Robert Lejeune

Chemiluminescence as diagnostic tool. A review.

The principles of chemiluminescence and its applications as diagnostic tool are reviewed. After an introduction to the theoretical aspects of luminescence and energy transfer, the different classes of chemiluminogenic labels including luminol, acridinium compounds, coelenterazine and analogues, dioxetanes, systems based on peroxyoxalic acid and their derivatives are described emphasizing the molecules which best fulfil the requirements of todays clinical chemistry. Applications of chemiluminescence and enhanced chemiluminescence to immunoassays, receptor assays, DNA probes, biosensors and oxygen metabolism are discussed as well as the role of enzymes in the selectivity and the sensitivity of these reactions.

Direct determination of tagitinin C in Tithonia diversifolia leaves by on-line coupling of supercritical carbon dioxide extraction to FT-IR spectroscopy by means of optical fibres

Supercritical fluid extraction (SFE) with carbon dioxide as extraction medium was on-line coupled to a FT-IR spectrometer equipped with a Mercury Cadmium Telluride (MCT) detector using a tailor-made high-pressure fibre optic flow cell. This method was optimised and developed for the monitoring in real time and the quantification of dynamic extractions of tagitinin C from Tithonia diversifolia leaves. In order to demonstrate the method ability to allow the direct quantification of tagitinin C in the extract medium the standard addition method was used. The area integration of curves obtained by plotting the absorbance of the highly specific C=O stretching vibration at 1668cm(-1) versus time (i.e. extractograms) was used as instrumental response. The SFE/FT-IR process was successfully validated using the accuracy profile as decision tool. On this basis, a linear regression model was chosen for the calibration curve. The relative standard deviation for repeatability and intermediate precision were between 0.8 and 3.1 %, respectively. Moreover, the method was found to be accurate as the two-sided 95% beta-expectation tolerance interval did not exceed the acceptance limits of 85 and 115% on the analytical range investigated (500-2500microg of added amount of tagitinin C). The proposed method allowed the non-destructive extraction of tagitinin C and its on-line quantitative determination in less than 25min thus facilitating the subsequent experiments or the pharmacological studies performed on this compound.

High stability and high efficiency chemiluminescent acridinium compounds obtained from 9‐acridine carboxylic esters of hydroxamic and sulphohydroxamic acids

A series of hydroxamic acids and sulphohydroxamic acids were prepared and linked to 9-acridinecarboxylic acid through a pseudo-ester function. After N-methylation of the heterocyclic ring, the different compounds were tested for their chemiluminescent properties. Substituents on the hydroxamic functions have shown various effects (steric or electronic) on the luminescence yield or stability of the molecule. The most interesting derivatives were selected in terms of chemical stability and chemiluminescence efficiency. 9-[(N-hydroxysuccinimidyl-4-oxo-4-N-phenylaminobutanoate)N-carb oxylat e]-10-methyl-acridinium (FA6), 9-(N-phenylpivalamide-N-carboxylate)-10-methylacridinium (FA17) and 9-(N-phenylpivalamide N-carboxylate)-10-carboxymethyl-acridinium (FA18) iodomercurates are very promising as chemiluminescent labels. These compounds can be detected at very low levels (10(-16)-10(-17) mol/L) and in our stability evaluation, FA6, FA17 and FA18 showed similar results to the acridinium ester DMAE. Their half-lives at 20 degrees C are greater than 2 weeks.

Contribution a l'Etude Electocimique de Derives de l'Acide Mercapto-2 Pyridinecarboxylique-5. I

Abstract 5-methoxycarbonoyl-1-methyl-2-thio-1,2-dihydropyridine and 1-metyl -2-thio-1,2-dihydropyridine-5-carboxylic acid heve been studied by recent electrochemical methods. The analysis of classical, alternative (A.C.) and pulse polarograms led to precise the nature of the observed currents. The pH variatiors and the supporting electrolyte modifications have an important influence on these currents.

Development and validation of a quantitative method for the selective determination of tin species in tin octoate by differential pulse polarography

Tin octoate is used as a catalyst in the synthesis of polydimethylsiloxane (PDMS), a room temperature vulcanizing (RTV) silicone rubber. This rubber is largely used in the medical field due to its great biocompatibility. In this framework, a high-speed and costless analytical method for the determination of stannic ions, Sn(IV), in the presence of stannous ions, Sn(II), has been developed. The separation of these two ions was carried out using differential pulse polarography (DPP). For this purpose, the tin species contents in the catalyst is quantitatively extracted under inert condition to avoid any changes in the ratio Sn(IV)/Sn(II). Polarography showed well-shaped oxidation and reduction peaks respectively at -650 and -860mV for stannous ions. The peak of the stannic ion was well separated and appeared at -1210mV. Many parameters such as extraction process, extraction time, pH, chelating agents and polarographic conditions were optimized. We have also demonstrated that no oxidation of the stannous ions occurred during the sample preparation. The dosing range considered in this study extends between 10 and 40mug/mL, corresponding to 6.8% and 27.2% of the degradation product (Sn(IV)) in the catalyst, regarding to the sampling. Finally this method was successfully validated using the total error concept.

