ABSTRACT
We developed a robust analytical method for quantification of malondialdehyde (MDA) in urine and serum samples using dansylhydrazine (DH) as a derivatizing reagent. The derivatization procedure was partially carried out using an autosampler injection program to minimize errors associated with the low-volume addition of reagents and was optimized to yield a stable hydrazone derivative of MDA and its labeled d2-MDA analogue. The target MDA-DH derivatives were separated on an Agilent Zorbax Eclipse Plus Phenyl-Hexyl (3.0 × 100 mm, 3.5 µm) column. The mass-to-charge ratios of the target derivatives [(M+H)+ of 302 and 304 for MDA-DH and d2-MDA-DH, respectively] were analyzed in single ion monitoring mode using a single quadrupole mass spectrometer operated under positive electrospray ionization. The method limits of quantification were 5.63 nM (or 0.405 ng/mL) for urine analysis and 5.68 nM (or 0.409 ng/mL) for serum analysis. The quantification range for urine analysis was 5.63-500 nM (0.405-36.0 ng/mL) while the quantification range for serum analysis was 5.68-341 nM (0.409-24.6 ng/mL). The method showed good relative recoveries (98-103%), good accuracies (92-98%), and acceptable precisions (relative standard deviations 1.8-7.3% for inter-day precision; 1.8-6.1% for intra-day precision) as observed from the repeat analysis of quality control samples prepared at different concentrations. The method was used to measure MDA in individual urine samples (n = 287) and de-identified archived serum samples (n = 22) to assess the overall performance of the method. The results demonstrated that our method is capable of measuring urinary and serum levels of MDA, allowing its future application in epidemiologic investigations.
Subject(s)
Dansyl Compounds/metabolism , Hydrazines/metabolism , Malondialdehyde/metabolism , Body Fluids , Chromatography, High Pressure Liquid , Chromatography, Liquid , Humans , Hydrazines/blood , Hydrazines/urine , Limit of Detection , Malondialdehyde/blood , Malondialdehyde/urine , Tandem Mass SpectrometryABSTRACT
Currently used on F-16 fighter jets and some space shuttles, hydrazine could be released at toxic levels to humans as a result of an accidental leakage or spill. Lower-level exposures occur in industrial workers or as a result of the use of some pharmaceuticals. A method was developed for the quantitation of hydrazine in human urine and can be extended by dilution with water to cover at least six orders of magnitude, allowing measurement at all clinically significant levels of potential exposure. Urine samples were processed by isotope dilution, filtered, derivatized and then quantified by HPLC-MS-MS. The analytical response ratio was linearly proportional to the urine concentration of hydrazine from 0.0493 to 12.3 ng/mL, with an average correlation coefficientRof 0.9985. Inter-run accuracy for 21 runs, expressed as percent relative error (% RE), was ≤14%, and the corresponding precision, expressed as percent relative standard deviation (% RSD), was ≤15%. Because this method can provide a quantitative measurement of clinical samples over six orders of magnitude, it can be used to monitor trace amounts of hydrazine exposure as well as industrial and environmental exposure levels.
Subject(s)
Carcinogens/analysis , Hydrazines/urine , Calibration , Chromatography, High Pressure Liquid , Environmental Exposure , Humans , Quality Control , Radioisotope Dilution Technique , Reproducibility of Results , Solutions , Spectrometry, Mass, Electrospray Ionization , Tandem Mass SpectrometryABSTRACT
In this paper, very simple and rapid sensor has been developed for the spectrophotometric determination of pico-molar level of hydrazine using Alizarin red. There was a decrease of optical intensity of the probe in the presence of hydrazine. The LOD is calculated from the linear graph between 5-100 pM as 0.66 pM of hydrazine which is well below the risk level proposed by Agency for Toxic Substance and Disease Registry. The probe selectivity for the detection of hydrazine was tested in the presence of commonly encountered metal ions and anions. The calibration curves showed good linearity for working ranges from 5-100 pM and 0.5-40 mM respectively, with R(2)=0.9911 and 0.9744, indicate the validity of the Beer-Lambert law. The binding constant and the free energy change values are determined by the Benesi-Hildebrand method. Determination of hydrazine in environmental water and human urine samples are successfully performed by the proposed method with the recovery of 100%.
