Search PubMed⌕ Search

Biomedical subjects

J K Nicholson

Publications and source records attributed to J K Nicholson.

At least 73 records · Page 4Linked to original sources
PubMed2000

Mass directed peak selection, an efficient method of drug metabolite identification using directly coupled liquid chromatography-mass spectrometry-nuclear magnetic resonance spectroscopy.

Mass spectrometry (both MS and MS-MS) has been used to determine which eluting chromatography peaks in an LC-MS-nuclear magnetic resonance (NMR) experiment should be selected for extended NMR spectroscopic measurement. This mass directed selection of chromatographic peaks has been applied to test mixtures and urine samples for identification of drug metabolites. It was used to simultaneously determine when drug-related material was eluting and provided molecular mass information on these components. Stop-flow LC-NMR was used to acquire data for structural characterisation of drug-related components. This work further serves to demonstrate the potential of coupling tandem mass spectrometry using an ion trap spectrometer with LC-NMR spectroscopy, to provide an extremely powerful tool in structural elucidation.

Chromatography, Liquid↗
PubMed2000

Directly coupled HPLC-NMR and HPLC-NMR-MS in pharmaceutical research and development.

The methodology for the direct coupling of HPLC with NMR spectroscopy and the simultaneous double coupling of HPLC with NMR and mass spectrometry (MS) is described. Indications of the necessary technical developments to achieve this are given, and the applications of these new techniques to studies of pharmaceutical relevance are reviewed. These include studies of combinatorial chemistry libraries, synthetic chemical impurities, characterisation of drug mixtures, identification of natural products of possible pharmaceutical interest and identification of xenobiotic metabolites in human, animal and in vitro systems. In addition, HPLC-NMR has been used to investigate xenobiotic metabolite reactivity. Finally, the potential future directions of the techniques are discussed.

Animals↗
PubMed2000

Identification and quantification of metabolites of 2,3,5,6-tetrafluoro-4-trifluoromethylaniline in rat urine using 19F nuclear magnetic resonance spectroscopy, high-performance liquid chromatography-nuclear magnetic resonance spectroscopy and high-performance liquid chromatography-mass spectrometry.

The urinary excretion profile and identity of the metabolites of 2,3,5,6-tetrafluoro-4-triflouromethylaniline, following i.p. administration to the rat at 50 mg kg(-1), were determined using a combination of 19F-NMR, HPLC-NMR and HPLC-MS. A total of 38% of the dose was eliminated in the urine as five metabolites. The major routes of metabolism were N-glucuronidation, sulfation and oxidation with a significant amount of metabolic defluorination to give a number of ortho-ring hydroxylated metabolites. The oxidised metabolites were excreted as glucuronide and/or sulfate conjugates.

Aniline Compounds↗
PubMed2000

Application of directly coupled LC-NMR-MS to the structural elucidation of metabolites of the HIV-1 reverse-transcriptase inhibitor BW935U83.

The human in vivo metabolism of the HIV-1 reverse transcriptase inhibitor 5-chloro-1-(2',3'-dideoxy-3'-fluoro-erythro-pentofuranosyl)uracil (BW935U83) was studied using 19F NMR spectroscopy, directly coupled LC-NMR and LC-NMR-MS. The number and relative proportions of the drug metabolites were obtained from 19F NMR spectra of whole human urine. The novel use of the continuous-flow 19F detected LC-NMR experiment yielded chromatographic retention times and 19F chemical shifts for the parent drug, the glucuronide conjugate of the parent and an early eluting polar metabolite. The parent drug and its glucuronide conjugate were easily characterised by directly coupled 1H LC-NMR spectroscopy and two-dimensional TOCSY experiments. The identification of the second metabolite was achieved using 19F NMR and directly coupled 1H LC-NMR-MS which furnished the molecular weight, and through the use of MS-MS techniques, information on the fragment ions. This species was identified as 3-fluoro-ribolactone.

