Giuliana Bianco

Determination of glycoalkaloids and relative aglycones by nonaqueous capillary electrophoresis coupled with electrospray ionization-ion trap mass spectrometry

Glycoalkaloids are naturally occurring nitrogen‐containing compounds present in many species of the family Solanaceae, including cultivated and wild potatoes (Solanum spp.), tomatoes (Lycopersicon spp.), etc. These compounds have pharmacological and toxicological effects on humans due to their significant anticholinesterase activity and disruption of cell membranes. Herein is reported the development of a capillary electrophoresis (CE) method using nonaqueous (NA) separation solutions in combination with ion trap mass spectrometry (MS and MS/MS) detection for the identification and quantification of glycoalkaloids and their relative aglycones. A mixture 90:10 v/v of MeCN‐MeOH containing 50 mM ammonium acetate and 1.2 M acetic acid (applied voltage of 25.5 kV) was selected as a good compromise for the separation and detection of these compounds. The electrospray MS measurements were carried out in the positive ionization mode using a coaxial sheath liquid, methanol‐water (1:1) with 1% of acetic acid at a flow rate of 2.5 νL/min. Under optimized experimental conditions, the predominant ion was the protonated molecular ion ([M+H]+) of solanidine (m/z = 398), tomatidine (m/z = 416), chaconine (m/z = 852), solanine (m/z = 868), and tomatine (m/z = 1034). MS/MS experiments were carried out systematically by changing the relative collisional energy and monitoring the intensities of the fragment ions that were not high enough to allow better quantification than with the mother ions. The method was used for analyzing glycoalkaloids in potato extracts.

Establishing the occurrence of major and minor glucosinolates in Brassicaceae by LC-ESI-hybrid linear ion-trap and Fourier-transform ion cyclotron resonance mass 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 linear ion trap (LTQ) and Fourier transform ion cyclotron resonance mass 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 C(4), C(5) and C(6), 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.

Determination of mono- and disaccharides in milk and milk products by high-performance anion-exchange chromatography with pulsed amperometric detection

A simple and sensitive liquid chromatographic method for the separation and quantification of mono- and disaccharides in raw- and processed-milk is described. Samples of cows’, buffalos’, sheeps’ and goats’ milk were analyzed upon clarification and appropriate dilution for the quantification of lactose, galactose, glucose and N-acetylglucosamine (GlcNAc). The separation was accomplished by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC–PAD), using a gold working electrode and dilute alkaline eluents modified by a millimolar concentration of barium acetate. The eluent composition employed was designed to provide optimum separation with respect to the selected sample, without interference from the matrix components. The analytical method was successfully employed for the determination of mono- and disaccharides naturally occurring in dairy milk, mozzarella cheese and whey samples, with high sensitivity and accuracy.

Accurate mass analysis of N‐acyl‐homoserine‐lactones and cognate lactone‐opened compounds in bacterial isolates of Pseudomonas aeruginosa PAO1 by LC‐ESI‐LTQ‐FTICR‐MS

N-acyl-homoserine-lactones (AHSLs) are widely conserved signal molecules present in quorum sensing systems of Gram-negative bacteria such as Pseudomonas aeruginosa. We present here the results obtained with a hybrid linear trap/Fourier transform ion cyclotron resonance (LTQ-FTICR) mass spectrometer used to investigate the occurrence of AHSLs and cognate N-acyl-homoserines (AHSs) in bacterial isolates of P. aeruginosa (strain PAO1). Two hydrolysed AHSs were found in significant amounts, most likely formed through the lactone opening of N-3-oxo-decanoyl-L-homoserine-lactone (3OC10-HSL) and N-3-oxo-dodecanoyl-L-homoserine-lactone (3OC12-HSL). Structure elucidation of these ring-opened molecules, i.e. N-3-oxo-decanoyl-L-homoserine (3OC10-HS), and N-3-oxo-dodecanoyl-L-homoserine (3OC12-HS), which are not detected by bacterial biosensors, was performed by high-resolution and accurate mass measurements upon liquid chromatography (LC) and confirmed by tandem MS in the LTQ analyser. Assignment of chemical formula, with mass spectra in the form of [M+H]+, was significantly expedited by extracted ion chromatograms (XICs) because the number of potentially plausible formulae for each protonated signalling molecule was considerably reduced a priori by the LC behaviour, the high mass measurement accuracy available in FTICR mass spectra and the isotopic patterns. At least two concentration levels were observed in spent culture supernatants of P. aeruginosa: compounds at a relatively high content (5-15 microM) that is C4-HSL, 3OC10-HS, and 3OC12-HS and those occurring at a lower content (<0.2 microM) that is C6-HSL and C8-HSL. The implications of this work extend to a great variety of Gram-negative bacteria.