Subject(s)
Anthraquinones/chemistry , Hydrazines/analysis , Hydrazines/urine , Spectrophotometry/methods , Water Pollutants, Chemical/analysis , Water Pollutants, Chemical/urine , Water/analysis , Environmental Monitoring/methods , Humans , Limit of DetectionABSTRACT
1. Laromustine (VNP40101M, also known as Cloretazine) is a novel sulfonylhydrazine alkylating (anticancer) agent. This article describes the use of quantitative whole-body autoradiography (QWBA) and mass balance to study the tissue distribution, the excretion mass balance and pharmacokinetics after intravenous administration of [(14)C]VNP40101M to rats. A single 10 mg/kg IV bolus dose of [(14)C]VNP40101M was given to rats. 2. The recovery of radioactivity from the Group 1 animals over a 7-day period was an average of 92.1% of the administered dose, which was accounted for in the excreta and carcass. Most of the radioactivity was eliminated within 48 h via urine (48%), with less excreted in feces (5%) and expired air accounted for (11%). The plasma half-life of [(14)C]laromustine was approximately 62 min and the peak plasma concentration (Cmax) averaged 8.3 µg/mL. 3. The QWBA study indicated that the drug-derived radioactivity was widely distributed to tissues through 7 days post-dose after a single 10 mg/kg IV bolus dose of [(14)C]VNP40101M to male pigmented Long-Evans rats. The maximum concentrations were observed at 0.5 or 1 h post-dose for majority tissues (28 of 42). The highest concentrations of radioactivity were found in the small intestine contents at 0.5 h (112.137 µg equiv/g), urinary bladder contents at 3 h (89.636 µg equiv/g) and probably reflect excretion of drug and metabolites. The highest concentrations in specific organs were found in the renal cortex at 1 h (28.582 µg equiv/g), small intestine at 3 h (16.946 µg equiv/g), Harderian gland at 3 h (12.332 µg equiv/g) and pancreas at 3 h (12.635 µg equiv/g). Concentrations in the cerebrum (1.978 µg equiv/g), cerebellum (2.109 µg equiv/g), medulla (1.797 µg equiv/g) and spinal cord (1.510 µg equiv/g) were maximal at 0.5 h post-dose and persisted for 7 days. 4. The predicted total body and target organ exposures for humans given a single 100 µCi IV dose of [(14)C]VNP40101M were well within the medical guidelines for maximum radioactivity exposures in human subjects.
Subject(s)
Antineoplastic Agents/administration & dosage , Antineoplastic Agents/pharmacokinetics , Hydrazines/administration & dosage , Hydrazines/pharmacokinetics , Metalloporphyrins/chemistry , Neoplasms/drug therapy , Sulfonamides/administration & dosage , Sulfonamides/pharmacokinetics , Animals , Antineoplastic Agents/blood , Antineoplastic Agents/urine , Humans , Hydrazines/blood , Hydrazines/urine , Injections, Intravenous , Male , Models, Animal , Rats , Rats, Long-Evans , Rats, Sprague-Dawley , Sulfonamides/blood , Sulfonamides/urine , Tissue DistributionABSTRACT
BACKGROUND: Accurate mass based LC-MS combined with statistical analysis is established as a core analytical technology for metabonomic studies. This is primarily due to the specificity, sensitivity and structural elucidation capabilities of the technology. The vast majority of these studies are performed using acidic-based mobile phases in combination with positive ESI mode LC-MS. Recent studies have investigated the use of highly basic pH mobile phases (>10 pH units) in bioanalytical studies that utilize positive ESI mode LC-MS. This non-traditional combination has been shown to improve analyte retention, chromatographic peak shape, and S/N for a variety of probe pharmaceutical compounds in biofluid samples. RESULTS: The incorporation of basic pH mobile phases resulted in increased retention for analytes that where comparatively weakly retained by a traditional acidic-modified mobile phase. Increased resolution of isomers, which otherwise co-eluted under acidic conditions, was observed. Moreover, the implementation of basic pH mobile phases further allowed for the detection of complementary marker ions. CONCLUSION: Basic pH mobile phases utilized with positive ESI mode LC-MS have the potential for producing increased information from metabonomic studies and could lead to the detection of analytes that may prove to be valid biomarkers.
