Investigation of the conformational influences on the estrogenic activity of 1,2-bis(2,6-dichloro-4-hydroxyphenyl)ethylenediamines and of their platinum(II) complexes, II: Synthesis and studies on the estrogenic activity of cis- and trans[bis(2,6-dichloro-4-hydroxybenzylamine)]dihaloplatinum(II)-c omplexes

2,6-Dichloro-4-hydroxybenzylamine (1) and its N-methyl (2) and N-ethyl (3) derivatives were synthesized and tested for estrogen receptor affinity as well as for estrogenic activity. In contrast to their related highly active 1,2-bis(2,6-dichloro-4-hydroxyphenyl)ethylenediamines (meso-4 - meso-6) none of the benzylamines showed hormonal activity. The coordination of the benzylamine 1 to platinum did not lead to an estrogenic compound. The reasons for the different activity of [meso- 1,2(bis-2,6-dichloro-4-hydroxyphenyl)ethylenediamine]dichloroplatinum(II ) (meso-4-PtCl2) and cis[bis(2,6-dichloro-4-hydroxybenzylamine)]dichloroplatinum(II) (cis-1-PtCl2), the latter of which can be considered as a ring-opened counterpart of the highly active meso-4-PtCl2, are thoroughly discussed under inclusion of conformational facts. The results of this and the preceding work show, that the pharmacophore meso-1,2-bis(2,6-dichloro-4-hydroxyphenyl)ethylenediamine (meso-4) which is exclusively responsible for the estrogenic activity of meso-4-PtCl2 causes comparable hormonal effects in two different conformations with O-O distances of about 8 A (complex) and of about 12 A (diamine). Therefore, we discuss two binding sites for estrogens in their receptor.     

A two-intermediate model for imidazole-promoted peroxyoxalate chemiluminescence

A mechanism is proposed for imidazole-catalysed peroxyoxalate chemiluminescence. The reaction model includes a sequential formation of 1-aroxalylimidazole and 1,1'-oxalyldiimidazole as light-producing reaction intermediates. The suggestion is supported by the kinetic data obtained for the reaction of imidazole with bis(4-nitrophenyl) oxalate and on the recently reported ability of 1,1'-oxalyldiimidazole to function as an efficient chemiluminescence reagent. The relative contributions of different catalytic pathways and hydrolytic side-reactions are discussed.     

Determination of C-21 ketosteroids in serum using trifluoromethanesulfonic acid catalyzed precolumn dansylation and 1,1'-oxalyldiimidazole postcolumn peroxyoxalate chemiluminescence detection

A new procedure for the quantitation of C-21 ketosteroids using trifluoromethanesulfonic acid-catalyzed precolumn dansylation and coupled column liquid chromatographic separation, followed by postcolumn 1,1'-oxalyldiimidazole peroxyoxalate chemiluminescence detection is presented. In the simultaneous optimization of chromatographic resolution and chemiluminescence intensity, a coupled column chromatographic system and a stopped-flow system were used. An eluent containing 20 mM phosphate buffer at pH 6.7 accomplished an efficient separation of 3 alpha-hydroxy-5 beta-pregnan-20-one from a mixture containing 10 C-21 ketosteroids. Phosphate buffer also proved to be the most advantageous, among the six buffers tested, for sensitive detection. Experimental design and multivariate data analysis were used to characterize and optimize the postcolumn reaction chemistry in the chromatographic system. A valid full factorial design with excellent predictability showed that the flow rates for both 1,1'-oxalyldiimidazole and hydrogen peroxide were the factors most strongly affecting the sensitivity of the system. The theoretical plate numbers were above 11,000 for all 10 dansylated ketosteroids. The 3 sigma detection limit estimated from 3 alpha-hydroxy-5 beta-pregnan-20-one calibration curve data was 1.6 pmol (n = 4, 125 microL injected) and spiked serum containing 0-74 pmol of this compound showed overall recoveries of 73 +/- 9% (n = 12). Quantitation of 3 alpha-hydroxy-5 beta-pregnan-20-one was finally carried out on 45 serum samples and the results compared to those from a radioimmunoassay (RIA) method. The data acquired with the procedure described in this work compare well with the results from RIA, which confirms the reliability of the new analytical procedure.     

