Automated quantitative determination of the new renin inhibitor CGP 60536 by high-performance liquid chromatography.

A fully automated high-performance liquid chromatography method with fluorescence detection for the determination of the renin inhibitor CGP 60536 in animal and human plasma and urine has been developed and validated. After addition of an internal standard, the compounds were automatically extracted from 400 microl of plasma or urine with methyl alcohol-acetic acid (99:1, v/v) on 100-mg Bond-Elut CN cartridges using the Gilson ASPEC system. The on-line chromatographic separation was performed on a LiChrospher 100 RP8 5-microm particle size packed analytical column (25x0.4 cm I.D.). The mobile phase consisted of acetonitrile-0.01 M potassium dihydrogenphosphate (65:35, v/v) at a flow-rate of 0.8 ml/min. The analytes were detected using a fluorescence detector at excitation and emission wavelengths of 280 and 330 nm, respectively. The limit of quantitation was established at 4.5 ng/ml in plasma (accuracy 106% and precision 1%), and 9.0 ng/ml in urine (accuracy 101% and precision 13%). The method was applied to the investigation of the pharmacokinetics of CGP 60536.

Effect of renal impairment on the pharmacokinetics and pharmacodynamics of desirudin.

OBJECTIVE: To investigate the pharmacokinetics and pharmacodynamics of desirudin in subjects with various degrees of renal impairment in comparison with subjects with normal renal function. METHODS: Eight subjects with normal renal function (creatinine clearance > 90 ml/min) received 0.5 mg/kg desirudin intravenously over 30 minutes. Four subjects with mild renal failure (creatinine clearance between 61 and 90 ml/min) received 0.5 mg/kg. Five subjects with moderate renal failure (creatinine clearance between 31 and 60 ml/min) received 0.25 mg/kg. Six subjects with severe renal failure (creatinine clearance < 31 ml/min) received 0.125 mg/kg. RESULTS: Specific maximum concentration values (maximum concentrations corrected to a dose of 1 mg/kg) increased slightly with decreasing creatinine clearance. Mean specific area under the plasma concentration-time curve increased by a factor of 1.15, 2.83, and 7.0 for subjects with mild, moderate, and severe renal failure, respectively, compared with healthy subjects. Total urinary excretion of desirudin was about 55% to 60% of the dose in all four groups; elimination was delayed for subjects with moderate and severe renal failure. Total and renal clearance of desirudin were proportional to creatinine clearance. Total plasma clearance of desirudin was proportional to renal clearance of the drug. Prolongation of activated partial thromboplastin time was increased among subjects with moderate and severe renal failure despite a dose reduction. Area under the dynamic activated partial thromboplastin time curve for subjects with moderate renal failure remained the same as that for healthy subjects despite a dose reduction by a factor of two. Area under the dynamic curve increased by a factor of about 1.5 for subjects with severe renal failure despite a dose reduction by a factor of four. CONCLUSION: A dose reduction by a factor of six is recommended for persons with severe renal failure.

Evaluation of the gastric absorption and emptying of drugs under various pH conditions using a simple intubation method: application to diclofenac.

Sodium diclofenac (50 mg) together with [14 C]-PEG as a non-absorbable marker were dissolved in 400 ml of water (A), phosphate buffer pH 7.5 (B) or a homogenized meal (C). Each of these was ingested in random order by six volunteers on 3 consecutive days. Some gastric absorption of the drug was established with C but the plasma drug concentration-time profiles mainly reflected the process of gastric emptying.

High-performance liquid chromatographic determination of the penem antibiotic (5R,6S)-2-aminomethyl-6-[(1R)-hydroxyethyl]-2-penem-3-carboxylic acid in human plasma and urine.

High-performance liquid chromatographic methods for the determination of (5R,6S)-2-aminomethyl-6-[(1R)-hydroxyethyl]-2-penem-3-carboxylic acid in plasma and urine have been developed, validated and applied to clinical samples. After addition of another penem, (5R,6S)-2-aminoethyl-6-[(1R)-hydroxyethyl]-2-penem-3-carboxylic acid, as an internal standard, plasma proteins are precipitated with a saturated solution of ammonium sulphate. A portion of the supernatant is injected on to a reversed-phase column (RP 8-10 micron) that is eluted with pH 6 phosphate buffer. The urine assay entails a 25-fold dilution with pH 6 buffer and addition of the internal standard prior to injection. The detector response at 320 nm is a linear function of concentration over the ranges 1.6-410 mumol/l (0.4-100 micrograms/ml) and 41-1025 mumol/l (10-250 micrograms/ml) for the plasma and urine assays, respectively. These methods have proved to be suitable for pharmacokinetic investigations in man.

High-performance liquid chromatographic determination of diclofenac and its monohydroxylated metabolites in biological fluids.

Sensitive and selective high-performance liquid chromatographic assays for diclofenac and its monohydroxylated metabolites in biological fluids are described. Using ultraviolet detection at 282 nm, diclofenac is assayed in plasma at concentrations down to 10 ng/ml; total (free + conjugated) diclofenac and its monohydroxylated metabolites (the sum of 3'- + 4'-hydroxydiclofenac and 5-hydroxydiclofenac) are assayed in urine after chemical hydrolysis at concentrations down to 200 ng/ml. The applicability of the described assays is shown.

Gas chromatographic determination of phentolamine (Regitine) in human plasma and urine.

A method for the determination of unconjugated phentolamine at concentrations down to 5 ng/ml in human plasma, and of free and total (free plus conjugated) phentolamine down to 25 ng/ml in urine is described. After addition of 2-[N-(p-chlorophenyl)-N-(m-hydroxyphenyl)-aminomethyl]-2-imidazoline as internal standard, both compounds are extracted into benzene-ethyl acetate (1:1, v/v) at pH 10, transferred into an acidic aqueous solution and back-extracted at pH 10 into benzene-ethyl acetate. They are then derivatized with N-heptafluorobutyrylimidazole. The derivatives are determined by gas chromatography using a 63Ni electron-capture detector. In urine, total (free plus conjugated) phentolamine is determined after enzymatic hydrolysis. The technique was applied for the study of the plasma concentrations and urinary elimination after oral administration to man.

Determination of clomipramine or imipramine and their mono-demethylated metabolites in human blood or plasma by high-performance liquid chromatography.

A high-performance liquid chromatographic method for the quantitative assay of clomipramine or imipramine and their mono-demethylated metabolites in human blood or plasma is described. After addition of the internal standards, imipramine for clomipramine, desipramine for desmethylclomipramine and conversely, the compounds are extracted from blood or plasma at pH 10 into heptane containing 1% isoamyl alcohol; they are then back-extracted into an acidic aqueous phase and re-extracted at a basic pH into heptane. After evaporation, the residue is dissolved in 300 microliters of mobile phase and 150 microliters is injected. Both drugs and their mono-demethylated metabolites are well separated from the blood or plasma components and the other metabolites on a silica gel column using ethanol--hexane-dichloromethane-diethylamine (30:62:8:5 . 10(-3)) as the mobile phase at a flow-rate of 1.5 ml/min. The limit of sensitivity is 5 ng/ml for clomipramine and imipramine and 10 ng/ml for the corresponding mono-demethylated metabolites. This method has been used to analyse plasma from subjects given therapeutic doses of clomipramine or imipramine.