Subject(s)
Chromatography, High Pressure Liquid , Metabolomics/instrumentation , Spectrometry, Mass, Electrospray Ionization , Administration, Oral , Animals , Biomarkers/urine , Hydrazines/metabolism , Hydrazines/urine , Hydrogen-Ion Concentration , Male , Principal Component Analysis , Rats , Rats, Sprague-Dawley , Solvents/chemistry , Toxins, Biological/metabolism , Toxins, Biological/urineABSTRACT
The metabolism and disposition of eltrombopag, the first-in-class small molecule human thrombopoietin receptor agonist, were studied in six healthy men after a single oral administration of a solution dose of [(14)C]eltrombopag (75 mg, 100 µCi). Eltrombopag was well tolerated. The drug was quickly absorbed and was the predominant circulating component in plasma (accounting for 63% of the total plasma radioactivity). A mono-oxygenation metabolite (M1) and acyl glucuronides (M2) of eltrombopag were minor circulating components. The predominant route of elimination of radioactivity was fecal (58.9%). Feces contained approximately 20% of dose as glutathione-related conjugates (M5, M6, and M7) and another 20% as unchanged eltrombopag. The glutathione conjugates were probably detoxification products of a p-imine methide intermediate formed by metabolism of M1, which arises through cytochrome P450-dependent processes. Low levels of covalently bound drug-related intermediates to plasma proteins, which could result from the reaction of the imine methide or acyl glucuronide conjugates with proteins, were detected. The bound material contributes to the longer plasma elimination half-life of radioactivity. Renal elimination of conjugates of hydrazine cleavage metabolites (mostly as M3 and M4) accounted for 31% of the radiodose, with no unchanged eltrombopag detected in urine.
Subject(s)
Benzoates/pharmacokinetics , Hydrazines/pharmacokinetics , Pyrazoles/pharmacokinetics , Receptors, Thrombopoietin/agonists , Administration, Oral , Adult , Benzoates/blood , Benzoates/metabolism , Benzoates/urine , Biotransformation , Blood Proteins/metabolism , Cytochrome P-450 Enzyme System/metabolism , Feces/chemistry , Glucuronides/blood , Glutathione/metabolism , Half-Life , Humans , Hydrazines/blood , Hydrazines/metabolism , Hydrazines/urine , Male , Middle Aged , Protein Binding , Pyrazoles/blood , Pyrazoles/metabolism , Pyrazoles/urine , Receptors, Thrombopoietin/metabolismABSTRACT
Atazanavir (ATV) is an antiretroviral drug of the protease inhibitor class. Multiple adverse effects of ATV have been reported in clinical practice, such as jaundice, nausea, abdominal pain, and headache. The exact mechanisms of ATV-related adverse effects are unknown. It is generally accepted that a predominant pathway of drug-induced toxicity is through the generation of reactive metabolites. Our current study was designed to explore reactive metabolites of ATV. We used a metabolomic approach to profile ATV metabolism in mice and human liver microsomes. We identified 5 known and 13 novel ATV metabolites. Three potential reactive metabolites were detected and characterized for the first time: one aromatic aldehyde, one α-hydroxyaldehyde, and one hydrazine. These potential reactive metabolites were primarily generated by CYP3A. Our results provide a clue for studies on ATV-related adverse effects from the aspect of metabolic activation. Further studies are suggested to illustrate the impact of these potential reactive metabolites on ATV-related adverse effects.
