Sample preparation including sol-gel immunoaffinity chromatography for determination of bisphenol A in canned beverages, fruits and vegetables.

The paper describes the development of a very simple method to prepare samples of canned food (beverages, fruits and vegetables) for the determination of bisphenol A by isocratic HPLC with fluorescence detection. The new sample preparation method makes use of the selectivity of bisphenol A antibodies immobilized in a silica matrix by an inexpensive and simple sol-gel technique. In spite of applying highly complex food matrices, immunoaffinity columns could be used for clean-up of at least 15 real samples. Limits of detection (S/N=3) ranged from 0.1 ng/ml for beverages to 4.3 ng/g for vegetables.

Determination of less polar heterocyclic aromatic amines in standardised beef extracts and cooked meat consumed in Austria by liquid chromatography and fluorescence detection.

An analysis method was developed for the determination of trace levels of less polar heterocyclic aromatic amines (HAs) in food samples. The development started from a frequently used sample pre-treatment scheme which was slightly improved to make it applicable with high-performance liquid chromatography (HPLC) with fluorescence detection. The method was applied for the analysis of a standardised beef extract containing 5-15 ng/g of HAs and the results are compared with those of the other participants in the same European project. In addition, the method was used for the analysis of less polar HAs in cooked meat consumed in Austria.

Determination of heterocyclic aromatic amines in beef extract, cooked meat and rat urine by liquid chromatography with coulometric electrode array detection.

This paper describes a method for the determination of heterocyclic aromatic amines (HAs; DMIP, IQ, MeIQ, MeIQx, 4,8-DiMeIQx, 7,8-DiMeIQx, AalphaC, PhIP) by high-performance liquid chromatography (HPLC) with coulometric electrode array detection. The compounds are separated on reversed phase columns (LiChroCart Superspher 60 RP-select B, 250 mm x 2 mm, 4 microm and LiChrospher 60 RP-select B, 250 mm x 4 mm, 5 microm) using mobile phases consisting of acetonitrile/buffer/distilled water and detected at eight working electrodes at potentials between +190 and +680 mV against modified palladium electrodes. In the context of an EU-interlaboratory exercise, the method was applied to analyse a standardised test solution and--after isolation of the analytes by several clean-up steps--for the analysis of standardised beef extract and grilled meat. Further, the method could be applied for the analysis of HAs in suspensions of bacteria and rat urine without any sample preparation step beyond sample dilution. The data obtained show that HPLC with coulometric electrode array detection gives accurate results.

Determination of fifteen nucleotides in cultured human mononuclear blood and umbilical vein endothelial cells by solvent generated ion-pair chromatography.

The paper describes the development of a method for the determination of 15 nucleotides in cultured mononuclear blood and umbilical vein endothelial cell lysates by solvent generated ion-pair chromatography. The phase system is generated via a mobile phase of 100 mM phosphoric acid adjusted to pH 6.2 with triethylamine. Nucleotides are eluted by applying a linear magnesium ion gradient. The method is robust, highly reproducible and easily adaptable to other cell lysates and allows the separation and quantitation of the nucleotides with detection limits in the range from 17 (ADP) to 126 (CDP) pmol in 20-microl aliquots.

In vitro effects of mycophenolic acid on the nucleotide pool and on the expression of adhesion molecules of human umbilical vein endothelial cells.

