Tonic action of endothelin type B and dopamine D3 receptors in spontaneously hypertensive and deoxycorticosterone acetate-salt hypertensive rats: effects of intrarenally applied selective antagonists.

Endothelins and renal dopamine contribute to control of renal function and arterial pressure in health and various forms of experimental hypertension, the action is mediated by tonic activity of specific receptors. We determined the action mediated by endothelin type B and by dopamine D3 receptors (ETB-R, D3-R) in anaesthetized spontaneously hypertensive (SHR) and in deoxycorticosterone acetate (DOCA)-salt hypertensive rats. In rats of both hypertension models infused during 60 min into the interstitium of in situ kidney were either ETB-R antagonist, BQ788 (0.67 mg kg-1 BW h-1) or D3-R antagonist, GR103691 (0.2 mg kg-1 BW h-1). Arterial pressure (MAP), renal artery blood flow (RBF, transonic probe) and renal medullary blood flow (MBF, laser-Doppler) were measured along with sodium, water and total solute excretion (UNaV, V, UosmV). Experiments with ETB-R blockade confirmed their tonic vasodilator action in the whole kidney (RBF) and medulla (MBF) in both hypertension models. In SHR only, the first evidence was provided that ETB-R specifically increases transtubular backflux of non-electrolyte solutes. In DOCA-salt rats ETB-R blockade caused an early decrease in water and salt transport whereas an increase was often reported from many previous studies. The most striking effect of D3-R blockade in SHR was a selective increase in MBF, which strongly suggested tonic vasoconstrictor action of these receptors in the renal medulla; this speaks against prevailing opinion that D3 receptors are virtually inactive in SHR. In our model variant of DOCA-salt rats of D3-R blockade clearly caused a rapid major increase in MAP in parallel with depression of renal haemodynamics.

GC-MS method for the simultaneous determination of ß-blockers, flavonoids, isoflavones and their metabolites in human urine.

A sensitive and selective method based on gas chromatography hyphenated to mass spectrometry (GC-MS) for the screening of 23 different compounds including ß-blockers, flavonoids, isoflavones and metabolites in human urine sample was developed and validated. The present paper reports, for the first time, the method for the simultaneous determination of ß-blockers, isoflavones, flavonoids and metabolites in human urine samples. When flavonoids are ingested in combination with drugs that have a narrow therapeutic range, interactions between flavonoids and drugs should be investigated. Substances of interest were extracted from urine samples by solid-phase extraction (SPE) employing a mixture of tert-butyl methyl ether:methanol:formic acid (4.5:4.5:1; v/v/v) as a mobile phase and Oasis HLB (Waters) as a stationary phase. Before extraction, urine samples were incubated with ß-glucuronidase/sulfatase in order to achieve enzymatic hydrolysis. Before GC-MS analysis the analytes had to be derivatized with N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA) into their trimethylsilyl derivatives by incubating for 60 min at 60 °C. Statistical central composite design and response surface analysis were used to optimize the derivatization reagent. These multivariate procedures were efficient in determining the optimal separation condition, using peak areas as responses. The calibration curves were indicative of high linearity (r² ≥ 0.9992) in the range of interest for each analyte. LODs (S/N=3) ranged between 0.6 and 9.7 ng/ml. Intra-day and inter-day precision (CV, %) was less than 4.96%, accuracy between 0.01 and 4.98% and recovery was found in the range from 70.20 to 99.55%. The developed method can be applied to the routine determination of examined compounds' concentrations in human urine. Moreover the method is suitable for detecting pharmaceutical compounds containing ß-blockers, isoflavones and flavonoids in urine after administration to humans.

Analysis of herbicides: demonstration of the utility of enzyme immunoassay verification by HPLC.

Evidence has accumulated that herbicides in the environment present a significant health hazard to the population. Therefore, the levels of heavily used substances such as atrazine and simazine and their metabolites need to be regularly assessed. The objective was to develop a rapid and simple tube ELISA procedure suitable for use in field studies and non-specialized laboratories. The antisera used were polyclonal antibodies raised in sheep against atrazine or simazine amido caproic acid conjugated to bovine serum albumin. The antibodies were first used to construct a two-step competitive ELISA procedure in 96-well microtitre plates. The 96-well format was then adapted to a coated-tube enzyme immunoassay, by immobilization of hapten-gelatine conjugates on polystyrene tubes. This enabled the colour to be read using a basic spectrophotometer. Soil samples were collected from agricultural and non-agricultural sites in Poland. Atrazine and simazine were extracted by liquid extraction from soil and assayed by tube ELISA. In addition, the samples were extracted by solid-phase extraction before analysis by HPLC. The immunoassays and chemical analysis were carried out by different individuals who were unaware of each other's results, which were then compared at the end of the study. Correlation of the two methods was excellent, with R=98.7 and 81.3 for atrazine and simazine, respectively. The immunoassay yielded the same order of results without having to perform solid-phase extraction before analysis. The study has demonstrated that the simple antigen-coated tube assay provides a cost-effective and valuable screening test. Comparison with the more elaborate, heavily labour-intensive HPLC analysis demonstrated that the results obtained by the simpler enzyme-immunoassay tests were within the same order.