Subject(s)
Aldehydes/metabolism , Cytochrome P-450 CYP3A/metabolism , HIV Protease Inhibitors/pharmacokinetics , Hydrazines/metabolism , Oligopeptides/pharmacokinetics , Pyridines/pharmacokinetics , Aldehydes/analysis , Aldehydes/chemistry , Aldehydes/urine , Animals , Atazanavir Sulfate , Biotransformation , Chromatography, High Pressure Liquid , Cytochrome P-450 CYP3A/genetics , Cytochrome P-450 CYP3A Inhibitors , Enzyme Inhibitors/pharmacology , Feces/chemistry , HIV Protease Inhibitors/analysis , HIV Protease Inhibitors/chemistry , HIV Protease Inhibitors/urine , Humans , Hydrazines/analysis , Hydrazines/chemistry , Hydrazines/urine , Ketoconazole/pharmacology , Metabolomics/methods , Mice , Microsomes, Liver/drug effects , Microsomes, Liver/metabolism , Oligopeptides/analysis , Oligopeptides/chemistry , Oligopeptides/urine , Pyridines/analysis , Pyridines/chemistry , Pyridines/urine , Recombinant Proteins/metabolism , Spectrometry, Mass, Electrospray Ionization , Tandem Mass SpectrometryABSTRACT
A cloud point extraction process using mixed micelle of the anionic surfactant sodium dodecyl sulfate and the nonionic surfactant Triton X-114 to extract hydrazine from aqueous solutions was investigated. The method is based on the condensation reaction of hydrazine with p-(dimethylamino)benzaldehyde, azine formation, and mixed micelle-mediated extraction of azine in the presence of NaCl electrolyte as an inducing phase separation. An azine product was concentrated in surfactant-rich phase after separation. The optimal extraction and reaction conditions (e.g., surfactant, reagent and electrolyte concentrations, and centrifuge time) were studied and the analytical characteristics of the method (e.g., limit of detection, linear range, preconcentration, and improvement factors) were obtained. Linearity was obeyed in the range of 0.50-110ngml(-1) of hydrazine and the detection limit of the method is 0.08ngml(-1). The interference effect of some cations, anions, and organic compounds was also tested. The method was successfully applied to the determination of hydrazine in water and biological samples.
Subject(s)
Electrolytes/chemistry , Hydrazines , Micelles , Water Pollutants, Chemical , Water Supply/analysis , Benzaldehydes/chemistry , Hydrazines/blood , Hydrazines/urine , Octoxynol , Polyethylene Glycols/chemistry , Sensitivity and Specificity , Sodium Dodecyl Sulfate/chemistry , Spectrum Analysis , Surface-Active Agents/chemistry , Water Pollutants, Chemical/blood , Water Pollutants, Chemical/urineABSTRACT
1H NMR spectra of biofluids provides a wealth of biochemical information on the metabolic status of an organism. Through the application of pattern recognition and classification algorithms, the data have been shown to provide information on disease diagnosis and the beneficial and adverse effects of potential therapeutics. Here, a novel approach is described for identifying subsets of spectral patterns in databases of NMR spectra, and it is shown that the intensities of these spectral patterns can be related to the onset and recovery from a toxic lesion in both a time-related and dose-related fashion. These patterns form a new type of combination biomarker for the biological effect under study. The approach is illustrated with a study of liver toxicity in rats using NMR spectra of urine following administration of a model hepatotoxin hydrazine.
Subject(s)
Drug-Related Side Effects and Adverse Reactions/classification , Magnetic Resonance Spectroscopy/methods , Animals , Bayes Theorem , Databases as Topic , Dose-Response Relationship, Drug , Drug-Related Side Effects and Adverse Reactions/urine , Hydrazines/administration & dosage , Hydrazines/toxicity , Hydrazines/urine , Liver/drug effects , Liver/metabolism , Male , Rats , Rats, Wistar , Reproducibility of Results , Structure-Activity Relationship , Time FactorsABSTRACT
Metabonomics can be viewed as the process of defining multivariate metabolic trajectories that describe the systemic response of organisms to physiological perturbations through time. We have explored the hypothesis that the homothetic geometry of a metabolic trajectory, i.e., the metabolic response irrespective of baseline values and overall magnitude, defines the mode of response of the organism to treatment and is hence the key property when considering the similarity between two sets of measurements. A modeling strategy to test for homothetic geometry, called scaled-to-maximum, aligned, and reduced trajectories (SMART) analysis, is presented that together with principal components analysis (PCA) facilitates the visualization of multivariate response similarity and hence the interpretation of metabonomic data. Several examples of the utility of this approach from toxicological studies are presented as follows: interlaboratory variation in hydrazine response, CCl(4) dose-response relationships, and interspecies comparison of bromobenzene toxicity. In each case, the homothetic trajectories hypothesis is shown to be an important concept for the successful multivariate modeling and interpretation of systemic metabolic change. Overall, geometric trajectory analysis based on a homothetic modeling strategy like SMART facilitates the amalgamation and comparison of metabonomic data sets and can improve the accuracy and precision of classification models based on metabolic profile data. Because interlaboratory variation, normal physiological variation, dose-response relationships, and interspecies differences are also key areas of concern in genomic and proteomic as well as metabonomic studies, the methods presented here may also have an impact on many other multilaboratory efforts to produce screenable "-omics" databases useful for gauging toxicity in safety assessment and drug discovery.
