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1.
Timothy G. Heath Joseph P. Mooney Robert Broersma 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》1997,688(2):1593
Characterizing components eluting from a HPLC column is enhanced when multiple detectors are incorporated in-line. The performance of a system consisting of a combination of two detectors - electrospray ionization mass spectrometry (and tandem mass spectrometry) and radioactivity monitoring, following gradient separation with a 250×2.1mm I.D. (Vydac Protein and Peptide C18, 5 μm, 300 Å) column - is evaluated with respect to chromatographic integrity and detection. The HPLC effluent was split (8:1) and a post-column make-up solvent was added to flow directed towards the radioactivity detector containing a solid glass cell. Trifluoracetic acid (0.1%) was added to the make-up flow solvent to prevent silanol interactions from degrading the profile displayed in the 14C trace. A 14C chromatographic peak representing 550 dpm was detected with signal-to-noise ratio of 3. This system was used for rapidly characterizing the biliary metabolites of an arginine fluoroalkyl ketone analog of d-MePhe-Pro-Arg, a potent thrombin inhibitor currently being evaluated as a drug candidate. These metabolites are shown to comprise of mono- and dihydroxylated drug as well as a reduced ketone form of the drug. Combining the radioactivity monitor in-line with the mass spectrometer ensured that all of the major metabolites (as evident from the 14C profile) were characterized by mass spectrometry. 相似文献
2.
《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》1997,688(2):281-289
Characterizing components eluting from a HPLC column is enhanced when multiple detectors are incorporated in-line. The performance of a system consisting of a combination of two detectors - electrospray ionization mass spectrometry (and tandem mass spectrometry) and radioactivity monitoring, following gradient separation with a 250×2.1mm I.D. (Vydac Protein and Peptide C18, 5 μm, 300 Å) column - is evaluated with respect to chromatographic integrity and detection. The HPLC effluent was split (8:1) and a post-column make-up solvent was added to flow directed towards the radioactivity detector containing a solid glass cell. Trifluoracetic acid (0.1%) was added to the make-up flow solvent to prevent silanol interactions from degrading the profile displayed in the 14C trace. A 14C chromatographic peak representing 550 dpm was detected with signal-to-noise ratio of 3. This system was used for rapidly characterizing the biliary metabolites of an arginine fluoroalkyl ketone analog of d-MePhe-Pro-Arg, a potent thrombin inhibitor currently being evaluated as a drug candidate. These metabolites are shown to comprise of mono- and dihydroxylated drug as well as a reduced ketone form of the drug. Combining the radioactivity monitor in-line with the mass spectrometer ensured that all of the major metabolites (as evident from the 14C profile) were characterized by mass spectrometry. 相似文献
3.
Complete separation by high performance liquid chromatography of metabolites of arachidonic acid from incubation with human and rabbit platelets 总被引:1,自引:0,他引:1
Mike VanRollins Samuel H.K. Ho James E. Greenwald Michael Alexander Nancy J. Dorman Lan K. Wong Lloyd A. Horrocks 《Prostaglandins & other lipid mediators》1980,20(3):571-577
Separations of all major cyclooxygenase and lipoxygenase metabolites of arachidonic acid were obtained by high performance liquid chromatography (HPLC). A C18 reverse-phase column was used in ion suppression mode to separate underivatized metabolites of arachidonic acid isolated from human and rabbit platelets. The metabolites were monitored by measuring radioactivity or ultraviolet light absorption at 192 nm (absorption by double bonds). Comparisons of TLC and HPLC separations demonstrated that the HPLC separation of metabolites of [1-14C]arachidonic acid was quantitative. HPLC also resolved several minor metabolites that were not detected by scanning of TLC separations. 相似文献
4.
