Hilary Major

Metabonomics with 1H-NMR spectroscopy and liquid chromatography-mass spectrometry applied to the investigation of metabolic changes caused by gentamicin-induced nephrotoxicity in the rat.

Abstract The model nephrotoxin gentamicin was administered to male Wistar-derived rats daily, for 7 days, at 60 mg kg−1 day−1, subcutaneously, twice daily. Conventional clinical chemistry urinalysis showed a significant increase in N-acetyl-β-D-glucosaminidase (NAG) activity from day 3. At necropsy on day 9, clear histological damage to the kidney was noted with all animals showing a generally severe nephropathy primarily focused on the proximal convoluted tubules. The urinary excretion pattern of endogenous metabolites over the time course of the study was studied using a combination of 1H-NMR spectroscopy and HPLC-TOF-MS/MS using electrospray ionization (ESI). Changes in the pattern of endogenous metabolites as a result of daily administration of gentamicin were readily detected by both techniques with significant perturbations of the urinary profile observed from day 7 onwards. The findings by 1H-NMR included raised glucose and reduced trimethylamine N-oxide (TMAO). Changes in metabonomic profiles were observed by HPLC-MS in both positive and negative ESI. The MS data showed reduced xanthurenic acid and kynurenic acid, whilst neutral loss experiments also revealed a changed pattern of sulphate conjugation on gentamicin administration.

A metabonomic investigation of the biochemical effects of mercuric chloride in the rat using 1H NMR and HPLC-TOF/MS: time dependant changes in the urinary profile of endogenous metabolites as a result of nephrotoxicity

The effects of the administration of a single dose of the model nephrotoxin mercuric chloride (2.0 mg kg(-1), subcutaneous) to male Wistar-derived rats on the urinary metabolite profiles of a range of endogenous metabolites has been investigated using (1)H NMR and HPLC-MS. Urine samples were collected daily for 9 days from both dosed and control animals. Analysis of these samples revealed marked changes in the pattern of endogenous metabolites as a result of HgCl(2) toxicity. Peak disturbances in the urinary metabolite profiles were observed (using both NMR and HPLC-MS) at 3 days post dose. Thereafter the urinary metabolite profile gradually returned to a more normal composition. Markers of toxicity identified by (1)H NMR spectroscopy were raised concentrations of lactate, alanine, acetate, succinate, trimethylamine (TMA), and glucose. Reductions in the urinary excretion of citrate and alpha-ketoglutarate were also seen. Markers identified by HPLC-MS, in positive ion mode, were kynurenic acid, xanthurenic acid, pantothenic acid and 7-methylguanine which decreased after dosing. In addition an ion at m/z 188, probably 3-amino-2-naphthoic acid, was observed to increase after dosing. As well as these identified compounds other ions at m/z 297 and 267 decreased after dosing. In negative ion mode a range of sulfated compounds were observed, including phenol sulfate and benzene diol sulfate, which decreased after dosing. As well as the sulfated components an unidentified glucuronide at m/z 326 was also observed to decrease after dosing. The results of this study demonstrate the complementary nature of the NMR and MS-based techniques for metabonomic analysis.

A multi-analytical platform approach to the metabonomic analysis of plasma from normal and zucker (fa/fa) obese rats

Plasma obtained from 20 week old normal Wistar-derived and Zucker (fa/fa) rats was analysed using a number of different analytical methodologies to obtain global metabolite profiles as part of metabonomic investigations of animal models of diabetes. Samples were analysed without sample pre-treatment using 1H NMR spectroscopy, after acetonitrile solvent protein precipitation by ultra-performance liquid chromatography-MS (UPLC-MS) and after acetonitrile protein precipitation and derivatisation for capillary gas chromatography-MS (GC-MS). Subsequent data analysis using principal components analysis revealed that all three analytical platforms readily detected differences between the plasma metabolite profiles of the two strains of rat. There was only limited overlap between the metabolites detected by the different methodologies and the combination of all three methods of metabolite profiling therefore provided a much more comprehensive profile than would have been provided by their use individually.

