On-line post-column photochemical derivatization in liquid chromatographic--diode-array detection analysis of binary drug mixtures

HPLC methods were developed for the analysis of pharmaceutical creams containing binary drug mixtures (betamethasone valerate-chlorocresol; hydrocortisone-miconazole nitrate; desonide pivalate-chlorhexidine; dexamethasone-clotrimazole; triamcinolone acetonide-econazole nitrate). The chromatographic separations were performed on C-18 and cyano columns under reversed-phase conditions. A post-column on-line photochemical reactor (irradiation at 254 nm) was arranged between the analytical column and the diode-array detector to enhance the performance of the method. Two UV spectra (photoreactor on and off) were obtained for each analyte and these additional sources of information proved to be useful for the unambiguous identification of the various analytes. The method was applied to the quality control of commercial creams using a solid-phase extraction procedure for the sample clean-up.     

Erythrocyte thiol status regulates band 3 phosphotyrosine level via oxidation/reduction of band 3-associated phosphotyrosine phosphatase

Oxidative stress-induced tyrosine phosphorylation has been ascribed to activation of phosphotyrosine kinase or to inhibition of phosphotyrosine phosphatase (PTP). We have previously identified a PTP associated with band 3 in the human erythrocyte membrane, a PTP that is normally highly active and prevents the appearance of band 3 phosphotyrosine. Here we show that treatment of erythrocytes with the thiol-oxidizing agent diamide leads to the formation of PTP disulfides (PTP-band 3 mixed disulfides) and inhibition of dephosphorylation, allowing the accumulation of band 3 phosphotyrosine. Upon reduction of the disulfides, the band 3 phosphotyrosine is dephosphorylated. Erythrocyte thiol alkylation by N-ethylmaleimide results in irreversible PTP inhibition and irreversible phosphorylation. The results are consistent with the notion that alterations in cellular thiol status affect the cell phosphotyrosine status and that oxidative stress-induced tyrosine phosphorylation involves inhibition of PTP.     

Plasma analysis of alpha-difluoromethylornithine using pre-column derivatization with naphthalene-2,3-dicarboxaldehyde/CN and multidimensional chromatography

A procedure for the plasma analysis of alpha-difluoromethylornithine (DFMO) has been developed that utilizes pre-column derivatization with naphthalene-2,3-dicarboxaldehyde/cyanide (NDA/CN) in pH 9.2 borate buffer. Selective derivatization of delta-amine of DFMO followed by quenching of the reaction results in the formation of a cyanobenz [f] isoindole (CBI) derivative that is stable for 24 h. Plasma was prepared for derivatization by a single step procedure which resulted in an ultrafiltrate compatible with derivatization and analysis. The DFMO derivative (CBI-DFMO) was separated from plasma interferences by multidimensional chromatography with an analysis time of 28 min. The response for DFMO in plasma was linear over the range of 2.1 x 10(-8) 2.1 x 10(-6) M after derivatization. This procedure encompasses a useful linear range and offers the advantages of minimal sample preparation and production of a stable fluorophor.     

A convenient derivatization method for gas chromatography/mass spectrometric determination of phenmetrazine in urine using 2,2,2-trichloroethyl chloroformate

The present experiment examined the effects of dopamine receptor antagonism on subjects' motivation to seek food. Rats were trained to discriminate between 2 olfactory cues predicting either the presence (S+) or absence (S-) of food reinforcement in the goal box of a straight-arm runway. Rats learned to traverse the alley quickly when presented with the S+ and much more slowly when presented with the S-. Haloperidol pretreatment was unable to alter this pattern of behavior (i.e., rats still ran quickly when presented with the scent that predicted food availability). Thus, it seems that the same dopamine antagonist treatments that have been shown to disrupt food reinforcement do not prevent the food-seeking behavior produced by presentation of food-predictive cues.     

Review of Behavioral analysis of drug dependence.

