A targeted GC-MS/MS approach for the determination of eight sterols in microgreen and mature plant material.

Over the past decades, a remarkable number of scientific studies supported the correlation between an adequate dietary intake of phytosterols (PS) and the reduced risk of cardiovascular diseases. PS are known to inhibit the intestinal absorption of cholesterol, thus promoting the reduction of the low-density lipoproteins (LDL) amount in the bloodstream. Despite the fact that a non-negligible atherogenicity was recognized to PS, thus requiring a careful risk-benefits assessment for plant sterol supplementation, the potential role of PS as cholesterol-lowering agents has been contributing to the spreading awareness of the health benefits associated with the consumption of plant-based foods. In recent years, this has been fueling the market of innovative vegetable products, such as microgreens. Surprisingly, the recent literature concerning microgreens exhibited the lack of studies focusing on the characterization of PS. To fill this gap, a validated analytical method based on the hyphenation of gas chromatography and tandem mass spectrometry is proposed here for the quantitative analysis of eight phytosterols, namely ß-sitosterol, campesterol, stigmasterol, brassicasterol, isofucosterol, and cholesterol, lathosterol and lanosterol. The method was exploited for the characterization of the PS content in 10 microgreen crops, i.e., chia, flax, soybean, sunflower, rapeseed, garden cress, catalogna chicory, endive, kale and broccoli raab. Finally, these results were compared to the PS content of mature forms of kale and broccoli raab. A remarkable amount of PS was detected in chia, flax, rapeseed, garden cress, kale, and broccoli raab microgreens. 100 g (wet weight) of these microgreen crops were found to contain from 20 to 30 mg of the investigated PS. Interestingly, in the case of kale and broccoli raab microgreens, the overall PS content was higher than the one measured in the edible parts of the corresponding mature forms. Additionally, a symmetric change of the PS inner profile was observed between the two growth stages of the latter two crops. Here, the overall decrease of the PS sterol content in the mature forms was associated with the increase of the relative amount of ß-sitosterol and campesterol at the expense of minor PS species, such as brassicasterol.

Insight into the Storage-Related Oxidative/Hydrolytic Degradation of Olive Oil Secoiridoids by Liquid Chromatography and High-Resolution Fourier Transform Mass Spectrometry.

The study of negative effects potentially exerted by the exposure to oxygen and/or light and, thus, also by the type of container on the quality of extra virgin olive oil (EVOO) during its prolonged storage requires an appropriate choice of analytical methods and components to be monitored. Here, reverse-phase liquid chromatography coupled to high-resolution/accuracy Fourier transform mass spectrometry with electrospray ionization was exploited to study oxidative/hydrolytic degradation processes occurring on the important bioactive components of EVOO known as secoiridoids, i.e., oleuropein and ligstroside aglycones, oleacin, and oleocanthal, during storage up to 6 months under controlled conditions. Specifically, isomeric oxidative byproducts resulting from the transformation of a carbonylic group of the original secoiridoids into a carboxylic group and compounds resulting from hydrolysis of the ester linkage of secoiridoids, i.e., elenolic and decarboxymethyl elenolic acids and tyrosol and 3-hydroxytyrosol, were monitored, along with their precursors. Data obtained from EVOO storage at room temperature in glass bottles with/without exposure to light and/or oxygen indicated that, although it was more relevant if a periodical exposure to oxygen was performed, a non-negligible oxidative degradation occurred on secoiridoids also when nitrogen was used to saturate the container headspace. In a parallel experiment, the effects of storage of the same EVOO (250 mL) for up to 6 months in containers manufactured with different materials/shapes were considered. In particular, a square dark glass bottle, a stainless-steel can, and a ceramic jar, typically used for EVOO commercialization, and a clear polyethylene terephthalate bottle, purposely chosen to prompt secoiridoid degradation through exposure to light and oxygen, were compared. Dark glass was found to provide the best combined protection of major secoiridoids from oxidative and hydrolytic degradation, yet the lowest levels of oxidized byproducts were observed when the stainless-steel can was used.

The occurrence of inositolphosphoceramides in spirulina microalgae.

