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)

The determination of ascorbic acid by liquid chromatography (LC) was improved by performing the analysis in the presence of solvents that had been purged with argon to reduce the concentration of oxygen. This methodological modification eliminated the oxidation of ascorbic acid during the chromatographic procedure and reduced the minimum detection level to 1 microgram. Solutions of ascorbic acid have been successfully stabilized for 67 days by addition of dithiothreitol to a deaerated solution of water-acetonitrile (25 + 75 v/v), sealed under argon in amber vials and stored at -20 degrees C. In a second independent study, a procedure for the extraction of ascorbic acid from nonfat dry milk in a single step was developed. The ascorbic acid content of Nonfat Dry Milk (SRM 1549) was determined by LC, using the method of standard additions. The mean ascorbic acid content was 54 +/- 5 micrograms/g of sample. Analysis of variance of the analytical results indicates that there is a significant continual increase in the content of the ascorbic acid in each bottle from first to last sample.
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PMID:Stabilization of ascorbic acid and its measurement by liquid chromatography in nonfat dry milk. 368 Jan 14

The 1-nitroacridine nitracrine [NC,1-nitro-9-(dimethylaminopropyl-amino)acridine] is a potent hypoxia-selective cytotoxic agent in culture, but lacks activity against hypoxic tumor cells in vivo at therapeutically accessible doses. To clarify reasons for this failure in vivo the metabolism of NC was investigated in stirred suspension cultures of Chinese hamster ovary cells, in EMT-6 spheroids, and in mice. One major low molecular weight metabolite (identical to that generated by NaBH4/Pd/C reduction) was observed in hypoxic (less than 10 ppm O2) single cell suspensions, while [G-3H-acridinyl]NC formed trichloroacetic acid- and acetonitrile-insoluble macromolecular adducts (MA) at a rate seven-fold higher than in aerobic (20% O2) cultures. Formation of these adducts correlated with cytotoxicity under air or nitrogen, and hence may provide a dosimeter for NC-induced damage. Autoradiographic investigation of the distribution of MA in spheroids equilibrated with 5% O2 showed that the label was restricted to the outer cell layers rather than being localized in the hypoxic central region. Thus metabolic activation is probably too rapid, even in well-oxygenated cells, to allow adequate distribution to hypoxic microenvironments in tumors. In mice, levels of MA were higher in liver, kidney, spleen and lung than in Lewis lung tumors, indicating that oxygen concentration does not exert a dominant influence on relative rates of metabolic activation in vivo. The development of nitroacridines with useful hypoxic selectivity in vivo will require identification of analogs for which reductive metabolism is more completely inhibited at oxygen concentrations found in normal tissues.
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PMID:Reductive metabolism and hypoxia-selective toxicity of nitracrine. 374 44

A rapid, selective, sensitive and reproducible HPLC-electrospray tandem mass spectrometric method has been developed for the analysis of novel triazole antifungal agents, SYN-2869 and its derivatives (SYN-2836, SYN-2903 and SYN-2921), in rat plasma using SYN-2506 as an internal standard. Isolation of these compounds from plasma and sample desalting were performed by a simple extraction procedure involving protein precipitation, vacuum-drying and reconstitution with acetonitrile. For all the agents, linearity was observed over the range of 10-10,000 ng/ml (r > or = 0.996) and the limit of quantitation was 10 ng/ml using a 100-microliter plasma volume. A measurement rate of 400-500 samples/day/instrument could be achieved using this method.
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PMID:Liquid chromatographic-electrospray tandem mass spectrometric determination of novel antifungal agents in plasma. 1021 73

