Determination of alpidem, an imidazopyridine anxiolytic, and its metabolites by column-switching high-performance liquid chromatography with fluorescence detection.

Alpidem, 6-chloro-2-(4-chlorophenyl)-N,N-dipropylimidazo[1,2-a]pyridine- 3-acetamide, is an anxiolytic imidazopyridine that undergoes a first-pass elimination after oral administration to humans; it is actively metabolized and three circulating metabolites have been identified in plasma due to N-dealkylation, oxidation or a combination of both processes. For the determination of the unchanged drug and its metabolites in human plasma, a column-switching HPLC method was developed. The method, based on solid-phase extraction (performed on-line), involves the automatic injection of plasma samples (200 microliters) on to a precolumn filled with C18 material, clean-up of the sample with water in order to remove protein and salts and transfer of the analytes to the analytical column (after valve switching) by means of the mobile phase. All the processes were performed in the presence of an internal standard, a compound chemically related to alpidem. During the analytical chromatography, the precolumn was flushed with different solvents and after regeneration with water, it was ready for further injections. The analytical column was a C8 type and the mobile phase was acetonitrile-methanol-phosphate buffer solution (45:15:45, v/v/v) at a flow-rate of 1.5 ml min-1. The column was connected to a fluorimetric detector operating at excitation and emission wavelengths of 255 and 423 nm, respectively. The limits of quantitation of alpidem and three metabolites were 2.5 and 1.5 ng ml-1, respectively, in human plasma.

Determination of zolpidem, a new sleep-inducing agent, and its metabolites in biological fluids: pharmacokinetics, drug metabolism and overdosing investigations in humans.

For the determination of zolpidem, a new sleep inducer, and its metabolites in human plasma and urine, three methods were developed that are suitable for pharmacokinetics, drug metabolism and overdosing investigations. The methods used for pharmacokinetic and drug metabolism studies are based on column-switching high-performance liquid chromatography; they do not require any sample manipulation because the plasma or diluted urine is injected into a pre-column where clean-up and preconcentration take place. The analytes are transferred by valve-switching to the C18 analytical column for chromatography. To investigate overdose cases, urine samples only are used: the method is simple, because the diluted urine can be injected directly into the analytical column (phenyl type). This allows the identification and quantification of the principal urinary metabolite of zolpidem, the unchanged drug being practically undetectable. All the methods use fluorescence detection, which affords high sensitivity and selectivity. It is necessary to use a method capable of the determination of metabolites even if these are apparently pharmacologically inactive, because in different physiopathological populations the qualitative and quantitative metabolic profiles of zolpidem could be different. The method designed for the investigation of (accidental or deliberate) overdose cases is, as required on such occasions, simple and rapid, with good selectivity with respect to commonly prescribed psychotropic drugs.

Stimulation of arachidonic acid metabolism by CCl4 in isolated rat hepatocytes.

Arachidonic acid metabolism was evaluated in isolated rat hepatocytes after CCl4 exposure. CCl4 induced dose-dependently the synthesis and release of prostacyclin (PGI2) and thromboxane (TXB2). Treatment with prostaglandin E2 (PGE2) 30 min after exposure to CCl4, significantly reduced the cell damage as well as the release of TXB2 from the cells.

The fate of leached di-(2-ethylhexyl)-phthalate (DEHP) in patients on chronic haemodialysis.

The fate of the plasticiser di-(2-ethylhexyl)-phthalate and/or its metabolites, phthalic acid esters (PAE), in 12 patients on chronic haemodialysis was studied. The total amount of PAE retained by the patients was estimated by monitoring the plasma concentrations from the inflow and outflow tubes of the dialyzer during 4-h dialysis sessions. There was an estimated uptake of 46 mg of PAE during a single dialysis session. The values for a volumetric factor (Vf) related to the increment in plasma PAE concentrations were found to increase during the first hour of treatment (72 litres at steady-state), and then to progressively decrease. The changes in the kinetic parameters during the dialysis session were grouped into three phases according to the fate of the plasticiser in the patient. We also monitored the plasma concentrations of PAE in the same patients for 40 days during dialysis with another kind of plasticised (tri-(2-ethylhexyl)-trimellitate [TOTM]) tubes. The PAE concentrations were similar to those found in healthy humans after about 5 weeks.