《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》1995,672(2):207-215
An isocratic reversed-phase high-performance liquid chromatographic (HPLC) method using an Ultrasphere IP column has been developed for the determination of testosterone and its metabolites after incubation of 4-14C-labelled or unlabelled testosterone with rat liver microsomes. Compounds were eluted with methanol-water-tetrahydrofuran (35:55:10, v/v, pH 4.0) and detected by ultraviolet (UV) absorption at 245 nm. UV or on-line radioactivity detection can be used although, due to differences in detector cell volumes, peak resolution is slightly better with UV detection. Selectivity was validated by collecting HPLC peaks and verifying their identity by gas chromatography-mass spectrometry after derivatization by N,O-bis(trimethylsily)trifluoroacetamide-trimethylchlorosilane. A three-day validation was performed to determine the linearity, repeatability, reproducibility and accuracy of the method, using corticosterone as internal standard. The method is applicable to the measurement of cytochrome P-450 isoenzyme activities in rat liver. 相似文献
5.
Incubation of [14C]benzene or [14C]phenol with liver microsomes from untreated rats, in the presence of a NADPH-generating system, gave rise to irreversible binding of metabolites to microsomal macromolecules. For both substrates this binding was inhibited by more than 50% by addition of superoxide dismutase to the incubation mixtures. The decrease in binding was compensated for by accumulation of [14C]hydroquinone, indicating superoxide-mediated oxidation of hydroquinone as one step in the activation of benzene to metabolites binding to microsomal macromolecules. Since our previous work had shown that binding occurred mainly with protein rather than ribonucleic acid and was virtually completely prevented by glutathione, suggesting identity of metabolite(s) responsible for binding to protein and glutathione, a conjugate was chemically prepared from p-benzoquinone and reduced glutathione (GSH) and identified by field desorption mass spectrometry (FDMS) as 2-(S-glutathionyl) hydroquinone. Microsomal incubations, containing an NADPH-generating system, with benzene, phenol, hydroquinone or p-benzoquinone in the presence of [3H]glutathione or, alternatively, with [14C]benzene or [14C]phenol in the presence of unlabeled glutathione, were performed. All of these incubations gave rise to a peak of radioactivity eluting from the high pressure liquid chromatograph (HPLC) at a retention time identical to that of the chemically prepared 2-(S-glutathionyl) hydroquinone, whilst microsomal incubation of catechol in the presence of [3H]glutathione led to a conjugate with a very different retention time which was not observed after incubation of benzene or phenol. The microsomal metabolites of p-benzoquinone, hydroquinone and phenol thus eluting from the HPLC were further identified as the 2-(S-glutathionyl) hydroquinone by field desorption mass spectrometry. The glutathione adduct formed from benzene during microsomal activation eluted from HPLC with the same retention time and its mass spectrum also contained the molecular ion (MH+) (m/e 416) of this conjugate as an intense peak, but the fragmentation patterns did not allow definite assignments probably due to the considerably smaller amounts of ultimate reactive metabolites formed from this pre-precursor and thus relatively larger amounts of impurities.The results indicate that rat liver microsomes activate benzene via phenol and hydroquinone to p-benzosemiquinone and/or p-benzoquinone as quantitatively important reactive metabolites. 相似文献
6.
《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》1999,735(1):17-24
The biological significance of non-acetylated metallothionein (MT) was investigated from the viewpoint of Nα-acetylation after induction of MT synthesis by metallic and non-metallic inducers, by partial hepatectomy and under physiological conditions. Nα-Acetylated and non-acetylated forms of MT-2 in liver supernatants and plasma were detected by the tandem size-exclusion and anion-exchange HPLC columns with in-line detection by mass spectrometry. The non-acetylated isoform of MT-2 (MT-2′) was present at a comparable level to the Nα-acetylated form of MT-2 (MT-2) at an early stage after induction by not only zinc but also cadmium, and by partial hepatectomy in the livers of rats. Plasma MT-2 in neonatal rats was similar to liver MT-2 in the composition of Nα-acetylated and non-acetylated forms, suggesting that there are no differences in the roles of Nα-acetylation of MT in the extracellular trafficking of MT. The column switching HPLC method with in-line detection by inductively coupled argon plasma mass spectrometry (ICP-MS) was shown to be a sensitive and powerful method to detect MT proteins at not only isoform level but also at acetylated and non-acetylated form levels. 相似文献
7.
