E. Pianezzola

Ratio of the R and S enantiomers of salsolinol in food and human urine

Salsolinol is present in human fluids and tissues as well as in some foods and beverages. It was found previously that the R enantiomer of salsolinol predominates in human urine whereas the S enantiomer predominates in Port wine. In this study a new methodology for measuring the proportion of the R and S salsolinol enantiomers in dried banana and human urine is described. In dried banana, a food particularly rich in salsolinol, the R/S ratio was found to be very near to 1. In urine from additional healthy volunteers, the presence of only the R enantiomer was detected. The origin of urinary salsolinol and its enantiomeric composition are discussed with respect to exogenous salsolinol.

Sensitive procedure for the determination of reboxetine enantiomers in human plasma by reversed-phase high-performance liquid chromatography with fluorimetric detection after chiral derivatization with (+)-1-(9-fluorenyl)ethyl chloroformate.

A sensitive and selective high-performance liquid chromatographic method for the determination of reboxetine enantiomers in human plasma was developed. Although two chiral centres are present in reboxetine, its stereospecific synthesis leads to two rather than four possible enantiomers. After extraction from plasma and reaction with (+)-1-(9-fluorenyl)ethyl chloroformate, reboxetine enantiomers were separated as diastereoisomeric derivatives by reversed-phase high-performance liquid chromatography (HPLC) and determined by fluorimetric detection. The HPLC analysis time was about 90 min. The linearity, precision, accuracy and limit of quantification of the method were evaluated. No interference from blank plasma sample was observed. The suitability of the method for in vivo samples was assessed by the analysis of plasma samples obtained from a healthy male volunteer who had received a single oral dose of 4 mg of reboxetine in tablet form.

Determination of ethambutol in human plasma and urine by high-performance liquid chromatography with fluorescence detection

A sensitive and selective HPLC method for the determination of ethambutol in human plasma and urine was developed. Ethambutol was extracted from basified plasma samples (0.2 ml) with diethyl ether, back-extracted into 0.01 M phosphoric acid and derivatized with 4-fluoro-7-nitrobenzo-2-oxa-1, 3-diazole. After 30 min at 80 degrees C and elimination of the reactive excess, the compound was determined by reversed-phase liquid chromatography. urine was analysed for ethambutol after dilution 1:200 with distilled water and derivatization as described for plasma. Quantification in plasma and urine was achieved by fluorescence detection of the eluate. The linearity, precision and accuracy of the method were evaluated. No interference from the constituents of human plasma and urine was observed. The limit of quantification was 10 ng/ml in plasma and 10 micrograms/ml in urine. The suitability of the method for in vivo samples was checked by analysis of plasma and urine samples drawn from healthy volunteers who had received a 1200-mg oral dose of the test compound.

A sensitive procedure for the quantitation of free and N-(2-hydroxypropyl)methacrylamide polymer-bound doxorubicin (PK1) and some of its metabolites, 13-dihydrodoxorubicin, 13-dihydrodoxorubicinone and doxorubicinone, in human plasma and urine by reversed-phase HPLC with fluorimetric detection☆

A high-performance liquid chromatographic assay has been developed and validated for the determination in plasma and urine of doxorubicin (DXR) and some of its metabolites released in vivo from an N-(2-hydroxypropyl)methacrylamide (HPMA) polymer containing DXR linked through its aminosugar moiety to the polymer via an oligopeptide spacer (PK1). The method also allows measurement of the DXR still bound to the polymer. Following addition of two internal standards, the free compounds were extracted twice with isopropanol-chloroform (25:75, v/v). The first extraction was performed at physiological pH and the second after buffering at pH 8.4, in order to extract the aglycones and the glycosides, respectively. Determination of total DXR (polymer-bound plus free DXR) was performed, after quantitative acid hydrolysis to release doxorubicinone from free or polymer-bound DXR, by extraction with the same solvent mixture at pH 7.4. In both cases the organic phase was evaporated to dryness; the compounds were then separated by reversed-phase high-performance liquid chromatography (HPLC) under isocratic conditions and quantitated by fluorimetric detection. In the chromatograms all the analytes appeared to be separated at the baseline and no interference from blank human plasma and urine was observed. The suitability of the method for in vivo samples was checked by the analysis of plasma and urine samples obtained from a cancer patient who had received a single intravenous dose of the test compound.

Sensitive high-performance liquid chromatographic method for the determination of the three main metabolites of selegiline (L-deprenyl) in human plasma

A high-performance liquid chromatographic (HPLC) method with fluorescence detection was developed for the determination in human plasma of the three main metabolites of selegiline (L-deprenyl): amphetamine, methamphetamine and norselegiline. The HPLC separation of the analytes was performed under isocratic conditions, after extraction from plasma and precolumn derivatization with 9-fluorenylmethyl chloroformate. The linearity, precision and accuracy of the method were evaluated; the limit of quantification for all three metabolites in plasma was 0.5 ng/ml.