Anti-HIV Agents↗
PubMed2000

Use of directly coupled ion-exchange liquid chromatography-mass spectrometry and liquid chromatography-nuclear magnetic resonance spectroscopy as a strategy for polar metabolite identification.

Ion-exchange LC-MS and LC-NMR have been successfully used to identify a novel N-acetyl metabolite of a highly polar drug candidate [2-(ethanimidoylamino)ethyl]sulfonyl alanine (GW273629) under development as a therapeutic agent. This has been achieved using a simple HPLC method without the need for complicated and time consuming pre- or post-column derivatisation. Ion-exchange chromatography using simple ionic strength buffer and organic solvent mobile phases, as applied here, should be suitable for the analysis of other charged polar species. Optimisation of the system described could result in the development of a rational generic HPLC approach specifically designed for the characterisation of polar drug molecules and their metabolites.

Animals↗
PubMed2000

The initial pathogenesis of cadmium induced renal toxicity.

The novel application of magic angle spinning 1H NMR spectroscopy, coupled with pattern recognition techniques, has identified biochemical changes in lipid and glutamate metabolism that precede classical nephrotoxicity. These changes occurred in the bank vole (Clethrionomys glareolus) after chronic dosing, at a low level of exposure and at a renal Cd(2+) concentration (8.4 microgram/g dry wt) that was nearly two orders of magnitude below the WHO critical organ concentration (200 microg/g wet wt). These early stage effects of Cd(2+) on the biochemistry of renal tissue may reflect adaptation mechanisms to the toxic insult or the preliminary stages of the toxicological cascade.

Acidosis, Renal Tubular↗
PubMed2000

High-resolution magic angle spinning (1)H NMR spectroscopy of intact liver and kidney: optimization of sample preparation procedures and biochemical stability of tissue during spectral acquisition.

High-resolution magic angle spinning (MAS) (1)H NMR spectroscopy has been used to investigate the biochemical composition of whole rat renal cortex and liver tissue samples. The effects of a number of sample preparation procedures and experimental variables have been investigated systematically in order to optimize spectral quality and maximize information recovery. These variables include the effects of changing the sample volume in the MAS rotor, snap-freezing the samples, and the effect of organ perfusion with deuterated saline solution prior to MAS NMR analysis. Also, the overall biochemical stability of liver and kidney tissue MAS NMR spectra was investigated under different temperature conditions. We demonstrate improved resolution and line shape of MAS NMR spectra obtained from small spherical tissue volume (12 microl) rotor inserts compared to 65 microl cylindrical samples directly inserted into the MAS rotors. D(2)O saline perfusion of the in situ afferent vascular tree of the tissue immediately postmortem also improves line shape in MAS NMR spectra. Snap-freezing resulted in increased signal intensities from alpha-amino acids (e.g., valine) in tissue together with decreases in renal osmolytes, such as myo-inositol. A decrease in triglyceride levels was observed in renal cortex following stasis on ice and in the MAS rotor (303 K for 4 h). This work indicates that different tissues have differential metabolic stabilities in (1)H MAS NMR experiments and that careful attention to sample preparation is required to minimize artifacts and maintain spectral quality.

Animals↗
PubMed2000

Mass spectrometrically detected directly coupled high performance liquid chromatography/nuclear magnetic resonance spectroscopy/mass spectrometry for the identification of xenobiotic metabolites in maize plants.

