore è preliminare lo sviluppo e l’ottimizzazione di una metodica di immobilizzazione enzimatica efficiente, in grado di assicurare un notevole grado di stabilità dell’enzima immobilizzato ed elevate attività. Diverse tecniche di immobilizzazione sono state esplorate per la lisina ossidasi quali ad es. l’immobilizzazione covalente su nylon [3] o membrane preattivate [4], l’intrappolamento in collagene [5], il crosslinking [6] o il co-crosslinking [7] dell’enzima su elettrodi di Pt modificati con 1,2-diaminobenzene. In particolare, il co-crosslinking è una tecnica di immobilizzazione particolarmente versatile e vantaggiosa in quanto applicabile ad una vasta gamma di enzimi e facilmente adattabile alle geometrie elettrodiche usualmente utilizzate nei rivelatori elettrochimici in batch ed in flusso. Biosensori per colina ed acetilcolina [8], ad es., sono stati realizzati immobilizzando il sistema bienzimatico acetilcolinesterasi/colina ossidasi su elettrodi di Pt mediante co-crosslinking con albumina di siero bovina e glutaraldeide; ancora, lo stesso approccio ha permesso la realizzazione di un biosensore interference and fouling-free per il glucosio [9] basato su un doppio strato costituito da glucosio ossidasi co-crosslinked e polipirrolo overossidato. Nel laboratorio degli autori è stato di recente messo a punto un nuovo biosensore per la determinazione della lisina in campioni di interesse farmacologico ed alimentare basato su co-crosslinking della lisina ossidasi con una proteina inerte quale l’albumina di siero bovino e la glutaraldeide come crosslinker. Uno studio delle concentrazioni ottimali di enzima, proteina inerte e crosslinker ha permesso la realizzazione su elettrodi di Pt di una membrana ad elevata attività enzimatica ed al tempo stesso meccanicamente stabile tanto da permetterne l’applicazione per analisi in flusso. Il sensore così realizzato ha evidenziato un valore di sensibilità relativamente elevato, pari a circa 1.4 µA/mM mm2, un range lineare esteso sino a circa 0.6 mM, un breve tempo di risposta (6-7 secondi) ed una stabilità tale da consentire un impiego in continuo per più di 40 giorni senza apprezzabile variazione della sensibilità. Una caratterizzazione elettroanalitica del biosensore ha consentito di stimare un valore di KM apparente pari a circa 2.1 ± 0.2 mM ed, al tempo stesso, ha evidenziato uno spiccato controllo diffusivo, difficilmente riscontrabile nei dispositivi basati sulla immobilizzazione elettrochimica. In particolare, questo studio ha messo in evidenza la possibilità di modulare il comportamento cinetico dell’elettrodo da diffusivo a enzimatico variando il pH dell’elettrolita di supporto. E’ possibile quindi nel presente caso ottimizzare le performances del biosensore senza modificare variabili complesse quali la concentrazione dell’enzima immobilizzato e lo spessore e la permeabilità al substrato della membrana enzimatica. Nonostante gli enzimi siano notoriamente specifici nei confronti di un singolo substrato, la lisina-ossidasi catalizza in soluzione, seppur con rese inferiori, anche l’ossidazione di altri aminoacidi [10], quali l’ornitina, la fenilalanina e l’arginina, che potrebbero interferire nella determinazione della lisina. Sebbene l’approccio più usuale per ovviare a questo inconveniente sia quello di selezionare la fonte dell’enzima [7] in base alla sua maggiore specificità, il presente studio ha evidenziato che un opportuno controllo cinetico del sensore, variando il pH e/o la velocità di flusso, permette con un comune enzima commerciale specificità ottimali se non migliori a quelle riscontrabili con enzimi selezionati da fonti opportune. La codeposizione sull’elettrodo di un polimero permselettivo elettrosintetizzato [9], ha infine permesso la realizzazione di un biosensore per L-lisina, scevro da interferenza ed avvelenamento, tramite elettrodeposizione di una membrana polimerica permselettiva basata su polipirrolo overossidato. Riferimenti 1. W. C. White, G. Guilbault, Anal. Chem., 50 (1978) 1481 2. E. Dempsey, J. Wang, V. Wollenberg, M. Ozsos, M. R. Smith, Biosensors and Bioelectronics, 7 (1992) 323 3. E. Vrbovà e M. Marek, Anal. Chim. Acta, 239 (1990) 263 4. M. G. Lavagnini, D. Moscone, G. Palleschi, D. Compagnone, C. Cremisini, Talanta, 40(8) (1993) 1301 5. E. Vrbovà e M. Marek, Collect. Czech. Chem. Commun., 55 (1990) 2568 6. A. Curulli, S. Kelly, C. O’Sullivan, G. G. Guilbaut, G. Palleschi, Biosensors and Bioelectronics 13 (1998) 1245 7. S. C. Kelly, P. J. O’Connell, C. K. O’Sullivan, G. G. Guilbaut, , Anal. Chim. Acta, 412 (2000) 111 8. A. Guerrieri, G. E. De Benedetto, F. Palmisano, P. G. Zambonin, Analyst, 120 (1995) 2731 9. A. Guerrieri, G. E. De Benedetto, F. Palmisano, P. G. Zambonin, Biosensors & Bioelectronics, 13 (1) (1998) 103 10. H. Kusakabe, K. Kodama, A. Kuminaka, H. Yoshimo, H. Misono, K. Soda, J. Biol. Chem., 255 (1980) 97

