Characterisation of commercially available linear alkylbenzenesulfonates by LC-SPE-NMR/MS (liquid chromatography-solid phase extraction-nuclear magnetic resonance spectroscopy-mass spectroscopy).

Commercially available linear alkylbenzenesulfonates (LASs) are a mixture of various homologues and isomers, leading to 20 major species. In this work we investigated the commercial product by liquid chromatography-solid phase extraction-nuclear magnetic resonance spectroscopy-mass spectrometry (LC-SPE-NMR/MS). The commercial product was separated into 17 fractions by liquid chromatography (LC). After chromatographic separation, 5% of the flow was split to a mass spectrometer (MS) while 95% was send to post-column solid phase extraction cartridges for enrichment of the analytes (LC-SPE). After elution from the SPE-cartridges a NMR-spectrometer equipped with a cryo-probe was used for the characterisation of the different LASs species. For the first time (1)H-1D and H-H-COSY spectra for 14 LASs species out of 20 major isomers are presented, whereas the 6 remaining species are detected as mixtures in 3 (1)H-1D and H-H-COSY spectra. These data were used to correlate the chromatographic retention of the LASs isomers to the substitution pattern of the alkyl chain.

In vitro metabolite identification of ML3403, a 4-pyridinylimidazole-type p38 MAP kinase inhibitor by LC-Qq-TOF-MS and LC-SPE-cryo-NMR/MS.

Prediction of the metabolic profile of a potential new drug is recommended at an early stage in industrial drug discovery process to determine whether or not any potentially reactive or toxic metabolites are formed. In the present study, we investigated the in vitro metabolism of ML3403 ({4- [5-(4-Fluorophenyl)-2-methylsulfanyl-3H-imidazol-4-yl]-pyridin-2-yl -(1-phenylethyl)-amine), a potent and selective p38 MAP kinase inhibitor using mouse liver microsomes. The combination of LC-ESI-Qq-TOF (tandem quadrupole time-of-flight)-MS (mass spectrometer) and LC-SPE (solid phase extraction)-cryo-NMR (nuclear magnetic resonance)/MS at 600 MHz has been applied for comprehensive and straightforward structural elucidation of ML3403 metabolites. It was possible to determine the metabolic profile of ML3403, revealing eight different metabolites formed by N-desalkylation, S-mono- and di-oxidation, aliphatic hydroxylation and pyridine-N-oxidation. The ESI-Qq-TOF-MS data yielded elemental compositions of all metabolites and their fragments by evaluation of the accurate mass and isotopic pattern information using the sigma-fit algorithm. Evaluation of 2D NMR spectra obtained from pure ML3403 an its major metabolite ML3603 allowed the unequivocal assignment of the resonances in 1D NMR spectra obtained directly from the microsomal incubation by LC-SPE-cryo-NMR/MS. The presented method significantly decreases the time required for a complete structural assignment of metabolites from microsomal in vitro assays.

Identification of phenacetin metabolites in human urine after administration of phenacetin-C2H3: measurement of futile metabolic deacetylation via HPLC/MS-SPE-NMR and HPLC-ToF MS.

The metabolism of acetyl-labelled phenacetin-C2H3 was investigated in man following a single (150 mg) oral dose. Urine samples were collected at predose, 0-2 h and >2-4 h post-dose, and samples from each time-point were then analysed directly using 1H-nuclear magnetic resonance (NMR) spectroscopy. The phenacetin metabolites acetaminophen (paracetamol) glucuronide, sulphate and the N-acetyl-L-cysteinyl conjugate were identified by this method, and all showed clear evidence of the loss of the original 2H3-acetyl label and its replacement with 1H3 (futile deacetylation). The observed percentage futile deacetylation by 1H-NMR spectroscopy was measured as approximately 20% in each metabolite (about 2% of the recovered dose). After sample preparation by solid-phase extraction on a C18 solid-phase extraction (SPE) cartridge, further profiling was performed using high-performance liquid chromatography/mass spectrometry-solid-phase extraction-nuclear magnetic resonance (HPLC/MS-SPE-NMR) confirming futile deacetylation had taken place as indicated by NMR spectroscopy on both the isolated acetaminophen glucuronide and L-cysteinyl-metabolites. Additional analysis by high-performance liquid chromatography-time-of-flight mass spectrometry (HPLC-ToF MS) identified further phenacetin metabolites, and from these data the mean percentage of futile deacetylation was measured as 31% +/- 2% for the acetylated phenacetin metabolites. A number of non-acetylated metabolites were also detected in the sample via HPLC-ToF MS. The results showed that phenacetin underwent a transient formation via a number of toxic intermediates to a much greater extent than had been observed in similar studies on acetaminophen. These results may contribute to the understanding of the analgesic nephropathy reported following chronic phenacetin consumption.