Leachability of a new plasticizer tri-(2-ethylhexyl)-trimellitate from haemodialysis tubing.

In two groups of patients on chronic haemodialysis treatment, the common PVC-DEHP blood tubing was replaced with tubing containing tri-(2-ethylhexyl)-trimellate (TOTM) as plasticizer. The aim of the present study was to measure the amount of TOTM and/or its metabolites (TAE s) in plasma, resulting from TOTM that might leach from the dialysis tubes. The arterial levels of TAE s at the start of the dialysis sessions were monitored by Selected Ion Monitoring (SIM) analysis once a week for a period of 42 days. A gradual decrease in TAE was observed during the first 21 days of the haemodialysis treatment with the PVC-TOTM tubes. After three weeks, the TAE levels remained constant until the end of the study. On day 120 of the haemodialysis treatment, the plasma TAE concentrations from the inflow and outflow tubes of the dialyzer were monitored during a single haemodialysis session at different times after starting dialysis. At the beginning of the dialysis session, the mean concentration of TAE was 55.81 +/- 14.98 ng/ml (mean +/- standard error), while at the end the levels were 73.34 +/- 17.05 ng/ml. There were no significant differences between venous and arterial sampling points in the trimellitic ester concentrations. Less TOTM is apparently leached from haemodialysis than DEHP. TOTM can be recommended as an alternative plasticizer to DEHP, but its possible toxicity in humans should be investigated before it can be used routinely.

Reduction rate of 14C-Carmoisine by resting cell bacterial suspension from human and rat faeces.

14C-Carmoisine (250/ug; 1.25 x 10(6) dpm) was incubated under strictly anaerobic conditions with resting cell suspension from stool specimens collected from male rats and human male healthy adults. The kinetics of azoreduction was determined as amount of naphthionic acid (NA), the stable metabolite of Carmoisine produced by the activity of the anaerobic bacteria. The analytical determinations were performed by radio-HPLC technique. There were no significant qualitative differences in the radiochromatographic profiles of samples obtained from human and rat flora suspensions. The calculated reduction rates were 5.03 +/- 0.18 nmoles of NA/250/ug protein/min, and 1.72 +/- 0.12 nmoles of NA/250/ug protein/min for rat and human faecal resting cells respectively. From our results it seems that the enzymatic reaction, following zero order kinetics, is similar for the two species and only the reduction rate is different.

Lack of correlation between TPA-induced prostaglandin biosynthesis and ornithine decarboxylase activity in Balb/c mouse 3T3 fibroblasts.

12-O-tetradecanoylphorbol-13-acetate (TPA) induced in Balb/c 3T3 cells an earliest prostaglandin biosynthesis and an ornithine decarboxylase activation, this time-relation being more evident if serum was added to incubation medium in low concentration (0.2%). However the two TPA-induced events can be almost totally dissociated by pharmacological means, such as indomethacin and calcium-ionophore A23187 which affected PG response to TPA, but did not influence ODC induction.

Similar profile of urinary and faecal metabolites of 14C-carmoisine in male and pregnant female rats after oral administration.

Pregnant rats received 14C-Carmoisine (200 mg kg-1; 25 microCi) by gavage on days 16-19 of gestation. The animals were killed and maternal tissues, amniotic fluid, placentae, foetal membranes and foetuses were analyzed for radioactivity. No evidence for the transplacental transfer of 14C-Carmoisine or its metabolites was obtained. Male rats were given a single oral administration of 14C-Carmoisine (200 mg kg-1; 25 microCi) and killed at different times after dosing. In both male and female animals, more than 90% of the radioactivity was excreted in faeces and urine within 64 h, and the results suggested that there was no significant absorption of the azodye and no preferential concentration of the red food colour or its metabolites in any particular tissue. Analyses by HPLC, combined with a radioactivity monitor (RAM), of urine and faeces of such animals show that five radioactive peaks were present in the radiochromatogram in addition to unmodified Carmoisine. The mean peak shows the retention time and the u.v. spectrum of authentic naphthionic acid. The results demonstrate that the pregnancy does not affect the kinetic and the metabolic profile of a single oral administration of the azodye Carmoisine given at different days of gestation.