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At least 109 records · Page 6Linked to original sources
PubMedDate not supplied

Direct detection of new flucytosine metabolites in human biofluids by 19F nuclear magnetic resonance.

19F nuclear magnetic resonance was used for the analysis of flucytosine (FC; 5-fluorocytosine) metabolites in biological fluids of a patient with cryptococcal meningitis who was intravenously injected with this drug at a daily dose of 7.5 g (2.5 g at 8-hr intervals). This method allows a direct, simultaneous, and quantitative determination of all the fluorinated metabolites of FC, in the range of sensitivity allowed by the spectrometer (sensitivity threshold, 0.01 mM). In urine, in addition to the already reported metabolites [unmetabolized FC and alpha-fluoro-beta-alanine (FBAL)], three new metabolites were identified: a glucuronide of FC (GLFC), 6-hydroxy-5-fluorocytosine (60HFC), and fluoride ion F-. The same metabolites (except F-) were found in plasma. In cerebrospinal fluid, only unchanged FC and GLFC were observed. The total urinary excretion during an 8-hr period between two injections of FC was 100.4% of the injected dose. Unchanged FC was the major excretory product accounting for 96.1% of the total. GLFC and 6OHFC made up, respectively, 2.7% and 1.2% of the excreted metabolites. The proportions of F- and FBAL were very low, respectively, 0.3% and 0.1% of the excreted metabolites. The global urinary excretion over a 24-hr period was 102% of the injected dose. The proportions of metabolites were very close to those obtained for the 8-hr period. In plasma, the proportions of metabolites were analogous to those determined in urine. In cerebrospinal fluid, GLFC represents 1% of the fluorinated metabolites.(ABSTRACT TRUNCATED AT 250 WORDS)

Body Fluids↗
PubMedDate not supplied

The simultaneous determination of pethidine and norpethidine in biofluids by nitrogen selective gas chromatography.

A rapid sensitive and selective gas chromatographic method has been developed for the simultaneous determination of pethidine and its major basic metabolite, norpethidine, using a nitrogen selective detector. The procedure involved a preliminary ethereal extraction of the drug, its metabolite and internal marker (lignocaine) from the alkalinised biological fluids (plasma or urine). The extract, after concentration, was analysed by a GC system (3 per cent OV 17 on Gas Chrom Q, 80-100 mesh) linked to a nitrogen selective detector. The calibration graphs (relating peak height ratios of the drug to internal marker and concentration) of pethidine and norepethidine were linear and reproducible over the ranges of 5 ng/ml to 100 ng/ml for plasma samples and 50 ng/ml to 1000 ng/ml for urine samples. The recovery of the drug and metabolite from plasma samples at 5 ng/ml and 100 ng/ml is 100 per cent and 84.9 per cent respectively for pethidine, and 100 per cent and 90.9 per cent respectively for norpethidine. Similar recovery from urine samples of pethidine and norpethidine is also achieved at 50 ng/ml and 1000 ng/ml levels. The reproducibility of the assay procedure for pethidine and norpethidine is 100 +/- 0.02 per cent and 100 +/- 0.04 per cent at 100 ng/ml level, and 100 +/- 0.15 per cent and 100 +/- 0.12 per cent at 5 ng/ml level.

Chromatography, Gas↗
PubMed2005

Standard line slopes as a measure of a relative matrix effect in quantitative HPLC-MS bioanalysis.

A simple experimental approach for studying and identifying the relative matrix effect (for example "plasma-to-plasma" and/or "urine-to-urine") in quantitative analyses by HPLC-MS/MS is described. Using as a database a large number of examples of methods developed in recent years in our laboratories, the relationship between the precision of standard line slopes constructed in five different lots of a biofluid (for example plasma) and the reliability of determination of concentration of an analyte in a particular plasma lot (or subject) was examined. In addition, the precision of standard line slopes was compared when stable isotope-labeled analytes versus analogs were used as internal standards (IS). Also, in some cases, a direct comparison of standard line slopes was made when different HPLC-MS interfaces (APCI versus ESI) were used for the assay of the same compound, using the same IS and the same sample preparation and chromatographic separation conditions. In selected cases, the precision of standard line slopes in five different lots of a biofluid was compared with precision values determined five times in a single lot. The results of these studies indicated that the variability of standard line slopes in different lots of a biofluid [precision of standard line slopes expressed as coefficient of variation, CV (%)] may serve as a good indicator of a relative matrix effect and, it is suggested, this precision value should not exceed 3-4% for the method to be considered reliable and free from the relative matrix effect liability. Based on the results presented, in order to assess the relative matrix effect in bioanalytical methods, it is recommended to perform assay precision and accuracy determination in five different lots of a biofluid, instead of repeat (n=5) analysis in the same, single biofluid lot, calculate standard line slopes and precision of these slopes, and to use <3-4% slope precision value as a guide for method applicability to support clinical studies. It was also demonstrated that when stable isotope-labeled analytes were used as internal standards, the precision of standard line slopes in five different lots of a biofluid was </=2.4% irrespective of the HPLC-MS interface utilized. This clearly indicated that, in all cases studied, the use of stable isotope-labeled IS eliminated relative matrix effect. Also, the utilization of the APCI interface instead of ESI led to the elimination of the relative matrix effect in all cases studied. When the precision of standard line slope values exceeds the 3-4% limit, the method may require improvements (a more efficient chromatography, a more selective extraction, a stable isotope-labeled IS instead of an analog as an IS, and/or a change in the HPLC-MS interface) to eliminate the relative matrix effect and to improve assay selectivity.