Reconstructed ion chromatograms have been used to identify relevant high performance liquid chromatography (HPLC) peaks in a directly coupled high performance liquid chromatography/nuclear magnetic resonance spectroscopy/mass spectrometry (HPLC/NMR/MS) experiment. This has been applied to a study of the metabolism of a model compound, 5-nitropyridone (2-hydroxy-5-nitropyridine), in maize plants grown hydroponically. By monitoring the on-flow reconstructed ion chromatogram corresponding to the 5-nitropyridone fragment at m/z 143, and additional molecular ions corresponding to metabolites identified as products from similar compounds, relevant peaks were identified rapidly for subsequent stopped-flow 1H NMR spectroscopic analysis. The combination of coupled HPLC/NMR/MS enabled the direct identification of three metabolites, namely the N-glucoside, N-malonylglucoside, and O-malonylglucoside. This work demonstrates the power of HPLC/NMR/MS for the structural elucidation of xenobiotic metabolites in complex biological matrices (such as plant material) with minimal sample preparation. In particular, using mass spectrometry for the initial identification of relevant HPLC peaks allows the analysis of complex samples without the necessity for other spectroscopic markers, such as 19F NMR signal for fluorinated compounds or UV spectroscopy for molecules with strong UV chromophores.

Chromatography, High Pressure Liquid↗
PubMed2000

Distinction between normal and renal cell carcinoma kidney cortical biopsy samples using pattern recognition of (1)H magic angle spinning (MAS) NMR spectra.

The technique of magic angle spinning (MAS) high resolution (1)H NMR spectroscopy applied to intact tissues provides excellent peak resolution and thus much biochemical information. The use of computer-based pattern recognition techniques to classify human renal cortex tissue samples as normal or tumour based on their (1)H MAS NMR spectra has been investigated. In this preliminary study of 22 paired control and tumour samples, exploratory data analysis using principal components based on NMR spectral intensities showed clear separation of the two classes. Furthermore, using the supervised method of linear discriminant analysis, based on individual data point intensities or on integrated spectral regions, it was possible to distinguish between the normal and tumour kidney cortex tissue with 100% accuracy, including a single example of a metastatic tumour from a primary lung carcinoma. A tumour sample from the collecting duct of the kidney showed a different NMR spectral profile, and pattern recognition indicated that this sample did not classify with the cortical tumours.

Amino Acids↗
PubMed2000

Directly coupled liquid chromatography with inductively coupled plasma mass spectrometry and orthogonal acceleration time-of-flight mass spectrometry for the identification of drug metabolites in urine: application to diclofenac using chlorine and sulfur detection.

We report the application of high-performance liquid chromatography (HPLC) linked to inductively coupled plasma mass spectrometry (ICPMS) and orthogonal acceleration time-of-flight mass spectrometry (oa-TOFMS) for the identification of phase I and II urinary metabolites of diclofenac. The metabolites were separated by reversed-phase HPLC monitored with a UV diode array detector (UV-DAD) after which 90% of the eluent was directed to an ICPMS source, with the remainder going to an oa-TOF mass spectrometer. Compounds containing (35)Cl, (37)Cl and (32)S were detected specifically using ICPMS and identified by oa-TOFMS. The metabolites detected and identified in this way included glucuronic acid and sulfate conjugates, mono- and dihydroxylated and free diclofenac. In addition a previously unreported in vivo metabolite, an N-acetylcysteinyl conjugate of diclofenac, was also characterised. This is the first application of the combination of HPLC/UV-DAD/ICPMS/oa-TOFMS for the investigation of the metabolic fate of chlorinated xenobiotics by direct biofluid analysis.

Animals↗
PubMed2000

High-resolution (1)H and (1)H-(13)C magic angle spinning NMR spectroscopy of rat liver.

High-resolution magic angle spinning (MAS) (1)H NMR spectra of small samples (ca. 8 mg) of intact rat liver are reported for the first time. One dimensional spectra reveal a number of large well-resolved NMR signals mainly from low to medium molecular weight compounds (generally <1000 Daltons) from a variety of chemical classes. A range of 2D MAS-NMR experiments were performed, including (1)H J-resolved (JRES), (1)H-(1)H total correlation spectroscopy (TOCSY) and (1)H-(13)C heteronuclear multiple quantum coherence (HMQC) to enable detailed signal assignment. Resonances were assigned from alpha- and beta-glucose, glycerol, alanine, glutamate, glycine, dimethylglycine, lysine, and threonine, together with phosphocholine, choline, lactate, trimethylamine-N-oxide (TMAO), and certain fatty acids. Well-resolved (1)H NMR signals from glycogen (poly 1-4 alpha-glucose) were observed directly in intact liver using MAS-NMR spectroscopy. In addition, the resonances from the glycogen C(1)H proton in alpha(1-->4) linked glucose units with either alpha(1-->4) units adjacent or alpha(1-->6) linked branches could be resolved in a high-resolution (1)H NMR experiment giving direct in situ information on the ratio of alpha(1-->4) to alpha(1-->6) units. This indicates that despite the relatively high MW (>1,000,000 Daltons) there is considerable segmental motion in the glycogen molecules giving long (1)H T(2) relaxation times. Magn Reson Med 44:201-207, 2000.

