Discovery of xanthine oxidase inhibitors from a complex mixture using an online,restricted-access material coupled with column-switching liquid chromatography with a diode-array detection system

To find potential lead compounds for antigout drug discovery, an automated online, restricted-access material coupled with column-switching liquid chromatography with a diode-array detection (RAM–LC–DAD) system was developed for screening of xanthine oxidase (XO) inhibitors and their affinity rankings in complex mixtures. The system was first evaluated by analyzing a mixture of six compounds with known inhibition of XO. Nonspecific binding to the denatured XO was investigated and used as the control for screening. Subsequently, the newly developed system was applied to screening of a natural product, Oroxylum indicum extract, and four compounds which could specifically interact with XO were found and identified as oroxin B, oroxin A, baicalin, and baicalein. The results were verified by a competitive binding test using the known competitive inhibitor allopurinol and were further validated by an inhibition assay in vitro. The online RAM–LC–DAD system developed was shown to be a simple and effective strategy for the rapid screening of bioactive compounds from a complex mixture.

Screening Dyrk1A inhibitors by MALDI-QqQ mass spectrometry: systematic comparison to established radiometric,luminescence, and LC–UV–MS assays

Enzyme-catalyzed reactions play key roles in disease pathology, thus making them relevant subjects of therapeutic inhibitor screening experiments. Matrix-assisted laser desorption/ionization (MALDI) assays have been demonstrated to be able to replace established screening approaches. They offer increased sample throughput, but care must be taken to avoid instrumental bias from differences in ionization efficiencies. We compared a MALDI-triple-quadrupole (QqQ) method for the Dyrk1A peptide substrate woodtide to LC–MS, liquid chromatography with ultraviolet detection (LC–UV), luminescence, and radiometric assays. MALDI measurements were performed on a MALDI-QqQ instrument in the multiple-reaction monitoring mode. Different MALDI conditions were investigated to address whether matrix type, sample support, and MRM- or SIM-based detection conditions can be used to accommodate the molar responses of substrate peptide and its phosphorylated form. UV detection served as a reference method. The impact of MALDI matrix on IC₅₀ values was small, even considering that matrix preparations were used that are known to alleviate response differences. IC₅₀ values determined by MALDI were ca. 2-fold lower than those determined by LC–UV. Although MALDI generated lower ion yields for the phosphorylated peptide than for the peptide substrate, we found that a correction of compound potencies was readily possible using correction factors based on unbiased LC–UV results. A thorough method development delivered a robust assay with excellent performance (Z′ > 0.91) that was close to that seen for LC–UV.

Comprehensive two-dimensional liquid chromatography coupled to the ABTS radical scavenging assay: a powerful method for the analysis of phenolic antioxidants

The on-line combination of comprehensive two-dimensional liquid chromatography (LC?×?LC) with the 2,2′-azino-bis(3-ethylbenzothiazoline)-6 sulphonic acid (ABTS) radical scavenging assay was investigated as a powerful method to determine the free radical scavenging activities of individual phenolics in natural products. The combination of hydrophilic interaction chromatography (HILIC) separation according to polarity and reversed-phase liquid chromatography (RP-LC) separation according to hydrophobicity is shown to provide much higher resolving power than one-dimensional separations, which, combined with on-line ABTS detection, allows the detailed characterisation of antioxidants in complex samples. Careful optimisation of the ABTS reaction conditions was required to maintain the chromatographic separation in the antioxidant detection process. Both on-line and off-line HILIC?×?RP-LC–ABTS methods were developed, with the former offering higher throughput and the latter higher resolution. Even for the fast analyses used in the second dimension of on-line HILIC?×?RP-LC, good performance for the ABTS assay was obtained. The combination of LC?×?LC separation with an on-line radical scavenging assay increases the likelihood of identifying individual radical scavenging species compared to conventional LC–ABTS assays. The applicability of the approach was demonstrated for cocoa, red grape seed and green tea phenolics.

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.

Detailed Study of Cyanobacterial Microcystins Using High Performance Tandem Mass Spectrometry

Microcystins (MC) are a large group of toxic cyclic peptides, produced by cyanobacteria in eutrophic water systems. Identification of MC variants mostly relies on liquid chromatography (LC) combined with collision-induced dissociation (CID) mass spectrometry. Deviations from the essential amino acid complement are a common feature of these natural products, which makes the CID analysis more difficult and not always successful. Here, both CID and electron capture dissociation (ECD) were applied in combination with ultra-high resolution Fourier transform ion cyclotron resonance mass spectrometry to study a cyanobacteria strain isolated from the Salto Grande Reservoir in Sao Paulo State, Brazil, without prior LC separation. CID was shown to be an effective dissociation technique for quickly identifying the MC variants, even those that have previously been difficult to characterize by CID. Moreover, ECD provided even more detailed and complementary information, which enabled us to precisely locate metal binding sites of MCs for the first time. This additional information will be important for environmental chemists to study MC accumulation and production in ecosystems.