The change in amino acid enrichment, an indicator of a change in protein synthesis and/or degradation, is usually measured using gas chromatography-mass spectrometry and/or (GC-combustion) isotope ratio mass spectrometry. Unfortunately, often a complex and sensitive derivatization procedure and/or a large amount of sample is required. Also, these techniques are less suited to study intermediary metabolism, in which the simultaneous application (and thus measurement) of multiple amino acid tracers is preferred. Alternatively, in this study the possibilities of the coupling of liquid chromatography and mass spectrometry were explored, resulting in the measurement of both the concentration and isotope enrichment of o-phthaldialdehyde (OPA)-derivatizated plasma amino acids in one run. This was achieved by the injection of OPA-derivatizated amino acids into an automated HPLC system. After the elution of buffer salts and reagent excess to drain using column switching, the column effluent was directed via a fluorescence detector into a Thermoquest Model LCQ benchtop LC-MS. Mass spectrometric measurements were performed in "zoom-scan" mode, employing multiple scan events if the target components were not baseline separated. Best signal-to-noise ratio's were obtained using the LCQ's electrospray probe in the negative mode. Still, when working under standard conditions the total ion current of OPA-amino acid derivatives eluting at the beginning of the chromatogram (e.g., citrulline, arginine and glycine) was by a factor of 5 lower, compared to components eluting in the last part of the chromatogram (leucine, valine, and ornithine). These differences could be minimized by increasing the temperature of the heated capillary to 260 degrees C and by applying 5% collision energy (between the skimmer and the first octapole) to the first eluting components. A further improvement could not be obtained by the addition of makeup liquids like ammonia, acetic acid, methanol, or acetonitrile (up to 25% of column effluent flow). Considering these results and the fact that the first eluting amino acid derivatives are the most polar ones, we hypothesized that hydration of these components interferes with the ionization process. A linear calibration curve was obtained for both fluorescent response and total ion current (TIC) for all amino acids in the range from 5 to 1000 pmol per injection. The coefficient of variation of the fluorescent response was typically on the order of 1-4%, for the TIC this was between 4 and 9%. However, measurement of isotope ratios requires not only the determination of the area of the base peak, but also of the area of the (enriched) isotopomeric peak(s), having a much lower abundance. Therefore, isotope ratio measurements require the injection of at least 25 pmol of the amino acid derivative of interest (except for ARG 50 pmol) to obtain true ratio's. The accuracy of the isotope enrichment measurement was determined by the injection of a standard containing all major physiological amino acids (400 pmol each) and a standard at physiological concentrations (ranging from 50 pmol (CIT) to 350 pmol (VAL). Standard deviation of the isotopic ratios ranged from 0.1 to 0. 5% for the high (400 pmol) standards and from 0.2 to 0.8% for the low (physiological) standard, which is comparable with GC-MS. A plot of the results against the theoretical values gave a linear curve for all isotopes studied (R2 ranged from 0.9984 to 0.9997). However, the [1-13C]-enriched amino acids measured (LEU, GLY, and VAL) gave a closer agreement to the expected values as was found for [ureido-13C-5,5-2H2]-enriched citrulline and [guanidino-15N2]-enriched arginine. We could not determine whether this was due to the measurement procedure itself or resulting from an instability of the tracers in solution. Nevertheless, the results were reproducible and the theoretical value could be calculated using the tangent of the enrichment curves. (ABSTRACT TRUNCATED)
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PMID:Determination of amino acid isotope enrichment using liquid chromatography-mass spectrometry. 1036 Sep 99

This paper presents liquid chromatography/mass spectrometry (LC/MS) and liquid chromatography/tandem mass spectrometry (LC/MS/MS) approaches for the rapid characterization of three urinary isomeric metabolites and their two precursor metabolites of SYN-2836, a novel antifungal agent, in dogs administered multiple oral doses of the agent (30 mg kg(-1) day(-1)). A collection of correlative data regarding the SYN-2836 metabolites was obtained by LC/MS and LC/MS/MS performed under complementary conditions such as the columns (C(18) vs cyano type), the mobile phase systems (acetonitrile-water-formic acid vs acetonitrile-water-ammonium acetate) and the electrospray ionization modes (positive vs negative). Metabolite identification was accomplished based on not only the LC/MS/MS data (product ion spectra) but also the LC/MS data indicating chromatographic behaviors of the metabolites. SYN-2836 and SYN-2869, an analog of the former, showed almost the same metabolic pathways following the same multiple-dose administration of the individual agents to the dogs. Therefore, correlation analysis in product ion spectra between corresponding metabolites of SYN-2836 and SYN-2869, and also in metabolic pathways between the two agents, was strategically used to facilitate the identification of the SYN-2836 (and SYN-2869 if necessary) metabolites. For the reason that various elucidation strategies were used complementarily, the chemical structures of the metabolites were unambiguously attained and the isomeric metabolites were explicitly differentiated without the use of other analytical methods. The methodologies used in this study may be applicable to metabolite screening of several structurally related agents simultaneously, promoting lead finding and optimization of drug candidates using a metabolism-based approach.
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PMID:Structure elucidation of three isomeric metabolites of SYN-2836, a novel antifungal agent, in dogs via liquid chromatography/mass spectrometry and liquid chromatography/tandem mass spectrometry methodologies. 1054 12

A simple reversed-phase high-performance liquid chromatography (HPLC) method with UV detection was developed and validated for the quantitation of SYN-2869, a novel triazole antifungal agent and its analogs in rat plasma. The method involved a simple precipitation of plasma protein with acetonitrile (1:10 ratio). The reconstituted sample after evaporation to dryness was injected onto a HPLC column. SYN-2869 and its analogs were separated from the matrix components on a symmetry C18 column using an aqueous mobile phase of acetonitrile and water with a flow rate of 1 ml min(-1). A step gradient of 40-80% acetonitrile eluted all four compounds. The run time was 30 min. The linear range was 0.5 10 microg ml(-1)(r2 > 0.999). The limit of quantitation was 0.5 microg ml(-1). The inter-day precision and accuracy for SYN-2869 standard concentration were from 1.9 to 8.5% and from 1.4 to +/- 4.40%, respectively. The precision and accuracy of intra-day quality control samples were from 4.6 to 5.2% and from 4.6 to 12%, respectively.
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PMID:High-performance liquid chromatographic analysis of new triazole antifungal agent SYN-2869 and its derivatives in plasma. 1070 87

