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)

A method was developed for the determination of endogenous steroids in urine using liquid chromatography-tandem mass spectrometry (LC-MS/MS) with methyltestosterone as internal standard. After enzymatic hydrolysis by beta-glucuronidase and liquid-liquid extraction, the urine sample was chromatographed on a Cosmosil C18 column with a mixture of methanol and ammonium acetate-formic acid (68:32, v/v) as mobile phase, then detected using MS/MS system with electrospray ionization (ESI) in multi-reaction monitoring (MRM) mode. The detection limits ranged from 0.01 ng/mL to 10 ng/mL. The recoveries ranged from 96.7% to 106.5%, and the intra- and inter-day precisions (measured as relative standard deviations) were less than 7% and 11%, respectively. With simple and fast sample preparation, the method was sensitive and specific for simultaneous determination of these 5 kinds of endogenous steroids in urine. The method has been successfully applied in pharmacokinetic study and is thus a potential alternative for gas chromatography-mass spectrometry (GC-MS) based procedures in routine analysis of endogenous steroids such as DHEA in human urine.
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PMID:[Determination of endogenous steroids in urine by liquid chromatography-tandem mass spectromretry]. 1843 17

A reliable liquid chromatography/tandem mass spectrometry has been developed for simultaneous evaluation of the activities of five cytochrome P450s (CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A) in rat plasma and urine. The five-specific probe substrates/metabolites include phenacetin/paracetamol (CYP1A2), tolbutamide/4-hydroxytolbutamide and carboxytolbutamide (CYP2C9), mephenytoin/4'-hydroxymephenytoin (CYP2C19), dextromethorphan/dextrorphan (CYP2D6), and midazolam/1'-hydroxymidazolam (CYP3A). Internal standards were brodimoprim (for phenacetin, paracetamol, midazolam and 1'-hydroxymidazolam), ofloxacin (for 4'-hydroxymephenytoin, dextromethorphan and dextrorphan) and meloxicam (for tolbutamide, 4-hydroxytolbutamide and carboxytolbutamide). Sample preparation was conducted with solid-phase extraction using Oasis HLB cartridges. The chromatography was performed using a C(18) column with mobile phase consisting of methanol/0.1% formic acid in 20 mM ammonium formate (75:25). The triple-quadrupole mass spectrometric detection was operated in both positive mode (for phenacetin, paracetamol, midazolam, 1'-hydroxymidazolam, brodimoprim, 4'-hydroxymephenytoin, dextromethorphan, dextrorphan and ofloxacin) and negative mode (for tolbutamide, 4-hydroxytolbutamide, carboxytolbutamide and meloxicam). Multiple reaction monitoring mode was used for data acquisition. Calibration ranges in plasma were 2.5-2500 ng/mL for phenacetin, 2.5-2500 ng/mL for paracetamol, 5-500 ng/mL for midazolam, and 0.5-500 ng/mL for 1'-hydroxymidazolam. In urine calibration ranges were 5-1000 ng/mL for dextromethorphan, 0.05-10 microg/mL for dextrorphan and 4'-hydroxymephenytoin, 5-2000 ng/mL for tolbutamide, 0.05-20 microg/mL for 4-hydroxytolbutamide and 0.025-10 microg/mL for carboxytolbutamide. The intra- and inter-day precision were 4.3-12.4% and 1.5-14.8%, respectively for all of the above analytes. The intra- and inter-day accuracy ranged from -9.1 to 8.3% and -10 to 9.2%, respectively for all of the above analytes. The lower limits of quantification were 2.5 ng/mL for phenacetin and paracetamol, 5 ng/mL for midazolam, 0.5 ng/mL for 1'-hydroxymidazolam, 5 ng/mL for dextromethorphan, 50 ng/mL for dextrorphan and 4'-hydroxymephenytoin, 5 ng/mL for tolbutamide, 50 ng/mL for 4-hydroxytolbutamide and 25 ng/mL for carboxytolbutamide. All the analytes were evaluated for short-term (24 h, room temperature), long-term (3 months, -20 degrees C), three freeze-thaw cycles and autosampler (24 h, 4 degrees C) stability. The stability of urine samples was also prepared with and without beta-glucuronidase incubation (37 degrees C) and measured comparatively. No significant loss of the analytes was observed at any of the investigated conditions. The current method provides a robust and reliable analytical tool for the above five-probe drug cocktail, and has been successfully verified with known CYP inducers.
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PMID:Liquid chromatography/tandem mass spectrometry method for simultaneous evaluation of activities of five cytochrome P450s using a five-drug cocktail and application to cytochrome P450 phenotyping studies in rats. 1861 8