A stable bacterial community absorbed and transformed the herbicide metolachlor [2-chloro-N-(2-ethyl- 6-methylphenyl)-N-(2-methoxy-1-methylethyl)-acetamide] from a liquid medium. About 80% of the added ring-[U-14C]metolachlor (50 μg/ml) disappeared from the medium and accumulated inside the cells. The ratio of cellular 14C to 14C in 1 mg of supernatant reached a value of 1.1 × 104 in a 10-day-old culture. 14C remaining in the medium consisted primarily of two dechlorinated products of metolachlor with m/z 233 and 263 as determined by mass spectrometry. The 14C-labeled material absorbed by the cells was strongly bound; only 2% of the 14C was released into deionized water after shaking for 3 h. Approximately 96% of the 14C associated with the biomass was extracted with acetone, and high-performance liquid chromatographic analysis of this fraction showed six peaks containing radioactivity. Since no metolachlor was detected by chromatographic analysis, it was concluded that the radioactivity recovered from the cells represented transformed products of metolachlor. Pure cultures isolated from the bacterial mixed culture were less effective in transforming and accumulating metolachlor. These results suggest that it may be advantageous to seed an aquatic environment with a mixture of microorganisms, rather than individual microbial species, as a method for removal or detoxification of metolachlor. 相似文献
8.
Column chromatography and spectroscopy have been employed in analyzing pyrimidine derivatives obtained from alkaline-treated 7-methylguanosine (7-meGuo). High performance liquid chromatography (HPLC) revealed that the alkaline generated products consist predominantly of two forms of ring opened 7-methylguanine (rom7Gua) in equal amounts. Material from both Dowex 50 and Sephadex LH-20 columns was readily resolvable into two HPLC peaks. The species in one peak appears to be composed of formylated and that in the other of deformylated rom7Gua. The presence of a deformylated species is supported by the absence of radioactivity in one of the two peaks obtained when ring opened [8-14C]guanosine was analyzed by HPLC. The formylated species was retained on the liquid chromatography column for 8 min with a 3% methanol, 0.01 M NH4H2PO4 (pH 5.1) solvent and for 6 min with a 6% methanol, 0.01 N NH4H2PO4 (pH 5.1) solvent system; the deformylated species was retained for 6.3 min with the first solvent and 4.5 min with the second solvent. Subsequent to Dowex 50 chromatography in an ammonium formate solvent, about 90% of the material was formylated. When stored at 24°C for 72 h in a solvent without formate ions, the material was shown by HPLC to consist of equal amounts of the formylated and deformylated species. These results indicate that the two species of rom7Gua are in equilibrium. The rom7Gua excised from DNA by formamidopyrimidine (FAPy)-DNA glycosylase was shown to coelute with the formylated species. 相似文献
9.
Lisa Yon Brian Faulkner Sumolya Kanchanapangka Narongsak Chaiyabutr Sompast Meepan Bill Lasley 《Zoo biology》2010,29(6):760-766
Noninvasive hormone assays provide a way to determine an animal's health or reproductive status without the need for physical or chemical restraint, both of which create unnecessary stress for the animal, and can potentially alter the hormones being measured. Because hormone metabolism is highly species‐specific, each assay must be validated for use in the species of interest. Validation of noninvasive steroid hormone assays has traditionally required the administration of relatively high doses of radiolabelled compounds (100 µCi or more of 14C labeled hormone) to permit subsequent detection of the excreted metabolites in the urine and feces. Accelerator mass spectrometry (AMS) is sensitive to extremely low levels of rare isotopes such as 14C, and provides a way to validate hormone assays using much lower levels of radioactivity than those traditionally employed. A captive Asian bull elephant was given 1 µCi of 14C‐testosterone intravenously, and an opportunistic urine sample was collected 2 hr after the injection. The sample was separated by HPLC and the 14C in the fractions was detected by AMS to characterize the metabolites present in the urine. A previously established HPLC protocol was used, which permitted the identification of fractions into which testosterone sulfate, testosterone glucuronide, and the parent compound testosterone elute. Results from this study indicate that the majority of testosterone excreted in the urine of the Asian bull elephant is in the form of testosterone sulfate. A small amount of testosterone glucuronide is also excreted, but there is no parent compound present in the urine at all. These results underscore the need for enzymatic hydrolysis to prepare urine samples for hormone assay measurement. Furthermore, they highlight the importance of proper hormone assay validation in order to ensure accurate measurement of the desired hormone. Although this study demonstrated the utility of AMS for safer validation of noninvasive hormone assays in nondomestic species, this methodology could also be applied to studies of nutrient metabolism and drug pharmakokinetics, both areas in great need of further study in wildlife species. Zoo Biol 29:760–766, 2010. © 2010 Wiley‐Liss, Inc. 相似文献
10.
