Characterization of grape seed procyanidins by comprehensive two-dimensional hydrophilic interaction × reversed phase liquid chromatography coupled to diode array detection and tandem mass spectrometry

In this work, the development and optimization of a new methodology to analyze grape seed procyanidins based on the application of two-dimensional comprehensive LC is presented. This two-dimensional method involves the use of a microbore column containing a diol stationary phase in the first dimension coupled to either a C₁₈ partially porous short column or a C₁₈ monolithic column in the second dimension. The orthogonal hydrophilic interaction?×?reversed phase liquid chromatography (HILIC×RP-LC) system is interfaced through a ten-port two-position switching valve. The optimized HILIC×RP-LC separation followed by diode array and tandem mass spectrometry detection (HILIC×RP-LC-DAD-MS/MS) made possible the direct analysis of a complex grape seed extract and allowed the tentative identification of 43 flavan-3-ols, including monomers and procyanidin oligomers till a polymerization degree of 7 units with different galloylation degrees. To the best of our knowledge, this is the first time that this powerful analytical technique is employed to characterize complex procyanidin samples. This work successfully demonstrates the great capabilities of the HILIC×RP-LC-DAD-MS/MS coupling for the direct analysis of very complex natural samples like grape seeds.

High-resolution monolithic columns—a new tool for effective and quick separation

This work describes a comparison of three types of commercial high-performance liquid chromatography silica monolithic columns with different inner diameters and generations of monolithic sorbent: a “classic” monolithic column, the first generation (Onyx? monolithic C₁₈, 100 mm?×?4.6 mm, Phenomenex); a “narrow” monolithic column for fast separation at lower flow rates (Chromolith® Performance RP-18e, 100 mm?×?3 mm, Merck); and a recently introduced “high-resolution” monolithic column, the next generation (Chromolith® HighResolution RP-18e, 100 mm?×?4.6 mm, Merck). Separation efficiency (number of theoretical plates, height equivalent to a theoretical plate and van Deemter curves), working pressure, the symmetry factor and resolution were critical aspects of the comparison in the case of the separation of ascorbic acid, paracetamol and caffeine. The separations were performed under isocratic conditions with a mobile phase consisting of 10:90 (v/v) acetonitrile–phosphoric acid (pH 2.80). Detailed comparison of the newest-generation monolithic column (Chromolith® HighResolution) with the previously introduced monolithic sorbents was performed and proved the advantages of the Chromolith® HighResolution column.

Continuous vs. segmented second-dimension system gradients for comprehensive two-dimensional liquid chromatography of sugarcane (Saccharum spp.)

A comprehensive two-dimensional liquid chromatography system in combination with photodiode array and mass spectrometry detection was developed for analysis of polyphenols in sugarcane (Saccharum spp.) leaf extracts. To achieve this, a micro cyano column and a partially porous octodecylsilica column were used in the first and the second dimension, respectively. The choice of the cyano column over other reversed-phase columns tested for the first-dimension separation was due to its lower correlation selectivity with respect to the octodecylsilica column, which was used for the second-dimension separation. Even when reversed-phase mode was used in both dimensions, a satisfactory degree of orthogonality was achieved by use of different gradient elution modes in the second dimension. By means of the setup investigated, 38 polyphenolic compounds were detected, and among them 24 were positively identified by means of complementary data from photodiode array and mass spectrometry detection and an in-house database. This is the first time such a powerful analytical technique has been used for polyphenolic characterization of sugarcane extracts.

Molecularly imprinted solid-phase extraction monolithic capillary column for selective extraction and sensitive determination of safranine T in wolfberry

A method was developed to sensitively determine safranine T in wolfberry by molecularly imprinted solid-phase extraction (MISPE) coupled with high-performance liquid chromatography and laser-induced fluorescence detection (HPLC-LIF). The MISPE capillary monolithic column was prepared by water-bath in situ polymerization, using safranine T, methacrylic acid (MAA), and ethylene dimethacrylate (EDMA) as template, functional monomer, and cross-linker, respectively. The properties of the homemade MISPE capillary monolithic column, including capacity and specificity, were investigated under optimized conditions and the morphologies of inner polymers were characterized by scanning electron microscopy (SEM). The mean recoveries of safranine T in wolfberry ranged from 91.2 % to 92.9 % and the intraday and interday relative standard deviation (RSD) values all ranged from 3.4 % to 4.2 %. Good linearity was obtained over 0.001–1.0 μg mL^–1 (r?=?0.9999) with a detection limit (S/N?=?3) of 0.4 ng g^–1. Under the selected conditions, enrichment factors of over 90-fold were obtained and the extraction on the monolithic column effectively cleaned up the wolfberry matrix. The results demonstrated that the proposed MISPE-HPLC-LIF method could be applied to sensitively determine safranine T in wolfberry.