A liquid chromatography tandem mass spectrometry (LC/MS(n)) method for the determination of 12 corticosteroids in bovine liver has been optimized and validated in accordance with the European Commission Decision 2002/657/EC. A bovine liver sample was deconjugated with beta-glucuronidase/sulfatase enzyme, extracted with diethyl ether and further cleaned up with Solid Phase Extraction (SPE) before analysis with LC/MS(n). Two different enzyme extracts (originating from Helix Pomatia and Keyhole Limpet) and three SPE elution solvents (ethyl acetate, acetonitrile and methanol) were compared during the optimization. Helix Pomatia is generally known as the enzyme most being used for enzymatic hydrolysis purposes. Nevertheless, when detecting corticosteroids in the low microg kg(-1) concentration range, the Helix Pomatia extract may lead to interferences in the final LC/MS(n) chromatogram. When using the Keyhole Limpet enzyme extract, no interferences were observed and therefore, this extract was the best choice for enzymatic hydrolysis tested in this case. Ethyl acetate was used as elution solvent during the validation procedure since SPE elution with acetonitrile resulted in higher chromatographic backgrounds, while elution with methanol showed less reproducible results. Validation of the optimized method was carried out for 10 of the 12 corticosteroids, giving mean recoveries between 91 and 109%, and repeatability and reproducibility coefficients of respectively maximum 13.7 and 18.0%. The working ranges for the linear calibration curves were 5-20 microg kg(-1) for prednisolone, methylprednisolone and prednisone and 0.5-4 microg kg(-1) for the other compounds (coefficients of determination R(2)> or =0.97). Specificity, decision limit (CCalpha) and detection capability (CCbeta) were for all compounds within the EC specified limits.
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PMID:Optimization and validation of a liquid chromatography tandem mass spectrometry (LC/MSn) method for analysis of corticosteroids in bovine liver: evaluation of Keyhole Limpet beta-glucuronidase/sulfatase enzyme extract. 1921 13

Diosmetin (3',5,7-trihydroxy-4'-methoxyflavone) is the aglycone of the flavonoid glycoside diosmin (3',5,7-trihydroxy-4'-methoxyflavone-7-ramnoglucoside). Diosmin is hydrolyzed by enzymes of intestinal micro flora before absorption of its aglycone diosmetin. A specific, sensitive, precise, accurate and robust HPLC assay for the simultaneous determination of diosmin and diosmetin in human plasma was developed and validated. Plasma samples were incubated with beta-glucuronidase/sulphatase. The analytes were isolated by liquid-liquid extraction with tert-butyl methyl ether at pH 2, and separated on a C(18) reversed-phase column using a mixture of methanol/1% formic acid (58:42, v/v) at a flow rate of 0.5ml/min. APCI in the positive ion mode and multiple reaction monitoring (MRM) method was employed. The selected transitions for diosmin, diosmetin and the internal standard (7-ethoxycoumarin) at m/z were: 609.0-->463.0, 301.2-->286.1 and 191, respectively. A good linearity was found in the range of 0.25-500ng/ml (R(2)>0.992) for both compounds. The intra-batch assay precision (CV) for diosmin and diosmetin ranged from 1.5% to 11.2% and from 2.8% to 12.5%, respectively, and the inter-batch precision were from 5.2% to 11.5% and 8.5% to 9.8%, respectively. The accuracy was well within the acceptable range the accuracies (from -2.7% to 4.2% and -1.6% to 3.5% for diosmin and diosmetin, respectively). The mean recoveries of diosmin, diosmetin and the internal standard were 87.5%, 89.2% and 67.2%. Stability studies showed that diosmin and diosmetin were stable in different conditions. Finally, the method was successfully applied to the pharmacokinetic study of diosmin in healthy volunteers following a single oral administration (Daflon).
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PMID:Simultaneous determination of diosmin and diosmetin in human plasma by ion trap liquid chromatography-atmospheric pressure chemical ionization tandem mass spectrometry: Application to a clinical pharmacokinetic study. 2065 32

The urine from mice exposed to styrene vapors (600 and 1200 mg/m(3), 6 h) was analyzed for ring-oxidized metabolites of styrene. To facilitate the identification of metabolites in urine, the following potential metabolites were prepared: 2-, 3-, and 4-vinylphenol (2-, 3-, and 4-VP), 4-vinylpyrocatechol, and 2-, 3-, and 4-vinylphenylmercapturic acid (2-, 3-, and 4-VPMA). For the analysis of vinylphenols beta-glucuronidase-treated urine was extracted and derivatized with acetanhydride/triethylamine before injection into GC/MS. Three isomers, 2-, 3-, and 4-VP, were found in the exposed urine using authentic standards. Additionally, three novel minor urinary metabolites, arylmercapturic acids 2-, 3-, and 4-VPMA, were identified by LC-ESI-MS(2) by comparison with authentic standards. Excretion of the most abundant isomer, 4-VPMA, amounted to 535 +/- 47 nmol/kg and 984 +/- 78 nmol/kg, representing approximately 0.047 and 0.043% of the absorbed dose for the exposure levels of 600 and 1200 mg/m(3), respectively. The ratio of 2-VPMA, 3-VPMA, and 4-VPMA was approximately 2:1:6. In model reactions of styrene 3,4-oxide (3,4-STO) with N-acetylcysteine in aqueous solutions and of its methyl ester in methanol, 4-vinylphenol was always the main product, while 3-vinylphenol has never been detected. No mercapturic acid was found in the reaction of 3,4-STO with N-acetylcysteine in aqueous solution at pH 7.4 or 9.7, but a small amount of 4-VPMA methyl ester was detected by LC-ESI-MS after the reaction of 3,4-STO with N-acetylcysteine methyl ester. In contrast, no mercapturic acid was found in the reaction of 3,4-STO with N-acetylcysteine in aqueous solution at pH 7.4 or 9.7. These findings indicate a capability of 3,4-STO to react with cellular thiol groups despite its rapid isomerization to vinylphenol in an aqueous environment. Moreover, the in vivo formation of 2- and 3-isomers of both VP and VPMA, neither of which was formed from 3,4-STO in vitro, strongly suggests that another arene oxide, styrene 2,3-oxide, might be a minor metabolic intermediate of styrene.
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PMID:New urinary metabolites formed from ring-oxidized metabolic intermediates of styrene. 2002 Jul 50

