Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.2.1.31 (beta-glucuronidase)
7,680 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have developed a rapid, sensitive and precise high-performance liquid chromatographic method using fluorescence detection for the simultaneous determination of thiabendazole and unconjugated 5-hydroxythiabendazole in serum. Sample pretreatment consists only of protein precipitation with acetonitrile containing the internal standard, 2-methylindole. Detection limits were found to be 0.1 microgram/ml serum for thiabendazole and 0.4 microgram/ml serum for 5-hydroxythiabendazole. Between-day analytical precision coefficients of variation for serum-based controls were 7% and 11% for thiabendazole levels of 1 and 5 micrograms/ml, respectively; and 43% and 8% for 5-hydroxythiabendazole levels of 6 and 60 micrograms/ml, respectively. We also devised a microenzymatic method for the conversion of the glucuronide and sulfate esters of 5-hydroxythiabendazole using beta-glucuronidase [EC 3.2.1.31] and sulfatase [EC 3.1.6.1]. Thus, quantitation of the separate metabolites was possible. We also utilized a special adaptation of the chromatographic procedure for the determination of the 5-hydroxythiabendazole metabolites in the sera of uremic patients, which can contain large amounts of interfering fluorescent substances. The method should be particularly useful for monitoring thiabendazole therapy in patients unable to eliminate the potentially toxic metabolites.
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PMID:Determination of thiabendazole and 5-hydroxythiabendazole in human serum by fluorescence-detected high-performance liquid chromatography. 710 70

To study the disposition kinetics of methamphetamine (MAP), we have developed a sensitive high-performance liquid chromatographic (HPLC) assay to quantitate the enantiomers of MAP and its major metabolites, amphetamine (AP), p-hydroxymethamphetamine (p-OH-MAP), and p-hydroxyamphetamine (p-OH-AP), the latter two of which are hydroxylated metabolites, in rat urine. To determine conjugated hydroxylated metabolites, urine samples were treated with beta-glucuronidase. Both hydrolyzed and nonhydrolyzed p-OH-MAP and p-OH-AP were extracted into ethyl acetate and back extracted with 0.05M HCl. To determine MAP and AP, urine samples were extracted with benzene, followed by back extraction into 0.05M HCl. The acid layer was collected, and to it was added (-)-1-(9-fluorenyl)ethyl chloroformate (FLEC) for the derivatization of MAP and its metabolites. Derivatization was allowed to proceed for 24 h at room temperature. The derivatized products were separated on a C18 column with a mobile phase consisting of acetate buffer (pH 3.6)-acetonitrile-tetrahydrofuran. Quantitation was achieved using a fluorescence detector at an excitation wavelength of 265 nm and an emission wavelength of 330 nm. Linear standard curves were obtained over the concentration range of 5-100 ng/mL. The interday and intraday coefficients of variation for the assay for all eight enantiomers at 10 and 75 ng/mL were less than 13%. The detection limit was 5 ng/mL or 0.5 ng on-column.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Separation and quantitation of the enantiomers of methamphetamine and its metabolites in urine by HPLC: precolumn derivatization and fluorescence detection. 756 90

In this assay of the nonsteroidal antiestrogen droloxifene and two metabolites in human serum, the serum samples were deproteinized with an equal volume of acetonitrile and then injected into an analytical octadecylsilane column. The analytical column was equilibrated with acetonitrile/water (1/1, vol/vol) containing acetic acid and diethyl amine and eluted isocratically with 66% acetonitrile in the same buffer. Droloxifene, N-desmethyldroloxifene, and 4-methoxdroloxifene were post-column converted to fluorophors by ultraviolet illumination while passing through a 10-m transparent knitted polytetrafluorethylene reaction coil. Analytical recovery was close to 100%. Within- and between-day precision corresponded to a coefficient of variation (CV) of 2-5% at serum concentrations of > or 25 ng/ml, except for 4-methoxydroloxifene (CV 7-10% at a concentration of 25 ng/ml). By increasing the injection volume from 50 to 250 microliters, the detection limits could be decreased from approximately 5 to 1 ng/ml. Conjugated droloxifene could be estimated in a second run after treatment of sample with an enzyme preparation containing beta-glucuronidase plus sulphatase. The recovery of droloxifene glucuronide was close to 100%. Sulphate conjugates have not been identified and were not accounted for.
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PMID:Determination of droloxifene and two metabolites in serum by high-pressure liquid chromatography. 762 22

