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:2.7.10.2 (focal adhesion kinase)
44,029 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

To investigate the ways in which different levels of exposure affect the metabolic activation pathways of benzene in humans, and to examine the relationship between urinary metabolites and other biological markers, we have developed two sensitive and specific liquid chromatography-tandem mass spectrometry (LC-MS/MS) assays for quantitation of the benzene metabolites trans,transmuconic acid (t,t-MA), S-phenylmercapturic acid (S-PMA), hydroquinone (HQ), catechol (CAT), and for estimation of 1,2,4-trihydroxybenzene (BT). In our first assay, urinary S-PMA and t,t-MA were measured simultaneously by liquid chromatography-electrospray ionization-tandem mass spectrometry-selected reaction monitoring (LC-ESI-MS/MS-SRM) in the negative ionization mode. In this assay, the metabolites [13C6]-S-PMA and [13C6]-t,t-MA were used as internal standards. The efficacy of this specific assay was evaluated in human urine specimens from 28 smokers and 18 nonsmokers serving as the benzene-exposed and nonexposed groups, respectively. The coefficient of variation (CV) of analyses on different days (n = 8) for S-PMA was 7% for samples containing 9.4 micrograms/L urine, and for t,t-MA was 10% for samples containing 0.07 mg/L. The mean levels of S-PMA and t,t-MA in smokers were 1.9-fold (p = 0.02) and 2.1-fold (p = 0.03) higher, respectively, than those in nonsmokers.
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PMID:Development of liquid chromatography-electrospray ionization-tandem mass spectrometry methods for determination of urinary metabolites of benzene in humans. 1050 Sep 79

Eleven beta-lactam antibiotics were analyzed in fortified and incurred beef kidney tissue using high-performance liquid chromatography/electrospray ionization/selective reaction monitoring-ion trap tandem mass spectrometry (LC/ESI-SRM-MS(n)). The analytes included: deacetylcephapirin, amoxicillin, cephapirin, desfuroylceftiofur cysteine disulfide (DCCD, a biomarker of ceftiofur), ampicillin, cefazolin, Pen G, oxacillin, cloxacillin, naficillin and dicloxicillin. Analytes were extracted with acetonitrile and water. Clean-up was performed by solid-phase extraction. Limits of confirmation in fortified tissue are as follows (tolerances or target levels in parentheses): deacetylcephapirin: 10-50 ng/g (100 ng/g); amoxacillin: 50-100 ng/g (10 ng/g); cephapirin: 10 ng/g (100 ng/g); DCCD: 500 ng/g (8000 ng/g); ampicillin: 10 ng/g (10 ng/g); cefazolin: 10 ng/g (10-50 ng/g); Pen G: 10 ng/g (50 ng/g); oxacillin: 10 ng/g (10-50 ng/g); cloxacillin: 10 ng/g (10 ng/g); naficillin: 10 ng/g (10-50 ng/g); dicloxacillin: 100-500 ng/g (10-50 ng/g). The present method was also tested on incurred kidney tissue that had previously been analyzed using a microbial assay. Good correspondence was found between the results from this new method and the bioassay. However, the present method is much more specific and, in several cases, more sensitive than the bioassay. In addition, the time of analysis is significantly shorter than the bioassay. We also found that SRM MS(n) was superior in the analysis of unknown incurred tissue than full spectrum MS(n). We also obtained an MS/MS spectrum of DCCD that is significantly at variance with previously published fragmentation spectra.
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PMID:Confirmatory analysis of beta-lactam antibiotics in kidney tissue by liquid chromatography/electrospray ionization selective reaction monitoring ion trap tandem mass spectrometry. 1266 Oct 18

New SRM (selected reaction monitoring) data dependent exclusion (MS)(n) measurement makes it possible to obtain MS(3) fragmentation data for all MS(2) fragments, useful for structural determination of drug metabolites using ESI ion trap. MS(2) fragments are produced by cleavage of all protonated molecules at the lone electron pairs of heteroatoms or the pi electrons of double and triple bonds, benzene rings and hetero-rings of drugs. Usually, data dependent MS(3) measurement cleaves only MS(2) fragment of highest intensity, that normally does not contain important metabolic sites. Fragmentation data from all parts of drug metabolites is required to determine structure. In addition to the usual basic measurement of protonated molecules and (MS)(n) fragmentation of drug metabolites, we demonstrate the use of SRM data dependent (MS)(n) measurement, plus new SRM data dependent exclusion (MS)(n) measurement for structural determination of metabolites.
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PMID:New SRM data dependent exclusion (MS)n measurement for structural determination of drug metabolites using LC/ESI/Ion trap MS. 1561 60

