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Biomedical subjects

I D Wilson

Publications and source records attributed to I D Wilson.

At least 73 records · Page 4Linked to original sources
PubMed1999

Studies on the metabolism of 4-fluoroaniline and 4-fluoroacetanilide in rat: formation of 4-acetamidophenol (paracetamol) and its metabolites via defluorination and N-acetylation.

1. The urinary metabolic fate of 4-fluoroaniline (4-FA) and 1-[13C]-4-fluoroacetanilide (4-FAA) has been studied using NMR-based methods after 50 and 100 mg kg(-1) i.p. doses respectively to the male Sprague-Dawley rat. 2. 4-FA was both ortho- and para-hydroxylated. The major metabolite produced by ortho-hydroxylation was 2-amino-5-fluorophenylsulphate accounting for approximately 30% of the dose. Of the dose, approximately 10% was excreted via para-hydroxylation and the resulting defluorinated metabolites were N-acetylated and excreted as sulphate (major), glucuronide (minor) and N-acetyl-cysteinyl (minor) conjugates of 4-acetamidophenol (paracetamol). 3. The major route of metabolism of 1-[13C]-4-FAA was N-deacetylation and the metabolites excreted in the urine were qualitatively identical to 4-FA. The paracetamol metabolites produced via para-hydroxylation were also a product of N-deacetylation and reacetylation, as the [13C]-label was not retained. 4. These studies demonstrate the value of [13C]-labelling in understanding the contribution of N-acetylation, and futile deacetylation-reacetylation reactions, in aniline metabolism. In addition, this work sheds new light on the metabolic lability of certain aromatic fluorine substituents.

Acetaminophen↗
PubMed1999

Quantitative structure-metabolism relationships (QSMR) using computational chemistry: pattern recognition analysis and statistical prediction of phase II conjugation reactions of substituted benzoic acids in the rat.

1. Quantitative relationships between molecular physico-chemical properties of 22 substituted benzoic acids and the extent of excretion of their metabolites in rat urine have been investigated using computational chemistry and multivariate statistics. 2. A data set of 34 theoretically derived physico-chemical descriptors calculated was used to classify the benzoic acids according to their predominant urinary metabolic fate. 3. Quantitative structure-metabolism relationships were obtained by linear regression using combinations of physico-chemical descriptors allowing the prediction of % urinary excretion of glycine (r = 0.73) and glucuronide conjugates (r = 0.82) and % urinary excretion of the parent compound (r = 0.91).

Animals↗
PubMed1999

Quantitative studies on the urinary metabolic fate of 2-chloro-4-trifluoromethylaniline in the rat using 19F-NMR spectroscopy and directly coupled HPLC-NMR-MS.

1. The metabolism and urinary excretion of 2-chloro-4-trifluoromethylaniline has been studied in the rat using 19F-NMR spectroscopy and directly coupled HPLC-NMR-MS methods. The compound was dosed to three male Sprague-Dawley rats (50 mg kg(-1) i.p.) and urine collected over 0-8, 8-24 and 24-48 h post-dosing. 2. A total urinary recovery of 56.3+/-2.2% of the dose was achieved up to 48 h after dosing. The major metabolite in the urine was identified as 2-amino-3-chloro-5-trifluoromethylphenylsulphate accounting for a total of 33.5+/-2.2% of the dose. 3. Further metabolites detected and characterized included 2-chloro-4-trifluoromethylphenylhydroxylamine glucuronide (13.2+/-0.5% of the dose), 2-amino-3-chloro-5-trifluoromethylphenylglucuronide (3.8+/-0.4% of the dose) and 2-chloro-4-trifluoromethylaniline-N-glucuronide (3.6+/-0.1% of the dose). Several minor metabolites were also found and identified, including 2-chloro-4-trifluoromethylphenylsulphamate, which together accounted for 2.1+/-0.4% of the dose. 4. Directly coupled HPLC-NMR-MS and 19F-NMR spectroscopy is shown to provide an efficient approach for the unequivocal and rapid determination of the quantitative urinary metabolic fate and excretion balance of a fluorinated xenobiotic without the necessity for specific radiolabelling.