Spirulina microalga (Arthrospira platensis) is an interesting phototrophic organism because of its high content of nutrients including proteins, lipids, essential amino acids, antioxidants, vitamins, polysaccharides, and minerals. Hydrophilic interaction liquid chromatography (HILIC) coupled to linear ion trap (LIT) and Orbitrap Fourier transform mass spectrometry (FTMS) via ESI was employed for the separation and characterization of lipid species in A. platensis. Inositolphosphoceramides (IPC) are minor but important constituents of spirulina; their investigation was accomplished by HILIC-ESI-MS including collision-induced dissociation (MS2 , MS3 ) of deprotonated molecules in the LIT analyzer and a schematic fragmentation pattern is described. All four commercial spirulina samples revealed the occurrence of the same IPC species at m/z 796.6 (d18:0/16:0;1), 810.6 (d18:0/17:0;1), 824.6 (d18:0/18:0;1), and 826.6 (d18:0/17:0;2) but in diverse relative abundance. This study sets the stage for future investigations on IPC in other algae and microalgae.

An easily transferable protocol for in-situ quasi-non-invasive analysis of protein binders in works of art.

Proteomic approaches based on mass spectrometry have become increasingly popular for protein binder's identification in works of art. The identification of the binder employed may offer key information on paintings and other polychrome objects and contribute to assess their historical and technical context, also providing useful hints for a proper restoration and/or conservation treatment. Usually, the protocols employed to this purpose are invasive and at least micro sampling is required. Here, we present a simple transferable method for a quasi-non-invasive analysis of binders in artworks based on the use of a very small poly (2-hydroxyethyl methacrylate)/poly (vinylpyrrolidone) hydrogel (3 mm × 3 mm) previously loaded with trypsin for the in-situ digestion of proteins and applied onto the objects' surface. Upon extraction of digested peptides from the hydrogel, they were examined by MALDI-TOF-MS and/or LC-ESI-MS/MS. The method was validated on fresh and aged model pictorial layers; optical microscope images, and spectrophotocolorimetry confirmed that neither damage nor color alteration of the painting layer occurred, and no hydrogel residue was left. X-ray photoelectron spectroscopy carried out on paint models confirmed that the treatment with trypsin-loaded gels did not modify the pigment composition, even on aged samples. The protocol was successfully applied to a painting on wood mockup aged thirty years, a statue dated XV century exposed in San Lorenzo church (Bisceglie, Bari, Apulia), and a liturgical scroll Benedictio ignis et fontis (Benedizionale) of the Museo Diocesano of Bari dated eleventh century; in all these objects the proteinaceous binder was readily and successfully identified.

MALDI-TOF mass spectrometry analysis of proteins and lipids in Escherichia coli exposed to copper ions and nanoparticles.

Escherichia coli (E. coli) is one of the most important foodborne pathogens to the food industry responsible for diseases as bloody diarrhea, hemorrhagic colitis and life-threatening hemolytic-uremic syndrome. For controlling and eliminating E. coli, metal nano-antimicrobials (NAMs) are frequently used as bioactive systems for applications in food treatments. Most NAMs provide controlled release of metal ions, eventually slowing down or completely inhibiting the growth of undesired microorganisms. Nonetheless, their antimicrobial action is not totally unraveled and is strongly dependent on metal properties and environmental conditions. In this work, we propose the use of matrix-assisted laser desorption ionization time-of-flight (MALDI TOF) mass spectrometry as a powerful tool for direct, time efficient, plausible identification of the cell membrane damage in bacterial strains exposed to copper-based antimicrobial agents, such as soluble salts (chosen as simplified AM material) and copper nanoparticles. E. coli ATCC 25922 strain was selected as 'training bacterium' to set up some critical experimental parameters (i.e. cell concentration, selection of the MALDI matrix, optimal solvent composition, sample preparation method) for the MS analyses. The resulting procedure was then used to attain both protein and lipid fingerprints from E. coli after exposure to different loadings of Cu salts and NPs. Interestingly, bacteria exposed to copper showed over-expression of copper binding proteins and degradation of lipids when treated with soluble salt. These findings were completed with other investigations, such as microbiological experiments. Copyright © 2016 John Wiley & Sons, Ltd.

Superbasic alkyl-substituted bisphosphazene proton sponges: a new class of deprotonating matrices for negative ion matrix-assisted ionization/laser desorption mass spectrometry of low molecular weight hardly ionizable analytes.

