Simultaneous detection of five different 2-hydroxyethyl-DNA adducts formed by ethylene oxide exposure, using a high-performance liquid chromatography/electrospray ionisation tandem mass spectrometry assay

A method has been developed for the simultaneous detection and quantitation of five different 2-hydroxyethyl-DNA (HE-DNA) adducts that could be formed as a result of exposure to ethylene oxide (EO). In addition to the major N7-HE-guanine (N7-HEG) adducts this assay can also measure the less prevalent but potentially more biologically significant N1-HE-2'-deoxyadenosine (N1-HEdA), O(6)-HE-2'-deoxyguanosine (O(6)-HEdG), N(6)-HE-2'-deoxyadenosine (N(6)-HEdA) and N3-HE-2'-deoxyuridine adducts (N3-HEdU). The method involves the isolation of HE adducts from the unmodified nucleosides by either neutral thermal hydrolysis or enzymatic digestion, followed by high-performance liquid chromatographic (HPLC) purification, before detection and quantification by liquid chromatography tandem mass spectrometry (LC/MS/MS) using selective reaction monitoring (SRM). The limits of detection were in the range 0.5-25 fmol for each individual adduct, making this one of the most sensitive assays available for the detection of N7-HEG. To illustrate the possible applications of the assay, it has been employed in the measurement of endogenous/background and EO-induced HE adducts in a variety of DNA samples.     

Rapid and sensitive quantification of urinary N7-methylguanine by isotope-dilution liquid chromatography/electrospray ionization tandem mass spectrometry with on-line solid-phase extraction

A rapid, highly specific and sensitive isotope-dilution liquid chromatography/tandem mass spectrometry (LC/MS/MS) method coupled with an on-line solid-phase extraction (SPE) system was developed to measure N7-methylguanine (N7-MeG) in urine. 15N5-Labeled N7-MeG was synthesized to serve as an internal standard, and an on-line SPE cartridge was used for on-line sample cleanup and enrichment. The urine sample can be directly analyzed within 15 min without prior sample purification. The detection limit for this method was estimated as 8.0 pg/mL (4.8 pmol) on-column. This method was further applied to study exposure to methylating agents arising from cigarette smoke. Sixty-seven volunteers were recruited, including 32 regular smokers and 35 nonsmokers. Urinary cotinine, a major metabolite of nicotine, was also determined using an isotope-dilution LC/MS/MS method. The results showed that urinary levels of N7-MeG observed in smokers (4215 +/- 1739 ng/mg creatinine) were significantly (P < 0.01) higher than those in nonsmokers (3035 +/- 720 ng/mg creatinine). It was further noted that the urinary level of N7-MeG was found to be correlated with that of cotinine for smokers, implying that cigarette smoking resulted in increased DNA methylation, followed by depurination and excretion of N7-MeG in urine. As a result of the on-line extraction system, this method is capable of routine high-throughput analysis and accurate quantitation of N7-MeG, and could be a useful tool for health surveillance of methylating agent exposure.     

Analysis of N7-(benzo[a]pyrene-6-yl)guanine in urine using two-step solid-phase extraction and isotope dilution with liquid chromatography/tandem mass spectrometry

Analysis of urinary N7-(benzo[a]pyren-6-yl)guanine (BP-6-N7Gua), a DNA adduct induced by benzo[a]pyrene, may serve as a risk-associated biomarker for exposure to polyaromatic hydrocarbons (PAHs). In this study a highly sensitive and specific analytical method, incorporating on-line sample preparation coupled with isotope-dilution liquid chromatography and tandem mass spectrometry (LC/MS/MS), was developed to quantitate this adduct in human urine. In order to achieve accurate quantitation, 15N5-labeled BP-6-N7Gua was synthesized to serve as the internal standard, and a two-step solid-phase extraction (SPE) procedure using C8 and SCX cartridges was used for sample cleanup. BP-6-7-N7Gua was analyzed using positive ion LC/MS/MS operated in multiple reaction monitoring (MRM) mode. The [M+H]+ ions at m/z 402 and 407 and the common fragment ion of [M+H]+ at m/z 252 were monitored for quantification. The recovery of this analyte after two-step SPE was 90%, and the limit of detection was 2.5 fmol/mL in 10 mL of urine. This highly specific and sensitive method for BP-6-N7Gua in urine may be applied to assess exposure to PAHs in coke-oven workers for future molecular epidemiology studies on health effects of PAHs.     