Aniline Compounds↗
PubMed1999

Investigation of the quantitative metabolic fate and urinary excretion of 3-methyl-4-trifluoromethylaniline and 3-methyl-4-trifluoromethylacetanilide in the rat.

The urinary metabolites of 3-methyl-4-trifluoromethylaniline in the rat were characterized and quantified using a combination of (19)F NMR, HPLC-NMR ((1)H and (19)F), and HPLC-mass spectrometry techniques. The major routes of metabolism were amine N-acetylation and methyl group C-oxidation to the benzyl alcohol (with subsequent glucuronide conjugation) and further to the corresponding benzoic acid derivative. Quantitatively only a small proportion of the urinary metabolites contained the free amino group, and these were products of ortho-hydroxylation (2 and 6 position) with additional conjugation to form the ether sulfates and glucuronides. An N-glucuronide of the parent compound was also identified. 3-Methyl-4-trifluoromethylacetanilide ((13)C-labeled in the acetyl group) gave virtually the same overall metabolite profile as 3-methyl-4-trifluoromethylaniline; however, a significant level of futile N-deacetylation and reacetylation occurred as ca. 50% of the excreted N-acetylated major metabolites contained no (13)C-label at the acetyl, having been replaced by an endogenous (12)C-acetyl source. This level of futile deacetylation is the highest yet reported for a substituted aniline/acetanilide and indicates a high degree of electronic activation of the amino group toward the acetyltransferase enzymes in vivo.

Anilides↗
PubMed1998

Comparison of the properties of polymeric and C8 based materials for solid phase extraction.

The extraction properties of two polymeric solid phase extraction materials, styryldivinyl benzene (SDB) and 'Oasis' have been compared with those of a base deactivated C8 bonded silica gel using a range of acidic and basic test analytes. In the case of the two polymer phases good extraction of all the test compounds from aqueous buffer was obtained over the pH range 2-10. On the C8 material, efficient extraction of the most polar acidic analyte, anisic acid, was only obtained between pH 2 and 6. The use of methanol water mixtures, or methanol water mixtures modified with either trifluoroacetic acid (TFA) or triethylamine (TEA) as eluents was investigated for the recovery of the analytes following extraction. The use of TFA or TEA as ionic modifiers strongly influenced the efficiency of the elution step. The effect of a plasma matrix on extraction efficiency was also investigated, with the result depending upon the analyte. An approach to assessing the performance of the three phases has been developed based on the percentages of methanol in the eluent resulting in the recovery of 50% of the analyte, and in determining the difference between eluents giving recoveries of 10 and 90%.

Chemistry Techniques, Analytical↗
PubMed1998

Directly coupled CZE-NMR and CEC-NMR spectroscopy for metabolite analysis: paracetamol metabolites in human urine.

Direct coupling of NMR spectroscopic detection with both capillary zone electrophoresis (CZE) and capillary electrochromatography (CEC) was applied to the separation of metabolites of the drug paracetamol in an extract of human urine. Continuous-flow CZE-NMR and CEC-NMR allowed the detection of the major metabolites, the glucuronide and sulfate conjugates of the drug and the endogenous material hippurate. Identification of these substances was achieved by examination of individual rows of the NMR chromatogram and this also gave estimates of the detection limits. For CEC-NMR, spectra were also obtained in the stopped-flow mode including a two-dimensional TOCSY NMR experiment which afforded confirmatory evidence for paracetamol glucuronide. Characterisation of drug metabolites using NMR spectroscopy is therefore possible with nanolitre sample volumes.

Acetaminophen↗
PubMed1998

Effect of diet on the urinary excretion of hippuric acid and other dietary-derived aromatics in rat. A complex interaction between diet, gut microflora and substrate specificity.