John B. Sutherland James P. Freeman Allison L. Selby Peter P. Fu Dwight W. Miller Carl E. Cerniglia 《Archives of microbiology》1990,154(3):260-266
The metabolism of phenanthrene, a polycyclic aromatic hydrocarbon (PAH), by Streptomyces flavovirens was investigated. When grown for 72 h in tryptone yeast extract broth saturated with phenanthrene, the actinomycete oxidized 21.3% of the hydrocarbon at the K-region to form trans-9,10-dihydroxy-9,10-dihydrophenanthrene (phenanthrene trans-9,10-dihydrodiol). A trace of 9-phenanthrol was also detected. Metabolites isolated by thin-layer and high performance liquid chromatography were identified by comparing chromatographic, mass spectral, and nuclear magnetic resonance properties with those of authentic compounds. Experiments using [9-14C]phenanthrene showed that the trans-9,10-dihydrodiol had 62.8% of the radioactivity found in the metabolites. Circular dichroism spectra of the phenanthrene trans-9,10-dihydrodiol indicated that the absolute configuration of the predominant enantiomer was (–)-9S,10S, the same as that of the principal enantiomer produced by mammalian enzymes. Incubation of S. flavovirens with phenanthrene is an atmosphere of 18O2, followed by gas chromatographic/mass spectral analysis of the metabolites, indicated that one atom from molecular oxygen was incorporated into each molecule of the phenanthrene trans-9,10-dihydrodiol. Cytochrome P-450 was detected in 105,000×g supernatants prepared from cell extracts of S. flavovirens. The results show that the oxidation of phenanthrene by S. flavovirens was both regio- and stereospecific.Abbreviations CD
circular dichroism
- DMF
N,N-dimethyl-formamide
- GC/MS
gas chromatography/mass spectrometry
- HPLC
high performance liquid chromatography
- NMR
nuclear magnetic resonance
- ODS
octadecylsilane
- PAH
polycyclic aromatic hydrocarbon
- TLC
thin-layer chromatography
- TMS
tetramethylsilane
- UV
ultraviolet 相似文献
11.
A sensitive and simple radiochemical assay is described to measure argininosuccinase activity in crude tissue homogenates and cultured cells. The method depends on the use of argininosuccinate labeled uniformly with 14C in the six carbons of the arginine moiety. On incubation in the presence of excess arginase, the [U-14C]arginine formed is measured as the sum of radioactivity in [U-14C]ornithine and [14C]urea. Separation from the substrate is accomplished on a small Domex 1-acetate column eluted with 25 mm acetic acid; ornithine and urea emerge in the first few milliliters while unutilized substrate remains on the column. [14C]Argininosuccinate was synthesized enzymatically from l-[U-14C]arginine and fumarate and isolated and purified as the barium salt. Development of a new purification step has brought the amino acid to a purity of 97% as judged by chromatographic and barium analysis. With the present specific radioactivity, as little as 5 to 10 nmol of product can be accurately measured under kinetically optimum conditions. 相似文献
12.