Behaviour of hexahydrobenzodipyrazolones towards chloroacetylation, aminoalkylation, Grignard reagent and their antimicrobial activity

Treatment of 2,3a,4,6,7a,8-hexahydrobenzo [1,2-c; 4,5-c] dipyrazole-3,7-dione (1) with chloroacetyl chloride gave the 2,6-bis (chloroacetyl) derivative (2), which on treatment with acetic anhydride pyridine afforded (3). Compound (2) when heated with pyridine afforded (1). Compound (1) underwent Mannich reaction with piperidine or morpholine and formaldehyde to give the 2,6-bis (piperidino or morpholinomethyl) derivatives (4a,b). Hydroxymethylation of (1) with formaldehyde gave the 2,6-bis (hydroxylmethyl) derivative (4), which on heating with piperidine afforded (4a), Reaction of 2,3a,4,6,7a,8-hexahydro- 2,6-bis (phenylsulphonyl) benzo [1,2-c; 4,5-?] dipyrazole-3,7-dione (7) with phenylmagnesium bromide gave dodecahydro-3,3,4a,7,7,8a-hexaphenyl-2,6- bis (phenylsulphonyl) benzo [1,2-c; 4,5-?] dipyrazole (8). Derivatives of hexahydrobenzodipyrazolone (9a-g) have been subjected to general screening for their antimicrobial activity.     

Post-column derivatization for fluorescence and chemiluminescence detection in capillary electrophoresis

Instrumental developments and applications of post-column derivatization for fluorescence and chemiluminescence detection in capillary electrophoresis (CE) are reviewed. Various systems to merge the reagent solution with the separation medium have been developed, including coaxial capillary reactors, gap reactors and free solution or end-column systems. For all reactor types the geometry of the system, as well as the method to propel the reaction mixture (by pressure or by voltage) appeared to be critical to preserve the separation efficiency. Plate numbers of over 100,000 could be realised with different reactors. The strict requirements on the rate of post-column derivatization reactions to be applied in CE limit the number of different reagents that have been used. For fluorescence detection, with laser or lamps as the excitation source, so far mainly o-phthalaldehyde and its naphthalene analogue have been used as reagent. Derivatization systems that are based on complexation reactions also showed good promise for application in CE. Detection limits could be obtained that were comparable to those obtained after pre-column derivatization. Various reagents for chemiluminescence detection (e.g. the luminol and peroxyoxalate systems) have been studied. The often complicated chemistry involved made application of these reagents in CE even more difficult. Results obtained so far, in terms of sensitivity, have not been up to expectation, with detection limits usually in the order of micromol l(-1).     

Nonenzymatic chemiluminescent detection and quantitation of total protein on Western and slot blots allowing subsequent immunodetection and sequencing

We have studied the light emission efficiency of proteins labeled with different fluorescent dyes chemically excited by the bis(2,4,6-trichlorophenyl)oxalate (TCPO)-H2O2 reaction. Using this peroxyoxalate chemiluminescence system, the best results were obtained with proteins covalently labeled with 2-methoxy-2,4-diphenyl-3(2H)-furanone (MDPF). Blotted proteins on polyvinylidene difluoride (PVDF) membranes can be labeled rapidly with MDPF. Our results demonstrate that energy from the excited intermediate produced in the TCPO-H2O2 reaction can be efficiently transferred to MDPF-labeled proteins in solution and on PVDF membranes. Although this nonenzymatic chemiluminescent system produces a background emission that reduces the sensitivity, the method developed in this work allows detection of 5 ng of protein in blots after 5 min exposure to X-ray film. Chemiluminescence of MDPF-labeled proteins on Western and slot blots may also be detected and quantified using a charge-coupled device (CCD) camera or a storage phosphor imaging system. This chemiluminescent method allows the staining of the total electrophoretic pattern but does not preclude further N-terminal sequencing and immunodetection of specific bands.     