1. A combined in vivo and in vitro study has been devised to investigate an observation, obtained by 1H NMR of urine, that Alp:AprSD (Wistar derived) rats kept under standard husbandry conditions did not excrete urinary hippuric acid (HA). meta-(hydroxyphenyl)-propionic acid ¿m-HPPA¿ was identified as the major aromatic component in urine samples lacking HA. 2. Examination of urine from Alp:APrSD and Zücker (obese negative) rats fed various diets showed that the lack of HA/presence of m-HPPA was due to diet and not to the strain of animal. This observation was reinforced by the demonstration that the administration of benzoic acid (BA) to rats not previously excreting urinary HA resulted in the return of this component to the urinary excretion profile. Thus rats receiving the standard diet were still capable of glycine conjugation. 3. Changing the diet of rats excreting m-HPPA led to the cessation of m-HPPA excretion and the return of HA urine excretion. Interestingly, switching back to the original diet did not cause the loss of HA and the re-emergence of m-HPPA. 4. In vitro studies on the two enzyme systems responsible for glycine conjugation (benzoyl CoA:synthetase and benzoyl CoA:glycine N-acyltransferase) in isolated liver mitochondria showed that m-HPPA did not inhibit either enzyme. However, m-HPPA was not found to be a substrate for the first reaction step explaining why it was found in the urine as the free acid and not as a glycine conjugate. 5. The absence and presence of m-HPPA and hippuric acid is suggested to be due to a combination of differences in dietary precursors of substrates for glycine conjugation and a dietary dependent redistribution of the intestinal microflora responsible for breakdown of plant phenolics and aromatic amino acids. Taken collectively this study emphasises how a simple diet change can cause a profound change in metabolism.

Acyltransferases↗
PubMed1998

19F-NMR and directly coupled HPLC-NMR-MS investigations into the metabolism of 2-bromo-4-trifluoromethylaniline in rat: a urinary excretion balance study without the use of radiolabelling.

1. The metabolic fate and urinary excretion of 2-bromo-4-trifluoromethylaniline has been studied in rat using 19F-NMR spectroscopic and directly coupled HPLC-NMR-MS methods. The compound was dosed to Sprague-Dawley rats (50 mg kg-1, i.p.) and urine collected over 0-8, 8-24 and 24-48 h post-dosing. 2. A total urinary recovery of 53.5 +/- 7.0% of the dose was achieved up to 48 h after dosing. The major metabolite in the urine was identified as 2-amino-3-bromo-5-trifluoromethylphenylsulphate accounting for a total of 35.7 +/- 6.2% of the dose. 3. Further metabolites detected were 2-bromo-4-trifluoromethylphenylhydroxylamine-1V-glucuronide (9.7 +/- 0.2% of the dose), 2-bromo-4-trifluoromethylaniline-N-glucuronide (3.0 +/- 0.3%) and 2-amino-3-bromo-5-trifluoromethylphenylglucuronide (2-St 0-4). Minor metabolites, including 2-bromo-4-trifluoromethylphenylhydroxylamine-O-glucuronide, 2-amino-3-bromo-5-trifluoromethylphenol and 2-bromo-4-trifluoromethylphenylsulphamate, in total accounted for 2.3 +/- 0.9% of the dose. 4. Directly coupled HPLC-NMR-MS and 19F-NMR spectroscopy proved to be efficient techniques for the unequivocal and rapid determination of the urinary metabolic fate and excretion balance of fluorinated xenobiotics without the need for radiolabelling.

Aniline Compounds↗
PubMed1997

Bioanalysis of p-trifluoromethylmandelic acid and Mosher's acid by chiral gas chromatography and fluorine nuclear magnetic resonance to study chiral inversion: application to rat urine samples.