A liquid chromatography/tandem triple-quadrupole mass spectrometry assay to quantify paclitaxel in rat tissue homogenates containing taxol or paclitaxel nanoliposome (PTX-NLP) was developed and validated. Liquid-liquid extraction with tert-butyl methyl ether was used for tissue sample preparation and docetaxel was used as the internal standard. Paclitaxel and docetaxel were separated on a 200 mm x 4.6 mm x 5 microm C(18) column and quantified using a triple-quadrupole mass spectrometer operating in positive ion electrospray selective reaction monitoring mode (ESI(+)-SRM) with a total run time of 6.0 min. The peak area of the m/z 876.3 --> 307.9 transition of paclitaxel is measured versus that of the m/z 830.3 --> 549.1 transition of docetaxel to generate the standard curves. The standard curves were linear over the concentration range of 0.2008-2008 ng/mL for different tissues. The method had high extraction recovery (>90%) and accuracy (>90%) with the intra-day and inter-day precision <15%. Frozen stability, freeze/thaw stability, extraction stability and solution stability at ambient temperature were examined, which indicated the tissue samples should be extracted within 5 days and avoid being frozen and thawed repeatedly over 5 times. Extracted samples after evaporation could be stored at -20 degrees C for 20 days without drug degradation and no degradation was also observed after solution samples were left to stand at ambient temperature for 24 h. This assay was used to support an in vivo biodistribution study of PTX-NLP in rats.
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PMID:Liquid chromatography/tandem triple-quadrupole mass spectrometry for determination of paclitaxel in rat tissues. 1671 70

Acetaldehyde is one of the most prevalent carcinogens in cigarette smoke. It is also a major metabolite of ethanol and is found widely in the human diet and environment. Acetaldehyde DNA adducts are critical for its carcinogenic properties. The role of acetaldehyde DNA adducts in human cancer related to tobacco and alcohol exposure could be investigated with a suitable biomarker. Therefore, in this study, we have developed a method for analysis of the major DNA adduct of acetaldehyde, N2-ethylidene-dGuo (1), in human leukocyte DNA. Leukocyte DNA was subjected to enzyme hydrolysis in the presence of NaBH3CN, which converts adduct 1 to N2-ethyl-dGuo (2). [15N5]N2-ethyl-dGuo was used as the internal standard. After solid-phase extraction, N2-ethyl-dGuo was quantified by LC-ESI-MS/MS-SRM. The method was sensitive, accurate, and precise, and applicable to low microgram amounts of DNA. It was applied to investigate the effect of smoking cessation on levels of adduct 1, measured as adduct 2. Twenty-five smokers who were only light drinkers were eligible for the study. Levels of adduct 2 were quantified at two baseline time points separated by one week and again after four weeks of abstinence from smoking and alcohol consumption. The mean (+/-S.D.) levels of adduct 2 measured in the leukocytes of the smokers were 1310 +/- 1720 (range 124-7700) and 1120 +/- 1140 (range 138-5760) fmol/micromol dGuo at the two baseline points and 705 +/- 438 (range 111-1530) fmol/micromol dGuo after 4 weeks of cessation. The median level of adduct 2 decreased significantly by 28% upon quitting smoking (P = 0.02). These results demonstrate that the major acetaldehyde DNA adduct can be reliably quantified by MS/MS methods in human leukocyte DNA and that cigarette smoking has a modest but significant effect on its levels.
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PMID:Quantitation of an acetaldehyde adduct in human leukocyte DNA and the effect of smoking cessation. 1722 33