The immunosuppressive drug mycophenolate mofetil (MMF) and its active metabolite mycophenolic acid (MPA) selectively inhibit inosine 5'-monophosphate dehydrogenase (IMPDH), and therefore interfere with cellular guanine nucleotide biosynthesis. IMPDH is additionally involved in the synthesis of membrane glycoproteins, some of which are adhesion receptors known to play an active part in the regulation of cell-cell contacts, which are crucial in the process of recruitment and transendothelial infiltration of activated leucocytes in the transplanted organ. As a consequence, MPA leads to a reduction of cellular infiltrates in the course of transplant rejection. In the present study, the effects of MPA on human umbilical vein endothelial cells (HUVEC) are investigated at both molecular and cellular levels. In our experiments, HUVECs are treated with tumor necrosis factor-alpha (TNF-alpha; 10 ng/ml) in order to mimic activation occurring at a rejection crisis. The dose-dependent influence of concomitant incubation with MPA (5-20 micromol/l; 48 h, 37 degrees C, 5% CO2) on their intracellular nucleotide profile is observed by determining the concentrations of purine and pyrimidine nucleotides, using a HPLC method based on solvent generated ion-exchange. The possibility of synergistic effects is investigated by incubating endothelial cells with mixtures of three different immunosuppressants (mycophenolic acid; cyclosporin A, 100 ng/ml; prednisolone, 1 micromol/l)--a combination commonly used after transplantation--varying the amount of MPA (5-20 micromol/l). Stimulation with TNFalpha does not significantly modulate the intracellular levels of nucleotides quantitated. In the presence of MPA concentrations of at least 5 micromol/l, GTP levels (68+/-12%) are significantly decreased compared to controls (100%). At a concentration of 20 micromol/l MPA, the GTP amount is reduced to 58+/-7%. In contrast to these observations, the levels of UDP and UTP are increasing significantly under coincubation with MPA concentrations greater than 5 micromol/l. At 20 micromol/l MPA, UDP and UTP are increased to 147+/-19% and 114+/-11%, respectively. All other nucleotides (CTP, ADP, ATP) reveal no significant alterations in their intracellular concentrations under the conditions applied. Incubation of TNFalpha-treated HUVEC monolayers, with a mixture of three immunosuppressive drugs varying the amount of MPA, show no significant differences compared with the data observed after incubation with MPA alone. In addition, the influence of MPA (10 micromol/l) on a cellular level is observed by measuring the cell surface expression of adhesion molecules on cytokine-stimulated HUVECs, using TNFalpha (10 ng/ml), interferon-gamma (100 ng/ml), interleukin-1beta (10 ng/ml) and interleukin-8 (20 ng/ml). Expression of the intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), endothelial leucocyte adhesion molecule-1 (ELAM-1) and platelet endothelial cell adhesion molecule-1 (PECAM-1) was assessed by flow cytometry. Activation of endothelial cell monolayers with TNFalpha significantly increases the mean fluorescence intensity of VCAM-1 (361+/-14%) and ICAM-1 (429+/-47%) surface expression, compared to controls, and additionally induces E-selectin expression (2919+/-134%). The same tendencies, but in a lesser degree, are observed under stimulation of cells with either IFNgamma or IL-1beta. Incubation with a combination of TNFalpha and MPA leads to a significant reduction in VCAM-1 (329+/-13%) and E-selectin (2613+/-167%) expression, compared to the values obtained for HUVEC incubated with the cytokine alone. Treatment of the cells with IL-1beta/MPA also reduces the expression of VCAM-1 to a level significantly lower than the level observed after stimulation with IL-1beta. Incubation with MPA alone reveals no significant modulation in the expression of all surface molecules tested compared to the values of unstimulated HUVECs. The experiments show that the immunosuppressive action of MPA not only inhibits lymphocyte proliferation but also decreases the expression of adhesion molecules on endothelial cells, which are the first target of the cellular rejection process.

Determination of the effects of mycophenolic acid on the nucleotide pool of human peripheral blood mononuclear cells in vitro by high-performance liquid chromatography.

Immunosuppressive drugs are needed to prevent the rejection of transplanted organs by the immune system. Immunosuppressive antimetabolites act by interrupting cell metabolism. Their mechanism of action can be studied in vitro by measuring the inhibition of biochemical activities which is reflected by changes in the nucleotide content. In our experiments, human peripheral blood mononuclear cells (PBMC) isolated from healthy volunteers were used. After PBMC stimulation with phytohaemagglutinin (PHA) to mimic activation occurring at a rejection crisis, cells were exposed to varying concentrations of different immunosuppressants (i.e., mycophenolic acid, cyclosporin A and prednisolone) for 68 h at 37 degrees C. Changes in nucleotide content were observed by determining the concentrations of 15 nucleotides using a newly developed HPLC method. The results obtained for mycophenolic acid (MPA; final concentrations in a range between 0.1 and 5 micromol/l), cyclosporin A (CsA; final concentrations between 100 ng/ml and 1 microg/ml) and prednisolone (final concentrations between 0.5 and 10 micromol/l) are given as percentage changes in nucleotide content versus controls and are expressed as mean +/- confidence interval. The possibility of synergistic effects was investigated by incubating the cells with mixtures of all three immunosuppressive drugs varying the amount of mycophenolic acid. In addition, we have shown the effects of MPA/guanosine co-incubation on the intracellular nucleotide levels. Stimulation of peripheral blood mononuclear cells with phytohaemagglutinin led to a significant increase of pyrimidine and purine nucleotides versus control values (100%). Pyrimidine (CTP, UDP, UTP) and purine nucleotides (GDP, GTP, ADP, ATP) were elevated up to 153+/-14% and 142+/-17%, respectively. Under co-incubation of cells with MPA, the GTP level decreased in a dose-related manner to 56+/-3% of control at a MPA final concentration of 5 micromol/l. Concomitantly, an increase of UTP values to 203+/-18% versus control was observed under co-incubation with 1 micromol/l MPA. Co-incubation of mononuclear cells with guanosine (50 micromol/l) compensated for the effects of MPA on intracellular GTP levels. Combination of MPA, CsA and prednisolone did not alter intracellular nucleotide profiles of PBMC compared to those under MPA incubation alone. The depletion of the guanine nucleotide pool and concomitant increase of uridine nucleotides under the influence of the immunosuppressive drug mycophenolic acid is caused by its inhibitory effects on the key enzyme of de novo purine biosynthesis, inosine 5'-monophosphate dehydrogenase (IMPDH).