Body Fluids↗
PubMed2006

HPLC-NMR with severe column overloading: fast-track metabolite identification in urine and bile samples from rat and dog treated with [14C]-ZD6126.

The subject of this study was the determination of the major urinary and biliary metabolites of [(14)C]-ZD6126 following i.v. administration to female and male bile duct cannulated rats at 10 mg/kg and 20 mg/kg, respectively, and male bile duct cannulated dogs at 6 mg/kg by HPLC-NMR spectroscopy. ZD6126 is a phosphorylated pro-drug, which is rapidly hydrolysed to the active metabolite, ZD6126 phenol. The results presented here demonstrate that [(14)C]-ZD6126 phenol is subsequently metabolised extensively by male dogs and both, male and female rats. Recovery of the dose in bile and urine was determined utilising the radiolabel, revealing biliary excretion as the major route of excretion (93%) in dog, with the majority of the radioactivity recovered in both biofluids in the first 6 h. In the rat, greater than 92% recovery was obtained within the first 24 h. The major route of excretion was via the bile 51-93% within the first 12 h. The administered phosphorylated pro-drug was not observed in any of the excreta samples. Metabolite profiles of bile and urine samples were determined by high performance liquid chromatography with radiochemical detection (HPLC-RAD), which revealed a number of radiolabelled components in each of the biofluids. The individual metabolites were subsequently identified by HPLC-NMR spectroscopy and HPLC-MS. In the male dog, the major component in urine and bile was the [(14)C]-ZD6126 phenol glucuronide, which accounted for 3% and 77% of the dose, respectively. [(14)C]-ZD6126 phenol was observed in urine at 1% of dose, but was not observed in bile. A sulphate conjugate of demethylated [(14)C]-ZD6126 phenol was identified in bile by HPLC-NMR and confirmed by HPLC-MS. In the rat, the bile contained two major radiolabelled components. One was identified as the [(14)C]-ZD6126 phenol glucuronide, the other as a glucuronide conjugate of demethylated [(14)C]-ZD6126 phenol. However, a marked difference in the proportions of these two components was observed between male and female rats, either due to a sex difference in metabolism or a difference in dose level. The glucuronide conjugate of the demethylated [(14)C]-ZD6126 phenol was present at higher concentration in the bile of male rats (4-34%), while the phenol glucuronide was present at higher concentration in the bile of female rats (8-70%) over a 0-6 h collection period. A third component was only observed in the bile samples (0-6 h and 6-12 h) of male rats. This was identified as being the same sulphate conjugate of demethylated [(14)C]-ZD6126 phenol as the one observed in dog bile. The rat urines contained two main metabolites in greatly varying concentrations, namely the demethylated [(14)C]-ZD6126 phenol glucuronide and the glucuronide of [(14)C]-ZD6126 phenol. Again, the differences in relative amounts between male and female rats were observed, the major metabolite in the urines from male rats being the demethylated [(14)C]-ZD6126 phenol (0-17% in 0-24 h), whilst the phenol glucuronide, accounting for 0.5-50% of the dose over 0-24 h, was the major metabolite in females. Methanolic extracts of the pooled biofluid samples were submitted for HPLC-NMR for the quick identification of the major metabolites. Following a single injection of the equivalent of 6-28 ml of the biofluids directly onto the HPLC-column with minimal sample preparation, the metabolites could be largely successfully isolated. Despite severe column overloading, the major metabolites of [(14)C]-ZD6126 could be positively identified, and the results are presented in this paper.