Reviews the book, Behavioral analysis of drug dependence edited by Steven R. Goldberg and Ian P. Stolerman (1986). This volume brings together the main findings of basic research in behavioral pharmacology that have direct relevance to issues in drug dependence. As the editors note, the book is unique in being organized around behavioral principles rather than specific drug classes. It is also unique in making accessible a series of clearly written, well-edited summaries of the experimental literature to professionals and students who have no special background in behavioral pharmacology. I would recommend the Goldberg and Stolerman collection to researchers as well as substance abuse professionals and I think any student of substance abuse will find much of value here. The book will make a wonderful seminar at the graduate or advanced undergraduate level. Goldberg and Stolerman have succeeded in making an excellent overview of the behavioral pharmacology literature on drug dependence available to us in a single volume. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Precolumn derivatization and capillary liquid chromatographic/frit-fast atom bombardment mass spectrometric analysis of cytokinins in Arabidopsis thaliana

New cytokinin derivatives with high surface activity were developed for capillary liquid chromatography/frit-fast atom bombardment (FAB) mass spectrometry. Propionyl ester derivatives of cytokinin nucleosides and glucosides and benzylamine derivatives of cytokinin bases gave stronger [M + H]+ ion currents than the underivatized compounds. In trace analysis by selective reaction monitoring, low (fmole) detection limits were found. In qualitative analysis by B/E-linked scanning, the derivatives also gave more spectral information, owing to the presence of fragment ions, diagnostic for the sugar moieties of nucleosides and glucosides, not present in the spectra of underivatized compounds. THe proposed FAB method was used to identify and quantify 10 isoprenoid cytokinins in Arabidopsis thaliana, including free bases, nucleosides, nucleotides and glucosides.     

Electron capture gas chromatographic analysis of the amine metabolites of pesticides: derivatization of anilines

A number of amines have been shown to result from metabolism of various pesticides. From an epidemiological standpoint, it may be possible to monitor human exposure to these pesticides through the excretion of their corresponding amines in urine. An investigation has been initiated to develop and apply methods of analysis of amines in human urine. The results of a survey of derivatization techniques involving several substituted anilines are presented. These include conditions for derivatization, utilizing a number of halo- and nitro- substituted reagents; electron capture and gas chromatographic properties of the derivatives; and stability of the derivatives to extraction and column chromatography for purposes of separation and cleanup. The recoveries of anilines from spiked water and urine samples at the 1.0 ppm and 0.1 ppm levels were between 85 and 90%. The advantages and disadvantages of the various derivatives and techniques are discussed and a rationale is presented for the preliminary selection of a particular derivative for application of the analysis of aniline metabolites in urine.     

Synthesis and characterization of BaCeO3 nanocrystals via solvothermal-based method

A facile approach to preparing well-dispersed nanocrystals of BaCeO3 was developed by a combination of solvothermal and annealing processes. The precursor consisted of amorphous BaCO3 and CeO2, and the conversion of the precursor to crystalline BafeO3 nanocrystals occurred upon heat treatment at a relatively low temperature. The as-processed BaCeO3 had an orthorhombic structure and the average size of such crystals was approximately 80 nm. The obtained products were characterized by Fourier Transform Infrared （FT-IR）, X-Ray Diffraction （XRD）, Laser Raman Spectroscopy （LRS）, Scanning Electron Microscopy （SEM）, Energy Dispersive X-Ray Spectrometry （EDS）, and Transmission Electron Microscopy （TEM）. This preparation process could also be used to synthesize doped barium cerate complex oxides Bafe0.95M0.05O3-δ （M=Y, Nd, Gd, and Sm）.     

Characterization of Escherichia coli thioredoxin variants mimicking the active-sites of other thiol/disulfide oxidoreductases

Thiol/disulfide oxidoreductases like thioredoxin, glutaredoxin, DsbA, or protein disulfide isomerase (PDI) share the thioredoxin fold and a catalytic disulfide bond with the sequence Cys-Xaa-Xaa-Cys (Xaa corresponds to any amino acid). Despite their structural similarities, the enzymes have very different redox properties, which is reflected by a 100,000-fold difference in the equilibrium constant (K(eq)) with glutathione between the most oxidizing member, DsbA, and the most reducing member, thioredoxin. Here we present a systematic study on a series of variants of thioredoxin from Escherichia coli, in which the Xaa-Xaa dipeptide was exchanged by that of glutaredoxin, PDI, and DsbA. Like the corresponding natural enzymes, all thioredoxin variants proved to be stronger oxidants than the wild-type, with the order wild-type < PDI-type < DsbA-type < glutaredoxin-type. The most oxidizing, glutaredoxin-like variant has a 420-fold decreased value of K(eq), corresponding to an increase in redox potential by 75 mV. While oxidized wild-type thioredoxin is more stable than the reduced form (delta deltaG(ox/red) = 16.9 kJ/mol), both redox forms have almost the same stability in the variants. The pH-dependence of the reactivity with the alkylating agent iodoacetamide proved to be the best method to determine the pKa value of thioredoxin's nucleophilic active-site thiol (Cys32). A pKa of 7.1 was measured for Cys32 in the reduced wild-type. All variants showed a lowered pKa of Cys32, with the lowest value of 5.9 for the glutaredoxin-like variant. A correlation of redox potential and the Cys32 pKa value could be established on a quantitative level. However, the predicted correlation between the measured delta deltaG(ox/red) values and Cys32 pKa values was only qualitative.     