N-Nitrosopyrrolidine (NPYR) is a hepatocarcinogen in rats. It is metabolically activated by cytochrome P450 enzymes in the liver leading to the formation of 4-oxobutanediazohydroxide (4) and related intermediates that react with DNA to form adducts. Because DNA adducts are thought to be critical in carcinogenesis by NPYR, we analyzed hepatic DNA of NPYR-treated rats for several adducts: N2-(tetrahydrofuran-1-yl)dGuo (N2-THF-dGuo, 13), N6-THF-dAdo (14), N4-THF-dCyd (17), and dThd adducts 15 and 16. The rats were treated with NPYR in the drinking water, 600 ppm for 1 week, or 200 ppm for 4 or 13 weeks. Hepatic DNA was isolated, enzymatically hydrolyzed, and analyzed by capillary LC-ESI-MS-SIM, which indicated the presence of adducts 13, 14, and 17. Because these adducts can be unstable at the deoxyribonucleoside level, further analyses were carried out using DNA treated with NaBH3CN, which converts adducts 13-17 to N2-(4-hydroxybut-1-yl)dGuo [N2-(4-HOB)dGuo, 18], N6-(4-HOB)dAdo (19), O2-(4-HOB)dThd (20), O4-(4-HOB)dThd (21), and N4-(4-HOB)dCyd (22). [15N]-Labeled analogues of adducts 18-20 and 22 were synthesized and used in this analysis, which was performed by capillary LC-ESI-MS/MS-SRM. Convincing evidence for the presence of adducts 18-22 was obtained. Levels of 18, 19, 20, and 21 were (mumol/mol dGuo): 3.41-5.39, 0.02-0.04, 2.56-3.87, and 2.28-5.05, respectively. Compound 22 was not quantified due to interfering peaks. These results provide the first evidence for tetrahydrofuranyl-substituted DNA adducts in the livers of rats treated with NPYR. The finding of dAdo and dThd adducts is of particular interest since previous studies have shown that NPYR causes mutations at AT base pairs in DNA of rat liver.
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PMID:Analysis of adducts in hepatic DNA of rats treated with N-nitrosopyrrolidine. 1739 61

Polycyclic aromatic hydrocarbons (PAHs) with four to six rings are potent carcinogens. This study analyzed ten of the sixteen US EPA priority PAHs using reversed-phase liquid chromatography/tandem mass spectrometry (LC/MS/MS) in selected reaction monitoring mode with two ionization sources: positive atmospheric pressure chemical ionization (APCI+) or positive elecrtrospray ionization (ESI+) with tropylium post-column derivatization. Several factors were investigated, including mobile phases, stationary phases of columns and chromatographic temperature, to determine how optimal separation and sensitivity might be achieved. Methanol used as an organic mobile phase provided better sensitivities for most PAHs than acetonitrile, although some PAHs co-eluted. Acidic buffers did not increase analyte signals. Use of Restek Pinnacle II PAH columns (250 x 4.6 mm or 250 x 2.1 mm, 5 microm) with water/acetonitrile gradient at 27 degrees C made possible a good separation of the ten analytes. [M]+. were the best precursor ions in both APCI and ESI, although fluoranthene could not be detected in ESI mode when tropylium post-column derivatization was performed. [M-28]+ and [M-52]+ were the major product ions of PAHs after collision-induced dissociation, a result of neutral losses of C(2)H(4) and (C(2)H(2))(2), respectively. Chromatographic separation for PAH isomers was crucial because the mass spectra were so similar that even MS/MS could not distinguish them from each other. The recoveries of sample preparations of PAHs spiked onto air-sampling filters ranged between 77.5 and 106% with relative standard deviations between 1.1 and 15.9%. This method was validated by analyzing NIST SRM 1649a (urban dust), producing results comparable with the certified PAH concentrations. The detection limits using APCI and ESI interfaces, defined as three times the noise levels, ranged between 0.23 and 0.83 ng and between 0.16 and 0.84 ng of on-column injection, respectively.
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PMID:Analysis of polycyclic aromatic hydrocarbons by liquid chromatography/tandem mass spectrometry using atmospheric pressure chemical ionization or electrospray ionization with tropylium post-column derivatization. 1793 49