Simultaneous detection of multiple nucleic acid targets in a homogeneous format

The acridinium ester 4-(2-succinimidyloxycarbonylethyl)phenyl-10-methylacridinium 9-carboxylate trifluoromethane sulfonate (AE), which reacts rapidly with alkaline hydrogen peroxide to produce light, has been used as a detection label in a number of assay procedures, including nucleic acid probe-based systems [Nelson et al. (1995) in Nonisotopic Probing, Blotting and Sequencing (Kricka, L. J., Ed.) pp 391-428, Academic Press, Inc., San Diego, CA]. We have synthesized a number of derivatives of this AE and characterized their chemiluminescent properties. These derivatives display significant differences in the kinetics of the chemiluminescence reaction as well as optimal pH for light production. These differences allow two or more derivatives to be simultaneously detected and quantitated in a single reaction vessel. Several of these derivatives have been covalently linked to nucleic acid probe molecules and have been further characterized in regard to chemiluminescence properties as well as hydrolysis of the ester bond in both single- and double-stranded conformations. On the basis of these properties, homogeneous assay formats utilizing DNA probes labeled with various AE derivatives were developed. Simultaneous detection and quantitation of Chlamydia trachomatis and Neisseria gonorrhoeae, the gag and pol regions of HIV, and wild-type and mutant HIV sequences was achieved with high sensitivity and discrimination.     

High-performance liquid chromatographic separation of microcystins derivatized with a highly fluorescent dienophile

Microcystins are potent hepatotoxins produced by cyanobacteria, and are also tumor promoters as well as potent inhibitors of the catalytic subunits of protein phosphatases 1 and 2A. In order to establish a physicochemical method for individual detection and determination of trace amounts of microcystins, we developed a derivatization method for fluorescence (FL) and chemiluminescence (CL) detection, in which a highly fluorescent dienophile, DMEQ-TAD (4-[2-(6,7-dimethoxy-4-methyl-3-oxo-3,4-dihydroquinoxalinyl) ethyl]-1,2,4-triazoline-3,5-dione), was used as the labeling reagent. DMEQ-TAD reacted smoothly with the conjugated diene of the Adda moiety to give 2 stereoisomers of the adducts. As a result of the extensive experiments, the following reaction conditions were optimized for the labeling: sample amount, 10 micrograms; reaction solvent, DMF:acetonitrile (1:1); reaction time, 15 minutes; reaction temperature, 70 degrees C; amount of DMEQ-TAD used relative to that of microcystin, 80 equivalent. The resulting 6 adducts from microcystins-LR, -YR, and -RR can be separated from one another using the following reversed phase HPLC conditions in combination with a clean-up using ODS silica gel: column, Cosmosil 5C18-AR (150 x 4.6 I.D. mm); mobile phase, methanol:0.05M phosphate buffer (pH 3) (1:1); flow rate, 1.0 ml/min; detection, FL lambda ex 370 nm, lambda em 440 nm. The detection limits of the DMEQ-TAD derivatives were estimated to be 100 and 500 pg for LR, and 65 and 2,500 pg for RR using FL and CL detections, respectively; and the detection behavior was different from that of the Dns-Cys derivatives, which were more sensitive to CL than FL.     

Determination of trace amounts of urea by using flow injection with chemiluminescence detection

Reaction between urea and hypobromite in alkaline solution was found to produce chemiluminescence with a maximum wavelength at 510 nm. A simple chemiluminescence detection method was used for the determination of urea in human urine and natural aqueous samples, which combined this chemiluminescence reaction with a flow injection analysis system. The relative standard deviation for 5 x 10(-7) mol dm-3 urea is 1.9% (n = 6), and the detection limit is 9.0 x 10(-8) mol dm-3 (3Sr). As this chemiluminescence reaction is very fast, a double concentric tube mixer connected directly to the chemiluminescence cell was used to mix urea solution and hypobromite solution. Alkylamines, carboxylic acids and most amino acids do not interfere in the determination. Ammonium ion interferes, but the sensitivity for ammonium ion is only 1% of that for urea. The interference from ammonium ion was removed sufficiently by using an on-line cation-exchange column.     