Polychlorinated biphenyls in contaminated soil samples evaluated by GC–ECD with dual-column and GC–HRMS ☆

We present and compare results obtained from the analysis of polychlorinated biphenyls (PCBs) of a limited number of contaminated soil samples collected in three areas of Basilicata region (south of Italy). The levels of PCBs were evaluated by using two analytical methods: (i) parallel dual-column gas-chromatography with dual electron capture detectors (GC-ECD) and (ii) gas-chromatography coupled to high-resolution mass spectrometry (GC-HRMS) via electron impact ionization (EI) in the multiple ion monitoring mode (MIM, two ions per compound). Two extraction methods prior to sample cleanup were also examined: microwave-assisted extraction (MAE) and ultrasonic-assisted extraction (UAE). The MAE was the extraction procedure adopted using acetone/n-hexane (1:1, v/v) as it is mainly characterized by higher sample throughput and allowed reduced consumption of organic solvents. While extraction and analysis of spiked soil samples showed the applicability of both methods, systematic differences between the results were obtained for the sum of PCBs as a result of some non-detected congeners by GC-ECD compared with GC-HRMS. Indeed, high resolution MS using EI mode (electron energy 40eV) with a resolving power of 10,000 provides additional information about the contamination pattern. The GC-ECD screening of 11 soil samples led to just one sample non-compliant to as it was close to the guide value for soils fixed by the Italian legislation (i.e., 60ppb for private or urban soil). Using GC-HRMS, the amount of all PCBs found ranged from 5.4 to 127ppb with five soil samples non-compliant to the guide value. The number of identified congeners ranged from 1 to 9 and 9 to 18 using dual-column GC-ECD and GC-HRMS, respectively.

Quantitative determination of taurine in real samples by high-performance anion-exchange chromatography with integrated pulsed amperometric detection.

As taurine is a very important compound involved in a large number of metabolic processes, it is naturally present in the mammal tissues and is often deliberately added in some foods as a fortifying component. A detailed knowledge of taurine metabolic roles in biological systems can be obtained only if a sensitive, reliable and rapid analytical method is available. This article describes the successful application of high-performance anion-exchange chromatography coupled with integrated pulsed amperometric detection (HPAEC-IPAD) for taurine determination in egg white and yolk samples, as well extracts of human serum and urine. Applications are shown for determination of taurine in soft drinks and pharmaceutical preparations where the taurine content was evaluated by standard additions. These results were achieved without prior derivatization of taurine.

Identification and fragmentation pathways of caffeine metabolites in urine samples via liquid chromatography with positive electrospray ionization coupled to a hybrid quadrupole linear ion trap (LTQ) and Fourier transform ion cyclotron resonance mass spectrometry and tandem mass spectrometry

Liquid chromatography (LC) with positive ion electrospray ionization (ESI+) coupled to a hybrid quadrupole linear ion trap (LTQ) and Fourier transform ion cyclotron resonance mass spectrometry (FTICRMS) was employed for the simultaneous determination of caffeine and its metabolites in human urine within a single chromatographic run. LC/ESI-FTICRMS led to the unambiguous determination of the molecular masses of the studied compounds without interference from other biomolecules. A systematic and comprehensive study of the mass spectral behaviour of caffeine and its fourteen metabolites by tandem mass spectrometry (MS/MS) was performed, through in-source ion trap collision-induced dissociation (CID) of the protonated molecules, [M+H](+). A retro-Diels-Alder (RDA) process along with ring-contraction reactions were the major fragmentation pathways observed during CID. The base peak of xanthine precursors originates from the loss of methyl isocyanate (CH(3)NCO, 57 Da) or isocyanic acid (HNCO, 43 Da), which in turn lose a CO unit. Also uric acid derivatives shared a RDA rearrangement as a common fragmentation process and a successive loss of CO(2) or CO. The uracil derivatives showed a loss of a ketene unit (CH(2)CO, 42 Da) from the protonated molecule along with the loss of H(2)O or CO. To assess the potential of the present method three established metabolite ratios to measure P450 CYP1A2, N-acetyltransferase and xanthine oxidase activities were evaluated by a number of identified metabolites from healthy human urine samples after caffeine intake.