RATIONALE: Here hardly ionizable and low molecular weight compounds are detected in negative ion mode by using novel superbasic proton sponges based on 1,8-bisphosphazenylnaphthalene (PN) as MALDI matrices. Among the selected proton sponges, 1,8-bis(trispyrrolidinophosphazenyl)naphthalene (TPPN) has shown the best behaviour as matrix since it allows the direct detection of intact cholesterol without derivatization also in real challenging samples. METHODS: Very weakly acidic compounds such as sterols, steroids, fatty alcohols and saccharides were detected in reflectron negative ion mode by a MALDI TOF/TOF system equipped with a neodymium-doped yttrium lithium fluoride (Nd:YLF) laser (345 nm) with typical mass accuracy of 10 ppm. MS/MS experiments were performed by using ambient air as the collision gas. RESULTS: Contrary to traditional MALDI matrices, superbasic proton sponges allowed the easy deprotonation of an alcohol functional group without a previous chemical derivatization step. Experimental evidence indicates that analyte deprotonation is achieved in the condensed phase, i.e. PN superbasic proton sponges operate according to a recently proposed model named matrix assisted ionization/laser desorption (MAILD). A detection limit of 3 pmol/spot of cholesterol (model compound) with a signal-to-noise ratio ≥ 10 was typically obtained. CONCLUSIONS: For the first time, the usefulness of novel superbasic proton sponges is demonstrated for MALDI detection of hardly ionizable compounds such as sterols, steroids, fatty alcohols and saccharides. The leading candidate TPPN has been successfully applied for negative ion MAILD-MS analysis of cholesterol, fatty acids and phospholipids in egg yolk and brain tissue extracts. Copyright © 2016 John Wiley & Sons, Ltd.

Boronic acid chemistry in MALDI MS: a step forward in designing a reactive matrix with molecular recognition capabilities.

A boronic analogue of the archetype matrix α-cyano-4-hydroxycinnamic acid (CHCA) has been synthesised providing a new "reactive matrix" that possesses molecular recognition properties. This matrix selectively recognizes vic-diols, α-hydroxyacids, aminols and first allowed the detection of anions as fluoride (unaffordable by usual matrices).

1,8-bis(dimethylamino)naphthalene/9-aminoacridine: a new binary matrix for lipid fingerprinting of intact bacteria by matrix assisted laser desorption ionization mass spectrometry.

The effectiveness of a novel binary matrix composed of 1,8-bis(dimethylamino)naphthalene (DMAN; proton sponge) and 9-aminoacridine (9AA) for the direct lipid analysis of whole bacterial cells by matrix assisted laser desorption ionization mass spectrometry (MALDI MS) is demonstrated. Deprotonated analyte signals nearly free of matrix-related ions were observed in negative ion mode. The effect of the most important factors (laser energy, pulse voltage, DMAN/9AA ratio, analyte/matrix ratio) was investigated using a Box-Behnken response surface design followed by multi-response optimization in order to simultaneously maximize signal-to-noise (S/N) ratio and resolution. The chemical surface composition of single or mixed matrices was explored by X-ray photoelectron spectroscopy (XPS). Moreover, XPS imaging was used to map the spatial distribution of a model phospholipid in single or binary matrices. The DMAN/9AA binary matrix was then successfully applied to the analysis of intact Gram positive (Lactobacillus sanfranciscensis) or Gram negative (Escherichia coli) microorganisms. About fifty major membrane components (free fatty acids, mono-, di- and tri-glycerides, phospholipids, glycolipids and cardiolipins) were quickly and easily detected over a mass range spanning from ca. 200 to ca. 1600 m/z. Moreover, mass spectra with improved S/N ratio (compared to single matrices), reduced chemical noise and no formation of matrix-clusters were invariably obtained demonstrating the potential of this binary matrix to improve sensitivity.

Fingerprinting of egg and oil binders in painted artworks by matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis of lipid oxidation by-products.

A matrix-assisted laser desorption ionization time-of-flight mass spectrometry-based approach was applied for the detection of various lipid classes, such as triacylglycerols (TAGs) and phospholipids (PLs), and their oxidation by-products in extracts of small (50-100 µg) samples obtained from painted artworks. Ageing of test specimens under various conditions, including the presence of different pigments, was preliminarily investigated. During ageing, the TAGs and PLs content decreased, whereas the amount of diglycerides, short-chain oxidative products arising from TAGs and PLs, and oxidized TAGs and PLs components increased. The examination of a series of model paint samples gave a clear indication that specific ions produced by oxidative cleavage of PLs and/or TAGs may be used as markers for egg and drying oil-based binders. Their elemental composition and hypothetical structure are also tentatively proposed. Moreover, the simultaneous presence of egg and oil binders can be easily and unambiguously ascertained through the simultaneous occurrence of the relevant specific markers. The potential of the proposed approach was demonstrated for the first time by the analysis of real samples from a polyptych of Bartolomeo Vivarini (fifteenth century) and a "French school" canvas painting (seventeenth century).

Solid phase microextraction--liquid chromatography (SPME-LC) determination of chloramphenicol in urine and environmental water samples.