Methods for the nuclear magnetic resonance and gas chromatographic analysis of the enantiomers of p-trifluoromethylmandelic acid (p-TFM) and Mosher's acid (alpha-methoxy-alpha-(trifluoromethyl)phenylacetic acid) present in rat urine samples are described. Gas chromartography was performed using cyclodextrin capillary columns with both compounds analysed following derivatisation with methanolic HCl. Nuclear magnetic resonance was performed directly on the untreated urine samples following addition of beta-cyclodextrin. The methods were suitable for the determination of the individual enantiomers of the analytes in urine. Analysis of the rat urine samples indicated that the p-TFM had undergone a unidirectional enantiomeric interconversion in vivo, while the enantiomers of Mosher's acid were excreted unchanged.

Animals↗
PubMed1997

NMR and HPLC-NMR spectroscopic studies of futile deacetylation in paracetamol metabolites in rat and man.

HPLC-NMR spectroscopy has been used to investigate the level of deacetylation followed by reacetylation (futile deacetylation) of metabolites of paracetamol detected in human and rat urine. This has been achieved through the synthesis and administration of paracetamol isotopically labeled at the acetyl group with C2H3, 13CH3 and 13CO-13CH3. Using paracetamol-C2H3 it had been shown that in the rat the sulphate metabolite present in the urine shows 10-13% futile deacetylation depending on the dose, whereas for paracetamol-13CO-13CH3 the corresponding value was about 8%. After solid phase extraction, it was also possible to determine the level of futile deacetylation in the glucuronide metabolite using directly-coupled HPLC-NMR. This approach was facilitated by the use of acetonitrile-d3 as an HPLC eluent and the HPLC-NMR analyses showed that the level of futile deacetylation in the sulphate and glucuronide metabolites were equal at about 9%. The glucuronide of paracetamol-C2H3 was the predominant metabolite in man and following separation using HPLC-NMR, the level of futile deacetylation was shown to be 1% for the glucuronide and 2% for the sulphate, these values being equal within experimental error. This work demonstrates the utility of NMR and HPLC-NMR spectroscopy for isotope exchange studies.

Acetaminophen↗
PubMed1997

NMR spectroscopic studies on the metabolism and futile deacetylation of phenacetin in the rat.

1. 1H-NMR spectroscopy of urine was used to determine the % deacetylation and re-acetylation of 2H-labelled (in the acetyl) phenacetin metabolites in the rat. 2. Male Sprague-Dawley rats were each dosed with either phenacetin or phenacetin-C2H3 at 50 mg kg-1. The total urinary recoveries for phenacetin and phenacetin-C2H3 were 47.6 +/- 16.7 and 50.1 +/- 16.2% respectively (not significantly different, p > 0.05). Paracetamol sulphate and glucuronide are the major urinary metabolites of both protio and deuteriophenacetin. 3. The futile deacetylation given by the urinary recovery of protio-acetyl metabolites of phenacetin-C2H3 was 29.6 +/- 0.9% for paracetamol sulphate and 36.6 +/- 3.1% for paracetamol glucuronide. These observations demonstrate a high level of futile deacetylation in the paracetamol conjugates formed by metabolism of phenacetin-C2H3 and this may indicate a high metabolic flux through the nephrotoxic intermediate 4-aminophenol. 4. The level of futile deacetylation for phenacetin was significantly higher than that found previously in studies of labelled paracetamol in rat or man, and may be important in understanding the higher nephrotoxicity of phenacetin as compared with paracetamol.

Acetaminophen↗
PubMed1996

Combined HPLC, NMR spectroscopy, and ion-trap mass spectrometry with application to the detection and characterization of xenobiotic and endogenous metabolites in human urine.

