Entecavir is a deoxyguanosine nucleotide antiviral agent with the activity against hepatitis B virus (HBV). The agent possesses a polar structure, which is predetermined for hydrophilic interaction chromatography (HILIC). Novel, fast and sensitive HILIC-UHPLC method developed in this study included separation from matrix component on BEH Amide stationary phase by isocratic elution using binary mobile phase composed of acetonitrile/5mM ammonium acetate pH 4.0 (75:25) at flow-rate 0.3 ml/min. Analysis under RP-UHPLC conditions was also possible on BEH C18 stationary phase with mostly aqueous binary mobile phase composed of (4:96) acetonitrile/0.01% formic acid. The comparison of sensitivity of the two UHPLC-MS/MS methods both using selected reaction monitoring (SRM) for quantitation revealed only slightly higher sensitivity for HILIC determination, however much better method linearity, repeatability and accuracy. HILIC separation mode provided also more convenient conditions for straightforward coupling with solid phase extraction (SPE). Entecavir was extracted on Oasis HLB cartridge (1 ml, 30 mg) and eluted by 75% acetonitrile in water, which is actually the HILIC mobile phase used in this study. Therefore the evaporation/reconstitution step was omitted, which substantially accelerated the sample preparation step. The method was validated using stable isotopically labeled internal standard entecavir-C(2)(13) N(15), which is the most appropriate internal standard. Validation results demonstrated good method accuracy (with < 5% error, and 26% at LOQ), recovery (87-114%), precision (<4% RSD), selectivity and sensitivity (LOQ=100 pg/ml). The matrix effects determined by both post-column infusion method as well as post-extraction addition method were negligible (<15%).
- MeSH
- extrakce na pevné fázi MeSH
- guanin analogy a deriváty farmakokinetika moč MeSH
- hydrofobní a hydrofilní interakce MeSH
- krysa rodu rattus MeSH
- limita detekce MeSH
- lineární modely MeSH
- reprodukovatelnost výsledků MeSH
- tandemová hmotnostní spektrometrie metody MeSH
- vysokoúčinná kapalinová chromatografie metody MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Among numerous adducts formed by reaction of DNA with p-benzoquinone (p-BQ), an electrophilic metabolite of benzene, only N2-(4-hydroxyphenyl)guanine (N2HPG) has been confirmed in vivo. If excreted in urine N2HPG would be a candidate non-invasive biomarker of the DNA damage caused by benzene. To test this hypothesis, biotransformation of N2HPG was studied in rats. Unchanged N2HPG in urine amounted to 8.0 ± 2.2% and 9.1 ± 1.7% (mean ± SE) at the dose of 2 mg/kg excreted within 1 and 2 days after ip dosage, respectively. After acidic hydrolysis of the urine a slight but consistent increase in urinary N2HPG to 9.5 ± 3.2% and 11 ± 2.6% of dose was found within 1 and 2 days, respectively, indicating formation of hydrolysable conjugates. An oxidised metabolite was detected by LC-ESI-MS and identified by comparison with authentic standard as N2-(4-hydroxyphenyl)-8-oxoguanine (N2HPOG). Its excretion amounted to 2.7 ± 0.2% of dose and increased to 12.0 ± 2.7% of dose when N2HPOG was released from its conjugates by acidic hydrolysis. Glucuronides and sulphates of both N2HPG and N2HPOG were confirmed in urine by LC-ESI-MS and by enzymatic treatment with glucuronidase/sulphatase. These results indicate an extensive metabolism of N2HPG in vivo, which must be taken into account when considering N2HPG as a possible biomarker of exposure to benzene.
