The analysis of acetaminophen (paracetamol) and seven metabolites in rat, pig and human plasma by U(H)PLC-MS.

A U(H)PLC-MS/MS method is described for the analysis of acetaminophen and its sulphate, glucuronide, glutathione, cysteinyl and N-acetylcysteinyl metabolites in plasma using stable isotope-labeled internal standards. P-Aminophenol glucuronide and 3-methoxyacetaminophen were monitored and semi-quantified using external standards. The assay takes 7.5 min/sample, requires only 5 µl of plasma and involves minimal sample preparation. The method was validated for rat plasma and cross validated for human and pig plasma and mouse serum. LOQ in plasma for these analytes were 0.44 µg/ml (APAP-C), 0.58 µg/ml (APAP-SG), 0.84 µg/ml (APAP-NAC), 2.75 µg/ml (APAP-S), 3.00 µg/ml (APAP-G) and 16 µg/ml (APAP). Application of the method is illustrated by the analysis of plasma following oral administration of APAP to male Han Wistar rats.

A comparison of collision cross section values obtained via travelling wave ion mobility-mass spectrometry and ultra high performance liquid chromatography-ion mobility-mass spectrometry: Application to the characterisation of metabolites in rat urine.

A comprehensive Collision Cross Section (CCS) library was obtained via Travelling Wave Ion Guide mobility measurements through direct infusion (DI). The library consists of CCS and Mass Spectral (MS) data in negative and positive ElectroSpray Ionisation (ESI) mode for 463 and 479 endogenous metabolites, respectively. For both ionisation modes combined, TWCCSN2 data were obtained for 542 non-redundant metabolites. These data were acquired on two different ion mobility enabled orthogonal acceleration QToF MS systems in two different laboratories, with the majority of the resulting TWCCSN2 values (from detected compounds) found to be within 1% of one another. Validation of these results against two independent, external TWCCSN2 data sources and predicted TWCCSN2 values indicated to be within 1-2% of these other values. The same metabolites were then analysed using a rapid reversed-phase ultra (high) performance liquid chromatographic (U(H)PLC) separation combined with IM and MS (IM-MS) thus providing retention time (tr), m/z and TWCCSN2 values (with the latter compared with the DI-IM-MS data). Analytes for which TWCCSN2 values were obtained by U(H)PLC-IM-MS showed good agreement with the results obtained from DI-IM-MS. The repeatability of the TWCCSN2 values obtained for these metabolites on the different ion mobility QToF systems, using either DI or LC, encouraged the further evaluation of the U(H)PLC-IM-MS approach via the analysis of samples of rat urine, from control and methotrexate-treated animals, in order to assess the potential of the approach for metabolite identification and profiling in metabolic phenotyping studies. Based on the database derived from the standards 63 metabolites were identified in rat urine, using positive ESI, based on the combination of tr, TWCCSN2 and MS data.

Ultrahigh-Performance Liquid Chromatography Tandem Mass Spectrometry with Electrospray Ionization Quantification of Tryptophan Metabolites and Markers of Gut Health in Serum and Plasma-Application to Clinical and Epidemiology Cohorts.

A targeted ultrahigh-performance liquid chromatography tandem mass spectrometry with electrospray ionization (UHPLC-ESI-MS/MS) method has been developed for the quantification of tryptophan and its downstream metabolites from the kynurenine and serotonin pathways. The assay coverage also includes markers of gut health and inflammation, including citrulline and neopterin. The method was designed in 96-well plate format for application in multiday, multiplate clinical and epidemiology population studies. A chromatographic cycle time of 7 min enables the analysis of two 96-well plates in 24 h. To protect chromatographic column lifespan, samples underwent a two-step extraction, using solvent protein precipitation followed by delipidation via solid-phase extraction (SPE). Analytical validation reported accuracy of each analyte <20% for the lowest limit of quantification and <15% for all other quality control (QC) levels. The analytical precision for each analyte was 2.1-12.9%. To test the applicability of the method to multiplate and multiday preparations, a serum pool underwent periodic repeat analysis during a run consisting of 18 plates. The % CV (coefficient of variation) values obtained for each analyte were <15%. Additional biological testing applied the assay to samples collected from healthy control participants and two groups diagnosed with inflammatory bowel disease (IBD) (one group treated with the anti-inflammatory 5-aminosalicylic acid (5-ASA) and one group untreated), with results showing significant differences in the concentrations of picolinic acid, kynurenine, and xanthurenic acid. The short analysis time and 96-well plate format of the assay makes it suitable for high-throughput targeted UHPLC-ESI-MS/MS metabolomic analysis in large-scale clinical and epidemiological population studies.

Drawing a different picture with pencil lead as matrix-assisted laser desorption/ionization matrix for fullerene derivatives.

