Effect of nonlinear protein binding on equilibration times for different initial conditions.

The dynamics in the equilibration of free drug in a two-compartment closed system, exhibiting nonlinear binding in one of the compartments, are elucidated. The dynamics of the free drug in the compartment containing the binding sites are first studied under the two initial conditions when the drug is added to the same and the other compartment; the dynamics of the drug in the compartment devoid of binding sites are then studied under the two initial conditions. Dynamic asymmetries are shown to exist among the four cases in the nonlinear region using an equilibrium limit which symmetrizes the dynamics for all four cases in the linear region. In the two cases where the dynamics are viewed in the compartment devoid of binding sites, the dynamic asymmetry is manifested by a reduction in the time required to reach the limit for the drug added to the compartment containing the binding sites compared with the other compartment. The time difference between these two cases becomes magnified when limits, reflective of the relative error in the estimate of the equilibrium value, are applied. In the linear region, application of these limits results in a time difference. This time difference again favors addition to the compartment containing the binding sites from a time-conservation perspective.

General method for evaluating the fraction of the irreversible organ clearance due to conversion of drug to a primary metabolite.

An expression for the fractional metabolic clearance of a drug across an organ has been developed. The expression is a function of areas under the temporal profiles of the drug and metabolite concentrations in the sampling compartment. Formation of the metabolite of interest is permitted throughout the linear systems analyzed. The expression is shown to be applicable to any linear system provided there is only a single direct feed from the organ of interest (compartment where formation of the particular metabolite is being investigated) to the drug-sampling compartment.

Electron-capture capillary gas chromatographic determination of phenylpropanolamine in human plasma following derivatization with trifluoroacetic anhydride.

A capillary gas chromatographic analysis of phenylpropanolamine in human plasma, following extraction and derivatization with trifluoroacetic anhydride, is presented. Using an electron-capture detector, the method was sensitive enough to quantitate as little as 1 ng of drug/mL of plasma. The coefficient of variation from 5-262 ng/mL varied between 5.6 and 1.6%, respectively. Plasma concentration data following one 25-mg dose of phenylpropanolamine hydrochloride in four healthy volunteers illustrates the suitability of this analytical method for monitoring plasma levels after oral administration of a typical dosage form.

Assay for acetazolamide in plasma.

A method for the analysis of acetazolamide, 5-acetamido-1,3,4-thiadiazole-2-sulfonamide, sensitive to 25 ng/ml in plasma, was developed. After extraction of acetazolamide and its propionyl analog, 5-propionamido-1,3,4-thiadiazole-2-sulfonamide, the internal standard, from plasma with ethyl acetate and removal of lipids from the residue of the ethyl acetate extract with methylene chloride, the sulfonamides were chromatographed on an octadecyl trichlorosilane bonded phase using high-pressure liquid chromatography. The method was developed to study plasma level profiles of different dosage forms of acetazolamide.

Submicrogram assay for scopolamine in plasma and urine.

A GLC-mass spectrometric method for scopolamine, sensitive to 50 pg/ml for a 4-ml plasma or urine sample, was developed. The method used a deuterated internal standard to minimize variability in absolute recovery in the extraction procedure. Scopoline and deuterated scopoline were formed from the base-catalyzed hydrolysis of scopolamine and the internal standard and were analyzed as the heptafluorobutyrates, using a GLC-mass spectrometric system by monitoring the m/e 138 and 141 fragments, respectively.

Acetazolamide binding to two carbonic anhydrase isoenzymes in human erythrocytes.

Acetazolamide binding to high activity and low activity carbonic anhydrase isoenzymes in red blood cells was studied. Inhibitory constants of 0.041 and 2.72 microM and maximum binding capacities of 17.2 and 155 microM, respectively, were found.

High-pressure liquid chromatographic method with postcolumn, in-line hydrolysis and fluorometric detection for indomethacin in biological fluids.

A rapid and sensitive method for the analysis of indomethacin in plasma and urine was developed using high-pressure liquid chromatography, postcolumn, in-line hydrolysis of indomethacin to a fluorophore, and detection of the fluorophore wih a fluorometer. The lower limit of detection was 1.5 ng/ml of plasma. The coefficient of variation at 30 ng/ml of plasma was 4.5% (n = 5). The nonconjugated metabolites of indomethacin, aspirin, and salicylate were resolved from indomethacin and the internal standard, alpha-methylindomethacin.

