Versatile lipid profiling by liquid chromatography-high resolution mass spectrometry using all ion fragmentation and polarity switching. Preliminary application for serum samples phenotyping related to canine mammary cancer.

Lipids represent an extended class of substances characterized by such high variety and complexity that makes their unified analyses by liquid chromatography coupled to either high resolution or tandem mass spectrometry (LC-HRMS or LC-MS/MS) a real challenge. In the present study, a new versatile methodology associating ultra high performance liquid chromatography coupled to high resolution tandem mass spectrometry (UHPLC-HRMS/MS) have been developed for a comprehensive analysis of lipids. The use of polarity switching and "all ion fragmentation" (AIF) have been two action levels particularly exploited to finally permit the detection and identification of a multi-class and multi-analyte extended range of lipids in a single run. For identification purposes, both higher energy collision dissociation (HCD) and in-source CID (collision induced dissociation) fragmentation were evaluated in order to obtain information about the precursor and product ions in the same spectra. This approach provides both class-specific and lipid-specific fragments, enhancing lipid identification. Finally, the developed method was applied for differential phenotyping of serum samples collected from pet dogs developing spontaneous malignant mammary tumors and health controls. A biological signature associated with the presence of cancer was then successfully revealed from this lipidome analysis, which required to be further investigated and confirmed at larger scale.

The response of Mucor plumbeus to pentachlorophenol: a toxicoproteomics study.

Pentachlorophenol (PCP) represents a critical concern worldwide due to its toxicity and recalcitrance to degradation. The capacity of Mucor plumbeus to transform PCP into several detoxification metabolites, including tetrachlorohydroquinone and several phase II conjugates, was observed by LC-HRMS. The data obtained support the degradation pathway proposed previously. PCP effects in M. plumbeus, an unsequenced species, were investigated using a proteomics approach (bidimensional gel electrophoresis followed by MALDI TOF/TOF analyses). The mycelial proteins identified in the differentially accumulated spots allowed the identification of PCP responsive proteins. The presence of PCP increased the energy demand, altered the cell wall architecture and cytoskeleton and induced a significant stress response. The latter was emphasised by the up-accumulation of protein species associated with defence mechanisms (e.g. HSP70 and cytochrome c peroxidase). Overall the data produced corroborate the capability of PCP to uncouple oxidative-phosphorylation in mitochondria. Importantly, one of the identified mycelial protein species, a NAD- and Zn-dependent ADH, is likely to be involved in PCP degradation. Amongst the fungal secretome analysed, no putative PCP degradative enzymes were detected. This work constitutes the first toxicoproteomic study involving a Zygomycota fungus and the very first concerning the effect of PCP in a fungal proteome.

Preventing false negatives with high-resolution mass spectrometry: the benzophenone case.

Benzophenone (BP) is one of the many contaminants reported as present in foodstuffs due to its migration from food packaging materials. Liquid chromatography/tandem mass spectrometry (LC/MS/MS) is acknowledged in the literature as the method of choice for this analysis. However, cases have been reported where the use of this methodology was insufficient to unambiguously confirm the presence of a contaminant. In previous work performed by the authors, the unequivocal identification of BP in packaged foods was not possible even when monitoring two m/z transitions (precursor ion - product ion), since ion ratio errors higher than 20% were obtained. In order to overcome this analytical problem a fast, sensitive and selective liquid chromatography/high-resolution mass spectrometry (LC/HRMS) methodology has been developed and applied to the analysis of BP in packaged foods. A direct comparison between LC/HRMS and LC/MS/MS data indicated better selectivity when working with LC/HRMS at a resolving power of 50,000 FWHM (full width at half maximum) than when monitoring two m/z transitions by LC/MS/MS. The resolving power used enabled the detection and identification of Harman as the compound impeding the confirmation of BP by LC-MS/MS. Similar quantitative results were obtained by an Orbitrap mass analyser (Exactive™) and a triple quadrupole mass analyser (TSQ Quantum Ultra AM™).