A head-space gas-chromatographic method for the determination of traces of ethylene oxide in sterilized medical devices

A head-space gas-liquid chromatography (HS-GLC) method for the determination of traces of ethylene oxide (EtO) in sterilized medical devices was developed. The method allows the determination of the amount of EtO by putting the samples directly into N,N-dimethylacetamide (DMA) and testing by means of HS-GLC. The procedure described is simple, sensitive, reproducible, and linear. The detection limit is 0.1 μg EtO/ml DMA, corresponding to 2 μg EtO/g sterilized material.     

Simultaneous determination of ethamsylate, tramadol and lidocaine in human urine by capillary electrophoresis with electrochemiluminescence detection

Ethamsylate, tramadol and lidocaine, partly excreted by the kidney, are generally used as hemostatic, analgesic and local anesthetic in surgery. We developed a simple and sensitive method for their simultaneous monitoring in human urine based on CE coupled with electrochemiluminescence detection by end-column mode. Under optimized conditions the proposed method yielded linear ranges from 5.0 x 10(-8) to 5.0 x 10(-5), 1.0 x 10(-7) to 1.0 x 10(-4) and 1.0 x 10(-7) to 1.0 x 10(-4) M with LODs of 8.0 x 10(-9) M (36 amol), 1.6 x 10(-8) M (72 amol) and 1.0 x 10(-8) M (45 amol) (S/N = 3) for ethamsylate, tramadol and lidocaine, respectively. The RSD for their simultaneous detection at 1.0 x 10(-6) M was 2.1, 2.8 and 3.2% (n = 7), respectively. For practical application an extraction step with ethyl acetate at pH 11 was performed to eliminate the influence of the sample ionic strength. The recoveries of ethamsylate, tramadol and lidocaine at different levels in human urine were between 87 and 95%. This method was used for simultaneous detection of ethamsylate, tramadol and lidocaine in clinic urine samples from two medicated patients. It was valuable in clinical and biochemical laboratories for monitoring these drugs for various purposes.     

Microdetermination of silicon in blood, serum, urine, and milk using furnace atomic absorption spectrometry

A simple, direct microanalytical method for quantitative determination of silicon in human whole blood, serum, urine, and milk by furnace atomic absorption technique has been developed. The method employs standard additions and combines the inherent specificity and simplicity of atomic absorption analysis with the greatly increased sensitivity possible with a heated graphite tube atomizer for the determination of silicon in microliter samples. The sensitivity of the method is 1.3 ng. The method is suitable for the direct analysis of silicon with no sample preparation other than dilution with deionized water, thereby minimizing contamination due to sample preparation. The relative standard deviation for 10 μl of blood (1:1), serum (1:1), urine (1:7), and milk (1:1) was 3.45% or less.     

Zur Umsetzung des Zweikomponentensystems Triethylphosphit/Tetrachlorkohlenstoff mit wasserstoffhaltigen Nucleophilen 1. Die Reaktion mit Säureamiden

Reaction of the Two-component System Triethylphosphite/Carbon Tetrachloride with Nucleophiles Containing Hydrogen. 1. Reaction with Acyl Amides Acyl amides react with the two-component system triethylphosphite/carbon tetrachloride yielding N-acyl phosphazenes, (EtO)₃P?N? Ac. In this way (EtO)₃P?N? P(O)(OEt)₂, (EtO)₃P?N? CN, (EtO)₃P?N? C(O)Ph, and (EtO)₃P?N? SO₂Ph were prepared. Ethyl esters of phosphoric acid and trichloromethane phosphonic acid were obtained as by-products.     

Determination of a guanosine-malonaldehyde adduct in urine by high-performance liquid chromatography with a thiobarbituric acid reaction detector.