Inspired by reports on the use of pencil lead as a matrix-assisted laser desorption/ionization matrix, paving the way towards matrix-free matrix-assisted laser desorption/ionization, the present investigation evaluates its usage with organic fullerene derivatives. Currently, this class of compounds is best analysed using the electron transfer matrix trans-2-[3-(4-tert-butylphenyl)-2-methyl-2-propenylidene] malononitrile (DCTB), which was employed as the standard here. The suitability of pencil lead was additionally compared to direct (i.e. no matrix) laser desorption/ionization-mass spectrometry. The use of (DCTB) was identified as the by far gentler method, producing spectra with abundant molecular ion signals and much reduced fragmentation. Analytically, pencil lead was found to be ineffective as a matrix, however, appears to be an extremely easy and inexpensive method for producing sodium and potassium adducts.

Identification of a novel human circulating metabolite of tenofovir disoproxil fumarate with LC-MS/MS.

BACKGROUND: Tenofovir disoproxil fumarate (TDF) is an antiretroviral drug used for the treatment of Human Immunodeficiency Virus and Hepatitis B infections. RESULTS: A metabolite that has previously not been observed in the circulation of humans was detected by LC-MS/MS in early time point plasma samples following administration of TDF to healthy volunteers. The metabolite was identified using a range of LC-MS/MS-based techniques as a monoester of TDF, derived from the partially hydrolyzed bis-ester prodrug. TDF, when spiked into plasma, was observed to degrade first to the putative monoester and subsequently to tenofovir. CONCLUSION: The presence of this unstable metabolite in some samples has implications for sample collection, handling and storage in studies of tenofovir where serum concentrations are determined.

Ion formation pathways of crown ether-fullerene conjugates in the gas phase.

The ion formation of crown ether-[60]fullerene conjugates of the type (crown - H)-C60-H with crown = 12cr4, 15cr5 and 18cr6 has been studied with matrix-assisted laser desorption/ionisation (MALDI) and electrospray ionisation mass spectrometry (ESI MS). In total five different ways of ion formation are presented, including metalation (MALDI, ESI), protonation and oxidation (both in MALDI) in the positive-ion mode and deprotonation (MALDI, ESI) and reduction (MALDI) in the negative-ion mode. In line with thermochemistry, the deprotonation and electron transfer processes involve the C60 moiety as the charge-carrying entity, while protonation and metalation occur at the crown ether. Particular emphasis has been placed on the study of metal cation attachment in MALDI varying the crown ether size in the conjugate and using different alkali metal chlorides in the target preparation. Dissociation reactions of the metalated conjugates are influenced by the interaction strength of the metal cation to the crown ether fullerene conjugate. The data confirm an increase in bond strength with smaller metal cations, supporting the notion of charge density-driven interactions.

Chemical and photochemical functionality of the first molecular bismuth vanadium oxide.

Anionic metal oxide clusters, so-called polyoxometalates, can be developed as molecular model compounds to mimic the chemical and photochemical reactivity of solid-state metal oxides on the molecular level. Inspired by the well-known visible-light photocatalyst BiVO(4), the first molecular bismuth vanadium oxide has been synthesized to investigate the chemical and photochemical similarities between the solid-state and molecular compounds. The cluster H(3)[(Bi(dmso)(3))(4)V(13)O(40)]·ca. 4 DMSO was obtained from simple precursors in almost quantitative yield. Structural analysis showed that the cluster shell is based on the unusual all-vanadium ε-Keggin framework [ε-V(12)O(40)](15-), which is stabilized by coordination of four Bi(III) centers. The acidic character of the three cluster protons was demonstrated by titration studies. The cluster shows promising photocatalytic properties in visible-light photooxidation reactions and has high activity (turnover number >1200), high quantum yield (Φ=7.6 %), and good recyclability, which make it a promising first example of a new class of heterometallic polyoxometalates.

Metal substitution in a Lindqvist polyoxometalate leads to improved photocatalytic performance.

The photochemical properties of homo- and heterometallic molybdate-based Lindqvist polyoxometalate clusters are investigated in a comparative study and it is shown that vanadium substitution can be used as a facile synthetic tool to optimize the visible light absorption and photocatalytic activity of the cluster. The mono-vanadium substituted unit, [VMo(5)O(19)](3-) shows light absorption up to 480 nm whereas the light absorption of the molybdate analogue [Mo(6)O(19)](2-) is mainly in the UV region below 400 nm. The electronic absorption properties of both clusters are further investigated using TD-DFT calculations which show that vanadium incorporation leads to the formation of low-energy O → V LMCT transitions. In comparative photochemical dye decomposition test reactions under UV and Vis irradiation, a higher reactivity is observed for [VMo(5)O(19)](3-) together with turnover numbers of more than 1600. In addition it is shown that under anaerobic conditions, the photoreaction proceeds faster than in the presence of oxygen, suggesting that oxygen acts as a quencher in one of the photoredox steps.