On-line coupling of sol-gel-generated immunoaffinity columns with high-performance liquid chromatography.

The paper demonstrates the possibility to use sol-gel-generated immunoaffinity columns as selective sample preparation step in on-line combination with HPLC. In the past sol-gel-generated immunoaffinity columns have only been included in off-line sample preparation schemes. Compared with conventional RP-materials on-line coupling of sol-gel-generated silica matrices with a pore structure designed to retain antibodies poses additional problems caused by their lower pressure tolerance and by the necessity to match the mobile phases not only to take into account the chromatographic properties but also the conformational stability of the antibodies. These problems have been overcome by an on-line system which can be regarded as a prototype for similar systems which exploit the selectivity of sol-gel immunoaffinity columns. The system consists of a sol-gel-generated immunoaffinity column coupled to an RP enrichment column and an analytical column. The practicality of such systems is demonstrated using the example of anti-pyrene immunoaffinity columns applied for the determination of pyrene in aqueous solutions.

Determination of 1-nitropyrene in herbs after selective enrichment by a sol-gel-generated immunoaffinity column.

Using the determination of 1-nitropyrene as an example the paper demonstrates the advantages of including a highly selective sol-gel-generated immunoaffinity column in the sequence of clean-up steps necessary to determine haptens in complex matrices. The sol-gel method to immobilise antibodies enlarges the variety of immunoaffinity columns available and leads to mechanically stable columns with constant retention characteristics. The sample preparation scheme proposed combines acetonitrile extraction, size-exclusion and immunoaffinity chromatography. 1-Nitropyrene is then separated by reversed-phase HPLC from interfering compounds and determined after catalytic on-line reduction to the corresponding amine by spectrofluorimetry. Concentrations in the range from 0.1 to 1.4 microg/kg 1-nitropyrene were detected in herbs.

Determination of true octanol-water partition coefficients by means of solvent generated liquid-liquid chromatography.

Compared to other methods for the determination of octanol-water partition coefficients chromatography offers a number of advantages: sample purification is unnecessary, the partition coefficients of the components of a mixture can be measured simultaneously and a minimum amount of sample is needed. In the past these determinations were almost exclusively carried out by liquid-solid chromatography (LSC) on alkyl bonded silica as stationary phase (conventionally described as 'reversed-phase liquid chromatography; RPLC). Such systems based on liquid-solid distribution are, however, a poor simulation of liquid-liquid partition. On the other hand liquid-liquid chromatographic columns loaded with high amounts of water-saturated octanol are unstable since they suffer from "column bleeding"--a loss of the stationary liquid octanol phase caused by erosion. It is shown that the technique of solvent generated liquid-liquid chromatography (SGLLC) leads to stable columns with liquid-liquid partition as the predominant retention mechanism, if systematic errors due to specific adsorption effects are avoided by the selection of an "inert" solid support. This is demonstrated by the comparison of LSC and SGLLC data. SGLLC significantly reduces the scattering of the retention data of calibration standards around the calibration line. The use of SGLLC thus significantly improves the accuracy and precision of the resulting octanol-water partition coefficients.