An LC method for the analysis of m-hydroxymandelic acid (MHMA) and m-hydroxyphenylglycol (MHPG) and their conjugates in human plasma was developed and validated. The method for the quantitation involved extraction of acidified plasma (subject to hydrolysis with beta-glucuronidase for 120 min with 500 units of enzyme/0.25 ml of plasma at 37 degrees C for the conjugates) with an organic phase (methyl-tert-butyl ether). Analysis of MHMA, MHPG and the internal standard (3-hydroxy-4-methoxymandelic acid) was carried out on an ODS stationary phase: 100 x 4.6 mm, 5 mu followed by a 75 x 4.6 mm, 3 mu using 1% acetonitrile in 0.1 M acetic acid as the mobile phase. An electrochemical detector operated at +1.15 V vs Ag/AgCl was employed for the detection. The standard curves were linear in the range of 10.0-250.0 ng ml-1 for MHMA and 5.0-125.0 ng ml-1 for MHPG. The limit of quantitation was 10.0 ng ml-1 for MHMA and MHPG. Acceptable accuracy and precision were obtained during the intra-batch and inter-batch analysis for both the assays.
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PMID:Determination of m-hydroxymandelic acid, m-hydroxyphenylglycol and their conjugates in human plasma using liquid chromatography with electrochemical detection. 798 25

An HPLC method for the determination of dextrorphan, an active metabolite of dextromethorphan, in plasma was established using column switching technique. The column switching system was equipped with a per-column of 30 mm x 5 mm ID, packed with mu Bondapak C18, 37-50 microns, and an analytical column of 150 mm x 5 mm ID, packed with YWG-C18, 5 microns. A 0.2% acetic acid solution was used as the pretreating mobile phase to wash out impurities from the per-column. The analytical mobile phase consisted of acetonitrile-water-acetic acid-triethylamine-dichloromethane (17:82:1:0.05:0.025). The plasma samples were directly injected into the HPLC system after enzymatic hydrolysis of dextrorphan glucuronide ester conjugate to free form with beta-glucuronidase. The dextrorphan was monitored with a fluorescence detector at 290 nm (excitation) and 315 nm (emission). The method was linear within the plasma concentration range of 20-640 ng/ml (r = 0.9987), and the detection limit was 4 ng/ml. The mean recoveries of the method averaged 103.8%. The relative standard deviations of the assay were less than 10% for both within-day and between-days.
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PMID:[Column switching HPLC method for determination of dextrorphan, an active metabolite of dextromethorphan, in plasma]. 823 84

A method for the simultaneous quantitation of imipramine and six metabolites (2- and 10-hydroxyimipramine, 2- and 10-hydroxydesipramine, didesmethylimipramine and desipramine) in human plasma and urine has been developed. The method is based on a three-step liquid-liquid extraction followed by isocratic, reversed-phase high-performance liquid chromatography with ultraviolet absorbance detection (detection wavelength: 220 nm). The chromatographic eluent consisted of 30% acetonitrile and 70% aqueous sodium perchlorate solution pH 2.5. Glucuronide conjugates in urine were deconjugated with beta-glucuronidase/arylsulphatase prior to extraction.
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PMID:High-performance liquid chromatography of imipramine and six metabolites in human plasma and urine. 845 8

Lamotrigine, a new antiepileptic drug, is analyzed by capillary zone electrophoresis. Samples were deproteinized with acetonitrile containing an internal standard, acidified with dilute acetic acid and injected into the capillary. The drug migrated rapidly with the cationic compounds in about 3.5 min far from any interfering substances. The test was linear between 0.5-10 mg/l. The analysis time was about 5 min. The CE values correlated well with an HPLC method (r = 0.97; n = 35). The mean serum concentration of 121 patients on this drug was 3.7 mg/l. Incubating the serum with beta-glucuronidase for 1 h increased the peak height of lamotrigine by about 24%.
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PMID:Serum lamotrigine analysis by capillary electrophoresis. 887 47

An HPLC method was developed and validated for the determination in human plasma and urine of the enantiomers of eliprodil, (+/-)-alpha-(4-chlorophenyl)-4[(4-fluorophenyl) methyl]piperidine-1-ethanol hydrochloride, a new anti-ischaemic agent administered as a racemate. Both enantiomers are present in human plasma in unchanged and glucuroconjugated form, whereas only the glucuroconjugated form is excreted into urine; as a consequence, such metabolites in human plasma and urine should be submitted to enzymatic deconjugation with beta-glucuronidase (Escherichia coli) before being extracted. The general method involves a liquid-liquid extraction of eliprodil and internal standard from alkalinized plasma or urine with n-hexane, evaporation of the organic phase and derivatization with (S)-(+)-naphthylethyl isocyanate to give carbamate diastereoisomeric derivatives of (S)-(+)- and (R)-(-)-eliprodil and internal standard; after evaporation of the derivatizing mixture and dissolution of the residue in a small volume of phosphate buffer-acetonitrile (60:40, v/v), an aliquot is injected into a column-switching HPLC system. The derivatized sample extract is purified on a precolumn filled with C8-bonded silica material, which is flushed with acetonitrile-water, then diastereoisomers of eliprodil and the internal standard are automatically transferred by the mobile phase to the analytical column. The analytical column is a C8 type, specially deactivated for basic compounds, the mobile phase is 0.025 M phosphate buffer (pH 2.6)-methanol-acetonitrile (42:2:56) at a flow-rate of 1.2 ml min-1 and fluorimetric detector operating at lambda ex = 275 nm and lambda em = 336 nm is used. The retention times, under these conditions, are about 16 and 17 min for (S)-(+)- and (R)-(-)-eliprodil diastereoisomers, respectively, and about 19 min for the first-eluted diastereoisomer of the internal standard. During the analysis time, the precolumn, reset in a different path from that of the analytical column, is back-flushed with different solvents, then re-equilibrated with acetonitrile-water before the next injection. Linearity in plasma, for unchanged eliprodil enantiomers, was assessed in the range 0.15-10 ng ml-1 and for total eliprodil enantiomers (unchanged + conjugated) in the range 0.75-500 ng ml-1; the limit of quantitation (LOQ) is 0.15 ng ml-1 for each unchanged enantiomer and 0.75 ng ml-1 for each total enantiomer. Linearity was also assessed in urine for total (conjugated) eliprodil enantiomers in the range 50-25 000 ng ml-1; the LOQ is 50 ng ml-1 for each enantiomer. The intra- and inter-day precision and accuracy of the method were investigated in plasma and urine and found to be satisfactory for pharmacokinetic studies. The method has been extensively used in pharamcokinetic studies in man treated with a 20-mg dose of eliprodil racemate and some results of this application are reported.
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PMID:Stereoselective determination of unchanged and glucuroconjugated eliprodil, a new anti-ischaemic drug, in human plasma and urine by precolumn derivatization and column-switching high-performance liquid chromatography with fluorescence detection. 900 57