A new method for the determination of a guanosine-malonaldehyde adduct, beta-D-ribofuranosylpyrimido[1,2-a]purin-10(3H)-one (GMA), in rat and human urine is described. The method involves rapid pretreatment using, in sequence, polyamide, ion-exchange and reversed-phase cartridges; determination is by means of high-performance liquid chromatography with a thiobarbituric acid reactor in series with a fluorescence detector. This device can quantitatively determine the adduct at the sub-picomole level. This rapid, selective and sensitive method is suitable for the determination of guanine-malonaldehyde adducts in biological samples, such as human and rat urine. A semi-preparative method for the extraction and purification of these adducts from rat urine and for their identification by mass spectrometry and high-performance liquid chromatography with ultraviolet detection is also reported.     

DEB-FAPy-dG Adducts of 1,3-Butadiene: Synthesis,Structural Characterization,and Formation in 1,2,3,4-Diepoxybutane Treated DNA**

Metabolic activation of the human carcinogen 1,3-butadiene (BD) by cytochrome 450 monooxygenases gives rise to a genotoxic diepoxide, 1,2,3,4-diepoxybutane (DEB). This reactive electrophile alkylates guanine bases in DNA to produce N7-(2-hydroxy-3,4-epoxy-1-yl)-dG (N7-DE-dG) adducts. Because of the positive charge at the N7 position of the purine heterocycle, N7-DEB-dG adducts are inherently unstable and can undergo spontaneous depurination or base-catalyzed imidazole ring opening to give N⁶-[2-deoxy-D-erythro-pentofuranosyl]-2,6-diamino-3,4-dihydro-4-oxo-5-N-1-(oxiran-2-yl)propan-1-ol-formamidopyrimidine (DEB-FAPy-dG) adducts. Here we report the first synthesis and structural characterization of DEB-FAPy-dG adducts. Authentic standards of DEB-FAPy-dG and its ¹⁵N₃-labeled analogue were used for the development of a quantitative nanoLC-ESI⁺-HRMS/MS method, allowing for adduct detection in DEB-treated calf thymus DNA. DEB-FAPy-dG formation in DNA was dependent on DEB concentration and pH, with higher numbers observed under alkaline conditions.     

Preferential intramolecular ring closure of aminoalcohols with diethyl carbonate to oxazolidinones

The closure by cyclization with diethyl carbonate (EtO)(2)CO from aminoalcohols 1 as starting material can lead to the oxazolidinones 2a, b and 2c, respectively. In the reaction of trans-isomer (6) and (EtO)(2)CO, isolated products were also only 5-membered oxazolidinone derivative (7), containing its dehydrated derivative 8. The preferential formation of the 5-membered oxazolidinone ring system apparently indicated that this process (5-Exo-Trig ring closure) is more favorable than that of 6- or 7-membered ring derivative (3 or 9) by 6- or 7-Exo-Trig ring closure.     

Characterisation of estrone-nucleic acid adducts formed by reaction of 3,4-estrone-o-quinone with 2'-deoxynucleosides/deoxynucleotides using capillary liquid chromatography/electrospray ionization mass spectrometry

Xenobiotic and endobiotic molecules can react with DNA leading to formation of so-called DNA adducts. This modified DNA can be repaired enzymatically, but, if not, these modifications are believed to be responsible for the initiation of carcinogenic processes. Hence, we studied the interaction of 2'-deoxynucleosides and 2'-deoxynucleotides with 3,4-estronequinone (3,4-E(1)Q), a metabolite of estrone (E(1)) and a supposed carcinogen. These estrone-nucleic acid adducts were analysed by capillary liquid chromatography (CapLC) coupled to electrospray ionization mass spectrometry (ESI-MS). Knowledge of their behaviour from in vitro studies is a prerequisite for detecting adducts in in vivo studies. Our initial attempts to synthesise nucleos(t)ide adducts of 3,4-E(1)Q in an aprotic solvent (dimethylformamide) yielded no adducts. However, under acidic aqueous conditions, adducts were obtained. With dGuo, a dGuo adduct was found in addition to a Gua adduct. Earlier publications on adduct formation in protic solvents failed to report formation of any adduct with dAdo. A N(3)-Ade adduct was reported upon reaction of 3,4-E(1)Q with Ade base and with DNA. With dAdo, we obtained two nucleoside adducts and six Ade adducts due to loss of 2'-deoxyribose. Thus, contrary to general belief that only 2,3-E(1)Q can form stable adducts, we showed formation of substantial amounts of intact DNA adducts with 3,4-E(1)Q in addition to deglycosylated adducts. Adducts were also obtained with dGMP and dAMP, but no phosphate alkylation was found. Adducts of dCyd, dCMP, dThd, and dTMP were not detected. Using chromatographic-MS data a structural relationship between the 2'-deoxynucleoside, 2'-deoxynucleotide and base adducts was found in the various reaction mixtures. The adducts of dGuo and dGMP reaction mixtures were alkylated at the same N(7)-position of the nucleobase, as indicated by the occurrence of a rapid deglycosylation reaction. In dAdo and dAMP reaction mixtures, 14 adducts were detected; their relationships from the LC and MS data reduced the number of structures to six adenine base alkylated adducts with respect to alkylation between N(1), N(3), N(7) and/or N(6) in the adenine and C(1), C(2) and/or C(6) in 3,4-E(1)Q. We could infer, in addition, whether they had an A ring attachment or a C(6) attachment on the estrone moiety.     