Quantitation of norfloxacin, a new antibacterial agent in human plasma and urine by ion-pair reverse-phase chromatography.

A specific and sensitive high-performance liquid chromatographic method for the analysis of norfloxacin in human plasma and urine is described. Norfloxacin was extracted from the sample matrix using dichloromethane under neutral conditions, followed by back extraction into dilute phosphoric acid for chromatographic analysis on a reverse-phase column with a mobile phase consisting of methanol, phosphate buffer, and ion-pairing reagent (pH 3.0) at a flow rate of 2.0 mL/min. The ability of this method to distinguish intact norfloxacin from its metabolites was demonstrated. The method is linear, quantitative, and reproducible for both plasma analysis (0.05-2.5 microgram/mL) and urinalysis (1.0-500 micrograms/mL) using peak area ratios (norfloxacin-internal standard) for quantitation. The stability of norfloxacin and its metabolites in dilute phosphoric acid was studied. To assess the presence of norfloxacin conjugates in the urine of dosed individuals, the effects of urine hydrolysis on drug quantitation were examined. Urine and plasma levels of norfloxacin at selected time points following the administration of single drug doses are presented.

A sensitive method for assay of a novel tricyclic compound using coulometric electrochemical detection.

A reversed-phase high-performance liquid chromatographic method using coulometric electrochemical detection in the oxidative mode has been developed for the analysis of 3-(9-chloro-5,6-dihydro-11-H-pyrrolo[2,1-b][3]benzazepine-11-ylidene- N,N-dimethyl-1-propanamine(E)-Z-butenedioate hydrogen maleate (1) in plasma of patients dosed with 2-8 mg/kg/d of the drug. Concentrations as little as 0.1 ng/mL of 1 in plasma can be estimated with a mean coefficient of variation of 7.4 +/- 1.08%. The utility of the procedure was demonstrated by the analysis of 500 patient samples from a rising multiple-dose study.

Subnanogram assay for pilocarpine in biological fluids.

A method for the determination of pilocarpine was developed in which the imidazole ring of pilocarpine was acylated with heptafluorobutyric anhydride, using triethylamine as a catalyst. After cleanup, the pilocarpine derivative was analyzed using GLC with electron-capture detection. The limit of sensitivity was 25-50 pg of pilocarpine, which had been subjected to the derivatization and cleanup procedures. The method was specific for pilocarpine, with the isopilocarpine derivative eluting prior to the pilocarpine derivative.

Stereoselective determination of (beta S,gamma R and beta R,gamma S)-4-[3-(4-acetyl-3-hydroxy-2-propylphenoxy) propylthio]-gamma-hydroxy-beta-methylbenzenebutanoic acid in human and rat plasma by normal-phase high-performance liquid chromatography.

A stereoselective high-performance liquid chromatographic method was developed for the determination of the enantiomeric composition comprising (beta S,gamma R and beta R,gamma S)-4-[3-(4-acetyl-3-hydroxy-2-propylphenoxy) propylthio]-gamma-hydroxy-beta-methylbenzenebutanoic acid in human and rat plasma. Both enantiomers, (beta S,gamma R)- and (beta R,gamma S)-1, were isolated from the plasma by liquid-liquid extraction, the organic phase was evaporated to dryness, and the residue was lactonized with p-toluenesulfonic acid followed by derivatization with R(+)-alpha-methylbenzylamine to form diastereomeric Schiff base adducts. Separation was performed on a silica column (Supelcosil) with a mobile phase composed of 97.8% hexane, 2% isopropyl alcohol, and 0.2% triethylamine. The column eluant was monitored with a variable wavelength UV detector set at 280 nm (0.005 AUFS). Based on the peak area ratios, the method shows excellent linear relationships with their corresponding concentrations and satisfactory intraday and interday assay precision and accuracy. The detection limit of this method is 25 ng/mL in plasma for each enantiomer. The method has been applied to the assay of plasma obtained from human subjects administered 1 in safety and tolerability studies and from rats administered 1 intravenously or per os.