A solid phase microextraction--liquid chromatography with ultraviolet detection (SPME-LC-UV) method for the determination of the antimicrobial agent chloramphenicol was developed. The performances of three commercially available fibers were compared; the Carbowax/TPR-100 was found to provide the most efficient extraction. All the aspects influencing the fiber adsorption (extraction time, temperature, pH, salt addition) and desorption (desorption and injection time, desorption solvent mixture composition) of the analyte were investigated. The method was eventually applied to the determination of the drug in both biological (urine) and environmental (tap and sea water) samples. The optimized procedure required a simple sample pretreatment, isocratic elution, and provided enough sensitivity for the analyte determination in the considered samples. The investigated linear ranges were 37-1000 ng/ml (urine), 0.1-10 ng/ml (tap water), 0.3-30 ng/ml (sea water). Within-day and between-days RSD% ranged between 5.5-6.2 and 8.7-9.0 (urine), 5.1-6.0 and 8.4-8.8 (tap water), 5.4-5.7 and 8.6-8.9 (sea water). Estimated LOD and LOQ were 37 and 95 ng/ml (urine), 0.1 and 0.3 ng/ml (tap water), 0.3 and 0.7 ng/ml (sea water).

Optimization of analytical and pre-analytical conditions for MALDI-TOF-MS human urine protein profiles.

Protein analysis in biological fluids, such as urine, by means of mass spectrometry (MS) still suffers for insufficient standardization in protocols for sample collection, storage and preparation. In this work, the influence of these variables on healthy donors human urine protein profiling performed by matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was studied. A screening of various urine sample pre-treatment procedures and different sample deposition approaches on the MALDI target was performed. The influence of urine samples storage time and temperature on spectral profiles was evaluated by means of principal component analysis (PCA). The whole optimized procedure was eventually applied to the MALDI-TOF-MS analysis of human urine samples taken from prostate cancer patients. The best results in terms of detected ions number and abundance in the MS spectra were obtained by using home-made microcolumns packed with hydrophilic-lipophilic balance (HLB) resin as sample pre-treatment method; this procedure was also less expensive and suitable for high throughput analyses. Afterwards, the spin coating approach for sample deposition on the MALDI target plate was optimized, obtaining homogenous and reproducible spots. Then, PCA indicated that low storage temperatures of acidified and centrifuged samples, together with short handling time, allowed to obtain reproducible profiles without artifacts contribution due to experimental conditions. Finally, interesting differences were found by comparing the MALDI-TOF-MS protein profiles of pooled urine samples of healthy donors and prostate cancer patients. The results showed that analytical and pre-analytical variables are crucial for the success of urine analysis, to obtain meaningful and reproducible data, even if the intra-patient variability is very difficult to avoid. It has been proven how pooled urine samples can be an interesting way to make easier the comparison between healthy and pathological samples and to individuate possible differences in the protein expression between the two sets of samples.

Silver nanofractals: electrochemical synthesis, XPS characterization and application in LDI-MS.

Silver nanofractals (Ag-NFs) have been electrosynthesized and characterized by means of morphological and spectroscopic analytical techniques. In particular, X-ray photoelectron spectroscopy has been used to assess the nanomaterial surface chemical state. Ag-NFs show interesting perspectives in bioanalytical applications, particularly as non-conventional desorption and ionization promoters in laser desorption ionization mass spectrometry.

Determination of clenbuterol in human urine and serum by solid-phase microextraction coupled to liquid chromatography.

A solid-phase microextraction (SPME)-LC-UV method for the determination of the beta-adrenergic drug clenbuterol in human urine and serum samples was developed for the first time using a polydimethylsiloxane/divinylbenzene (PDMS/DVB) coated fiber. The procedure required very simple sample pretreatments, isocratic elution, and provided highly selective extractions. All the aspects influencing fiber adsorption (extraction time, temperature, pH, salt addition) and desorption (desorption and injection time, desorption solvent mixture composition) of the analyte have been investigated. The linear ranges investigated in urine and serum were 10-500 and 5-500 ng/ml, respectively (that covers the typical clenbuterol concentration observed in biological fluids). Within-day and between-days R.S.D.% in urine ranged between 5.0-5.3 and 8.5-8.7, respectively, while in serum ranged between 5.5-5.9 and 8.7-9.1, respectively. Estimated LOD and LOQ were 9 and 32 ng/ml (spiked urine), respectively, and 5 and 24 ng/ml (spiked serum), respectively, well below the usual clenbuterol urinary and serum level.