Aspects of the inorganic chemistry of rubber vulcanisation. Part 5(1) complexes of zinc,nickel and cobalt derived from 2-mercaptobenzothiazole and its 5-ethoxy- and ethyl-derivatives

Summary Metal complexes of 2-mercaptobenzothiazole (5-RC₇H₃-NS₂H) (R=H) and its 5-ethoxy (R=EtO) and 5-ethyl (R=Et) derivatives which are reasonably soluble in common organic solvents are described. The species prepared include M(5-RC₇H₃NS₂)₂(pyr)₂ (pyr=pyridine; M=Zn, Ni or Co; R=Et or EtO) and [M(5-RC₇H₃NS₂)₂]_n (M=Zn or Ni; R= Et or EtO; M=Co, R=EtO). Addition of [Me₄N]-[5-RC₇ H₃NS₂] to [Ni(5-RC₇H₃NS₂)₂]_n afforded green, paramagnetic [Me₄N][Ni(5-RC₇H₃NS₂)₃] (R=H or EtO). Attempts to oxidise [Ni(5-EtOC₇H₃NS₂)₂] to nickel(IV) species are described. The formation of Co(5-EtC₇H₃NS₂)₃-(pyr)₂ and Co(C₇H₄NS₂)₃I₂, which may contain cobalt(III), is reported.     

Head-to-head right-handed cross-links of the antitumor-active bis(mu-N,N'-di-p-tolylformamidinato)dirhodium(II,II) unit with the dinucleotides d(GpA) and d(ApG)

Reactions of cis-[Rh(2)(DTolF)(2)(NCCH(3))(6)](BF(4))(2) with the dinucleotides d(GpA) and d(ApG) proceed to form [Rh(2)(DTolF)(2){d(GpA)}] and [Rh(2)(DTolF)(2){d(ApG)}], respectively, with bridging purine bases spanning the Rh-Rh unit in the equatorial positions. Both dirhodium adducts exhibit head-to-head (HH) arrangement of the bases, as indicated by the presence of H8/H8 NOE cross-peaks in the 2D ROESY NMR spectra. The guanine bases bind to the dirhodium core at positions N7 and O6, a conclusion that is supported by the absence of N7 protonation at low pH values and the notable increase in the acidity of the guanine N1H sites (pK(a) approximately 7.4 in 4:1 CD(3)CN/D(2)O), inferred from the pH-dependence titrations of the guanine H8 proton resonances. In both dirhodium adducts, the adenine bases coordinate to the metal atoms through N6 and N7, which induces stabilization of the rare imino tautomer of the bases with a concomitant substantial decrease in the basicity of the N1H adenine sites (pK(a) approximately 7.0-7.1 in 4:1 CD(3)CN/D(2)O), as compared to the imino form of free adenosine. The presence of the adenine bases in the rare imino form is further corroborated by the observation of DQF-COSY H2/N1H and ROE N1H/N6H cross-peaks in the 2D NMR spectra of [Rh(2)(DTolF)(2){d(GpA)}] and [Rh(2)(DTolF)(2){d(ApG)}] in CD(3)CN at -38 degrees C. The 2D NMR spectroscopic data and the molecular modeling results suggest the presence of right-handed variants, HH1R, in solution for both adducts (HH1R refers to the relative base canting and the direction of propagation of the phosphodiester backbone with respect to the 5' base). Complete characterization of [Rh(2)(DTolF)(2){d(GpA)}] and [Rh(2)(DTolF)(2){d(ApG)}] by 2D NMR spectroscopy and molecular modeling supports anti-orientation of the sugar residues for both adducts about the glycosyl bonds as well as N- and S-type conformations for the 5'- and 3'-deoxyribose residues, respectively.     