Time-course studies on the distribution and metabolism of valproate (VPA) in rat brain, liver, and kidney, after intraperitoneal injection of a mixture of [14C]VPA and [3H]VPA, showed that: (1) maximal amount of radioactivity in the various tissues was observed after 30 min from the time the drug was administered; (2) at 30 min the distribution of labeled VPA in brain, liver, and kidney was 17%, 64%, and 19% of the total radioactivity, respectively; (3) at 24 hr more than 97% of the total radioactivity was lost from the tissues and the14C/3H ratios increased significantly with time. Studies on the regional distribution of the drug showed that it is relatively homogeneously distributed. Studies on the subcellular distribution of the drug showed that it is associated mostly with the soluble and mitochondrial fractions, with little radioactivity in the myelin and synaptosomal fractions. Radiochromatography of VPA metabolites in perchloric acid extracts from brain, liver, and kidney revealed the presence of four metabolites. VPA was not incorporated into phospholipids of the neuronal membranes. Furthermore, it had no significant effects on Mg2+-ATPase and (Na++K+)-ATPase in synaptosomes and microsomes obtained either from control or from rats injected with VPA. It was concluded that this antiepileptic drug does not appear to act through its incorporation into neuronal membrane or through its action on the Na+ pump.Contribution No. 0601 from the Department of Cell and Molecular Biology, the Medical College of Georgia, Augusta, Georgia 30912. 相似文献
13.
Reverse-phase high-performance liquid chromatography was used to analyse 14C-labelled metabolites of indole-3-acetic acid (IAA) formed in the cortical and stelar tissues of Zea mays roots. After a 2-h incubation in [14C]IAA, stelar segments had metabolised between 1–6% of the methanol-extractable radioactivity compared with 91–92% by the cortical segments. The pattern of metabolites produced by cortical segments was similar to that produced by intact segments bathed in aqueous solutions of [14C]IAA. In contrast, when IAA was supplied in agar blocks to stelar tissue protruding from the basal ends of segments, negligible metabolism was evident. On the basis of its retention characteristics both before and after methylation, the major metabolite of [14C]IAA in Zea mays root segments was tentatively identified by high-performance liquid chromatography as oxindole-3-acetic acid.Abbreviations HPLC
High-performance liquid chromatography
- IAA
Indole-3-acetic acid 相似文献
14.
Background
Dihydroartemisinin (DHA), a powerful anti-malarial drug, has been used as monotherapy and artemisinin-based combination therapy (ACT) for more than decades. So far, however, the tissue distribution and metabolic profile of DHA data are not available from animal and humans.Methods
Pharmacokinetics, tissue distribution, mass balance, and elimination of [14C] DHA have been studieded in rats following a single intravenous administration. Protein binding was performed with rat and human plasma. Drug concentrations were obtained up to 192 hr from measurements of total radioactivity and drug concentration to determine the contribution by the parent and metabolites to the total dose of drug injected from whole blood, plasma, urine and faecal samples.Results
Drug was widely distributed after 1 hr and rapidly declined at 24 hr in all tissues except spleen until 96 hrs. Only 0.81% of the total radioactivity was detected in rat brain tissue. DHA revealed a high binding capacity with both rat and human plasma proteins (76–82%). The concentration of total radioactivity in the plasma fraction was less than 25% of that in blood total. Metabolism of DHA was observed with high excretion via bile into intestines and approximately 89–95% dose of all conjugations were accounted for in blood, urine and faeces. However, the majority of elimination of [14C] DHA was through urinary excretion (52% dose). The mean terminal half-lives of plasma and blood radioactivity (75.57–122.13 h) were significantly prolonged compared with that of unchanged DHA (1.03 h).Conclusion
In rat brain, the total concentration of [14C] was 2-fold higher than that in plasma, indicating the radioactivity could easily penetrate the brain-blood barrier. Total radioactivity distributed in RBC was about three- to four-fold higher than that in plasma, suggesting that the powerful anti-malarial potency of DHA in the treatment of blood stage malaria may relate to the high RBC binding. Biliary excretion and multiple concentration peaks of DHA have been demonstrated with high urinary excretion due to a most likely drug re-absorption in the intestines (enterohepatic circulation). The long lasting metabolites of DHA (> 192 hr) in the rats may be also related to the enterohepatic circulation. 相似文献15.