Investigation of the quantitative properties of the quadrupole orthogonal acceleration time-of-flight mass spectrometer with electrospray ionisation using 3,4-methylenedioxymethamphetamine

This paper describes the investigation of the potential of a quadrupole orthogonal acceleration time-of-flight mass spectrometer (Q-TOF) equipped with an atmospheric pressure ionisation interface for quantitative measurements of small molecules separated by reversed phase liquid chromatography. To this end, the detection limits and linear dynamic range in particular were studied in an LC/MS/MS experiment using 3,4-methylenedioxymethamphetamine standards and 3,4-methylenedioxyethylamphetamine for internal standardisation. In a second phase, the experiment was repeated with real biological extracts (whole blood, serum, and vitreous humour). A calibration for 3,4-methylenedioxymethamphetamine and its metabolite 3,4-methylenedioxyamphetamine was prepared in each of these matrices again using 3,4-methylenedioxyethylamphetamine as internal standard. The resulting quantitative data were compared with those obtained by liquid chromatography with fluorescence detection for the same extracts. The Q-TOF results revealed excellent sensitivity and a linear dynamic range of nearly four decades (2-10 000 pg on-column, r(2) = 0.9998, 1/x weighting). Furthermore, all the calibration curves prepared in biological material were superimposable, LC/MS/MS and LC-fluorescence, and the quantitative results for actual samples compared very favourably. It was concluded that the Q-TOF achieves a linear dynamic range for quantitative LC/MS/MS work exceeding that of fluorescence detection and at much better absolute sensitivity. Copyright 1999 John Wiley & Sons, Ltd.

Molecular Ions and Protonated Molecules Observed in the Atmospheric Solids Analysis Probe Analysis of Steroids

Atmospheric pressure chemical ionisation (APCI) has often been used to ionise steroids in mass spectrometry, usually when interfaced to high-performance liquid chromatography (HPLC). However, in positive ion mode, a dehydrated protonated molecule is often observed with a loss of structural information. The recently introduced technique of atmospheric solids analysis probe (ASAP) has the advantage that the sample can be analysed directly and does not need to be interfaced to HPLC. Existing ionisation sources such as direct analysis in real time (DART) and desorption electrospray ionisation (DESI) have shown the advantage of direct analysis techniques in a variety of applications. ASAP can be performed on commercial atmospheric pressure ionisation (API) mass spectrometers with only simple modifications to API sources. The samples are vaporised by hot nitrogen gas from the electrospray desolvation heater and ionised by a corona discharge. A range of commercially available steroids were analysed by ASAP to investigate the mechanism of ionisation. ASAP analysis of steroids generally results in the formation of the parent molecular ion as either the radical cation M+• or the protonated molecule MH+. The formation of the protonated molecule is a result of proton transfer from ionised water clusters in the source. However, if the source is dry, then formation of the radical cation is the primary ionisation mechanism.

Metabolomics as a diagnostic tool for hepatology: validation in a naturally occurring canine model

Human hepatopathies are a diagnostic challenge, with many distinct diseases having similar clinical signs and laboratory findings. Naturally occurring canine hepatic disease provides an excellent model for human diseases and similar diagnostic dilemmas exist; differentiating canine congenital portosystemic vascular anomalies (PVA) from acquired hepatopathies is difficult and traditionally requires invasive diagnostic procedures. The emerging post-genomic science of metabolomics is concerned with detecting global changes of populations of endogenous low molecular weight metabolites in biological samples and offers the possibility of identifying surrogate profiles of disease. Metabolomics couples sensitive metabolite analysis with sophisticated pattern recognition techniques. In this study, a metabolomic strategy has been employed to assess metabolite changes in the plasma of dogs with congenital PVA and acquired hepatic disease. Plasma samples were collected from 25 dogs, comprising 9 dogs with congenital PVA, 6 with acquired hepatopathy and 10 with non-hepatic disorders. Low molecular weight metabolites were analyzed by liquid chromatography-mass spectrometry (LC-MS). Following identification of metabolites, multivariate data analysis was used to compare profiles amongst groups. The analysis demonstrated significant disturbances in the plasma bile acid and phospholipid profiles of dogs with portovascular anomalies. In contrast to traditional laboratory parameters, the metabolomic strategy was able to produce a clear segregation between all three study groups. In conclusion, this study demonstrates the potential of metabolomics as a diagnostic tool for naturally occurring hepatic disease. With further validation, this approach will improve diagnostic capabilities, provide an insight into pathogenetic mechanisms, and ultimately inform therapeutic decision making in clinical hepatology.