A simple, rapid and sensitive method was developed for determining the presence of seven anabolic steroids (boldenone, nandrolone, testosterone, methyltestosterone, epiandrosterone, androsterone, and atnozolol) in human urine. Glucuronide-conjugates of these compounds were hydrolyzed with beta-glucuronidase. The anabolic steroids were analyzed by on-line in-tube solid-phase microextraction (SPME) coupled with liquid chromatography-mass spectrometry (LC-MS). The steroids were separated within 14 min by high performance liquid chromatography using a Chromolith RP-18e column and 5 mM ammonium formate/methanol (35/65, v/v) as a mobile phase at a flow rate of 1.0 mL/min. Electrospray ionization conditions in the positive ion mode were optimized for the MS detection of these compounds. The optimum in-tube SPME conditions were 20 draw/eject cycles with a sample size of 40 microL using a Supel-Q PLOT capillary column for the extraction. The extracted compounds could be desorbed readily from the capillary column by flow of the mobile phase, and no carryover was observed. Using the in-tube SPME LC-MS with SIM mode detection, good linearity of the calibration curve (r>0.995) was obtained in the concentration range of 0.5-20 ng/mL, except for stanozolol. The detection limits (S/N=3) of anabolic steroids were in the range 9-182 pg/mL and the proposed method showed 20-33-fold higher sensitivity than the direct injection method. The within-day and between-day precisions were below 4.0% and 7.3% (n=5), respectively. This method was applied successfully to the analysis of urine samples without the interference peaks. The recovery rates of anabolic steroids spiked into urine samples were above 85%. This method is useful to analyze the urinary levels of these compounds in anti-doping tests.
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PMID:Determination of anabolic steroids in human urine by automated in-tube solid-phase microextraction coupled with liquid chromatography-mass spectrometry. 2023 87

A highly sensitive method using ultra-fast liquid chromatography coupled with quadrupole/linear ion trap mass spectrometry (UFLC-Q/Trap MS) was developed to simultaneously screen and confirm nine beta-blockers (BBs) in porcine tissues (porcine muscle, liver and kidney). The method was used for trace determination of atenolol, pindolol, acebutolol, metoprolol, carazolol, labetalol, bisoprolol, propranolol and penbutolol. The homogenized tissues were hydrolyzed by beta-glucuronidase/aryl sulfatase and extracted with acetonitrile, followed by continuous purification procedures of disperse solid phase extraction (d-SPE) with diatomaceous earth and BondElut cartridge. The ultra-fast chromatographic separation was conducted on a Kinetex C18-XB column (150 mm x 2.1 mm, 2.6 microm) using 0.1% (v/v) formic acid aqueous solution and methanol as mobile phases in gradient elution. The optimized ion transitions were mployed in the mixed-mode of scheduled multiple reaction monitoring (sMRM) -information dependent acquisition (IDA)-enhanced product ion (EPI) scan. Qualification analysis was performed through spectra-matching with on-line lab-built MS/MS library. For quantification stable isotope-labelled analogues of the analytes were used as internal standards. As a result, in porcine liver, kidney and muscle, the nine BBs showed good linearity with all the correlation coefficients (r) more than 0.995 in the range of 0.1-20 microg/L. The limits of quantification (LOQ, S/N > or = 10) were 0.5 kg/kg for all the analytes. The developed method gave average recoveries of 87.5%-111.8% spiked at 0.5, 1.0 and 5.0 microg/kg with the relative standard deviations of 4.0%-12. 5%. The proposed method can be used to screen and confirm the nine BBs in a single run, which makes it effective in surveillance and detection of the BBs residues in porcine tissues.
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PMID:[Simultaneous determination of nine beta-blockers in porcine tissues by ultra-fast liquid chromatography coupled with quadrupole/linear ion trap mass spectrometry]. 2526 53


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