Animals↗
PubMed2000

NMR spectroscopy based metabonomic studies on the comparative biochemistry of the kidney and urine of the bank vole (Clethrionomys glareolus), wood mouse (Apodemus sylvaticus), white toothed shrew (Crocidura suaveolens) and the laboratory rat.

The metabolic profiles of three wild mammals that vary in their trophic strategies, the herbivorous bank vole (Clethrionomys glareolus), the granivorous wood mouse (Apodemus sylvaticus), and the insectivorous white-toothed shrew (Crocidura suaveolens), were compared with that of a widely used strain of laboratory rat (Sprague Dawley). In conjunction with NMR spectroscopic investigations into the urine and blood plasma composition for these mammals, high resolution magic angle spinning (HRMAS) 1H-nuclear magnetic resonance (NMR) spectroscopy was applied to investigate the composition of intact kidney samples. Adaptation to natural diet affects both renal metabolism and urinary profiles, and while these techniques have been used to study the metabolism of the laboratory rat little is known about wild small mammals. The species were readily separated by their urinary profiles using either crude metabolite ratios or statistical pattern recognition. Bank vole urine contained higher concentrations of aromatic amino acids compared with the other small mammals, while the laboratory rats produced relatively more hippurate. HRMAS 1H-NMR demonstrated striking differences in both lipid concentration and composition between the wild mammals and Sprague Dawley rats. Bank voles contained high concentrations of the aromatic amino acids phenylalanine, tyrosine and tryptophan in all tissue and biofluids studied. This study demonstrates the analytical power of combined NMR techniques for the study of inter-species metabolism and further demonstrates that metabolic data acquired on laboratory animals cannot be extended to wild species.

Animals↗
PubMed2000

The metabolism and excretion of [14c] 2- and 4-chlorobenzoic acids in the rat.

The metabolic fate of [14C]-labelled 2 and 4-chlorobenzoic acids (2- and 4-CBA) has been determined in the rat following intraperitoneal (i.p.) administration at 100 mg/kg to male rats. The major route of elimination for both 2-and 4-CBA was urine with > 80%, of the dose recovered in the initial 0-24 h after administration. Glycine conjugation was found to be the dominant metabolic fate for both [14C] 2- and 4-CBA however, the position of chloro substitution had a clear effect on the extent of metabolism via this route with ortho substitution reducing the extent of metabolism via this pathway.

Animals↗
PubMed2000

Application of directly coupled HPLC-NMR-MS/MS to the identification of metabolites of 5-trifluoromethylpyridone (2-hydroxy-5-trifluoromethylpyridine) in hydroponically grown plants.

Directly coupled HPLC-NMR-MS was used to characterize two major metabolites of 5-trifluoromethylpyridone (2-hydroxy-5-trifluoromethylpyridine), a model compound for herbicides, after it had been dosed into hydroponically grown maize plants. The combination of NMR and MS data allowed the identification of both of these metabolites, namely, the N-glucoside and O-malonylglucoside conjugates of the parent pyridone. This work demonstrates the efficiency and the potential application of HPLC-NMR-MS to the investigation of the metabolism of agrochemicals. The work also indicates that combination of the use of hydroponically grown plants and directly coupled HPLC-NMR-MS allows rapid identification of metabolites with little sample preparation.