Stereoselective disposition of the geometric isomers of a novel lipoxygenase cyclo-oxygenase inhibitor in dog and photochemical interconversion of its isomers.

A sensitive (10 ng/mL) and specific high-performance liquid chromatographic (HPLC) assay, with electrochemical (EC) detection, for the geometric isomers of 3-hydroxy-N-(2-phenyl-2-(2-thienyl)ethenyl-5-(trifluoromethyl)benzo(b) thiophene-2-carboxamide in dog and human plasma has been developed. Both isomers strongly absorb light, leading to an efficient E in equilibrium Z photoisomerization. After iv administration of a single isomer (Z) to a dog, only the Zisomer was detected in plasma; no in vivo conversion to the E isomer was observed. However, when a mixture of the E and Z isomers (58.6:41.4) was administered in the same manner to the same dog, the E:Z ratio decreased significantly to 47.5:52.5 six hours after drug administration, indicating stereoselective disposition of the isomers. The elimination of the E isomer was found to be faster than that of the Z isomer.

Determination of submicrogram quantities of clonidine in biological fluids.

A sensitive and specific GLC method using electron-capture detection was developed for clonidine in plasma and urine. Di-perfluoroacyl derivatives of both clonidine and the 4-methyl analog of clonidine (used as an internal standard) were formed, and an extraction process was developed for the removal of excess derivatization reagent and endogenous biological compounds; the assay permitted quantification of 25 pg of clonidine/ml in a 4-ml plasma sample. The assay was used to elucidate the time course of plasma concentrations in a normotensive subject following oral administration of 50, 100, and 200 microgram of clonidine hydrochloride and also to determine unchanged drug excreted in the urine.

Time course and disposition of methazolamide in human plasma and red blood cells.

Methazolamide was determined in plasma, whole blood, and urine by a GLC-mass spectrometric method. Temporal patterns of methazolamide concentrations in plasma and red blood cells were obtained following single- and multiple-dose oral administration of the drug. The nonlinearity in the binding of the drug to the red blood cell carbonic anhydrase was evident from a comparison of plasma and red blood cells concentrations. The drug was cleared slowly from the red blood cells. The binding constants to the two isoenzymes of carbonic anhydrase were determined from the plasma and red blood cell concentrations and were in agreement with those determined by previous measurements. The half-life of elimination was 7.5 hr. The urinary recovery of unchanged drug was approximately 25% of the administered dose.

A sensitive analytical method for the determination of a novel lipoxygenase inhibitor, 4-bromo-2,7-dimethoxy-3H-phenothiazin-3-one, in plasma using reductive electrochemical detection.

A reversed-phase high-performance liquid chromatographic assay using electrochemical detection in the reductive mode has been developed for the analysis of 4-bromo-2,7-dimethoxy-3H-phenothiazin-3-one (1) in plasma to determine drug absorption. Free drug in plasma in concentrations as little as 0.25 ng/mL can be estimated with a mean coefficient of variation (CV) of 6.3 +/- 2.6%. Metabolites which can be converted to the parent drug by acid hydrolysis can be quantified in concentrations of 10 ng/mL or more, with a mean CV of 4.3 +/- 1.9%. To test the procedure, plasma was obtained from dogs receiving 14C-labeled 1. After acid hydrolysis of plasma, the electrochemical assay for parent drug showed good agreement with the radioactive equivalents in plasma, suggesting that parent drug and metabolites can be satisfactorily analyzed by this procedure.

Bioanalysis and disposition of alpha-fluoromethylhistidine, a new histidine decarboxylase inhibitor.

A sensitive, selective, and rapid high-performance liquid chromatographic procedure was developed for the determination of alpha-fluoromethylhistidine (alpha-FMH) in human biological samples. The plasma assay required isolation of the drug using a weak cation-exchange resin prior to HPLC analysis with UV detection. The urine assay employed postcolumn derivatization with o-phthalaldehyde (without a thiol) and fluorescence detection. The extent of metabolism of alpha-FMH in humans was studied in four healthy volunteers using tritium-labeled material. No significant differences in the plasma and urine concentrations of radioactivity and unchanged drug were detected. In addition, the radiochromatograms of selected urine samples revealed a single peak with a retention time corresponding to the unchanged drug. The evidence presented suggests negligible biotransformation of alpha-FMH in humans.