Animals↗
PubMed1981

Determination of acetylmethadol and metabolites by use of high-performance liquid chromatography.

A method is described for the simultaneous determination of 1, alpha-acetylmethadol (LAAM) and five active metabolites--noracetylmethadol, dinoracetylmethadol, methadol, normethadol, and dinormethadol--in biofluids by high-performance liquid chromatography using a normal-phase column and a UV detector at 218 nm. The compounds are recovered from biofluids by a multistep liquid--liquid extraction. The mobile phase is methanol--acetonitrile (70:30, v/v) containing 0.015% ammonium hydroxide as the modifier. Retention times can be varied by adjusting the composition of the mobile phase to maximize peak height for quantitation using l-propranolol as the internal standard or peak separation for the collection of fractions. Using a UV detector the lower limit of sensitivity is 10 ng/ml of biofluid. Using fraction collection of radiolabeled drug and metabolites followed by liquid scintillation counting the lower limit of sensitivity is 1.0 ng/ml. Commonly used or abused narcotics including morphine, heroin, meperidine, methadone and propoxyphene do not interfere with the analysis. The method has been applied to plasma and urine samples from humans, sheep and rats. Extracts of urine from patients receiving maintenance treatment with LAAM contain LAAM and each of the five active metabolites.

Animals↗
PubMed1996

High-resolution diffusion and relaxation edited one- and two-dimensional 1H NMR spectroscopy of biological fluids.

A new approach to the characterization of biomolecules in whole biological fluids is presented based on simplification of 1H NMR spectra by utilizing differences in molecular diffusion coefficients alone and combinations of relaxation and diffusion parameters. New NMR pulse sequences incorporating both spectral editing features together with solvent water resonance elimination are presented. The methods are exemplified using whole human blood plasma, and it is shown that it is possible to obtain NMR spectra of the slowly diffusing species (generally large molecules) by diffusion editing, the slowly relaxing species (generally small molecules) by spin relaxation editing, or spectra showing any range of molecular mobility using a combination of the two methods. The diffusion-based editing methods are also applicable to the selection of resonances in two-dimensional NMR spectroscopy of biofluids, and we show this for the first time by the production of 1H-1H diffusion-edited TOCSY spectra of human blood plasma where the resonance intensities are weighted according to the molecular diffusion coefficient. In this case, by measuring a diffusion-edited 1H-1H TOCSY NMR spectrum of plasma, it is possible to obtain signals from only the macromolecular components, and this may be of benefit in the analysis of blood lipoproteins. In complex biofluids, the combination of diffusion and relaxation editing brings about considerable spectral simplification leading to an easier resonance assignment process. We also demonstrate the production of 1H NMR spectra with intensities corresponding to diffusion coefficient rather than number of protons, and this opens up new possibilities for pattern recognition classification of samples based on altered molecular mobility features of biofluid components.

Blood↗
PubMed1997

Measurement of biomolecular diffusion coefficients in blood plasma using two-dimensional 1H-1H diffusion-edited total-correlation NMR spectroscopy.

The use of two-dimensional diffusion-edited total-correlation NMR spectroscopy (DETOCSY) to measure diffusion coefficients of molecules in biofluids is demonstrated. The method takes advantage of the increased peak dispersion in a two-dimensional spectrum and allows the measurement of diffusion coefficients of individual molecules in complex biofluid samples in a condition close to their natural state. The NMR spectra can be edited on the basis of diffusion coefficient, and hence, approximate molecular size and this provides a qualitative means of simplifying the spectra for assignment purposes. The use of two-dimensional DETOCSY NMR spectra increases the probability that the individual cross-peaks arise from single components and hence allows single-exponential fitting of the magnetic field gradient dependence of cross-peak volumes. Interpretation of the derived diffusion coefficients improves the NMR resonance assignment process in complex biofluid mixtures by giving additional information on the molecular size. The DETOCSY approach also provides information on small molecule-macromolecule interactions such as the binding of drugs or endogenous species to serum proteins such as albumin.

Diffusion↗
PubMed2005

Interaction of fluorescent molecular rotors with blood plasma proteins.