Liquid chromatographic-mass spectrometric analysis of coal tar fractions

Conventionally, coal-derived tars are separated by solvent fractionation into pentane solubles, asphaltenes (pentane insoluble, benzene soluble) and preasphaltenes (benzene insoluble) with further separation of the pentane solubles into saturated, aromatic and polar fractions by open-column chromatography on silica.Normal-phase HPLC, using hexane with a gradient to dichloromethane-methanol (95 + 5) coupled to a mass spectrometer by a moving-belt interface, has indicated that the so-called aromatic fraction of a low-temperature hydropyrolysis coal tar contains some saturates (alkanes and cycloalkanes including hopanes, up to C64) and an appreciable amount of polar materials which may represent 50% of the aromatic fraction, in addition to aromatics from alkylbenzenes to alkylrubicenes as extensive homologous series.Mass spectrometry with electron impact and chemical ionisation using ammonia and isobutane has given new structural information on the polar materials present in this “aromatic” fraction. Fast atom bombardment of asphaltenes and benzene-insolubles indicated that their structures resemble those of the polar compounds and differ from those of the aromatics. Size-exclusion chromatography confirmed the relative molecular mass ranges detected by mass spectrometry, GC-MS with a capillary column proved inadequate for defining many of the aromatics. The relevance of the work to the structure of coal is considered.

Application of Capillary Electrophoresis–Mass Spectrometry to the Analysis of Urine Samples From Animals and Man Containing Paracetamol and Phenacetin and Their Metabolites

Capillary electrophoresis, in combination with electrospray mass spectrometry (CE–ESMS), has been applied to the analysis of phenacetin and paracetamol and its metabolites in freeze dried urine, and solid phase extracts of urine, following administration to rat and man. Using CE–ESMS the detection of 2 pg of d 3 -paracetamol on column was possible. Phenacetin, paracetamol and paracetamol glucuronide and sulfate were all readily detectable in the samples. The CE–ESMS method was also used to determine the extent of deacetylation/reacetylation of d 3 -acetyl-labelled paracetamol in vivo.

Identification of non-steroidal anti-inflammatory drugs and their metabolites in solid phase extracts of human urine using capillary electrophoresis–mass spectrometry

The use of capillary electrophoresis (CE) in combination with mass spectrometry (MS) for the identification of a number of non-steroidal anti-inflammatory drugs (NSAIDs) and their metabolites in solid phase extracts of human urine is described. A variety of MS techniques were employed, including tandem mass spectrometry (MS–MS), to profile the extracts. In urine obtained following administration of ibuprofen, the drug itself, its glucuronide metabolite, the hydroxylated metabolite and its corresponding glucuronide, and the carboxylic acid metabolite as the glucuronide were all identified. Similarly, a range of metabolites of flurbiprofen and aspirin, were identified in urine extracts using the hyphenated CE–MS system. These results clearly demonstrate the value of CE–MS for the rapid identification of such substances following their isolation from a biological matrix such as urine.

Metalloporphyrins in coal 5. Analysis of porphyrins in coal extracts using liquid chromatography-mass spectrometry

Abstract The application of liquid chromatography-mass spectrometry to the separation and identification of porphyrin homologues from coal is demonstrated using a moving belt interface. In this way C 32 , C 31 , C 30 and C 29 polyalkylporphyrins are shown to be present as ligands in the haem fraction isolated from an immature lignite (Canakkale-Can).