Detection and determination of aromatic amines as products of reductive splitting from selected azo dyes.

The current environment-friendly regulations concerning textile products ban the marketing of textiles dyed with azo dyes capable of reductively splitting carcinogenic aromatic amines. The study analyzes seven azo dyes whose chemical structure determines various quantities of splitting aromatic amines, such as benzidine. For tests, seven commercially available azo dyes with aromatic amines in their structure were selected. These included two hazardous dyes: Acid Red 85 and Direct Blue 6, both capable of reductively splitting carcinogenic benzidine. Of the remaining five azo dyes, three--Ponceau SS, Sudan II, and Disperse Yellow 7--are capable of splitting p-phenylenediamine and aniline, while Mordant Orange 1 and Disperse Orange 3 can split only p-phenylenediamine. For Acid Red 85 and Direct Blue 6, the quantity of benzidine split from them was analyzed, depending on the conditions of the reduction process (e.g., in the HPLC method, 104 g/kg of dye for reduction in NaOH, and 41 g/kg of dye for reduction in acetate buffer). The spectrophotometric method proved useful for preliminary analysis of amine content in examined samples. Spectrophotometric analysis may be used to determine the total content of amines counted as aniline. A full qualitative and quantitative analysis of amines released from azo dyes is possible using high-performance liquid chromatography (HPLC).

Quantitative structure-retention relationships of xanthines in RP HPLC systems with the new Chromolith RP-18e stationary phases.

The structural descriptors with the best potential for predicting the reversed-phase retention of xanthines and their derivatives on the new Chromolith RP-18e columns have been examined, as has the molecular mechanism of the separation. QSRR analysis was performed by simultaneous solution of a set of linear equations. The logarithms of the capacity factors were the dependent variables. Structural descriptors calculated by means of semi-empirical methods of quantum chemistry were chosen as independent variables.

Liquid chromatography in the analysis of neurotransmitters and alkaloids.

Several thin-layer chromatographic (TLC) and high-performance liquid chromatographic (HPLC) systems for the determination of biogenic amine neurotransmitters, alkaloids, and the metabolites of these two groups of compounds are developed. The possibility of the interference of these compounds is investigated. In all the systems developed for neurotransmitters, the retention of alkaloids is examined and vice versa. In TLC because of different visualization procedures (a dyeing reagent for alkaloids and visible light for neurotransmitters), compounds of both the groups may be determined simultaneously without the risk of interference. In HPLC, similarly, the possibility of false diagnosis through simultaneously employing the fluorimetric and diode-array detectors can be excluded.

Application of derivative spectrophotometry to determination of flavonoid mixtures.

Derivative spectrophotometry (DS) was applied to determination of flavonols and a flavon in two-compound mixtures by means of 'zero-crossing' technique. Selected mixtures were, quercetin-kaempferol, quercetin-myricetin and quercetin-luteolin. The developed method was used as a complementary technique to a HPLC system, which does not separate quercetin and luteolin.

Determination of selected herbicides and phenols in water and soils by solid-phase extraction and high-performance liquid chromatography.

A high-performance liquid chromatography procedure or the determination of the herbicides simazine, propazine, bromacil, metoxuron, and hexazinone is elaborated. Stationary phases RP8 and RP18 and mixtures of methanol-water (2:1 and 1:1, v/v) as a mobile phase are applied for this purpose. The conditions for solid-phase extraction are established, allowing the separation of phenols and herbicides in their mixtures and the extraction of phenols (from river and coke plant water) and herbicides (from the soil samples).

Derivative spectrophotometry in the analysis of mixtures of phenols and herbicides.

Derivative spectrophotometry (zero-crossing technique) was applied to the determination of selected phenols and herbicides in two-component mixtures. Methyl- and chlorophenols (3-methylphenol, 2,3- and 3,4-dimethylphenol, 2,5-, 2,6- and 3,4-dichlorophenol and 2,4,5-trichlorophenol) and triazine, uracil and urea herbicides (simazine, propazine, hexazinone, bromacil and metoxuron) were examined. The RSD values ranged between 0.05 and 4% and the recoveries obtained were between 97 and 110%. The developed derivative spectrophotometric method was also applied as a complementary technique for the separation of overlapping peaks of sample compounds obtained by HPLC with diode-array detection. Metoxuron and 3-methylphenol, metoxuron and 2,5-dichlorophenol and simazine and 2,6-dichlorophenol were determined simultaneously by this method at the level of 1 x 10(-3) g l-1.

Determination of nicotine, cotinine and caffeine in meconium using high-performance liquid chromatography.

A high-performance liquid chromatographic method with diode-array detection for the determination of nicotine and its metabolites, cotinine and caffeine, in meconium is described. This method is suitable to assess foetus exposure to tobacco smoke. The analytes were extracted by solid-phase extraction before chromatography. From among 30 meconium samples 11 were positive for cotinine (20-86 ng/g) and 27 for caffeine (10-45 ng/g). No nicotine was present in the samples because of its rapid metabolism into cotinine.

Separation and identification of metals in human bones, placenta and milk and in air by adsorption and ion-exchange thin-layer chromatography.