A method of evaluating drug efficacy by statistical analysis of healing speed of peptic ulcer

At present, the evaluation of anti-ulcer drugs is generally accomplished simply by calculating the cumulative healing rate at a certain point of time during treatment, which does not implicate any analysis of the healing speed of the ulcer. If the cumulative healing rate of an ulcer is expressed as a function of drug administration time, t, then it will be possible to calculate parameters concerning the healing speed of ulcers and thus evaluate drug efficacy as the time series analysis of the cumulative healing rate. A new method of evaluating anti-ulcer drugs by a statistical analysis of healing speed is proposed. A non-linear regression analysis was performed between two variables, t (time of drug administration: week) and y (non-healing rate: %), to obtain the exponential function y = Ae-kt. The theoretical values calculated from the exponential equation were in close proximity to the observed values. With this analysis, four parameters concerning the healing speed were defined, namely the healing rate constant, the initiation time of healing, the half-life of non-healing rate and the time necessary for 50% healing. With this method, the efficacy of drugs on peptic ulcer healing was dynamically analysed, the non-healing rate (y) being expressed as an exponential function of length of time (t) of treatment, thus obtaining digital parameters for healing speed.     

A novel derivatization method with 5-bromonicotinic acid N-hydroxysuccinimide for determination of the amino acid sequences of peptides

We have developed a novel method that effectively identifies the N-terminal product ions produced in the tandem mass spectrometry (MS/MS) analysis of peptides done in conjunction with the specific derivatization of the N-terminal amino group using 5-bromonicotinic acid N-hydroxysuccinimide ester (BrNA-NHS). Electrospray ionization with low-energy collision-induced dissociation (CID) MS/MS clearly differentiated the N-terminal product ions labeled with the 5-bromonicotinyl group from other ions, on the basis of the appearance of CID peaks with a doublet pattern characteristically separated by 2 mass units produced by the equal natural abundances of 79Br and 81Br. The tracing of a series of these bromine-containing product ions allows the easy amino acid sequencing of peptides. Using Gln-Arg-Leu-Gln-Ser-Asn-Gln-Leu-Lys as the test peptide, we found that within 30 minutes at pH 6.5 and 37 degrees C its alpha-amino group was completely acylated with BrNA-NHS (peptide: BrNA-NHS = 1:40; mol/mol). The epsilon-amino group of the C-terminal lysine residue was less likely to be acylated under these conditions, being only partly modified (about 20%). This suggests the possibility of keeping the epsilon-amino group free from acylation. The method was successfully applied to the determination of the amino acid sequences of peptides from porcine kidney aminoacylase I produced by digestion with lysyl endopeptidase and with Staphylococus aureus V8 protease.     

Development of a simple high-performance capillary electrophoretic method with on-line mode in capillary derivatization for the determination of spermidine