Multi-technique characterization of pictorial organic binders on XV century polychrome sculptures by combining microand non-invasive sampling approaches

A stony sculptural composition of the Nativity Scene is preserved in Altamura’s Cathedral (Apulia, Italy). This commonly called Apulian “presepe”, attributed to an unknown stonemason, is composed of polychrome carbonate white stone sculptures. While earlier stratigraphic tests have unveiled a complex superimposition of painting layers—meaning that several editions of the sculptures succeeded from the 16th to 20th century—a chemical investigation intended to identify the organic binding media used in painting layers was undertaken. Drawing on current literature, two strategies were exploited: a non-invasive in situ digestion analysis and an approach based on microremoval of painting film followed by the Bligh and Dyer extraction protocol. Both peptide and lipid mixtures were analyzed by matrix-assisted laser desorption/ionization-mass spectrometry (MALDIMS) and reversed-phase liquid chromatography coupled to mass spectrometry by electrospray ionization (RPLC-ESI-MS). Attenuated total reflectance Fourier-transform infrared spectroscopy (ATR-FTIR) examinations were also performed on micro-samples of painting films before lipids and proteins extraction. While human keratins were found to be common contaminants of the artwork’s surfaces, traces of animal collagen, siccative oils, and egg white proteins were evidenced in different sampling zones of the sculptures, thus suggesting the use of non-homogeneous painting techniques in the colored layers

Validation of an analytical method for nitrite and nitrate determination in meat foods for infants by ion chromatography with conductivity detection

Nitrate and nitrite as sodium or potassium salts are usually added to meat products to develop the characteristic flavor, to inhibit the growth of microorganisms (particularly Clostridium botulinum), and effectively control rancidity by inhibiting lipid oxidation. However, both nitrate and nitrite ions need to be monitored for ensuring the quality and safety of cured meats. In this work, for the first time the content of nitrite and nitrate ions in homogenized meat samples of baby foods was determined by a validated method based on ion chromatography (IC) coupled with conductivity detection. Recoveries of nitrate and nitrite ions in meat samples were not lower than 84 ± 6%. The detection limits of nitrate and nitrite were 0.08 mg L−1 and 0.13 mg L−1, respectively. Five commercial samples of homogenized meat, namely lamb, rabbit, chicken, veal, and beef, for infant feeding were investigated; while nitrite content was below the detection limit, nitrate ranged from 10.7 to 21.0 mg kg−1. The results indicated that nitrate contents were below the European (EU) fixed value of 200 mg kg−1, and an acceptable daily intake of 3.7 mg kg−1 was estimated