Gas chromatographic method for the determination of N-acetyl-S-(N-methylcarbamoyl)cysteine, a metabolite of N,N-dimethylformamide and N-methylformamide, in human urine

A simple method has been developed for the determination of N-acetyl-S-(N-methylcarbamoyl)cysteine in human urine. Treatment of a urine sample (1 ml) with ethanol (2 ml) and potassium carbonate (1.5 g) produces ethyl N-methylcarbamate, which is extracted into ethanol and measured by packed column gas chromatography with nitrogen-sensitive detection. The limit of quantitation in human urine is 1 microgram/ml and the between-sample coefficient of variation is 5-11%. Simultaneously, N,N-dimethylformamide, N-methylformamide and formamide can also be determined.     

Round-robin evaluation of a solid-phase microextraction-gas chromatographic method for reliable determination of trace level ethylene oxide in sterilized medical devices

Medical devices that are sterilized with ethylene oxide (EtO) retain small quantities of EtO residuals, which may cause negative systemic and local irritating effects, and must be accurately quantified to ensure non-toxicity. The goal of this round-robin study is to investigate the capability of a novel solid-phase microextraction-gas chromatographic (SPME-GC) method for trace-level EtO residuals analysis: three independent laboratories conducted a guided experiment using this SPME-GC method, in assessing method performance, ruggedness and the feasibility of SPME fibers. These were satisfactory across the independent laboratories, at the 0.05-5.00 ppm EtO range. This method was then successfully applied to analyze EtO residuals in several sterilized/aerated medical devices of various polymeric composition, reliably detecting and quantifying the trace levels of EtO residuals present ( approximately 0.05 ppm EtO). SPME is a feasible alternative for quantifying trace-level EtO residuals in sterilized medical devices, thereby lowering the limit of quantification (LOQ) by as much as two to three orders of magnitude over the current GC methodology of direct liquid injection.     

On-line identification of depurinating DNA adducts in human urine by capillary electrophoresis--fluorescence line narrowing spectroscopy

The benzo[a]pyrene (BP)-derived 7-(benzo[a]pyren-6-yl)guanine (BP-6-N7Gua) depurinating one-electron oxidation adduct was identified in the urine extracts of coal-smoke-exposed humans for the first time. Urine samples were prepared by solid-phase extraction and reversed-phase high-performance liquid chromatography. Subsequently, the BP-6-N7Gua adduct was identified on-line with capillary electrophoresis-- fluorescence line narrowing spectroscopy (CE-FLNS) at 4.2 K. The daily excretion of BP-6-N7Gua in human urine of individuals exposed to coal smoke was approximately 226 pmol per micromol of creatinine. Due to the high level of excretion we propose that BP-6-N7Gua adducts found in urine could serve as effective biomarkers for risk assessment of BP exposure. The results demonstrate that CE-FLNS allows for on-line separation and DNA adducts identification in complex fluid extracts.     

一种蛋白同化激素新药的成分分析及其尿样监控

利用红外光谱、核磁共振氢谱、紫外光谱以及质谱等表征手段对一种新型蛋白同化激素(AAS)口服药物的主成分进行了研究和鉴定,推定主成分为甲基-1-睾酮(methyl-1-testosterone, M1T, 17β-hydroxy-17α-methyl-5α-androst-1-en-3-one)。在此基础上,建立了M1T的气相色谱-质谱联用检测方法。方法的检出限（信噪比（S/N)为3)为2 ng/mL,定量限（S/N=10）为10 ng/mL;7次平行测定前处理后的加内标尿样的相对标准偏差为9.8%。用该方法测定了该药物在尿样中的排泄曲线。该方法的建立为AAS新药的发现、检测和监控做了很有意义的基础研究工作。     