- MeSH
- adukty DNA chemie metabolismus farmakokinetika moč MeSH
- benzochinony metabolismus MeSH
- biologické markery chemie metabolismus moč MeSH
- biotransformace MeSH
- glukuronidasa metabolismus MeSH
- glukuronidy chemie metabolismus moč MeSH
- guanin analogy a deriváty chemie metabolismus farmakokinetika moč MeSH
- hmotnostní spektrometrie s elektrosprejovou ionizací MeSH
- hydrolýza MeSH
- karcinogeny metabolismus MeSH
- koncentrace vodíkových iontů MeSH
- krysa rodu rattus MeSH
- oxidace-redukce MeSH
- potkani Wistar MeSH
- sírany chemie metabolismus moč MeSH
- sulfatasy metabolismus MeSH
- tandemová hmotnostní spektrometrie MeSH
- vysokoúčinná kapalinová chromatografie MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
DNA integrity was investigated in the lymphocytes of 50 bus drivers, 20 garagemen and 50 controls using the comet assay with excision repair enzymes. In parallel, 8-oxo-7,8-dihydro-2'-deoxyguanosine and 15-F(2t)-isoprostane levels in the urine and protein carbonyl levels in the plasma were assessed as markers of oxidative damage to DNA, lipids and proteins. Exposure to carcinogenic polycyclic aromatic hydrocarbons (cPAHs) and volatile compounds was measured by personal samplers for 48 and 24h, respectively, before the collection of biological specimens. Both exposed groups exhibited a higher levels of DNA instability and oxidative damage to biological macromolecules than the controls. The incidence of oxidized lesions in lymphocyte DNA, but not the urinary levels of 8-oxodG, correlated with exposure to benzene and triglycerides increased this damage. Oxidative damage to lipids and proteins was associated with exposure to cPAHs and the lipid peroxidation levels positively correlated with age and LDL cholesterol, and negatively with vitamin C. The carriers of at least one variant hOGG1 (Cys) allele tended to higher oxidative damage to lymphocyte DNA than those with the wild genotype, while XPD23 (Gln/Gln) homozygotes were more susceptible to the induction of DNA strand breaks. In contrast, GSTM1 null variant seemed to protect DNA integrity.
- MeSH
- dinoprost analogy a deriváty moč MeSH
- DNA-glykosylasy genetika metabolismus MeSH
- DNA účinky léků MeSH
- genetická predispozice k nemoci MeSH
- glutathiontransferasa genetika MeSH
- guanin analogy a deriváty moč MeSH
- karbonylace proteinů účinky léků MeSH
- kometový test MeSH
- látky znečišťující vzduch v pracovním prostředí toxicita MeSH
- lidé MeSH
- lymfocyty chemie účinky léků MeSH
- oxidační stres účinky léků MeSH
- peroxidace lipidů účinky léků MeSH
- polycyklické aromatické uhlovodíky analýza toxicita MeSH
- polymorfismus genetický MeSH
- poškození DNA MeSH
- těkavé organické sloučeniny analýza toxicita MeSH
- vazokonstriktory moč MeSH
- výfukové emise vozidel toxicita MeSH
- xeroderma pigmentosum - protein skupiny D genetika MeSH
- znečištění ovzduší MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
New urinary adenine adducts, 3-(2-hydroxy-1-phenylethyl)adenine (N3alphaA), 3-(2-hydroxy-2-phenylethyl)adenine (N3betaA), were found in the urine of mice exposed to styrene vapour. These styrene 7,8-oxide derived adenine adducts as well as previously identified guanine adducts, 7-(2-hydroxy-1-phenylethyl)guanine (N7alphaG) and 7-(2-hydroxy-2-phenylethyl)guanine (N7betaG) were quantified by HPLC-ESI-MS(2) and the excretion profile during and after a repeated exposure to 600mg/m(3) or 1200mg/m(3) of styrene for 10 consecutive days (6h/day) was determined. The excretion was dose dependent. Total N3 adenine adducts (N3alphaA+N3betaA) excreted amounted to nearly 0.8x10(-5)% of the absorbed dose while urinary N7 guanine adducts (N7alphaG+N7betaG) amounted to nearly 1.4x10(-5)% of the dose. No accumulation of the adducts was observed. Due to rapid depurination from the DNA, the excretion of both N3 adenine and N7 guanine adducts ceased shortly after finishing the exposure. Both N3 adenine and N7 guanine adducts may be used as non-invasive biomarkers of effective dose reflecting only a short time exposure to styrene.
- MeSH
- adenin analogy a deriváty moč MeSH
- adukty DNA analýza moč MeSH
- aplikace inhalační MeSH
- financování organizované MeSH
- guanin analogy a deriváty moč MeSH
- myši MeSH
- styren aplikace a dávkování metabolismus MeSH
- vysokoúčinná kapalinová chromatografie MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- myši MeSH
- zvířata MeSH