A new high-performance capillary electrophoretic (HPCE) method with an on-line mode in-capillary derivatization (ICD) procedure for determinations of some amines using 20 mmol/L sodium dodecyl sulfate (SDS) - 2 mmol/L o-phthalaldehyde (OPA) - 2 mmol/L N-acetylcysteine (NAC) - 20 mmol/L phosphate-borate buffer [9] has previously been shown. Although this technique offers direct fluorescence detection of free amines without any derivatization procedures before or after HPCE separation, the presence of spermidine (Spd) is difficult to detect due to low fluorescence intensity. The purpose of this study is to improve the detection sensitivity of Spd by reoptimizing this method with regard to the run buffer; the reoptimized method was applied to the determination of Spd in human plasma. To enhance the fluorescence intensity of the Spd signal, it is effective to use the run buffer in the presence of both beta-cyclodextrin (beta-CD: 8.8 mmol/L) and NAC at high concentration (16 mmol/L). By contrast, the intensity was remarkably decreased when SDS was used in the presence of beta-CD. After ultrafiltrating (UF) spiked human plasma with Spd, UF plasma was directly analyzed using the reoptimized method. Spd peak was detected and separated from the other peaks of blank plasma. The present method gave good linearity (r = 0.999), reproducibility (3.85% coefficient of variation at 5 micromol/L level; n = 10) and specificity. The detection limit and lower limit of quantitation is for 0.2 micromol/L and 1 micromol/L, respectively.     

Fluorescence correlation spectroscopy (FCS)--a highly sensitive method to analyze drug/target interactions

Fluorescence Correlation Spectroscopy (FCS) a new analytical technology, allows binding properties to be determined very accurately in biological assays at the level of single molecules. At concentrations of > or = 10(-12) M, binding constants, on/off-rates, and even reaction/enzyme kinetics can be determined in real-time, and in sample volumes as low as 10(-9) microliters. The FCS technology can be applied to study molecular and cellular interactions in homogeneous assays. Assay times in the range of seconds in combination with nanoliter sample volumes allow FCS to be used for high throughput screening to identify new pharmaceutical lead structures or new pharmacological targets. FCS is fully compatible with standard microtiter plate formats. However, for high throughput screening, specially designed sample carriers containing many thousand sub-microliter sample wells may be used in combination with a nanopipetting and sample retrieval system.     

沉淀法制备BAO-ZrO2(BSZ)纳米粉体研究

通过采用氯氧化锆(ZrOCl₂)、BaCl₂水溶液与NaOH共沉淀反应,生成钡稳定氧化锆的前驱体,该前驱体经过预烧结后得到BaO-ZrO₂粉体.研究了搅拌速率、pH值、反应温度、预烧结温度对粉体结晶度的影响.通过分析BSZ粉体的X射线衍射（XRD）和扫描电镜（SEM）结果得出,搅拌速率为280 rad/min、pH值为10、反应温度为80 ℃、预烧结温度为800 ℃是合成BSZ粉体的最佳工艺条件.      

Structure evolution and thermal stability of La2O3-doped mullite fibers via sol-gel method

The lanthana-doped mullite fibers were prepared via a sol-gel method from the aluminum isopropoxide-aluminum nitrate-tetraethyl orthosilicate system. The structure evolution and thermal stability of the mullite fibers were investigated by means of X-ray diffraction (XRD), Fourier transform infrared (FT-IR) spectroscopy, differential scanning calorimetry (DSC), scanning electron microscopy (SEM) and trans-mission electron microscopy (TEM). The results showed that the fibers obtained after calcination at 1000℃ was in the form of Al2O3-rich mullite, which tended to transform to stoichiometric mullite with the lanthana content increasing. The lattice parameter a exhibited a decline trend with the lanthana content increasing, while b showed an upward tendency. Correspondingly, the redshift of Si-O-Si bond from 1255 to 1245 cm-1 was observed. The grain growth was inhibited for the doped fibers, among which the 5% La2O3-doped sample presented a mini-mum crystallite size (17.2 nm) after calcination at 1000 ℃ for 1 h.     

Psychosocial antecedents of needle/syringe disinfection by drug users: a theory-based prospective analysis

Working from the AIDS risk reduction model and other theories of behavior change, we tested psychosocial antecedents of needle/syringe disinfection by 136 injection drug users. High perceived self-efficacy for risk reduction exerted a positive effect on needles/syringe disinfection attempts 1 year later. Self-efficacy was, in turn, related to lower perceived infection risk, peer norms more favorable to risk reduction, and greater knowledge of AIDS. Behavioral intention had no significant effect on subsequent disinfection attempts. These results suggest that disinfecting needles/syringes is partly non-volitional; that high perceived infection risk may be counterproductive to injection risk reduction; and that perceive self-efficacy, but not intention to change behavior, may be a useful leverage point for AIDS preventive intervention.