Novel hydrophilic cyclic monomers in hydrogel synthesis

A range of hydrogels was prepared by the copolymerisation of 2-hydroxyethyl methacrylate (HEMA) with various hydrophilic cyclic monomers (CM) derived from cis-1,2-dihydroxy-3,5-cyclohexadiene (DHCD). The specific systems investigated were the cis-1,2-bis(2,3-epoxybutanoyloxy)-, cis-1,2-bis(10,11-epoxyundecanoyloxy)-, cis-1,2-bis(trimethylsiloxy)- and cis-1,2-bis(tert-butyldimethylsiloxy)-3,5-cyclohexadienes. The EWC, tensile strength and initial modulus of the poly HEMA-CM hydrogels derived from the epoxy compounds showed lower EWCs but similar mechanical properties compared with polyHEMA itself. Treatment of the hydrogel derived from cis-1,2-bis(2,3-epoxybutanoyloxy)-3,5-cyclohexadiene with 20% sodium hydroxide increased the EWC value markedly but produced highly fragile materials.     

Chemiluminescence: sensitive detection technology for reporter gene assays

A series of enzyme-activated chemiluminescence-based assays of reporter gene expression, useful in many biomedical applications, has been developed. The chemiluminescence detection systems for beta-galactosidase, beta-glucuronidase (GUS), and secreted placental alkaline phosphatase (SEAP) reporter enzymes are all based on use of 1,2-dioxetane substrates. This detection technology also permits the combined luminescence detection of two different reporter enzymes in the same tube, e.g., a dual assay for beta-galactosidase and luciferase. The sensitivity of these chemiluminescence assays is several orders of magnitude greater than that of conventional colorimetric or fluorometric detection methods; e.g., the detection limit for beta-galactosidase by the chemiluminescence assay is 8 fg and by a fluorometric assay is 2 pg. Furthermore, chemiluminescence enables detection of beta-galactosidase, GUS, and SEAP enzyme concentrations over a dynamic range of more than five to six orders in magnitude. These assays offer highly sensitive, quantitative, rapid, nonisotopic detection of reporter enzymes that are widely used in both mammalian cells and plant cells.     

Modification of the beta- and alpha2-adrenergic sensitivity of rat submandibular glands by environmental stimuli and stress

In man, the rate of resting salivary secretion can be influenced by environmental stimuli related to light dark cycles or by noxious stimuli (stressors) of psychological origin. The sympathetic branch of the autonomic nervous system and the adrenal medulla play an important part in homeostatic responses. Previous observations have shown that chronic exposure of rats to constant light promotes degranulation of parotid acini and desensitization of submandibular beta-adrenergic receptors. Now the submandibular secretory response elicited by beta- and alpha2-adrenergic agonists was studied in rats chronically exposed to environmental conditions that modified the activities of sympathetic efferents to the pineal, salivary and adrenal glands. Adult male rats were exposed to constant light (LL) or constant darkness (DD) for 20 days, or to stress (2 h daily immobilization) for 14 days. Control animals were kept under the usual lighting conditions and without immobilization. Dose response curves to isoproterenol (i.v), before and after administration (i.v.) of a dose (20 microg/kg) of clonidine were obtained. Beta-adrenergic desensitization was observed in all the experimental groups, while alpha2-adrenergic desensitization was only observed in the stress and LL groups. The results suggest that circulating catecholamines could mediate light and stress effects on submandibular beta-adrenergic secretory responses. Extrasynaptic alpha2-adrenoceptors might modulate the submandibular secretory response when predictable environmental stimuli (daily light phase) or unpredictable stressors raise the concentrations of circulating catecholamines.     