The direct coupling of HPLC with NMR spectroscopy has been extended by splitting the HPLC eluent after conventional UV detection and sending part to a NMR spectrometer and part to an ion-trap mass spectrometer in a "triplehyphenated" HPLC-NMR-MS system. Combined UV, 1H NMR, and positive-ion electrospray MS detection was achieved in the continuous-flow mode using whole human urine from a subject dosed with acetaminophen. By means of HPLC-NMR-MS, the structural information available from the complementary spectroscopic techniques provided rapid confirmation of the identity of the acetaminophen glucuronide and sulfate metabolites, together with a number of endogenous metabolites. In particular, the HPLC-NMR-MS approach allowed the unequivocal identification of phenylacetylglutamine in human urine, an endogenous metabolite not previously observed in 1H NMR spectra of urine because of extensive overlap with resonances from other metabolites. The analytical advantages and complementarity of NMR and MS techniques in direct hyphenation with HPLC are discussed. The new technique of HPLC-NMR-MS will provide the scope for more comprehensive and fully automated analysis of biofluids and other complex mixtures than was previously available from single hyphenation of these instruments.

Acetaminophen↗
PubMed1996

Development of a simple liquid chromatographic method for the separation of mixtures of positional isomers and anomers of synthetic 2-, 3- and 4-fluorobenzoic acid glucuronides formed via acyl migration reactions.

Many drugs containing carboxylate groups form beta-1-O-acyl glucuronides as their major phase II metabolites in vivo. These ester glucuronides are potentially reactive due to the susceptibility of the acyl group to nucleophilic reactions resulting in hydrolysis, acyl migration or covalent adduct formation. In the present study, a number of synthetic fluorobenzoic acid glucuronide conjugates were chosen as models for chromatographic studies. A high-performance liquid chromatography method is presented for the simultaneous determination of the 1-, 2-, 3- and 4-positional isomers of the acyl glucuronides, and their alpha- and beta-anomers for the 2-, 3- and 4-fluorobenzoic acids as well as each aglycone formed as a result of hydrolysis. The same elution order was found for the acyl migrated glucuronide isomers of the three fluorobenzoic acids in their equilibrium mixtures. The alpha-4-O-acyl isomer eluted first followed by the beta-4-O-acyl isomer, then the beta-1-O-acyl, the beta-3-O-acyl, the alpha-3-O-acyl, the alpha-2-O-acyl and finally the beta-2-O-acyl isomer eluted. The method was used to determine the overall degradation rates, the acyl migration rates and the hydrolysis rates of 1-O-(2-fluorobenzoyl)-beta-D-glucopyranuronic acid 1-O-(3-fluorobenzoyl)-beta-D-glucopyranuronic acid and 1-O-(4-flurobenzoyl)-beta-D-glucopyranuronic acid in a buffer system pH 7.4 at 25 degrees C. It was found that the order of beta-1-glucuronide acyl migration rates was 2-fluorobenzoyl > 3-fluorobenzoyl > 4-fluorobenzoyl. Both the acyl migration rates and the elution order were interpreted in terms of electronic effect of the fluorine substituent on the carbonyl carbon.

Acylation↗
PubMed1996

Direct characterization of drug glucuronide isomers in human urine by HPLC-NMR spectroscopy: application to the positional isomers of 6,11-dihydro-11-oxodibenz[b,e]oxepin-2-acetic acid glucuronide.