On-line solid-phase microextraction of triclosan, bisphenol A, chlorophenols, and selected pharmaceuticals in environmental water samples by high-performance liquid chromatography–ultraviolet detection

A method using on-line solid-phase microextraction (SPME) on a carbowax-templated fiber followed by liquid chromatography (LC) with ultraviolet (UV) detection was developed for the determination of triclosan in environmental water samples. Along with triclosan, other selected phenolic compounds, bisphenol A, and acidic pharmaceuticals were studied. Previous SPME/LC or stir-bar sorptive extraction/LC-UV for polar analytes showed lack of sensitivity. In this study, the calculated octanol–water distribution coefficient (log D) values of the target analytes at different pH values were used to estimate polarity of the analytes. The lack of sensitivity observed in earlier studies is identified as a lack of desorption by strong polar–polar interactions between analyte and solid-phase. Calculated log D values were useful to understand or predict the interaction between analyte and solid phase. Under the optimized conditions, the method detection limit of selected analytes by using on-line SPME-LC-UV method ranged from 5 to 33 ng?L^?1, except for very polar 3-chlorophenol and 2,4-dichlorophenol which was obscured in wastewater samples by an interfering substance. This level of detection represented a remarkable improvement over the conventional existing methods. The on-line SPME-LC-UV method, which did not require derivatization of analytes, was applied to the determination of TCS including phenolic compounds and acidic pharmaceuticals in tap water and river water and municipal wastewater samples.

Rapid on-site TLC–SERS detection of four antidiabetes drugs used as adulterants in botanical dietary supplements

A novel facile method has been established for rapid on-site detection of antidiabetes chemicals used to adulterate botanical dietary supplements (BDS) for diabetes. Analytes and components of pharmaceutical matrices were separated by thin-layer chromatography (TLC) then surface-enhanced Raman spectroscopy (SERS) was used for qualitative identification of trace substances on the HPTLC plate. Optimization and standardization of the experimental conditions, for example the method used for preparation of silver colloids, the mobile phase, and the concentration of colloidal silver, resulted in a very robust and highly sensitive method which enabled successful detection when the amount of adulteration was as low as 0.001 % (w/w). The method was also highly selective, enabling successful identification of some chemicals in extremely complex herbal matrices. The established TLC–SERS method was used for analysis of real BDS used to treat diabetes, and the results obtained were verified by liquid chromatography–triple quadrupole mass spectrometry (LC–MS–MS). The study showed that TLC–SERS could be used for effective separation and detection of four chemicals used to adulterate BDS, and would have good prospects for on-site qualitative screening of BDS for adulterants.

Radical polymerization of methyl methacrylate with 2,2,3-triphenylpropanoic acid as an initiator

An unsymmetrical compound, 2,2,3-triphenylpropanoic acid (TPPA), was successfully prepared from phenyllithium, 1,1-diphenylethylene (DPE), gas carbon dioxide (CO₂), and aqueous standard solution of hydrochloric acid with LiCl deprivation. Characterization of the compound was performed by ¹H and ¹³C nuclear magnetic resonance (NMR) spectroscopy. The polymerization of methyl methacrylate (MMA) was performed in the presence of TPPA at 95 °C. The free radicals obtained were characterized by ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC/Q-TOF MS). Gel permeation chromatography (GPC) traces of the average molecular weight of poly(MMA) (PMMA) showed a series of translations with increasing time. The average molecular weight of PMMA indicated narrow polydispersity, and an approximately linear relationship was found between ln ([M]₀/[M]) and polymerization time.

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.

Development of liquid chromatography atmospheric pressure chemical ionization tandem mass spectrometry for analysis of halogenated flame retardants in wastewater

Until recently, atmospheric pressure photoionization (APPI) has typically been used for the determination of non-polar halogenated flame retardants (HFRs) by liquid chromatography (LC) tandem mass spectrometry. In this study, we demonstrated the feasibility of utilizing liquid chromatography atmospheric pressure chemical ionization (APCI) tandem mass spectrometry (LC-APCI-MS/MS) for analysis of 38 HFRs. This developed method offered three advantages: simplicity, rapidity, and high sensitivity. Compared with APPI, APCI does not require a UV lamp and a dopant reagent to assist atmospheric pressure ionization. All the isomers and the isobaric compounds were well resolved within 14-min LC separation time. Excellent instrument detection limits (6.1 pg on average with 2.0 μL injection) were observed. The APCI mechanism was also investigated. The method developed has been applied to the screening of wastewater samples for screening purpose, with concentrations determined by LC-APCI-MS/MS agreeing with data obtained via gas chromatography high resolution mass spectrometry.