Regulation of adenylate cyclase coupled beta-adrenergic receptors by beta-adrenergic catecholamines

Injection of frogs with beta-adrenergic catecholamines produced a selective desensitization (loss of responsiveness) of the erythrocyte membrane adenylate cylase to subsequent stimulation in vitro by isoproterenol. Basal, prostaglandin E1- and fluoride-sensitive enzyme activities were unaffected. A 77% (p less than 0.001) decline in isoproterenol-responsive enzyme activity in the cells from the treated animals was observed with no change in the Km for isoproterenol stimulation of the enzyme (concentration causing 1/2 maximal enzyme activation). The decrease in catecholamine-sensitive adenylate cyclase was accompanied by a parallel 68% (p less than 0.001) fall in the apparent number of beta-adrenergic receptors in the erythrocyte membranes, assessed by (-) (3H)alprenolol binding studies. There was no change in the affinity of the receptor binding sites. The catecholamine-induced desensitization and fall in the beta-adrenergic receptor number were both concentration and time-dependent and displayed beta-adrenergic specificity. Isoproterenol was more potent in desensitizing cells and in lowering the receptor number than was norepinephrine. The beta-adrenergic antagonist propranolol, but not the alpha-adrenergic antagonist phentolamine, blocked the desensitizing effects of isoproterenol. Propranolol itself, however, did not cause desensitization. Cells became resensitized to the stimulatory effects of catecholamines, in association with a return in beta-receptor number, when propranolol was injected into previously desensitized animals. The changes in receptor number in membranes from desensitized and resensitized animals were also reflected in soluble receptor preparations. The protein synthesis inhibitor cycloheximide did not affect either desensitization, resensitization, or the changes in receptor number which accompanied the changes in adenylate cyclase sensitivity to catecholamines. These findings suggest that the chronic occupancy of beta-adrenergic receptors by beta-adrenergic agonists (but not antagonists) decreases the number of functional beta-adrenergic receptor binding sites and, hence, lowers the responsiveness of adenylate cylase to catecholamine stimulation. The lack of effort of cycloheximide on these regulatory effects suggests that "inactivation" and subsequent "reactivation" of the receptors, rather than changes in receptor turnover, are involved.     

Phenol-degrading denitrifying bacteria in wastewater sediments

The simultaneous determination of amino and organic acids in plant tissue extracts using capillary gas chromatography is described. Plant leaves were extracted in 5% (w/v) perchloric acid and neutralized extracts were purified using C18 cartridges. The amino and organic acids in purified extracts were then converted to tert-butyldimethylsilyl (TBDMS) derivatives prior to separation and detection by capillary gas chromatography (GC) with flame ionization detection. Conditions required for optimal derivatization were investigated. Amino and organic acids were readily converted to their TBDMS derivatives using N-methyl-N-tert-butyldimethylsilyltrifluoroacetamide in dimethylformamide solvent 1:6 (v/v) with an average recovery of 90% and a reproducibility of about 5%. The characteristic [M-57] and [M-159] fragment ion of the TBDMS derivatives was confirmed using GC-MS. The proposed method was demonstrated by the determination of amino and organic acids in extracts of Acacia and Eucalyptus leaves, where detection limits were 1-20 ng.     

An economic synthesis of 1,2,3,4-tetra-O-acetyl-5-thio-D-xylopyranose and its transformation into 4-substituted-phenyl 1,5-dithio-D- xylopyranosides possessing antithrombotic activity