Fast liquid chromatography-tandem mass spectrometry for the analysis of bisphenol A-diglycidyl ether, bisphenol F-diglycidyl ether and their derivatives in canned food and beverages.

In this work a fast liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) method using a C18 Fused Core™ column, was developed for the simultaneous analysis of bisphenol A diglycidyl ether (BADGE), bisphenol A (2,3-dihydroxypropyl) glycidyl ether (BADGE·H(2)O), bisphenol A bis(2,3-dihydroxypropyl) ether (BADGE·2H(2)O), bisphenol A (3-chloro-2-hydroxypropyl) glycidyl ether (BADGE·HCl), bisphenol A bis(3-chloro-2-hydroxypropyl) ether (BADGE·2HCl) and bisphenol A (3-chloro-2-hydroxypropyl)(2,3-dihydroxypropyl ether) (BADGE·HCl·H(2)O) and bisphenol F diglycidyl ether (BFDGE), bisphenol F bis(2,3-dihydroxypropyl) ether (BFDGE·2H(2)O), bisphenol F bis(3-chloro-2-hydroxypropyl) ether (BFDGE·2HCl). The LC method was coupled with a triple quadrupole mass spectrometer, using an ESI source in positive mode and using the [M+NH(4)](+) adduct as precursor ion for tandem mass spectrometry experiments. The method developed was applied to the determination of these compounds in canned soft drinks and canned food. OASIS HLB solid phase extraction (SPE) cartridges were used for the analysis of soft drinks, while solid canned food was extracted with ethyl acetate. Method limits of quantitation ranged from 0.13 µgL(-1) to 1.6 µgL(-1) in soft drinks and 1.0 µgkg(-1) to 4.0 µgkg(-1) in food samples. BADGE·2H(2)O was detected in all the analyzed samples, while other BADGEs such as BADGE·H(2)O, BADGE·HCl·H(2)O, BADGE·HCl and BADGE·2HCl were also detected in canned foods.

Analysis of bisphenols in soft drinks by on-line solid phase extraction fast liquid chromatography-tandem mass spectrometry.

In this study, an automated on-line solid-phase extraction coupled to fast liquid chromatography-tandem mass spectrometry (on-line SPE fast LC-MS/MS) method was developed for the simultaneous analysis of bisphenol A (BPA), bisphenol F (BPF), bisphenol E (BPE), bisphenol B (BPB) and bisphenol S (BPS) in canned soft drinks without any previous sample treatment. A C18 (12 µm particle size) loading column was used for the SPE on-line preconcentration before the liquid chromatography baseline separation of bisphenol compounds using a C18 Fused-Core™ (50 mm × 2.1 mm i.d.) column, which took less than 3 min. Gradient elution and heated electrospray were used to reduce matrix effect and improve ionization efficiency. To select the most intense and selective transitions, fragmentation studies were performed by multiple-stage mass spectrometry in an ion trap mass analyzer and tandem mass spectrometry in a triple quadrupole instrument, this latter instrument being used for quantitation in SRM mode. Quality parameters of the method were established and we obtained a simple, fast, reproducible (RSD values lower than 10%) and accurate (precision higher than 93%) method for the analysis of bisphenols in canned soft drinks at the ng L(-1) level using matrix-matched calibration.

Multiple-stage mass spectrometry analysis of bisphenol A diglycidyl ether, bisphenol F diglycidyl ether and their derivatives.

The fragmentation of bisphenol A diglycidyl ether (BADGE), bisphenol F diglycidyl ether (BFDGE) and their derivatives was studied by electrospray ionization tandem mass spectrometry. Multiple-stage mass spectrometry and accurate mass measurements were combined to establish the fragmentation pathways. BADGEs and BFDGEs tend to form ammonium adducts under electrospray conditions which fragmented easily. The fragmentation of [M+NH(4)](+) for BADGEs started with the cleavage of the phenyl-alkyl bond, which was followed by the α-cleavage of the ether group to generate the characteristic product ions at m/z 135, [C(9)H(11)O](+), and m/z 107, [C(7)H(7)O](+). The fragmentation of the BFDGE isomer mixtures was studied by on-line reversed-phase liquid chromatography coupled to multiple-stage mass spectrometry (LC/MS(n)). Information obtained from product ion spectra for each BFDGE isomer and its comparison with the fragmentation pathway of BADGE allowed each isomer and the chromatographic elution order to be identified.