FT–IR measurement of tagitinin C after solvent extraction from Tithonia diversifolia

Tagitinin C, an antiplasmodial compound, identified as one major compound of the subtropical medicinal plant, Tithonia diversifolia, was determined by FT-IR spectroscopy method. The crude ether extracts from aerial parts of the plant were evaporated to dryness and re-dissolved in tetrachloroethylene (C(2)Cl(4)) before analysis. The magnitude of the absorbance of the very specific CO stretching vibration (nu(CO)) at 1664.8cm(-1) was exploited in order to quantify tagitinin C. The determination coefficient (r(2)) of the calibration scale was 0.9994, the detection limit was lower than 3mugml(-1) and the quantification limit was lower than 10mugml(-1). Recovery values from 100.5 to 101.7% were found for spiked concentration levels from 19.91 to 89.95mugml(-1). The main characteristics of the curves obtained from the calibration standards and from the standard addition technique were not statistically different (Student t-test) suggesting that matrix effects were negligible. The results obtained for the determination of tagitinin C in the crude ether extract from aerial parts of T. diversifolia by LC and FT-IR spectroscopic method agreed well: 0.76+/-0.02 and 0.773+/-0.009, of tagitinin C in dried plant respectively.

Contribution à l'étude Électrochimique de Dérivés de l'Acide Mercapto-2 Pyridinecarboxylique-5. II

Abstract Electrochemical caracteristics of 2-mercaptopyridine-5-carboxylic acid and its methylic ester have been determined using recent polarographic techniques. Measurements have been carried out as a function of parameters such as frequency, others surimposed signals and as a function of concentration and pH. Results are compared to those obtained for selectively methylated on sulfur or nitrogen atoms derivatives. Interpretations of the data are given. The optimal conditions for a quantitative determination by D.C. and differential pulse polarography are fixed.

Coupling of biologically active steroids to conjugating arms through ether linkages for use in immunochemistry

Conjugation of haptens through ether linkages avoids leakage problems in immunoassays, but this procedure is not easily applied to most steroids that bear low reacting hydroxyls. A new technique allowing the ether coupling of biologically active steroids with conjugating arms in mild conditions compatible with thermosensitive protecting groups is presented. In the first step, the solvent (an aromatic hydrocarbon) was dehydrated by azeotropic distillation in a soxhlet apparatus using a cartridge filled with 0.3 nm and 0.4 nm molecular sieves. In this protected medium, a thallium steroid alkoxide was completely formed by reaction of the steroid with thallium ethoxide and by the continuous elimination of ethanol. The halogenated chain was then introduced into the same medium and reacted in the absence of moisture to give the ether. 17beta-Hydroxy and 11alpha-hydroxy derivatives were involved in this reaction. The coupling was effective for all of the compounds tested after 2-36 h of reaction time and at temperatures between 80 and 140 degrees C. The conjugates were at least 95% pure, and yields ranged from 15 to 95%.

Determination of Zn, Cu, Ni and Co by Voltammetry in Plasma, without Mineralisation

Abstract Procedures are presented to determine successively Zn, Cu, Ni and Co in biological fluids by stripping voltammetry. Description of a desorption technique to separate proteins from metals is given and the efficiency is checked up on certified serum (seronormR). Comparison with wet mineralisation and oxygen burning is made. The program of differential pulse anodic stripping voltammetry (for Zn and Cu) and of adsorptive cathodic stripping voltammetry (for Ni and Co) is described.

Electrochemical Study Of 2-Mercaptopyridin-4-Caroxylic Acid and its Derivatives

Abstract The N-methylated derivatives of 2-mercapto-4-pyridincarboxylic acid have been studied by voltammetry as a function of polarographic parameters and pH. Their electrochemical behaviour has led us to propose a monoelectronic reduction of these compounds followed by a dimerization and a loss of sulfur compounds. The best conditions for the N-methylated derivatives determination by direct current and differential pulse polarography are also described.