D-Xylose was converted via 1,2-O-isopropylidene-alpha-D-xylofuranose (4) into 3-O-benzoyl-5-S-benzoyl-1,2-O-isopropylidene-alpha-D-xylofuranose which, after methanolysis, acetylation and subsequent acetolysis afforded 1,2,3,4-tetra-O-acetyl-5-thio-alpha-D-xylopyranose (14) in an overall yield of 36%. Reaction of 4 with thionyl chloride gave a mixture of the diastereomeric cyclic sulfites, the structures of which were established by X-ray crystallography. Their oxidation with sodium periodate afforded the corresponding cyclic sulfate 23. Treatment of 23 with potassium thioacetate gave the potassium salt of 5-S-acetyl-1,2-O-isopropylidene-alpha-D-xylofuranose 3-O-sulfonic acid (26) which, after methanolysis, acetylation and subsequent acetolysis afforded 14 in an overall yield of 56%. Treatment of 4 with sulfuryl chloride gave a mixture containing 5-chloro-3-O-chlorosulfonyl-5-deoxy-1,2-O-isopropylidene-alpha-D- xylofuranose, 3,7,9,11-tetraoxa-4-thia-10-dimethyl-tricyclo[6,3,0, 0(2,6)]undecane S-dioxide and 23 in a 2:3:7 ratio. Tetraacetate 14 was converted into the alpha-1-bromide 18 as well as into the alpha-1-O-trichloroacetimidate 17. These three compounds were used as donors for the glycosylation with 4-cyanothiophenol, affording the 4-cyanophenyl 2,3,4-tri-O-acetyl-1,5-dithio-alpha- (29) and beta-D-xylopyranoside (30) in different ratios, depending on the reaction conditions. When donor 18 was used in the presence of potassium carbonate, besides 29 and 30 two aryl C-glycosylated-thioglycosides, i.e. 4-cyano-2-(2,3,4-tri-O-acetyl-5-thio-beta-D-xylopyranosyl)phenyl 2,3,4-tri-O-acetyl-1,5-dithio-alpha- and beta-D-xylopyranoside (32 and 33) as well as 4-cyano-2-(2,3,4-tri-O-acetyl-5-thio-beta-D-xylopyranosyl)phenyl disulfide 34 could be isolated as byproducts. Deacetylation of 30 with sodium methoxide in methanol afforded, besides 4-cyano-phenyl 1,5-dithio-beta-D-xylopyranoside (1), the corresponding 4-[(methoxy)(imino)methyl]phenyl glycoside 2. The 4-cyano group of 1 was converted into the 4-aminothiocarbonyl, the 4-(methyl-thio)(imino)methyl, the 4-amidino and the 4-(imino)(hydrazino)methyl group. All of these glycosides showed a significant antithrombotic activity on rats.     

A lea-class gene of tomato confers salt and freezing tolerance when expressed in Saccharomyces cerevisiae

Although less cytotoxic, the new platinum complex [meso-1,2-bis(2,6-difluoro-4-hydroxyphenyl)-ethylenediamine]sulfatopl atinum (II) (2) is equipotent to cisplatin (1) in the oestrogen-dependent MXT mammary tumour of the mouse. As this may be due to oestrogen level-lowering properties, we compared the effect of 1 and 2 on steroidogenesis in the rat. A single dose of 1 and 2 (20 mumol/kg s.c.) decreased plasma testosterone level in male rats by 90% (1, day 3) and 80% (2, day 7). Luteinizing hormone level remained unchanged in intact and in ovariectomized rats. The activities of the following testicular enzymes were decreased (day 7): cholesterol side-chain cleavage enzyme (1: 33%; 2: 36%), 3 beta-hydroxysteroid dehydrogenase/delta 4,delta 5-isomerase (1: 31%; 2: 48%) and 17 alpha-hydroxylase/17,20-lyase (1: 21%; 2: 15%). Testicular microsomal cytochrome P450 content was also diminished (1: 60%; 2: 49%, day 7). Corticosterone level in plasma and biosynthesis in adrenal explants was not affected, indicating the selectivity of action at the gonadal level. In vitro, neither 1 nor 2 (2 and 20 microM) influenced binding of human chorionic gonadotropin to testis interstitial cells during an observation period up to 21 h. These results suggest that 1 and 2 act at the gonadal level by inhibiting the expression of the steroidogenic enzymes. They do not, however, inactivate the luteinizing hormone receptor.     

Morphology of M-cell axon arbors in striate cortex of monkeys reared with monocular aphakia

A human prostate cancer cell line (PC3) with abundant neurotensin (NT) receptors was used to demonstrate that NT potentiated 3',5'-cyclic adenosine monophate (cAMP) accumulation in response to a variety of stimuli, including both direct forskolin (F) and indirect (prostaglandin, (PGE2), isoproterenol (ISO) and cholera toxin (CTx)) activators of adenylyl cyclase. Several mechanisms were investigated and our results indicated an effect on the rate of cAMP formation and not on degradation or extrusion. For each stimulus, NT enhanced efficacy without altering EC50. The effect of NT did not involve stimulatory G-protein (Gs)-activation or interference with a tonic inhibitory G-protein (Gi)-mediated inhibition. A similar response was obtained when NT was added with the stimulus or given as a two minute pulse which was removed prior to addition of stimulus. The potentiating activity disappeared with a t1,2 of approximately 15 min. NT transiently elevated cellular [Ca2+]i and its effects on cAMP could be mimicked by [Ca2+]i-elevating agents (uridine triphosphate (UTP), thapsigargin and ionomycin). Buffering cellular [Ca2+]i with 1,2-bis (2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester (BAPTA-AM) inhibited cAMP responses to ISO and F in presence and absence of NT. These data support the idea that NT potentiated cAMP formation in response to a variety of stimuli by facilitating the activation of Ca2+ -dependent adenylyl cyclases.     