Monolithic molecularly imprinted solid-phase extraction for the selective determination of trace cytokinins in plant samples with liquid chromatography–electrospray tandem mass spectrometry

Cytokinins (CTKs) are a class of growth-regulating hormones involved in various physiological and developmental processes. More novel analytical methods for the accurate identification and quantitative determination of trace CTKs in plants have been desired to better elucidate the roles of CTKs. In this work, a novel method based on monolithic molecularly imprinted solid-phase extraction followed by liquid chromatography–electrospray tandem mass spectrometry (mMI-SPE-LC-MS/MS) was developed for accurate determination of four CTKs in plant samples. The molecularly imprinted polymer monolith was prepared by using kinetin as the template in syringes and exhibited specific recognition ability for the four CTKs in comparison with that of non-imprinted polymer monolith. Several factors affecting the extraction performance of mMI-SPE, including the pH of loading sample solution, the nature and volume of elution solvent, the flow rate of sample loading, and sample volume, were investigated, respectively. Under the optimized conditions, the proposed mMI-SPE-LC-MS/MS method was successfully applied in the selective extraction and determination of four CTKs in plant tissues, and it offers detection limits (S/N?=?3) of 104, 113, 130, and 89 pg/mL and mean recoveries of 85.9%, 79.3%, 73.5%, and 70.1% for kinetin, kinetin glucoside, trans-zeatin, and meta-topolin (mT), respectively, with the corresponding RSDs less than 15%.

N-Glycosidase treatment with 18O labeling and de novo sequencing argues for flagellin FliC glycopolymorphism in Pseudomonas aeruginosa

In prokaryote organisms, N-glycosylation of proteins is often correlated to cell–cell recognition and extracellular events. Those glycoproteins are potential targets for infection control. To date, many surface-glycosylated proteins from bacterial pathogens have been described. However, N-linked Pseudomonas surface-associated glycoproteins remain underexplored. We report a combined enrichment and labeling strategy to identify major glycoproteins on the outside of microorganisms. More precisely, bacteria were exposed to a mix of biotinylated lectins able to bind with glycoproteins. The latter were then recovered by avidin beads, digested with trypsin, and submitted to mass spectrometry. The targeted mixture of glycoproteins was additionally deglycosylated in the presence of H₂ ¹⁸O to incorporate ¹⁸O during PNGase F treatment and were also analyzed using mass spectrometry. This approach allowed us to identify a few tens of potential N-glycoproteins, among which flagellin FliC was the most abundant. To detect the possible sites of FliC modifications, a de novo sequencing step was also performed to discriminate between spontaneous deamidation and N-glycan loss. This approach led to the proposal of three potential N-glycosylated sites on the primary sequence of FliC: N26, N69, and N439, with two of these three asparagines belonging to an N-X-(S/T) consensus sequence. These observations suggest that flagellin FliC is a heterogeneous protein mixture containing both O- and N-glycoforms.

Selective microextraction of carbaryl and naproxen using organic–inorganic monolithic columns containing a double molecular imprint

We report on an organic–inorganic hybrid material that was double imprinted with the insecticide carbaryl and the anti-inflammatory drug naproxen by a single-step method and that can serve for selective microextraction of the two analytes. The materials, in the form of monolithic columns, were characterized by scanning electron microscopy and Fourier transform IR spectra. A simple, rapid and sensitive method was then developed for the simultaneous determination of carbaryl and naproxen in lettuce and river water using these columns for microextraction, HPLC for separation, and a diode array for UV detection. The limits of detection (at S/N?=?3) and quantification (at S/N?=?10) are in the ranges of 2.5 – 8.8 μg kg^?1 and 2.3 – 8.0 μg L^?1 for lettuce and Yangtze River water, respectively. The recoveries of this method range from 93.0 to 108 % (in case of analyzing lettuce and river water), and relative standard deviations are <8.9 %.