A rapid and rugged solid-phase extraction-liquid chromatographic-electrospray tandem mass spectrometric method has been developed to quantitate two novel human leukocyte elastase inhibitors, SYN-1390 and SYN-1396, in rat plasma. A reversed-phase column and an isocratic mobile phase consisting of acetonitrile-water-formic acid (70:30:0.2, v/v/v) were used. The mass spectrometer was operated in the multiple reaction monitoring mode. For both analytes, standard curves were linear over a working range of 0.1-20 microg ml(-1) (r> or =0.995) and the limit of quantitation was 0.1 microg ml(-1) with a 150 microl plasma volume. This assay proved to be useful for the determination of SYN-1390 and SYN-1396 in plasma samples from pharmacokinetic study.
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PMID:A rapid and reliable solid-phase extraction--LC/MS/MS assay for the determination of two novel human leukocyte elastase inhibitors, SYN-1390 and SYN-1396, in rat plasma. 2017 52

A simple, efficient procedure was developed for the preparation of urine samples, which greatly facilitated the identification of the urinary metabolites of a new antifungal agent SYN-2836. The urine samples following dilution with acetonitrile (ACN) formed distinct upper (ACN) and lower (aqueous) solution phases. The SYN-2836 metabolites were concentrated in the upper solution except that two glucuronides were concentrated in the lower solution. The upper solutions, containing concentrated metabolites and significantly reduced endogenous polar species, were ideally suitable for the metabolite identification. This novel sample preparation procedure would be applicable in identification of urinary metabolites of other drugs and drug candidates.
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PMID:Novel sample preparation method facilitating identification of urinary drug metabolites by liquid chromatography-tandem mass spectrometry. 1071 44

Argatroban is a peptidomimetic inhibitor of thrombin that is currently undergoing extensive clinical trials as a heparin substitute for thrombotic complications. Argatroban is readily metabolized into a major derivative, M1, that has pharmacological characteristics distinct from its parent compound. The currently available clot-based assays measure the cumulative anticoagulant effect of argatroban and its metabolite(s). Available HPLC methods do not differentiate between argatroban and M1-metabolite. A modified method was developed to simultaneouly quantitate M1-metabolite and argatroban in biological fluids. Initial validation studies for the method included clinical trials of argatroban in patients with heparin-induced thrombocytopenia, (ARG 911 Study) and coronary interventional procedures (ARG 310 Study). Plasma samples were extracted with acetonitrile and reconstituted in a mobile phase. Calibration curves were prepared by running known standards of argatroban and M1-metabolite in normal human plasma. Ultraviolet detection was made at 320 nm. The retention times for argatroban and M1-metabolite peaks were found to be 10.5 +/- 0.3 minutes and 3.9 +/- 0.1 minutes, respectively. The extraction efficiency was > 95% (r2 = 0.99). In heparin-induced thrombocytopenia patients with major bleeding complications (n = 30), the relative increase in M1-metabolite compared to argatroban varied widely (two- to eight-fold). The mean concentration of argatroban during the steady infusion period was found to be 0.7 +/- 0.35 microgram/mL, and for M1-metabolite, it was 5.5 +/- 2.8 micrograms/mL. Proportionate results were not seen when higher dosages of argatroban were administered (coronary angioplasty studies). Argatroban and M1-metabolite levels also compared well with the results in global clotting assays. Owing to the simultaneous quantitation of argatroban and M1-metabolite, this method provides a rapid assessment of the pharmacokinetics and pharmacodynamics of argatroban. The differential quantitation may be useful in the assessment of relative metabolic turnover of argatroban that can be related to the hepatic and renal functions in a given patient.
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PMID:Simultaneous monitoring of argatroban and its major metabolite using an HPLC method: potential clinical applications. 1072 23

A simple and rugged reversed-phase high-performance liquid chromatographic method with ultraviolet absorbance detection at 263 nm was developed and validated for the analysis of novel triazole antifungal agents SYN-2869 and its derivatives in tissues. The method involved homogenization with 0.01 M phosphate buffer (pH 7.8) for lung, brain and spleen tissues. The liver and kidneys were homogenized with acetonitrile:acetone (1:1). The plasma proteins were precipitated with ice-cold acetonitrile and supernatent was evaporated to dryness. The reconstituted samples were injected onto an HPLC system. SYN-2869 was separated from the matrix components on a symmetry C(18) column using a aqueous mobile phase of acetonitrile and water with a flow rate of 1 mL/min. A step gradient of 40-80% acetonitrile eluted SYN-2869 and the internal standard (SYN-2506). The linear range was 0.5-10 microgram/g (r(2) > 0.99). The limit of quantitation was 0.5 microgram/g. The inter-day precision and accuracy for SYN 2869 standard concentration were from 2.6 to 7.4% and from -1.56 to +3.29%, respectively. The method was applied to tissue samples collected from single intravenous administration to mice to evaluate the distribution of these novel antifungal agents to different tissues.
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PMID:High-performance liquid chromatographic assay for the determination of novel triazole antifungal agents in tissue. Application to tissue distribution studies. 1096 Aug 32


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