Many disease states have associated blood viscosity changes. Molecular rotors, fluorescent molecules with viscosity sensitive quantum yields, have recently been investigated as a new method for biofluid viscosity measurement. Current viscometer measurements are complicated by proteins adhering to surfaces and forming air-surface layers. It is unknown at this time what effects proteins may have on biofluid viscosity measurements using molecular rotors. To answer this question, binding affinities to blood plasma proteins were investigated by equilibrium dialysis for four hydrophilic molecular rotors. Aqueous solutions of 9-[(2-cyano-2-hydroxy-carbonyl)vinyl]julolidine (CCVJ) and three derivatives were prepared and dialyzed against solutions of bovine source albumin, fibrinogen and immunoglobulin G approximating normal physiologic concentrations and fresh-frozen human plasma. After equilibration, dye concentration on each side of the dialysis membrane was assessed by spectrophotometry. The relative binding affinity of the four dyes to the proteins and to the plasma was compared. Affinity of all dyes was highest for albumin. The bound dye fraction showed little change in relation to protein concentration in the physiological concentration range. Diol, the most hydrophilic molecular rotor tested showed the lowest affinity for albumin. This study indicates that hydrophilic molecular rotors are well-suited for biofluid viscosity measurement.

Animals↗
PubMed2001

The development of a staggered parallel separation liquid chromatography/tandem mass spectrometry system with on-line extraction for high-throughout screening of drug candidates in biological fluids.

A new parallel liquid chromatography/tandem mass spectrometry (LC/MS/MS) system has been developed, in which the mass detector was shared between two staggered parallel chromatographic runs. Since the chromatography for biofluids assay generally requires good analyte retention and thus tends to leave large blank chromatographic windows, this parallel system allowed the efficient use of the mass detector during these blank windows, resulting in significantly improved sample throughput. Also, in order to remove the bottleneck in sample extraction for this parallel separation system, a high-flow extraction device was used to perform on-line extraction. This allowed for the direct injection of biofluids onto the system. The performance and capability of this system was evaluated in tests that contained a single analyte (oxazepam) and multiple analytes (12-in-1). The results indicated that the data generated from this system were comparable to those obtained on a conventional single-column system. An application of the system for high-throughput pharmacokinetic screening of drug candidates was also demonstrated.

Chromatography, Liquid↗
PubMed1994

Solid-phase derivatization reactions for biomedical liquid chromatography.

Polymeric reagents have been developed for performing off- and on-line derivatizations of numerous organic analytes in HPLC-detection modes. Such reagents utilize ionic or covalent attachment of labile tags that possess specific detector enhancement properties: ultraviolet, electrochemical, fluorescence, and so forth. Specific synthetic procedures have evolved to generate various linkages of the tag to the underlying, polymeric support, usually involving activated ester connections (leashes). The polymer itself may play a number of roles in the nature of the overall reactions, such as hydrophobic-hydrophillic exclusion, pore size restriction, stabilization of the attachment leashes, and protection of the tags from hydrolysis in aqueous media. The basic, underlying chemistry of polymeric reagents has evolved to the point where it is possible to engineer the polymer support itself, the attachment leash, and the various tags that are then transferred to the analyte molecules. These procedures have now reached the stage of commercialization and practical applicability for real-world drugs and bioorganics in complex biofluid type samples. Polymer supported reagents can now be used for direct injection of biofluids with solid-phase (hydrophobic) extraction of the analytes of interest, followed by sample cleanup, derivatization, elution onto the HPLC column, peak compression, gradient HPLC elution, multiple detection, and final data interpretation with quantitation. This review summarizes much or most of what has been described in the scientific literature over the past decade in the various areas where polymeric reagents are being used for derivatization in HPLC and in capillary electrophoresis as well.

Chemistry Techniques, Analytical↗
PubMed2006

Metabolomic analysis using optimized NMR and statistical methods.

NMR-based metabolomics requires robust automated methodologies, and the accuracy of NMR-based metabolomics data is greatly influenced by the reproducibility of data acquisition and processing methods. Effective water resonance signal suppression and reproducible spectral phasing and baseline traces across series of related samples are crucial for statistical analysis. We assess robustness, repeatability, sensitivity, selectivity, and practicality of commonly used solvent peak suppression methods in the NMR analysis of biofluids with respect to the automated processing of the NMR spectra and the impact of pulse sequence and data processing methods on the sensitivity of pattern recognition and statistical analysis of the metabolite profiles. We introduce two modifications to the excitation sculpting pulse sequence whereby the excitation solvent suppression pulse cascade is preceded by low-power water resonance presaturation pulses during the relaxation delay. Our analysis indicates that combining water presaturation with excitation sculpting water suppression delivers the most reproducible and information-rich NMR spectra of biofluids.

Animals↗