Multi-analytical platform metabolomic approach to study miltefosine mechanism of action and resistance in Leishmania

Miltefosine (MT) (hexadecylphosphocholine) was implemented to cope with resistance against antimonials, the classical treatment in Leishmaniasis. Given the scarcity of anti- Leishmania (L) drugs and the increasing appearance of resistance, there is an obvious need for understanding the mechanism of action and development of such resistance. Metabolomics is an increasingly popular tool in the life sciences due to it being a relatively fast and accurate technique that can be applied either with a particular focus or in a global manner to reveal new knowledge about biological systems. Three analytical platforms, gas chromatography (GC), liquid chromatography (LC) and capillary electrophoresis (CE) have been coupled to mass spectrometry (MS) to obtain a broad picture of metabolic changes in the parasite. Impairment of the polyamine metabolism from arginine (Arg) to trypanothione in susceptible parasites treated with MT was in some way expected, considering the reactive oxygen species (ROS) production described for MT. Importantly, in resistant parasites an increase in the levels of amino acids was the most outstanding feature, probably related to the adaptation of the resistant strain for its survival inside the parasitophorous vacuole.

Rapid and sensitive detection of bacteria via platinum-labeled antibodies and on-particle ionization and enrichment prior to MALDI-TOF mass spectrometry

We introduce a rapid and sensitive approach to study the interactions of an affinity probe with the bacterial wall. Immunoglobulin was immobilized on platinum nanoparticles, and the resulting probe nanoparticles bind to bacterial walls as confirmed by transmission electron microscopy. A MALDI-MS assay was developed that can detect ~10⁵ cfu mL^?1 of S. marcescens and E. coli. This approach enables simple, rapid and straightforward detection of bacterial proteins, with high resolution and sensitivity, and without the requirement for tedious washing/separation steps.

Identification and quantification of flavonoids and ellagic acid derivatives in therapeutically important Drosera species by LC-DAD, LC-NMR, NMR, and LC-MS

Droserae herba is a drug commonly used for treatment of convulsive or whooping cough since the seventeenth century. Because of the contribution of flavonoids and ellagic acid derivatives to the therapeutic activity of Droserae herba, an LC?CDAD method has been developed for quantification of these analytes in four Drosera species used in medicine (Drosera anglica, D. intermedia, D. madagascariensis, and D. rotundifolia). During elaboration of the method 13 compounds, including three substances not previously described for Drosera species, were detected and unambiguously identified by means of extensive LC?CMS and LC?CNMR experiments and by off-line heteronuclear 2D NMR after targeted isolation. The most prominent component of D. rotundifolia and D. anglica, 2??-O-galloylhyperoside, with myricetin-3-O-??-glucopyranoside and kaempferol-3-O-(2??-O-galloyl)-??-galactopyranoside, were identified for the very first time in this genus. The LC?CDAD method for quantification was thoroughly validated, and enables, for the first time, separation and precise analysis of these analytes in Droserae herba. Simple sample preparation and use of a narrow-bore column guarantee low cost and simplicity of the suggested system, which is excellently suited to quality control of the drug or herbal medicinal products containing this drug.

Microfluidic robotic device coupled with electrochemical sensor field for handling of paramagnetic micro-particles as a tool for determination of plant mRNA

The expression of genes responsible for the biosynthesis of stress proteins corresponds to the exposition of an organism to abiotic and/or biotic stress. We utilize two types of paramagnetic particles for isolation of total mRNA from early somatic embryos of Norway Spruce (Picea abies /L./ Karst.) and maize plants (Zea mays L.) treated with cadmium(II) ions. The paramagnetic particles were evaluated for analysis of real samples, and poly-adenine was used as a model mRNA. Various approaches (from non-automatic to fully automatic) were tested in terms of handling the particles.