Physical properties of the solvent mixtures triethylphosphate+hexamethylphosphoric triamide at 25°C

Densities and viscosities have been determined, from 25 to 45°C, as well as the refractive indices and dielectric constants at 25°C, for mixtures of triethylphosphate (TEP) and hexamethylphosphoric triamide (HMPT), in the complete mole fraction interval. As far as steric considerations are concerned, comparison has been made with the properties of the two intermediate organophosphorus compounds, ethoxytetramethylphosphorodiamide (EtO)(Me₂N)₂PO and diethoxydimethylphosphoramide (EtO)₂(Me₂N)PO.     

Capillary electrophoretic determination of triamterene, methotrexate, and creatinine in human urine

A capillary zone electrophoresis (CZE) method using a fused-silica capillary (60.2 cm x 75 microm ID) was investigated for the determination of triamterene (TRI), methotrexate (MTX), and creatinine (CREA) in human urine. The separation was performed using a hydrodynamic injection time of 7 s (0.5 psi), a voltage of 25 kV, a capillary temperature of 30 degrees C, and 40 mM phosphoric acid adjusted to pH 2.25 by addition of triethanolamine as separation electrolyte. Under these conditions, analysis takes about 15 min. A linear response over the 0.5-15.0 mg L(-1) concentration range was found for TRI and MTX, and 0.5-80.0 mg L(-1) for CREA. Dilution of the sample (water:urine, 1:1 for TRI and MTX, and 1:25 for CREA determination) was the only step necessary prior to analysis by electrophoresis. The developed method is easy, rapid, and sensitive and has been applied to determine triamterene,methotrexate, and creatinine in urine samples with satisfactory results.     

Selectivity of purine alkylation by a quinone methide. Kinetic or thermodynamic control?

The alkylation reaction of 9-methyladenine and 9-methylguanine (as prototype substrates of deoxy-adenosine and -guanosine), by the parent o-quinone methide (o-QM), has been investigated in the gas phase and in aqueous solution, using density functional theory at the B3LYP/6-311+G(d,p) level. The effect of the medium on the reactivity, and on the stability of the resulting adducts, has been investigated by using the C-PCM solvation model to assess which adduct arises from the kinetically favorable path, or from an equilibrating process. The calculations indicate that the most nucleophilic site of the methyl-substituted nucleobases in the gas phase is the guanine oxygen atom (O(6)) (DeltaG()(gas) = 5.6 kcal mol(-)(1)), followed by the adenine N1 (DeltaG)(gas) = 10.3 kcal mol(-)(1)), while other centers exhibit a substantially lower nucleophilicity. The bulk effect of water as a solvent is the dramatic reduction of the nucleophilicity of both 9-methyladenine N1 (DeltaG)(solv) = 14.5 kcal mol(-)(1)) and 9-methylguanine O(6) (DeltaG)(solv) = 17.0 kcal mol(-)(1)). As a result there is a reversal of the nucleophilicity order of the purine bases. While O(6) and N7 nucleophilic centers of 9-methylguanine compete almost on the same footing, the reactivity gap between N1 and N7 of 9-methyladenine in solution is highly reduced. Regarding product stability, calculations predict that only two of the adducts of o-QM with 9-methyladenine, those at NH(2) and N1 positions, are lower in energy than reactants, both in the gas phase and in water. However, the adduct at N1 can easily dissociate in water. The adducts arising from the covalent modification of 9-methylguanine are largely more stable than reactants in the gas phase, but their stability is markedly reduced in water. In particular, the oxygen alkylation adduct becomes slightly unstable in water (DeltaG(solv) = +1.4 kcal mol(-)(1)), and the N7 alkylation product remains only moderately more stable than free reactants (DeltaG(solv) = -2.8 kcal mol(-)(1)). Our data show that site alkylations at the adenine N1 and the guanine O(6) and N7 in water are the result of kinetically controlled processes and that the selective modification of the exo-amino groups of guanine N2 and adenine N6 are generated by thermodynamic equilibrations. The ability of o-QM to form several metastable adducts with purine nucleobases (at guanine N7 and O(2), and adenine N1) in water suggests that the above adducts may act as o-QM carriers.