Polybrominated diphenyl ethers (PBDEs) in Mediterranean mussels (Mytilus galloprovincialis) from selected Apulia coastal sites evaluated by GC–HRMS

Samples of Mytilus galloprovincialis were examined to investigate the levels of polybrominated diphenyl ethers (PBDEs) in the Apulias marine environment, a region in the South of Italy. The levels of nine PBDE congeners were measured in 40 mussel samples taken from the aquaculture farms in the South of Adriatic Sea and North Ionian Sea along the Apulia coast. While accelerated solvent extraction (ASE) was the extraction technique adopted using acetone/n-hexane (1:1, v/v), the content of PBDEs was evaluated by using gas chromatography coupled with high-resolution mass spectrometry (GC-HRMS) via electron ionization (EI) in the multiple ion monitoring (MIM) mode (two ions for each compound). The (13)C mass-labeled compounds were used to establish the levels of PBDEs in M. galloprovincialis samples. The total concentration of PBDEs (ΣPBDEs) ranged from 0.2 to 6.9 ng/g dry mass, with the highest concentrations found around coastal areas of Salento. With regard to the composition of PBDE congeners, BDE-47, BDE-99 and BDE-100 were the predominant congeners in most of the samples. The present study is the first to report levels of PBDEs occurring in aquatic organisms living in aquaculture farms of Apulia region.

Validation of an analytical method for simultaneous high-precision measurements of greenhouse gas emissions from wastewater treatment plants using a gas chromatography-barrier discharge detector system.

Wastewater treatment plants (WWTPs) emit CO2 and N2O, which may lead to climate change and global warming. Over the last few years, awareness of greenhouse gas (GHG) emissions from WWTPs has increased. Moreover, the development of valid, reliable, and high-throughput analytical methods for simultaneous gas analysis is an essential requirement for environmental applications. In the present study, an analytical method based on a gas chromatograph (GC) equipped with a barrier ionization discharge (BID) detector was developed for the first time. This new method simultaneously analyses CO2 and N2O and has a precision, measured in terms of relative standard of variation RSD%, equal to or less than 6.6% and 5.1%, respectively. The methods detection limits are 5.3ppmv for CO2 and 62.0ppbv for N2O. The methods selectivity, linearity, accuracy, repeatability, intermediate precision, limit of detection and limit of quantification were good at trace concentration levels. After validation, the method was applied to a real case of N2O and CO2 emissions from a WWTP, confirming its suitability as a standard procedure for simultaneous GHG analysis in environmental samples containing CO2 levels less than 12,000mg/L.

Perceiving the chemical language of Gram-negative bacteria: listening by high-resolution mass spectrometry

AbstractGram-negative bacteria use N-acylhomoserine lactones (AHLs) as their command language to coordinate population behavior during invasion and colonization of higher organisms. Although many different bacterial bioreporters are available for AHLs monitoring, in which a phenotypic response, e.g. bioluminescence, violacin production, and β-galactosidase activity, is exploited, mass spectrometry (MS) is the most versatile detector for rapid analysis of AHLs in complex microbial samples, with or without prior separation steps. In this paper we critically review recent advances in the application of high-resolution MS to analysis of the quorum sensing (QS) signaling molecules used by Gram-negative bacteria, with much emphasis on AHLs. A critical review of the use of bioreporters in the study of AHLs is followed by a short methodological survey of the capabilities of high-resolution mass spectrometry (HRMS), including Fourier-transform ion cyclotron resonance (FTICR) MS and quadrupole time-of-flight (qTOF) MS. Use of infusion electrospray ultrahigh-resolution FTICR MS (12 Tesla) enables accurate mass measurements for determination of the elemental formulas of AHLs in Acidovorax sp. N35 and Burkholderia ubonensis AB030584. Results obtained by coupling liquid chromatography with a hybrid quadrupole linear ion trap-FTICR mass spectrometer (LC–LTQ-FTICRMS, 7-T) for characterization of acylated homoserine lactones in the human pathogen Pseudomonas aeruginosa are presented. UPLC–ESI-qTOF MS has also proved to be suitable for identification of 3O-C10HSL in Pseudomonas putida IsoF cell culture supernatant. Aspects of sample preparation and the avoidance of analytical pitfalls are also emphasized. FigureLC separation and FTICR MS identification of 3-oxo-C12-HSL in bacterial isolates of P. aeruginosa (strain ATCC 9027)