An assay was developed for the simultaneous measurement of cyclohexene oxide and its metabolites (cyclohexanol, trans-cyclohexane-1,2-diol, cyclohexane-1,2-diol-O-glucuronide, and N-acetyl-S-(2-hydroxycyclohexyl)-L-cysteine) in rat urine and plasma using gas chromatography. A mixture of ethyl acetate-acetonitrile (70:30) was used as the extracting solvent for both matrices. This liquid-liquid extraction procedure was followed by the separation of cyclohexene oxide and its metabolites on an HP-FFAP fused-silica capillary column. In order to determine the amount of cyclohexane-1,2-diol-O-glucuronide, samples were incubated at 37 degrees C with beta-glucuronidase and the amount of cyclohexane-1,2-diol formed from the reaction determined. The extraction efficiencies of cyclohexene oxide and cyclohexanol were greater than 90% both in urine and plasma. However, recovery from the plasma and urine for trans-cyclohexane-1,2-diol (60-68%) and N-acetyl-S-(2-hydroxycyclohexyl)-L-cysteine (approximately 76%) were considerably less. Long term stability studies showed that urine samples spiked with cyclohexene oxide and trans-cyclohexane-1,2-diol are stable at -20 degrees C for up to 9 weeks. However, plasma samples are only stable for up to 2 weeks under the same conditions. The calibration curves for all analytes were linear over the range of 12.5 to 400 micrograms/ml and correlation coefficients (r2) were greater than 0.990. The limit of detection for cyclohexene oxide, cyclohexanol, and N-acetyl-S-(2-hydroxycyclohexyl)-L-cysteine is 1.56 micrograms/ml, while the limit of detection for trans-cyclohexane-1,2-diol is 3.12 micrograms/ml. This method has been used for the determination of the disposition and metabolism of cyclohexene oxide, and may be applied in environmental monitoring, as well as in microbiological studies for other epoxide materials.
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PMID:Simultaneous determination of cyclohexene oxide and its metabolites in rat plasma and urine by gas chromatography. 930 Sep 9

The production and storage of explosives has resulted in the environmental accumulation of the mutagen 2,4,6-trinitrotoluene (TNT). In order to characterize the production of mutagenic urinary metabolites, 6-week old male Fischer 344 rats were administered 75 mg of TNT/kg or DMSO vehicle by gavage. The animals were placed into metabolism cages, and urine was collected for 24 hr. Following filtration, metabolites in the urine were deconjugated with sulfatase and beta-glucuronidase and concentrated by solid phase extraction. The eluate was fractionated by reverse-phase high-performance liquid chromatography (HPLC) using acetonitrile/water, and the fractions, were solvent exchanged in DMSO by nitrogen evaporation. Each HPLC fraction was bioassayed in strains TA98, TA98NR, TA100, and TA100NR without metabolic activation using a microsuspension modification of the Salmonella histidine reversion assay. Fractions 3, 5-18, 21, 22, and 24-26 contained mutagens detected by strain TA98. In the nitroreductase-deficient strain TA98NR, some mutagenic activity was lost; however, fractions 3, 6, 9-11, 15, and 25 clearly contained direct-acting mutagens. Fewer fractions were positive in strain TA100 (9-16, 19, 20, and 25) with less activity observed in the nitroreductase deficient strain TA100NR (fractions 3, 12, 14, 15, and 25). Although some mutagenic activity coeluted with known TNT metabolite standards, there were still many unidentified mutagenic peaks.
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PMID:Mutagenicity of HPLC-fractionated urinary metabolites from 2,4,6-trinitrotoluene-treated Fischer 344 rats. 936 8


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