Zwitterionic clusters with dianion core produced by electrospray ionisation of Brønsted acidic ionic liquids.

New Brønsted acidic ionic liquids (BAILs) are prepared by treating zwitterions, which are composed of an imidazolium cation and a sulfonate anion, with an alkanedisulfonic acid. Acidification of the zwitterions produces the cation and deprotonation of the alkanedisulfonic acid forms the anion of the new BAILs. Direct laser desorption/ionisation (LDI), matrix-assisted LDI (MALDI) and electrospray ionisation (ESI) are employed to transfer ions into the gas-phase for detection by mass spectrometry and for dissociation studies by tandem mass spectrometry. The components of the BAILs are confirmed by LDI and MALDI by the detection of the respective cation and anion and by ESI by the observation of the cation and the dianion. A prominent feature of ESI is the formation of aggregates (cluster ions). Positively charged cluster ions are formally composed of multiple zwitterions plus one additional proton. In the negative-ion mode the clusters also incorporate the zwitterions which are, however, linked with the alkanedisulfonate dianion. In collision-induced dissociations (CID), the cationic aggregates show the evaporation of zwitterions until the protonated zwitterion is reached. Similarly, the cluster dianions release zwitterions until the free alkane disulfonate dianion is reached. However, the 1:1 adduct of dianion and zwitterion also displays proton transfer and Coulomb explosion into the mono-protonated disulfonic mono-anion and an imidazole-based carbene with sulfonate mono-anion.

Water exchange reactivity and stability of cobalt polyoxometalates under catalytically relevant pH conditions: insight into water oxidation catalysis.

Water exchange on a molecular, purely inorganic cobalt-based water oxidation catalyst, [Co(4)(II)(H(2)O)(2)(α-P(1)W(9)O(34))(2)](10-) (1), in the catalytically relevant pH region (pH 6-10) is studied using (17)O-NMR spectroscopy and ultrahigh-resolution electrospray ionization mass spectrometry. The results are compared with those of the inactive [Co(II)(H(2)O)(1)Si(1)W(11)O(39)](6-) (2), which is stable in the same pH region. The results obtained provide mechanistic details of the elementary reaction step related to the water oxidation on homogeneous metal oxide catalysts under catalytically relevant conditions. It is shown that the structural integrity of 1 and 2 is maintained, no deprotonation of the aqua ligands on the Co(II) centers occurs, and the water exchange does not undergo any mechanistic changeover at the catalytic pH conditions. We have demonstrated that the water exchange process is influenced by the cluster environment surrounding the water binding sites and is fast enough to not be rate-limiting for the water oxidation catalysis.

Solution and solid-state interactions in a supramolecular ruthenium photosensitizer-polyoxometalate aggregate.

The intermolecular interactions between a ruthenium-based photosensitizer ([Ru(tbbpy)(2)(biH(2))](2+)) and a molecular metal oxide ([ß-Mo(8)O(26)](4-)) are investigated in solution and in the solid state. The supramolecular interactions were studied using (1)H-NMR, UV-Vis and emission spectroscopy, ESI mass spectrometry and single-crystal X-ray diffraction. The formation of supramolecular aggregates was observed both in the crystal lattice and in solution. In addition, it is shown that aggregation in solution can be controlled by the competitive formation of ion pairs.

Dynamic HPLC study of C70 chlorination reveals a surprisingly selective synthesis of C70Cl8.

A dynamic HPLC study of C(70) chlorination led to the discovery, isolation, characterization, and development of the efficient preparatory procedures for two previously unknown soluble chlorofullerenes C(70)Cl(8) and C(70)Cl(6) and for insoluble [C(70)Cl(8)](n). A novel synthesis of 99% pure C(70)Cl(10) with a nearly quantitative yield was also developed. The first stability study of C(70)Cl(10,8,6) in solution showed that these compounds are very light-sensitive.

One-step synthesis of fullerene hydride C(60)H2 via hydrolysis of acylated fullerenes.

The hitherto unexplored class of acylated fullerene compounds has been shown to be excellent C(60)H2 precursors. Upon a simple treatment with basic Al2O3, they are hydrolyzed quantitatively into C(60)H2. This key feature led to the development of a new, straightforward protocol for the selective synthesis of the simplest [60]fullerene hydride, C(60)H2. This protocol may offer an advantageous alternative to previously known methods for the synthesis of C(60)H2 allowing for a rapid access to C(60)H2 in good yield and high purity without tedious separating processes.