Natural products have historically been a rich source of lead molecules in drug discovery. However, natural products have been de-emphasized as high throughput screening resources in the recent past, in part because of difficulties in obtaining high quality natural products screening libraries, or in applying modern screening assays to these libraries. In addition, natural products programs based on screening of extract libraries, bioassay-guided isolation, structure elucidation and subsequent production scale-up are challenged to meet the rapid cycle times that are characteristic of the modern HTS approach. Fortunately, new technologies in mass spectrometry, NMR and other spectroscopic techniques can greatly facilitate the first components of the process - namely the efficient creation of high-quality natural products libraries, bimolecular target or cell-based screening, and early hit characterization. The success of any high throughput screening campaign is dependent on the quality of the chemical library. The construction and maintenance of a high quality natural products library, whether based on microbial, plant, marine or other sources is a costly endeavor. The library itself may be composed of samples that are themselves mixtures - such as crude extracts, semi-pure mixtures or single purified natural products. Each of these library designs carries with it distinctive advantages and disadvantages. Crude extract libraries have lower resource requirements for sample preparation, but high requirements for identification of the bioactive constituents. Pre-fractionated libraries can be an effective strategy to alleviate interferences encountered with crude libraries, and may shorten the time needed to identify the active principle. Purified natural product libraries require substantial resources for preparation, but offer the advantage that the hit detection process is reduced to that of synthetic single component libraries. Whether the natural products library consists of crude or partially fractionated mixtures, the library contents should be profiled to identify the known components present - a process known as dereplication. The use of mass spectrometry and HPLC-mass spectrometry together with spectral databases is a powerful tool in the chemometric profiling of bio-sources for natural product production. High throughput, high sensitivity flow NMR is an emerging tool in this area as well. Whether by cell based or biomolecular target based assays, screening of natural product extract libraries continues to furnish novel lead molecules for further drug development, despite challenges in the analysis and prioritization of natural products hits. Spectroscopic techniques are now being used to directly screen natural product and synthetic libraries. Mass spectrometry in the form of methods such as ESI-ICRFTMS, and FACS-MS as well as NMR methods such as SAR by NMR and STD-NMR have been utilized to effectively screen molecular libraries. Overall, emerging advances in mass spectrometry, NMR and other technologies are making it possible to overcome the challenges encountered in screening natural products libraries in today's drug discovery environment. As we apply these technologies and develop them even further, we can look forward to increased impact of natural products in the HTS based drug discovery.
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PMID:High impact technologies for natural products screening. 1808 16

Simultaneous quantification method of three major metabolites of cortisone and cortisol, tetrahydrocortisol, allotetrahydrocortisol and tetrahydrocortisone by liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) was investigated in a positive mode using a recently developed picolinyl derivatization. Conversion of each steroid into the corresponding picolinyl derivatives (1b, 2b or 3b) was performed by mixed anhydride method using picolinic acids and 2-methyl-6-nitrobenzoic anhydride. Derivatization proceeded smoothly to afford the corresponding 3, 21-dipicolinyl derivatives. Positive ion-ESI mass spectra of the picolinyl derivatives were dominated by an appearance of [M+H](+) as base peaks in all cases. The picolinyl derivatives provided 15 to 80-fold higher ESI response in the LC-ESI-MS/MS (selected reaction monitoring: SRM) when compared to those of underivatized molecules in a positive LC-ESI mode. The use of the picolinyl ester, solid-phase extraction, and deuterium labeled internal standards enabled the concentrations of these metabolites in human urine to be determined simultaneously by LC-ESI-MS/MS (SRM) with a small sample volume of less than 1microl urine.
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PMID:Simultaneous determination of tetrahydrocortisol, allotetrahydrocortisol and tetrahydrocortisone in human urine by liquid chromatography-electrospray ionization tandem mass spectrometry. 1839 66

Recently, cobalt (Co) is reported to be taken as a supplement by athletes for improving anaerobic performance. For the diagnosis of abuse, the limit of detection (LOD) of Co in the analysis should be lower than the concentrations of Co in plasma and urine of normal persons. A simple, rapid and sensitive method has been developed for the determination of Co in urine. Co was complexed with diethyldithiocarbamate (DDC) and extracted with isoamyl alcohol in the presence of citric acid. The detection of Co was achieved by injecting a 1-microL aliquot of isoamyl alcohol containing Co-DDC complex directly into an electrospray ionization tandem mass spectrometric (ESI-MS-MS) instrument without chromatographic separation. The quantification was performed using selected reaction monitoring at m/z 291 of the product ion Co(C(4)H(10)NCS)(2)(+) which was produced by collision-induced dissociation from the precursor ion Co(DDC)(2)(+) at m/z 355. ESI-MS-MS data were obtained in less than 10 min with an LOD of 0.05 microg L(-1) and a linear calibration range of 0.1-100 microg L(-1) using 10 microL of urine. The procedure was validated with certified reference materials (SRM 2670a and SRM 1643e). This method is suitable for the analysis of Co in the laboratories already equipped with an ESI-MS-MS instrument.
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PMID:Application of electrospray ionization tandem mass spectrometry for the rapid and sensitive determination of cobalt in urine. 1842 46


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