Chromatography, High Pressure Liquid↗
PubMed2000

Chemometric models for toxicity classification based on NMR spectra of biofluids.

1H NMR spectroscopic and pattern recognition (PR)-based methods were used to investigate the biochemical variability in urine obtained from control rats and from rats treated with a hydrazine (a model hepatotoxin) or HgCl(2) (a model renal cortical toxin). The 600 MHz (1)H NMR spectra of urine samples obtained from vehicle- or toxin-treated Han-Wistar (HW) and Sprague-Dawley (SD) rats were acquired, and principal components analysis (PCA) and soft independent modeling of class analogy (SIMCA) analysis were used to investigate the (1)H NMR spectral data. Variation and strain differences in the biochemical composition of control urine samples were assessed. Control urine (1)H NMR spectra obtained from the two rat strains appeared visually similar. However, chemometric analysis of the control urine spectra indicated that HW rat urine contained relatively higher concentrations of lactate, acetate, and taurine and lower concentrations of hippurate than SD rat urine. Having established the extent of biochemical variation in the two populations of control rats, PCA was used to evaluate the metabolic effects of hydrazine and HgCl(2) toxicity. Urinary biomarkers of each class of toxicity were elucidated from the PC loadings and included organic acids, amino acids, and sugars in the case of mercury, while levels of taurine, beta-alanine, creatine, and 2-aminoadipate were elevated after hydrazine treatment. SIMCA analysis of the data was used to build predictive models (from a training set of 416 samples) for the classification of toxicity type and strain of rat, and the models were tested using an independent set of urine samples (n = 124). Using models constructed from the first three PCs, 98% of the test samples were correctly classified as originating from control, hydrazine-treated, or HgCl(2)-treated rats. Furthermore, this method was sensitive enough to predict the correct strain of the control samples for 79% of the data, based upon the class of best fit. Incorporation of these chemometric methods into automated NMR-based metabonomics analysis will enable on-line toxicological assessment of biofluids and will provide a tool for probing the mechanistic basis of organ toxicity.

Animals↗
PubMed2000

High-performance liquid chromatography and inductively coupled plasma mass spectrometry (HPLC-ICP-MS) for the analysis of xenobiotic metabolites in rat urine: application to the metabolites of 4-bromoaniline.

The use of HPLC-ICP-MS for the profiling and quantification of the metabolites of 4-bromoaniline following reversed-phase gradient chromatography is demonstrated. In the 0-8 h post dose sample, which contained the highest concentrations of compound-related material, it was possible to detect at least 16 metabolites of the compound. The methodology described offers the possibility of obtaining metabolite profiles and quantification for drugs and other xenobiotics in biological fluids and excreta without the requirement for radiolabelled tracers.

Aniline Compounds↗
PubMed2000

Investigation of the metabolism of 14C/13C-practolol in rat using directly coupled radio-HPLC-NMR-MS.

1. The metabolic fate of 14C/13C-practolol was investigated using on-line HPLC-NMR-MS following oral administration to rat. The major route of elimination for the radiolabel was via the urine with the principal biotransformation products confirmed as the 2-hydroxy- and 2-hydroxyglucronide metabolites. 2. In addition, futile deacetylation, determined by the replacement of 13C-labelled acetyl groups with endogenous 12C-acetyls accounted for approximately 7-10% of the urinary metabolites, corresponding to approximately 5% of the dose undergoing N-deacetylation. 3. Evidence for chiral metabolism was sought via NMR of isolated metabolites using beta-cyclodextrin as a chiral shift agent. Practolol was excreted as a racemate. However, some enantioselective metabolism/excretion had occurred as the hydroxy- and hydroxyglucuronide were not excreted as racemic mixtures. 4. Directly coupled radio-HPLC-NMR-MS is extremely effective for the identification of the metabolites of radiolabelled xenobiotics in urine samples.

Adrenergic beta-Antagonists↗