Subject(s)
Toxicology/methods , Animals , Bromobenzenes/metabolism , Databases, Factual , Dose-Response Relationship, Drug , Hydrazines/metabolism , Hydrazines/urine , Multivariate Analysis , Pattern Recognition, Automated , Principal Component Analysis , Rats , Rats, Sprague-Dawley , Reproducibility of Results , Toxins, Biological/urineABSTRACT
Metabonomic analysis of biofluids and tissues utilizing high-resolution NMR spectroscopy and chemometric techniques has proven valuable in characterizing the biochemical response to toxicity for many xenobiotics. To assess the analytical reproducibility of metabonomic protocols, sample preparation and NMR data acquisition were performed at two sites (one using a 500 MHz and the other using a 600 MHz system) using two identical (split) sets of urine samples from an 8-day acute study of hydrazine toxicity in the rat. Despite the difference in spectrometer operating frequency, both datasets were extremely similar when analyzed using principal components analysis (PCA) and gave near-identical descriptions of the metabolic responses to hydrazine treatment. The main consistent difference between the datasets was related to the efficiency of water resonance suppression in the spectra. In a 4-PC model of both datasets combined, describing all systematic dose- and time-related variation (88% of the total variation), differences between the two datasets accounted for only 3% of the total modeled variance compared to ca. 15% for normal physiological (pre-dose) variation. Furthermore, <3% of spectra displayed distinct inter-site differences, and these were clearly identified as outliers in their respective dose-group PCA models. No samples produced clear outliers in both datasets, suggesting that the outliers observed did not reflect an unusual sample composition, but rather sporadic differences in sample preparation leading to, for example, very dilute samples. Estimations of the relative concentrations of citrate, hippurate, and taurine were in >95% correlation (r(2)) between sites, with an analytical error comparable to normal physiological variation in concentration (4-8%). The excellent analytical reproducibility and robustness of metabonomic techniques demonstrated here are highly competitive compared to the best proteomic analyses and are in significant contrast to genomic microarray platforms, both of which are complementary techniques for predictive and mechanistic toxicology. These results have implications for the quantitative interpretation of metabonomic data, and the establishment of quality control criteria for both regulatory agencies and for integrating data obtained at different sites.
Subject(s)
Magnetic Resonance Spectroscopy/methods , Specimen Handling , Toxicology/methods , Urinalysis/instrumentation , Animals , Dose-Response Relationship, Drug , Hydrazines/metabolism , Hydrazines/urine , Magnetic Resonance Spectroscopy/instrumentation , Male , Metabolism/drug effects , Observer Variation , Rats , Rats, Sprague-Dawley , Reproducibility of Results , Urine/chemistryABSTRACT
Cryogenic probe technology can significantly compensate for the inherently low sensitivity of natural abundance 13C NMR spectroscopy. This now permits its routine use in NMR spectroscopy of biofluids, such as urine or plasma, with acquisition times that enable a high throughput of samples. Metabonomic studies often generate numerous samples in order to characterize fully the time-dependent biochemical response to stimuli, but until now, they have been largely conducted using 1H NMR spectroscopy because of its high sensitivity and hence efficient data acquisition. Here, we demonstrate that information-rich 13C NMR spectra of rat urine can be obtained using appropriately short acquisition times suitable for biochemical samples when using a cryogenic probe. Furthermore, these data were amenable to automated pattern recognition analysis, which produced a profile of the metabolic response to the model hepatotoxin hydrazine that was consistent with earlier studies. Thus, a new source of detailed and complementary information is available to metabonomics using cryogenic probe 13C NMR spectroscopy.