In this work, 400 and 600 MHz 1H HPLC-NMR spectroscopic methods were developed and applied to separate and identify the positional glucuronide isomers and anomers of the model nonsteroidal antiinflammatory drug, 6,11-dihydro-11-oxodibenz[b,e]oxepin-2-acetic acid, in whole human urine. The HPLC methods utilized either an isocratic system, comprising 30% acetonitrile in water at pH 2.5, or a gradient elution system increasing from 30% to 60% acetonitrile, in order to achieve improved separation of the 2-, 3-, and 4-O-acylglucuronide isomers from the faster eluting endogenous urinary metabolites. Directly coupled stop-flow 1H HPLC-NMR spectroscopic measurements were made at the retention times indicated by the UV-monitored chromatographic peaks. The glucuronide isomers were identified from the 1H NMR spectra on the basis of their chemical shifts and spin-spin coupling patterns. The elution order was 4-O-acyl-, 3-O-acyl-, and finally 2-O-acylglucuronide, with tR values of 10.04, 11.68, and 12.64 min, respectively. Although the alpha- and beta-anomers of each of the positional isomers could not be separated in these solvent systems, they could be identified in the individual 1H NMR spectra. This work shows for the first time that directly coupled HPLC-NMR spectroscopy can be used directly to isolate and characterize acyl-migrated isomers of drug glucuronides in whole urine. This approach will be of value in the study of glucuronide acyl migration reactions of nonsteroidal antiinflammatory drugs and other xenobiotic ester glucuronides in whole biofluids.

Acetates↗
PubMed1996

750 MHz HPLC-NMR spectroscopic studies on the separation and characterization of the positional isomers of the glucuronides of 6,11-dihydro-11-oxodibenz[b,e]oxepin-2-acetic acid.

Ester glucuronides (beta-1-O-acyl-D-glucopyranuronates) of many drugs can undergo a series of acyl migration reactions, resulting in positional isomers and anomers which can react with serum proteins with possible toxicological consequences. We have investigated the acyl migration of the ester glucuronides of the model drug 6,-11-dihydro-11-oxodibenz[b,e]oxepin-2-acetic acid in pH 7.4 buffer using directly coupled 750 MHz stopped-flow HPLC-NMR spectroscopy. Using a reversed phase isocratic HPLC method with 21% acetonitrile and 79% D2O in the mobile phase, it was possible to separate and hence identify the individual positional isomers of the model drug glucuronide by 750 MHz HPLC-NMR. The order of elution of the isomers from the C18 column was 4alpha-, 4beta-, aglycon, 1beta-, 3beta-, 3alpha-, 2alpha-, 2beta- (alpha- and beta- referring to the anomerization state at C1 on the glucuronide ring and the numbers referring to the carbon number on the glucuronide ring to which the drug moiety has migrated). It is shown that directly coupled ultra-high-field HPLC-NMR spectroscopy offers a unique analytical advantage for obtaining structural information of interconverting compounds in equilibrium mixtures, and this method will be of value in the study of reactive drug glucuronides of toxicological importance.

Acetates↗
PubMed1996

The detection and characterization of analgesics and anti-inflammatory drugs on high performance thin-layer chromatography plates using tandem mass spectrometry: application to drugs and metabolites in urine.

The application of tandem mass spectrometry to the analysis and identification of analgesics and non-steroidal anti-inflammatory drugs such as paracetamol, ibuprofen and indomethacin following thin-layer chromatography (TLC) is described. TLC was combined successfully with mass spectrometry and with tandem mass spectrometry using silica gel and diol-bonded silica gel high performance TLC plates. The diol-bonded phase was found to be superior for use with biological samples and enabled the identification of paracetamol, ibuprofen and salicylhippuric acid (the major metabolite of acetylsalicylic acid) in human urine extracts following normal therapeutic doses.

Analgesics↗
PubMed1996

An investigation of the properties of a "shielded" phase for the solid phase extraction of acidic and basic compounds.

The extraction properties of Hisep, a novel bonded phase for solid phase extraction, have been investigated using a number of acidic and basic analytes and compared with those of a conventional ODS bonded material. Marked differences in extraction properties for the test analytes propranolol, anisic acid and ICI 128436 from aqueous buffer were noted between the two materials. Thus, extraction onto the ODS material was by both ionic and reversed-phase mechanisms whilst retention on the Hisep phase appeared to be mediated predominantly by reversed-phase hydrophobic interactions. The presence of dog plasma had no effect on the extraction of propranolol but did reduce the extraction efficiencies observed for anisic acid and ICI 128436 in a pH dependent manner.

Adrenergic beta-Antagonists↗