Analysis of stem cell lipids by offline HPTLC-MALDI-TOF MS

MALDI-TOF MS is traditionally used for “proteomics”, but is also a useful tool for lipid analysis. Depending on the applied matrix, however, some lipid classes are more sensitively detected than other ones and this may even lead to suppression effects if complex mixtures are analyzed. Therefore, a previous separation into the individual lipid classes is necessary. Using artificial lipid mixtures or easily available tissue extracts, it has been already shown that HPTLC-(High Performance Thin-Layer Chromatography)-separated lipids can be conveniently analyzed by MALDI-TOF MS directly on the TLC plate. Here we present an initial TLC-MALDI study of the lipid composition of ovine mesenchymal stem cells. Due to the complex composition of these cells, data are also compared to lipids extracted from human erythrocytes. It will be shown that even very minor lipid classes can be easily detected and with much higher sensitivity than by common staining protocols. Additionally, MS images of the developed TLC plates will be shown and potential applications, new methods of data analysis as well as problems discussed.

Data-Independent Microbial Metabolomics with Ambient Ionization Mass Spectrometry

Atmospheric ionization methods are ideally suited for prolonged MS/MS analysis. Data-independent MS/MS is a complementary technique for analysis of biological samples as compared to data-dependent analysis. Here, we pair data-independent MS/MS with the ambient ionization method nanospray desorption electrospray ionization (nanoDESI) for untargeted analysis of bacterial metabolites. Proof-of-principle data and analysis are illustrated by sampling Bacillus subtilis and Pseudomonas aeruginosa directly from Petri dishes. We found that this technique enables facile comparisons between strains via MS and MS/MS plots which can be translated to chemically informative molecular maps through MS/MS networking. The development of novel techniques to characterize microbial metabolites allows rapid and efficient analysis of metabolic exchange factors. This is motivated by our desire to develop novel techniques to explore the role of interspecies interactions in the environment, health, and disease. This is a contribution to honor Professor Catherine C. Fenselau in receiving the prestigious ASMS Award for a Distinguished Contribution in Mass Spectrometry for her pioneering work on microbial mass spectrometry.

A New Splitting Method for Both Analytical and Preparative LC/MS

This paper presents a novel splitting method for liquid chromatography/mass spectrometry (LC/MS) application, which allows fast MS detection of LC-separated analytes and subsequent online analyte collection. In this approach, a PEEK capillary tube with a micro-orifice drilled on the tube side wall is used to connect with LC column. A small portion of LC eluent emerging from the orifice can be directly ionized by desorption electrospray ionization (DESI) with negligible time delay (6~10 ms) while the remaining analytes exiting the tube outlet can be collected. The DESI-MS analysis of eluted compounds shows narrow peaks and high sensitivity because of the extremely small dead volume of the orifice used for LC eluent splitting (as low as 4 nL) and the freedom to choose favorable DESI spray solvent. In addition, online derivatization using reactive DESI is possible for supercharging proteins and for enhancing their signals without introducing extra dead volume. Unlike UV detector used in traditional preparative LC experiments, this method is applicable to compounds without chromophores (e.g., saccharides) due to the use of MS detector. Furthermore, this splitting method well suits monolithic column-based ultra-fast LC separation at a high elution flow rate of 4 mL/min.

Rapid enrichment of phosphopeptides by BaTiO3 nanoparticles after microwave-assisted tryptic digest of phosphoproteins, and their identification by MALDI-MS

We show that BaTiO₃ nanoparticles (NPs) can be used as a novel substrate for the rapid enrichment of phosphopeptides from microwave tryptic digests of α-casein and non-fat milk prior to their identification by MALDI-MS. Protein digestion is achieved by microwave tryptic digest for 50?s, and the resulting phosphopeptides can be effectively adsorbed on the surfaces of the NPs. The phosphopeptides were selectively detected via MALDI-MS. Digestion, enrichment and detection are accomplished within ～60?min. The method was applied to the indentification of 24 phosphopeptides from α-casein and of 21 phosphopeptides (of the α-casein type) from nonfat milk.