Qualitative metabolism assessment and toxicological detection of xylazine, a veterinary tranquilizer and drug of abuse, in rat and human urine using GC–MS, LC–MS n , and LC–HR-MS n

Xylazine is used in veterinary medicine for sedation, anesthesia, and analgesia. It has also been reported to be misused as a horse doping agent, a drug of abuse, a drug for attempted sexual assault, and as source of accidental or intended poisonings. So far, no data concerning human metabolism have been described. Such data are necessary for the development of toxicological detection methods for monitoring drug abuse, as in most cases the metabolites are the analytical targets. Therefore, the metabolism of xylazine was investigated in rat and human urine after several sample workup procedures. The metabolites were identified using gas chromatography (GC)–mass spectrometry (MS) and liquid chromatography (LC) coupled with linear ion trap high-resolution multistage MS (MS ⁿ ). Xylazine was N-dealkylated and S-dealkylated, oxidized, and/or hydroxylated to 12 phase I metabolites. The phenolic metabolites were partly excreted as glucuronides or sulfates. All phase I and phase II metabolites identified in rat urine were also detected in human urine. In rat urine after a low dose as well as in human urine after an overdose, mainly the hydroxy metabolites were detected using the authors’ standard urine screening approaches by GC–MS and LC–MS ⁿ . Thus, it should be possible to monitor application of xylazine assuming similar toxicokinetics in humans.

Simultaneous determination of water-soluble vitamins in SRM 1849 Infant/Adult Nutritional Formula powder by liquid chromatography–isotope dilution mass spectrometry

Assessing dietary intake of vitamins from all sources, including foods, dietary supplements, and fortified foods, would be aided considerably by having analytical methodologies that are capable of simultaneous determination of several vitamins. Vitamins naturally present in foods may occur in different chemical forms, with levels ranging over several orders of magnitude. Vitamins in dietary supplements and fortified foods, however, are typically added in a single chemical form, and matrix issues are usually not as complex. These sources should thus be relatively amenable to approaches that aim for simultaneous determination of multiple vitamins. Our recent work has focused on development of liquid chromatography (LC)–UV/fluorescence and LC–tandem mass spectrometry methods for the simultaneous determination of water-soluble vitamins (thiamine, niacin, pyridoxine, pantothenic acid, folic acid, biotin, and riboflavin) in dietary supplement tablets and fortified foods, such as formula powders and breakfast cereals. As part of the validation of our methods and collaboration in characterization of a new NIST SRM 1849 Infant/Adult Nutritional Formula powder, we report data on SRM 1849 using isotope dilution mass spectrometric methods. Use of available NIST Standard Reference Materials® as test matrices in our method development and validation gives a benchmark for future application of these methods. We compare three chromatographic approaches and provide data on stability of vitamin standard solutions for LC-based multiple vitamin determinations.

Hydrophilic molecularly imprinted polymers for bisphenol A prepared in aqueous solution

We have prepared a hydrophilic molecularly imprinted polymer (MIP) for the hydrophobic compound bisphenol A (BPA) in aqueous solution using 3-acrylamido-N,N,N-trimethylpropan-1-aminium chloride (AMTC) as the functional monomer. Under redox-polymerization conditions, BPA forms an ion-pair with AMTC, which was confirmed by ¹H-NMR titration. The imprinting effect in aqueous solution was evaluated by comparison of this material with the corresponding non-imprinted polymer (NIP) and with a control polymer (CP) bearing no AMTC. The MIP showed the highest activity among the three polymers, and the imprinting factors as calculated from the amount of BPA bound to the MIP divided by the amounts bound to NIP and CP, respectively, are 1.8 and 6.0. The MIP was selective for BPA in aqueous solution, while structurally related compounds are not recognized. Such a selectivity for a hydrophobic compound is rarely observed in aqueous medium because non-specific binding of BPA inevitably leads to hydrophobic interaction.

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.

Chemical imaging of trichome specialized metabolites using contact printing and laser desorption/ionization mass spectrometry

Cell transfer by contact printing coupled with carbon-substrate-assisted laser desorption/ionization was used to directly profile and image secondary metabolites in trichomes on leaves of the wild tomato Solanum habrochaites. Major specialized metabolites, including acyl sugars, alkaloids, flavonoids, and terpenoid acids, were successfully detected in positive ion mode or negative ion mode, and in some cases in both modes. This simple solvent-free and matrix-free sample preparation for mass spectrometry imaging avoids tedious sample preparation steps, and high-spatial-resolution images were obtained. Metabolite profiles were generated for individual glandular trichomes from a single Solanum habrochaites leaf at a spatial resolution of around 50 μm. Relative quantitative data from imaging experiments were validated by independent liquid chromatography–mass spectrometry analysis of subsamples from fresh plant material. The spatially resolved metabolite profiles of individual glands provided new information about the complexity of biosynthesis of specialized metabolites at the cellular-resolution scale. In addition, this technique offers a scheme capable of high-throughput profiling of metabolites in trichomes and irregularly shaped tissues and spatially discontinuous cells of a given cell type.