Nitroarene concentrations and direct-acting mutagenicity of diesel exhaust particulates fractionated by silica-gel column chromatography

Diesel exhaust particulates were extracted with benzene-ethanol (3:1, v/v) and separated into five fractions by silica-gel column chromatography. Direct-acting mutagenic activity was assayed by the Ames test using the Salmonella typhimurium YG1024 strain. The total activity of five fractions was about four times greater than that of the crude extract, suggesting that the activities in the fractions were suppressed in the crude extract. Strong activity was observed in fraction 4 which was eluted with dichloromethane (61.5% of the total activity) and fraction 5 which was eluted with ethanol (35.3%). Nitropolycyclic aromatic hydrocarbons (NPAHs) were determined by high-performance liquid chromatography with chemiluminescence detection. They were found mainly in fraction 4, although one NPAH was in fraction 3 which was eluted with n-hexane-dichloromethane (3:1, v/v). Based on these results, 53.1% of the activity in fraction 4 was attributed to NPAHs. The contribution of 1-nitropyrene and 1,3-, 1,6- and 1,8-dinitropyrenes was great and that of the other NPAHs was small. The mutagenic compound in fraction 5 was not identified. Fractions 1 and 2, which were eluted with n-hexane, and fraction 3 suppressed the activity of fraction 4. Polycyclic aromatic hydrocarbons in fractions 2 and 3 were considered as possible suppressors of NPAHs.     

Chemiluminescence analysis of captopril: comparison between luminol and rhodamine B-sensitized cerium(IV) methods

Two chemiluminescence methods for the determination of captopril are compared in the present paper. The first method is based on the reaction of captopril with the luminol-hydrogen peroxide-copper(II) system, the copper(II) ion acting as a key species in the light-emitting process. The catalytic activity of copper(II) decreases due to complex formation with the sulphhydrylic captopril analyte. Application of this indirect method allows captopril determinations in the 8.0-1.0 microM range as starting concentrations. Similarly, captopril was capable of generating chemiluminescence from acid cerium(IV) solutions, a reaction that could be analytically exploited by the inclusion of the rhodamine B fluorophore yielding a sensitized-type of chemiluminescence emission that allowed quantitation of captopril concentrations in the 0.1-6.0 microM range with a detection limit of 0.037 microM (original concentration). For both types of reaction the experimental conditions were optimized and a direct application was carried out on a commercial drug formulation.     

鲁米诺-H2O2-Cu（Ⅱ）化学发光法测定镍（Ⅱ）

基于Ni（Ⅱ）能置换出Cu（Ⅱ）-EDTA络合物中的Cu（Ⅱ）,与鲁米诺-H2O2-Cu（Ⅱ）化学发光体系相偶合,建立了测定Ni（Ⅱ）的置换偶合反应流动注射化学发光法测定Ni（Ⅱ）的新方法。考察了Cu（Ⅱ）-EDTA络合比、反应介质与pH、氧化剂的选择及其浓度以及鲁米诺浓度对体系相对发光强度的影响,并采用酒石酸掩蔽剂消除了干扰组分对测定的影响。在最佳的实验条件下,该方法测定Ni（Ⅱ）的线性范围为5.0×10-8～5.0×10-6 g/mL,检出限为2.0×10-8 g/mL (3σ)。对环境水样中的镍（Ⅱ）进行测定,其结果同原子吸收光谱法的测定值相一致,相对标准偏差为3.7%～4.6%。