A Nakagawa A Kitagawa M Asami K Nakamura D A Schoeller R Slater M Minagawa I R Kaplan 《Biomedical mass spectrometry》1985,12(9):502-506
Isotope ratio (IR) mass spectrometry was evaluated for the study of drug metabolism and balance using 13C, 15N2-labelled antipyrine (AP) as a test drug. Rats were given 40 mg kg-1 (13C,15N2)AP intraperitoneally. Breath, urine, faeces and blood were collected. Except for breath, samples were combusted in sealed quartz tubes. The resulting CO2 and N2 were analysed for excess 13C and 15N, relative to pre-dose samples, by IR mass spectrometry. In addition, blood levels of AP and cumulative excretion of urinary AP metabolites were determined by gas chromatography/mass spectrometry/selected ion monitoring (GC/MS/SIM) and high-performance liquid chromatography (HPLC) respectively. Excess 13C and 15N levels in blood were comparable with observed levels of AP, and urinary recoveries of 13C (42%) were in good agreement with those calculated from HPLC data (45%). N-Demethylation, one of the important pathways of AP metabolism, was most rapidly determined by excess 13CO2 excretion in breath (8%). The IR mass spectral analysis complemented gas chromatographic/mass spectrum and HPLC analyses, and was less complex. 相似文献
16.
A Tjernberg P.O Edlund B Norén 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》1998,715(2):111
Strong anion-exchange (SAX) chromatography and reversed-phase liquid chromatography (RPLC) followed by different mass spectrometric techniques for the separation and identification of conjugated and unconjugated 14C-labelled eltanolone (5β-Pregnan-3α-ol-20-one) metabolites in biological fluids are presented. Conjugates of estradiol were used as model compounds for the development of a SAX based group separation of neutral steroids, glucuronides, sulfates and di-conjugated steroids. The usefulness of the technique is demonstrated by the analysis of 14C-labelled eltanolone metabolites in dog urine. The analytical SAX column used prior to RPLC improved the capacity to separate the metabolites from each other and from endogenous components, compared to a single reversed-phase system. Liquid chromatography negative ion electrospray-mass spectrometry (LC–ESI-MS) was used for the molecular mass determination of conjugated eltanolone metabolites. Unconjugated metabolites and hydrolysed conjugates were identified using gas chromatography–mass spectrometry with an electron impact ion source (GC–MS) after trimethylsilyl (TMS) derivatization. An unexpected finding in dog urine was the diglucuronide formation of eltanolone (presumably after enolisation of its carbonyl group). 相似文献
17.
《Phytochemistry》1987,26(6):1607-1611
Helminthosporium sacchari, a fungus that causes disease in sugarcane, produces oligosaccharide-sesquiterpene toxins (HS toxins A, B, and C) that are required for infection and disease development. Two free sesquiterpenes were isolated from mycelium but not from culture fluids of the fungus. One sesquiterpene was identified by HPLC and mass spectrometry as an aglycone of HS toxin C and could be obtained by enzymatic hydrolysis of this toxin. The other sesquiterpene appeared to be the 2-keto form of the first compound. The aglycone from toxin C hydrolysis was labelled with tritium by successive treatments with active manganese dioxide, sodium boro[3H]hydride, and lithium aluminium hydride. The labelled compound was fed to cultures of H. sacchari, radioactivity was incorporated into HS toxin C and into lower molecular weight homologues. The results suggest a metabolic route (aglycone → metabolite Y, → HS toxin → metabolite X) for the biosynthesis of HS toxin; metabolites X and Y are lower molecular weight homologues of the toxin. 相似文献
18.