A TLC method for the separation of heavy metals and their complexes with dithizone, 4-(2-pyridylazo)resorcinol and ethylenediaminetetraacetic acid was devised and conditions for the solid-phase extraction of heavy metal ions in human bones, placenta and milk after microwave mineralization and in air after alkaline absorption were elaborated.

TLC separation and derivative spectrophotometry of some amino acids.

Chromatographic systems for the separation of amino acid mixtures on RP-18 as a stationary phase have been elaborated. The best results were obtained using methanol-water (1:1, v/v; 1:3, v/v; 1:5, v/v) as a mobile phase. The following amino acids have been examined: asparagine, arginine monohydrochloride, cystine, cysteine chloride, glycine, histidine monohydrochloride, hydroxyproline, isoleucine, leucine, lysine monochloride, methionine, ornithine monohydrochloride, phenylalanine, proline, threonine, tryptophan, tyrosine, serine, valine. Histidine (as monohydrochloride) and methionine were determined by first-, second- and third-derivative spectrophotometry in a mixture of several amino acids.

Lead and cadmium in human placentas and maternal and neonatal blood (in a heavily polluted area) measured by graphite furnace atomic absorption spectrometry.

OBJECTIVE: To measure the concentrations of the trace elements lead and cadmium in human placenta and in maternal and neonatal (cord) blood. To assess the influence of the strongly polluted environment on the content of metals in tissues and on the permeability of placenta to cadmium and lead. Various methods of mineralisation were tested before analysis. METHODS: Graphite furnace atomic absorption spectrometry was used for the determination of lead and cadmium. The samples for analysis were prepared by mineralisation under pressure in a Teflon bomb (HNO3, 110 degrees C), by wet ashing under normal pressure (HNO3 + H2O2 for 12 hours), and by microwave digestion in concentrated nitric acid. RESULTS: In analysed samples the following mean concentrations of cadmium and lead were found: in venous blood Pb = 72.50 ng/ml, Cd = 4.90 ng/ml; in placenta Pb = 0.50 microgram/g, Cd = 0.11 microgram/g; in cord blood Pb = 38.31 ng/ml, Cd = 1.13 ng/ml. CONCLUSION: High concentrations of lead and cadmium were found in placentas and in maternal blood whereas in neonatal blood there was an increased concentration of lead and only traces of cadmium. It is concluded that the placenta is a better barrier for cadmium than for lead. Among the examined methods of mineralisation, microwave digestion was the best.

The analysis of lead, cadmium, zinc, copper and nickel content in human bones from the upper Silesian industrial district.

The concentration of lead, cadmium, zinc, copper and nickel in autopsy samples of bones from adults living in the Upper Silesian industrial district (Poland)--an ecological disaster region--was determined by atomic absorption spectrometry (flame and flameless GF AAS). Lead concentrations ranged from 20 micrograms/g to 200 micrograms/g bone wet weight, cadmium from 0.4 microgram/g to 1.5 micrograms/g bone wet weight. About one-fourth of the bones examined from Silesia, contained lead in the range from 100 micrograms/g to 200 micrograms/g. The were no significant differences in zinc, copper and nickel concentration between the control groups. The samples were mineralized in a microwave digestion system. To avoid anomalous results caused by the influence of the matrix Ca3 (PO4)2--the procedure of lead determination was carried out at a temperature of 2000 degrees C, the cadmium determination at a temperature of about 1200 degrees C.

UV spectrophotometric investigations of biologically active maleic anhydride copolymers.

UV spectra and absorption coefficients of biologically active copolymers of maleic anhydride with carbohydrates, terpenes and 1,3-dioxane derivatives were measured. The quantitative relationships of absorbance vs concentrations of polymers were determined as well. The possibility of determining the copolymer contamination due to monomers and oligomeric compounds was shown.

Spectrophotometric and volumetric molybdenum determination with 2,2'-biquinoxalyl.

Volumetric and spectrophotometric methods for molybdenum determination based on reaction with 2,2'-biquinoxalyl (2,2'-BQx) in concentrated hydrochloric acid media have been developed. Absorption spectra of the 1,1'-dihydro 2,2'-biquinoxalylene complex shows the most intensive absorption band at 685 nm with molar absorptivity = 3.3 x 10(4) 1. mole(-1). cm(-1). The compound is characterized by good durability to high temperatures and concentrated acid media. The mentioned indicator gives distinct colour changes at the titration end-point. The spectrophotometric method for molybdenum determination is based on the use of the difference in absorbance between the oxidized and reduced forms of 2,2'-BQx. The indicator is reduced with Sn(II) and then part of it is reoxided as a result of addition of Mo(VI). The difference in absorbance between the blank determination and molybdenum sample increases linearily in the concentration range 0.2-2.0 mug Mo/cm(3). 2,2'-Biquinoxalyl was used as an indicator in the volumetric method for the determination of molybdenum concentrations in steel alloy. The interfering ions FE(III) and Cr(III) are easily eliminated as the precipitate of hydroxides. The mineral acids, hydrochloric sulphuric and perchloric acids, have been tested as reaction media.