Application of directly coupled HPLC-NMR-MS to the identification and confirmation of quercetin glycosides and phloretin glycosides in apple peel.

Directly coupled HPLC-NMR-MS was used to identify and confirm the presence of quercetin O-glycosides and phloretin O-glycosides in an extract of apple peel. From the MS and MS/MS data, the molecular weights of the intact molecules as well as those of quercetin and phloretin and their sugar moieties were deduced. The NMR data provided information on the identity of the compounds as well as the alpha and beta conformations and the position of the glycosides on quercetin and phloretin. The following O-glycosides of quercetin could be identified: quercetin-3-alpha-L-rhamnosyl-(1-->6)-beta-D-glucoside (rutin), quercetin-3-beta-D-galactoside (hyperin), quercetin-3-beta-D-glucoside (isoquercitrin), quercetin-3-beta-D-xyloside (reynoutrin), quercetin-3-alpha-L-arabinofuranoside (avicularin), and quercetin-3-alpha-L-rhamnoside (quercitrin). Phloretin was present as phloretin-2'-beta-D-glucoside (phloridzin) and the 2'-beta-D-xylosyl-(1-->6)-beta-D-glucoside. Concentrations were between 0.2 and 5 mg/g of apple peel.

Comparative studies of the leachate of an industrial landfill by gas chromatography-mass spectrometry, liquid chromatography-nuclear magnetic resonance and liquid chromatography-mass spectrometry.

Nowadays, the need to have a realistic characterization of industrial effluents in the environment has become more and more recognized. A palette of different analytical methods both for sample extraction and instrumental analysis are available today, some older, others introduced more recently. The aim of this research is to compare a number of these techniques. To do this we studied a real leachate from an industrial landfill and carried out chemical analyses for organic pollutants, using different extraction methods based on solid-phase extraction and solid-phase microextraction and different instrumental techniques such as GC-MS, LC-MS, NMR and LC-NMR. Results show the performances of the different techniques, which are complementary.

Quantitative Measurements in Continuous-Flow HPLC/NMR.

Two methods for the quantitative determination of compounds in continuous-flow HPLC/NMR are described. The first method uses an internal standard (caffeine) of known concentration directly mixed into the mobile phase, while with the second method, a known amount of internal standard is injected onto the column during the chromatographic run. The latter method was validated using several nitroaromatic compounds and explosives. Deviations between the injected and calculated amounts of analytes are usually below 10% while the relative standard deviation ranges from 2% in the upper microgram range to 40% at the limit of detection.

Application of Continuous-Flow HPLC-Proton-Nuclear Magnetic Resonance Spectroscopy and HPLC-Thermospray-Mass Spectroscopy for the Structural Elucidation of Phototransformation Products of 2,4,6-Trinitrotoluene.

An aqueous solution of 2,4,6-trinitrotoluene (TNT) was irradiated by natural sunlight for a period of 1 month to generate phototransformation products of this compound. After solid-phase extraction on a poly(styrene-divinylbenzene) copolymer at pH 1, the structures of several acidic nitroaromatic compounds were identified by means of continuous-flow HPLC/(1)H NMR and HPLC/TSP-MS measurements of this extract. By interpretation of both NMR and MS spectra, it was even possible to characterize noncommercially available phototransformation products of TNT. The results obtained by continuous-flow HPLC/(1)H NMR were compared with those obtained by the investigation of a groundwater sample of a former ammunition site near Elsnig, Germany. The results show that several identified phototransformation products of TNT are also present in this groundwater sample.

Application of on-line HPLC-1H NMR to environmental samples: analysis of groundwater near former ammunition plants.

Coupling of HPLC to NMR was applied for the first time to the analysis of environmental samples, i.e., water samples from an ammunition hazardous waste site. Using the continuous flow mode at very low flow rates (< or = 0.017 mL/min) and large volume injection (400 microL), the confirmation of many nitroaromatic compounds could be achieved down to the microgram-per-liter level after solid phase extraction of a groundwater sample from a former ammunition production site. At a flow rate of 0.006 mL/min, it is possible to detect less than 29 nmol (5 micrograms) of 1,3-dinitrobenzene injected on a 75 mm x 4 mm reversed phase C-18 column (particle size, 5 microns). The results obtained by HPLC-NMR are compared to those obtained by HPLC-PDA (photodiode array) of the same sample, demonstrating that many more compounds can be identified by the former compared to the latter method as a result of coelution of major and minor components in the HPLC chromatogram.