Arlene D. Bayley Johannes Van Staden Jennifer A. Mallett Siegfried E. Drewes 《Plant Growth Regulation》1989,8(3):193-204
The metabolic fate of [8-14C]benzyladenine applied to the excised organs of tomato (Lycopersicon esculentum Mill. cv. Heinz 1370) was investigated after 2 and 6 h of feeding. Although the roots were the most effective at uptake of the cytokinin the leaves metabolised it the most efficiently. The predominant metabolite in all of the tissues was an unknown compound which did not have a retention time corresponding with any of the standards used. The roots contained the most extensive range of metabolites which included the unknown metabolite and compounds co-eluting with adenine, and the riboside, nucleotide and 9-glucoside of benzyladenine. The 9-glucoside was detected only in the root material. The stem yielded the highest levels of radioactivity at the retention times of benzyladenosine-5-monophosphate and benzyladenosine. The radioactivity associated with these two cytokinins was transient in the leaf extract. This organ ultimately yielded radioactivity only at the retention times of the unknown metabolite and adenine. Since only the roots and leaves contained relatively large peaks of radioactivity at the elution volume of adenine it seems that degradative metabolism was more predominant in these organs than in the stem.Abbreviations Ade
adenine
- Ado
adenosine
- BA
benzyladenine
- BAR
benzyladenosine
- BA3G
3-glucosylbenzyladenine
- BA9G
9-glucosylbenzyladenine
- BARMP
benzyladenosine monophosphate
- HPLC
high performance liquid chromatography
- MS
mass spectrometry 相似文献
19.
Michael A. Heitkamp James P. Freeman Dwight W. Miller Carl E. Cerniglia 《Archives of microbiology》1991,156(3):223-230
The metabolism of 14C-labeled 1-nitropyrene in microcosms containing nonsterile estuarine sediments, and in cultures of a Mycobacterium sp. previously isolated from oil-contaminated sediments was investigated. Although mineralization of 1-nitropyrene by pure cultures of the Mycobacterium sp. totaled only 12.3% after 10 days of incubation, over 80% of the ethyl acetate extractable 14C-labeled compounds consisted of 1-nitropyrene metabolites. High pressure liquid chromatographic analysis of 1-nitropyrene degradation products indicated that two major metabolites were formed. They were identified as 1-nitropyrene cis-9,10-and 4,5-dihydrodiols, based on their UV-visible, mass and NMR spectra. Time course studies in microcosms showed that 1-nitropyrene was degraded slowly under aerobic and anaerobic conditions in estuarine sediments. Less than 1% had been converted to 14CO2 after 8 weeks of aerobic incubation. The addition of 1-nitropyrene to anaerobic sediments resulted in no 14CO2 evolution; however, the nitro group of 1-nitropyrene was reduced to form 1-aminopyrene. Although the mineralization of 1-nitropyrene in sediments was slow, the Mycobacterium sp. metabolized 1-nitropyrene in pure culture. This bacterium appears promising for the bioremediation of this ubiquitous pollutant in contaminated waste.Abbreviations DEP
Direct exposure probe
- HPLC
high pressure liquid chromatography
- GC/MS
gas chromatography/mass spectrometry
- Nitro-PAHS
nitropolycyclic aromatic hydrocarbons
- TLC
thin-layer chromatography
- UV
ultraviolet 相似文献
20.
Soraya Madani William N. Howald Ross F. Lawrence Danny D. Shen 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2000,741(2):301
This report describes an assay for the H1-receptor antagonist, terfenadine, and its two primary metabolites, terfenadine alcohol (TOH) and azacyclonol (AZ), using positive-ion, electrospray ionization–liquid chromatography–mass spectrometry. The assay was developed in support of kinetic studies of terfenadine oxidative metabolism in human liver and intestinal microsomes, which required quantification of incubate metabolites at low nanomolar concentrations. Terfenadine metabolites were extracted from basified microsomal incubates into methylene chloride. Reconstituted extracts were subject to liquid chromatographic separation on a cyano-reverse phase column. The [M+H]+ ions of terfenadine, terfenadine metabolites, and internal standard were monitored in the effluent by quadrupole mass spectrometry. The assay demonstrated linearity over an incubate concentration range of 5–250 and 12.5–1250 ng/ml for the metabolites and the parent drug, respectively. The respective limits of detection and quantitation for all three analytes were 1.5 and 5 ng/ml of microsomal incubate. Replicate analysis of quality control samples exhibited intra-day coefficients of variation ranging from 3.3% to 7.8% for the three analytes. The corresponding inter-day coefficients of variation ranged from 4.2% to 8.6%. The reproducibility and sensitivity of the assay, combined with the selectivity of mass spectrometric detection, should allow an accurate kinetic characterization of terfenadine oxidation mediated by the high affinity CYP3A enzymes in human liver and intestinal microsomes. 相似文献