Rapid comprehensive characterization of crude oils by thermogravimetry coupled to fast modulated gas chromatography–single photon ionization time-of-flight mass spectrometry

Comprehensive multi-dimensional hyphenation of a thermogravimetry device (i.e. a thermobalance) to gas chromatography and single photon ionization–time-of-flight mass spectrometry (TG–GC×SPI–MS) has been used to investigate two crude oil samples of different geographical origin. The source of the applied vacuum ultraviolet radiation is an electron beam pumped rare gas excimer lamp (EBEL). The soft photoionization favors the formation of molecular ions. Introduction of a fast, rapidly modulated gas chromatographic separation step in comparison with solely TG–SPI–MS enables strongly enhanced detection especially with such highly complex organic matrices as crude oil. In contrast with former TG–SPI–MS measurements, separation and identification of overlying substances is possible because of different GC retention times. The specific contribution of isobaric compounds to one mass signal is determined for alkanes, naphthalenes, alkylated benzenes, and other compounds.

Optimization in multidimensional gas chromatography applying quantitative analysis via a stable isotope dilution assay

Trace level analyses in complex matrices benefit from heart-cut multidimensional gas chromatographic (MDGC) separations and quantification via a stable isotope dilution assay. Minimization of the potential transfer of co-eluting matrix compounds from the first dimension (¹D) separation into the second dimension separation requests narrow cut-windows. Knowledge about the nature of the isotope effect in the separation of labeled and unlabeled compounds allows choosing conditions resulting in at best a co-elution situation in the ¹D separation. Since the isotope effect strongly depends on the interactions of the analytes with the stationary phase, an appropriate separation column polarity is mandatory for an isotopic co-elution. With 3-alkyl-2-methoxypyrazines and an ionic liquid stationary phase as an example, optimization of the MDGC method is demonstrated and critical aspects of narrow cut-window definition are discussed.

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

Gram-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.

Enzyme-Coupled Nanoparticles-Assisted Laser Desorption Ionization Mass Spectrometry for Searching for Low-Mass Inhibitors of Enzymes in Complex Mixtures

In this report, enzyme-coupled magnetic nanoparticles (EMPs) were shown to be an effective affinity-based tool for finding specific interactions between enzymatic targets and the low-mass molecules in complex mixtures using classic MALDI-TOF apparatus. EMPs used in this work act as nonorganic matrix enabling ionization of small molecules without any interference in the low-mass range (enzyme-coupled nanoparticles-assisted laser desorption ionization MS, ENALDI MS) and simultaneously carry the superficial specific binding sites to capture inhibitors present in a studied mixture. We evaluated ENALDI approach in two complementary variations: ‘ion fading’ (IF-ENALDI), based on superficial adsorption of inhibitors and ‘ion hunting’ (IH-ENALDI), based on selective pre-concentration of inhibitors. IF-ENALDI was applied for two sets of enzyme–inhibitor pairs: tyrosinase–glabridin and trypsin–leupeptin and for the real plant sample: Sparrmannia discolor leaf and stem methanol extract. The efficacy of IH-ENALDI was shown for the pair of trypsin–leupeptin. Both ENALDI approaches pose an alternative for bioassay-guided fractionation, the common method for finding inhibitors in the complex mixtures.

Multiplex Mass Spectrometric Imaging with Polarity Switching for Concurrent Acquisition of Positive and Negative Ion Images

We have recently developed a multiplex mass spectrometry imaging (MSI) method which incorporates high mass resolution imaging and MS/MS and MS³ imaging of several compounds in a single data acquisition utilizing a hybrid linear ion trap-Orbitrap mass spectrometer (Perdian and Lee, Anal. Chem. 82, 9393–9400, 2010). Here we extend this capability to obtain positive and negative ion MS and MS/MS spectra in a single MS imaging experiment through polarity switching within spiral steps of each raster step. This methodology was demonstrated for the analysis of various lipid class compounds in a section of mouse brain. This allows for simultaneous imaging of compounds that are readily ionized in positive mode (e.g., phosphatidylcholines and sphingomyelins) and those that are readily ionized in negative mode (e.g., sulfatides, phosphatidylinositols and phosphatidylserines). MS/MS imaging was also performed for a few compounds in both positive and negative ion mode within the same experimental set-up. Insufficient stabilization time for the Orbitrap high voltage leads to slight deviations in observed masses, but these deviations are systematic and were easily corrected with a two-point calibration to background ions.