Subject(s)
Nuclear Magnetic Resonance, Biomolecular/methods , Urine/chemistry , Animals , Carbon Isotopes , Cold Temperature , Hydrazines/urine , Rats , Rats, Sprague-DawleyABSTRACT
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.
Subject(s)
Drug-Related Side Effects and Adverse Reactions/classification , Hydrazines/urine , Mercuric Chloride/urine , Animals , Biotransformation , Drug-Related Side Effects and Adverse Reactions/urine , Factor Analysis, Statistical , Hydrazines/chemistry , Hydrazines/toxicity , Kidney Cortex/drug effects , Kidney Cortex/pathology , Liver/drug effects , Liver/pathology , Magnetic Resonance Spectroscopy , Mercuric Chloride/chemistry , Mercuric Chloride/toxicity , Models, Chemical , Pattern Recognition, Automated , Rats , Rats, Sprague-Dawley , Rats, Wistar , Reproducibility of Results , Structure-Activity RelationshipABSTRACT
The basic principle of derivatization of a hydrazide moiety with an aldehyde as applied in the method developed by Lacroix et al. [J. Chromatogr., 307 (1984) 137-144] for the quantitation of isoniazid and acetylisoniazid was improved by modification, standardization and extension to allow quantitation of hydrazine in patient samples. It could be shown that 40 microliters of 1% methanolic cinnamaldehyde per 200 microliters of deproteinized analysate gave maximal chromophoric isoniazid-cinnamaldehyde conjugate, read at 340 nm. The hydrolytic loss of isoniazid, crucial to the quantitation of acetylisoniazid, could be compensated for by introduction of an appropriate set of calibration curves. Although the method described here allows quantitation of monoacetylhydrazine and diacetylhydrazine, in addition to hydrazine, in mono-spiked samples, the method cannot be used for the quantitation of the acetylated metabolites of hydrazine in patient samples because of a lack of specificity. Linear calibration curves in the range 1-25 micrograms/ml for isoniazid and acetylisoniazid, 10-400 ng/ml for hydrazine and 50-1000 ng/ml for monoacetylhydrazine and diacetylhydrazine, could be constructed; analyte recoveries approaching 100% could be achieved in all instances.
Subject(s)
Antitubercular Agents/analysis , Chromatography, High Pressure Liquid/methods , Hydrazines/analysis , Isoniazid/analysis , Chromatography, High Pressure Liquid/statistics & numerical data , Drug Stability , Humans , Hydrazines/blood , Hydrazines/urine , Hydrolysis , Isoniazid/analogs & derivatives , Isoniazid/blood , Isoniazid/urine , Reproducibility of Results , Sensitivity and SpecificityABSTRACT
Plasma and liver levels of hydrazine were determined at 10, 30, 90 and 270 min in rats given 0.09, 0.27, 0.84 and 2.53 mmol of hydrazine per kg body weight orally by capillary gas chromatography-mass spectrometry of its pentafluorobenzaldehyde adduct (DFBA, m/z 388) using selected ion monitoring with 15N2-labelled hydrazine as the internal standard (adduct, m/z 390). The mean half-life for hydrazine in the plasma was approximately 2 h but varied with dose. Urinary excretion (0-24 h) of hydrazine and its metabolite acetylhydrazine were determined employing nitrogen-phosphorus detection of the adducts utilising a novel internal standard, pentafluorophenylhydrazine, the adduct of which structurally resembles DFBA. The fraction of the original dose excreted as hydrazine (and acetylhydrazine) declined with increasing dose.
Subject(s)
Hydrazines/metabolism , Liver/chemistry , Animals , Chromatography, Gas , Gas Chromatography-Mass Spectrometry , Hydrazines/blood , Hydrazines/urine , Male , Rats , Rats, Inbred StrainsABSTRACT
The metabolism and disposition of hydrazine and its effects on endogenous metabolites has been studied in rats by the use of high resolution proton NMR spectroscopy of urine. Several metabolites of hydrazine were detected, notably acetyl- and diacetylhydrazine and a cyclised metabolite which results from a hydrazone formed from 2-oxoglutarate and hydrazine. Effects of hydrazine on endogenous metabolites in urine and plasma were also observed; notably a dose-related increase in urinary taurine, a dose-related increase in urinary and plasma lactate, increases in urinary alpha-alanine, beta-alanine, methylamine and a decrease in urinary 2-oxoglutarate. This study has indicated the utility of using high resolution proton NMR spectroscopy to analyse urine for both metabolites and endogenous compounds after exposure of animals to toxic substances.