Determination of exemestane, a new aromatase inhibitor, in plasma by high-performance liquid chromatography with ultraviolet detection

A sensitive and selective high-performance liquid chromatographic method for the determination of 6-methylen-androsta-1,4-diene-3,17-dione (exemestane) and its 17-dihydro metabolite in human plasma has been developed. The analytes and internal standard (Norgestrel) were extracted from plasma samples with a methylene chloride-iso-octane mixture; the organic phase was dried and the residue was reconstituted with an acetonitrile-water mixture, then analyzed by reversed-phase liquid chromatography. Quantification was achieved by ultraviolet detection of the eluate. The linearity, precision and accuracy of the method were evaluated. No interference from the constituents of human blank plasma was observed. The lower limit of quantification was 10 ng/ml plasma. The suitability of the method for in vivo samples was checked by analysis of plasma samples drawn from healthy male volunteers who had received a 200-mg single oral dose of the test compound.

Determination of cabergoline in plasma and urine by high-performance liquid chromatography with electrochemical detection.

A sensitive and selective high-performance liquid chromatographic method for the determination of cabergoline in plasma and urine has been developed. After buffering plasma and urine samples, cabergoline was extracted with a methylene chloride-isooctane mixture, back-extracted into 0.1 M phosphoric acid, then analysed by reversed-phase high-performance liquid chromatography. Quantitation was achieved by electrochemical detection of the eluate. The linearity, precision and accuracy of the method were evaluated. No interference from the biological matrices (human plasma and urine) was observed. The assay was still inadequate in terms of sensitivity for the quantitation of cabergoline plasma concentrations after a single oral dose of 1 mg of the drug to humans, but was successfully used in the determination of the urinary excretion of the drug.

STEREOSELECTIVITY OF IDARUBICIN REDUCTION IN VARIOUS ANIMAL SPECIES AND HUMANS

1. The (13S)-dihydro derivative of idarubicin, (13S)-idarubicinol, is the major urinary metabolite of idarubicin in humans. Idarubicinol epimers were quantified by h.p.l.c. in urine from rats, mice, rabbits, dogs and man after i.v. administration of idarubicin, and in man after oral dosing. The (13R)- and (13S)-epimers of idarubicinol were determined in rat bile. 2. After i.v. injection of idarubicin. (13R)-idarubicinol was not detectable in mice and rabbit urine and no more than 0.5% of the dose was present in the urine of other species. In man, the proportion of (13R)-idarubicinol in total idarubicinol was similar after i.v. (4.1%) and oral (3.8-5.0%) administration of idarubicin; the same applies to rat bile and urine. 3. Reduction of idarubicin in vivo is dependent upon ketone reductases, and proceeds more stereoselectively than that of most ketones giving rise to the (13S)-epimer almost exclusively. The high stereospecificity in idarubicin reduction might result from chiral induction due to the presence of asymmetric centres near to the carbonyl group in idarubicin.

A STUDY OF THE EFFECTS OF RIFABUTIN ON ISONIAZID PHARMACOKINETICS AND METABOLISM IN HEALTHY VOLUNTEERS

The effect of repeated administration of rifabutin on the pharmacokinetics and metabolism of isoniazid was evaluated in 6 healthy volunteers. The subjects received on day 1 and 9 a single oral dose of 300 mg isoniazid and from day 2 to 8 a single daily oral dose of 300 mg rifabutin. Two out of 6 subjects were shown to be rapid acetylators. No significant modification of the plasma pharmacokinetic profiles of isoniazid and acetylisoniazid was found. Evidence exists in the present study for autoinduction of rifabutin metabolism; this is shown by the lower plasma concentrations obtained 24 h after the seventh dose as compared to the theoretical concentrations.

Radioimmunoassay for the synthetic ergoline derivative cabergoline in biological fluids.

An antiserum against cabergoline, a powerful dopamine-agonist under clinical trials for the treatment of Parkinsons disease and hyperprolactinemia, has been raised in rabbits by immunization with an immunogen produced by conjugation of cabergoline to bovine serum albumin. The antiserum was able to bind a derivative of cabergoline labelled with tritium and was able to distinguish the drug molecule from some of its close related compounds and from other agents that could be simultaneously present in plasma from patients undergoing treatment with cabergoline. The antiserum and the tritium labelled hapten were used to develop a radioimmunoassay for cabergoline determination in human plasma and urine. A linear relationship between cabergoline added and % radioactivity bound was found in the range 1.9-500 pg/tube. The addition in the assay of 200 microliters human plasma or 25 microliters urine did not affect the specific and the non-specific binding of the radiolabelled hapten so enabling us to obtain a final sensitivity of about 12 pg/ml plasma and 120 pg/ml urine. The assay was validated in terms of reproducibility, precision and accuracy over the whole range of concentrations tested both in plasma and urine. The plasma concentrations at the steady state in a patient with Parkinsons disease who had received the drug at single oral daily doses of 3, 5 and 7 mg were determined using the assay.