Chiral separation of racemic mandelic acids by use of an ionic liquid-mediated imprinted monolith with a metal ion as self-assembly pivot

A new chiral stationary phase based on molecularly imprinted polymers (MIP) was prepared in ionic liquid by use of the metal pivot concept. Imprinted monoliths were synthesized by use of a mixture of R-mandelic acid (template), 4-vinylpyridine, ethylene glycol dimethacrylate, and several metal ions as pivot between the template and functional monomer. A ternary mixture of dimethyl sulfoxide–dimethylformamide–[BMIM]BF₄ containing metal ions was used as the porogenic system. Separation of the enantiomers of rac-mandelic acid was successfully achieved on the MIP thus obtained, with resolution of 1.87, whereas no enantiomer separation was observed on the imprinted monolithic column in the absence of metal ions. The effects of polymerization conditions, including the nature of the metal ion and the ratios of template to metal ions and template to functional monomer, on the chiral separation of mandelic acid were investigated. The results reveal that use of metal ions as a pivot, in combination with ionic liquid, is an effective method for preparation of a highly efficient MIP stationary phase for chiral separation.

Normal silica gel and reversed phase thin-layer chromatography coupled with UV spectroscopy and IR-MALDI-o-TOF-MS for the detection of tetracycline antibiotics

Tetracyclines (TCs) form a group of bacteriostatic antibiotics with closely related structures and similar chemical and physicochemical properties. They are widely employed as therapeutics in human and veterinary medicine. Here, we introduce the combination of UV spectroscopic detection of high-performance thin-layer chromatography (HPTLC)-separated TCs with direct analysis on solid phase using infrared matrix-assisted laser desorption/ionization orthogonal time-of-flight mass spectrometry (IR-MALDI-o-TOF-MS). Normal silica gel phase- and water-wettable hybrid C₁₈ reversed phase layers (RP-18W) both allowed HPTLC separation and sensitive UV spectroscopic detection followed by MS analysis of distinct TCs bands using the liquid matrix glycerol. The novel approach of direct IR-MALDI-o-TOF-MS analysis resulted in the unequivocal identification of four structurally different TCs employed in this study, and linear calibration curves were produced for analyte amounts from 20 ng to 1 μg. MS analysis of TCs from RP-18W HPTLC plates was found to be superior when compared to the spectra acquired from the silica gel layer. Ionic analytes obtained from the RP-18W surface are mainly detected as protonated species of high abundance accompanied by a reduced formation of adducts as well as background ions arising from the matrix or the stationary phase. This results in decreased complexity of the spectra and enhanced sensitivity of the combinatorial method. An approximate detection limit of 5 ng of individual TCs in mixtures by combining RP-18W HPTLC with IR-MALDI-o-TOF-MS offers a novel timely and cost-efficient method for tracing TCs.

Development of an LC-MS/MS method for aromatase inhibitor screening

Aromatase (CYP 19A1) is a key steroidogenic enzyme that catalyzes the conversion of androgen to estrogen. In this study, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for aromatase inhibitor screening was developed and validated. The substrate androstenedione was incubated with human CYP 19A1 supersomes in the presence of NADPH for 30 min, and estrone formation was determined by LC-MS/MS analysis. Cortisone was used as internal standard. The incubation mixture was extracted using a liquid-liquid extraction method with ethyl acetate. Chromatographic separation was achieved using a C₁₈ column (3.0?×?50 mm, 2.7 μm) with a mobile phase consisting of 0.1 % formic acid/acetonitrile adopting gradient elution at a flow rate of 0.4 mL/min. The mass spectrometer was operated in positive electrospray ionization mode. The precursor-product ion pairs used for multiple reaction monitoring were m/z 287→97 (androstenedione), m/z 271?→?159 (estrone), and m/z 361?→?163 (IS, cortisone). The developed method met the required criteria for the validation of bioanalytical methods. The validated method was successfully applied to evaluate aromatase inhibitory activity of plants extracts of Simaroubaceae.