On-line solid phase extraction fast liquid chromatography-tandem mass spectrometry for the analysis of bisphenol A and its chlorinated derivatives in water samples.

In this study an on-line column-switching fast LC-MS/MS method was developed to analyze bisphenol A (BPA) and its chlorinated derivatives in water. Fast liquid chromatographic separation was performed on a C18 reversed phase column based on fused-core particle technology (2.7 microm particle size) providing analysis times shorter than 3 min and high peak efficiencies. The main benefit of this LC system is that it can easily be hyphenated to a conventional on-line preconcentration device allowing the direct analysis of water samples without any pretreatment at concentrations levels down to 60 ng L(-1) and preventing contaminations frequently reported in the analysis of BPA. This on-line SPE fast LC system was coupled to a triple quadrupole mass spectrometer operating in enhanced mass resolution mode (Q1 FWHM=0.7 Th, Q3 FWHM=0.1 Th) in order to minimize interferences and chemical noise. This highly sensitive and selective method was successfully employed to analyze BPA and its chlorinated derivatives in water samples.

Recent advances in mass spectrometry analysis of phenolic endocrine disruptors and related compounds.

This article reviews recent literature on current methodologies based on chromatography coupled to mass spectrometry to analyze phenolic compounds with endocrine-disrupting capabilities. For this review we chose alkylphenol ethoxylates, bisphenol A, bisphenol F, and their degradation products and halogenated derivatives, which are considered important environmental contaminants. Additionally, some related compounds such as bisphenol diglycidylethers were included. Growing attention has been paid to the mass spectrometric characterization of these compounds and the instrumentation and strategies used for their quantification and confirmation. The current use of gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS) methodologies with different mass spectrometers and ionization and monitoring modes is discussed. Practical aspects with regards to the use of these analytical techniques, such as derivatizing reagents in GC-MS, ion suppression in LC-MS, and the most problematic aspects of quantification, are included in the discussion.

Liquid chromatography/tandem mass spectrometry (highly selective selected reaction monitoring) for the analysis of isopropylthioxanthone in packaged food.

Isopropylthioxanthone (ITX), usually applied as a mixture of 2- and 4-isomers, is a common photo-initiator in UV inks used in paper- or plastic-based packaging materials. In this work a pentafluorophenylpropyl column (HS F5) has been used to achieve the chromatographic separation of the two isomers. A gradient elution with acetonitrile and a 25mM formic acid-ammonium formate at pH 3.75 are required to provide an Rs of 1.3 between the two compounds. The fragmentation pattern of ITX was studied using two mass analyzers, an ion trap (IT) (multi-stage fragmentation) and a triple quadrupole mass analyzer of hyperbolic rods (accurate mass (AM) measurement). The protonated molecule [M+H](+) observed in the mass spectrometry (MS) spectrum lost an isopropyl group, [M+H-C(3)H(6)](+). Later, this ion fragmented, yielding the radical ion [M+H-C(3)H(6)-CHO](+). The elemental composition of these product ions was confirmed by AM measurement. Electrospray ionization (ESI) was used as an ionization source to couple liquid chromatography (LC) to MS. Instrumental quality parameters of three acquisition modes provided by the triple quadrupole mass analyzer were studied and good run-to-run precision (relative standard deviation, RSD, lower than 10%) and limits of detection (LODs) down to 0.8pg injected in the LC-MS/MS system were obtained. Finally the LC-MS/MS method using H-SRM Q1 acquisition mode was used to analyze 2- and 4-ITX in a range of food samples. The use of highly selective selected reaction monitoring (H-SRM on Q1) resulted in improved selectivity without sensitivity loss.