Subject(s)
Hydrazines/urine , Animals , Hydrazines/pharmacology , Magnetic Resonance Spectroscopy/methods , Male , Protons , Rats , Rats, Wistar , Spectrum Analysis/methodsABSTRACT
Quantitative analyses of iproniazid (IPN) and deuterated analogue (IPN-d6) and of isopropylhydrazine (IP-Hy) and deuterated analogue (IP-Hy-d6) after conversion to pyrazole derivatives (IDP) were carried out by gas chromatography. The complete separation of protio- from deutero-forms of IPN and IDP was achieved by using a fused-silica CBP1 capillary column (50 m). The resolution coefficients between two isotopic molecules were 1.10 for IPN and 1.62 for IDP, respectively. The present isotopic fractionation procedure was applied to the isotope dilution analyses of IPN and IP-Hy. By the measurement of the samples prepared by the addition of known amounts of IPN and IPN-d6 to the control plasma and urine of rat, a linear relationship between peak height ratio and added amount ratio was observed. The correlation coefficients obtained by regression analysis were 0.9990 for the plasma and 0.9999 for the urine, respectively. In the case of IP-Hy, a linear relationship was also observed, and the correlation coefficients were 0.9998 for the plasma and 0.9997 for the urine, respectively. The present method was compared with the gas chromatography-mass spectrometry method in urinary samples from rats treated with IPN. The results of these parallel determinations were comparable.
Subject(s)
Chromatography, Gas/methods , Hydrazines/isolation & purification , Iproniazid/isolation & purification , Animals , Deuterium , Hydrazines/urine , Iproniazid/urine , Isotope Labeling , Rats , Rats, Inbred StrainsABSTRACT
15N-NMR has been used to study the metabolism of hydrazine in rats in vivo. Single doses of [15N2]hydrazine (2.0 mmol/kg: 98.6% g atom) were administered to rats and urine collected for 24 hr over ice. A number of metabolites were detected by 15N-NMR analysis of lyophilized urine. Ammonia was detected as a singlet at 0 ppm and unchanged [15N2]hydrazine was present in the urine detectable as a singlet at 32 ppm. Peaks were observed at 107 and 110 ppm which were identified as being due to the hydrazido nitrogen of acetylhydrazine and diacetylhydrazine, respectively. A resonance at 85 ppm was ascribed to carbazic acid, resulting from reaction of hydrazine with carbon dioxide. A singlet detected at 316 ppm was thought to be due to the hydrazono nitrogen of the pyruvate hydrazone. The resonance at 56 ppm was assigned to 15N-enriched urea, this together with the presence of ammonia indicates that the N-N bond of hydrazine is cleaved in vivo, possibly by N-oxidation, and the resultant ammonia is incorporated into urea. A doublet centred at 150 ppm and a singlet at 294 ppm were assigned to a metabolite which results from cyclization of the 2-oxoglutarate hydrazone. Therefore 15N-NMR spectroscopic analysis of urine has yielded significant new information on the metabolism of hydrazine.
Subject(s)
Hydrazines/metabolism , Ammonia/urine , Animals , Hydrazines/urine , Magnetic Resonance Spectroscopy/methods , Male , Rats , Rats, Inbred StrainsABSTRACT
The derivatization of urinary dicarboxylic acids with 2-nitrophenylhydrazine hydrochloride produced corresponding monohydrazides, which were separated from monocarboxylic acid hydrazides by two step extraction with ethyl acetate at different pH values. Monohydrazides of 11 straight- and branched-chain dicarboxylic acids were eluted isocratically on reversed-phase ion-pair chromatography within 24 min by the combination of pH, the polarity of mobile phase, and the size of counter ion. The analytical results showed good recovery and reproducibility using 3,3-dimethyglutaric acid as an internal standard. The present method provides a notable HPLC method with precolumn derivatization for the analysis of urinary dicarboxylic acids.