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An HPLC method for the quantification of ketoprofen enantiomers in human plasma is described. Following extraction with a disposable C 18 solid-phase extraction column, separation of ketoprofen enantiomers and I.S. (3,4-dimethoxy benzoic acid) was achieved using a chiral column [Chirex 3005; ( R )-1-naphthylglycine 3,5-dinitrobenzoic acid] with the mobile phase, 0.02 M ammonium acetate in methanol, set at a flow-rate of 1.2 ml/min. Baseline separation of ketoprofen enantiomers and I.S., free from interferences, was achieved in less than 20 min. The calibration curves ( n = 14) were linear over the concentration range of 0.16 to 5.00 μg/ml per enantiomer [mean r 2 of 0.999 for both enantiomers, root mean square error were 0.015 for R (−) and 0.013 for S (+)]. The inter-day coefficient of variation for duplicate analysis of spiked samples was less than 7% and the accuracy was more than 93% over the concentration range of 0.2 to 4.0 μg/ml for individual enantiomer using 1 ml of plasma sample. This method has been applied to a pharmacokinetic study from healthy human volunteers following the administration of a ketoprofen extended release product (200 mg). This method is simple, fast and should find wide application in monitoring pharmacokinetic studies of ketoprofen.

The metabolism of phenazopyridine was studied in the filamentous fungus Cunninghamella echinulata (synonym C. bainieri) ATCC 9244. Metabolic products were initially isolated by HPLC and TLC, with further characterization achieved by a combination of GLC, NMR, mass spectrum (MS), and GC–MS analyses. Selected samples of the incubation broths were treated with (β-glucuronidase–arylsulfatase. In addition to the reported mammalian metabolites p-aminophenol, acetaminophen, 2′-hydroxyphenazopyridine, 4′-hydroxyphenazopyridine, and parent drug, a novel metabolite was unequivocally identified as the sulfate monoester of 4′-hydroxyphenazopyridine.para-Hydroxylation of the aromatic ring with subsequent sulfoconjugation were observed as the major routes of metabolism. Ethanol, carbon monoxide, and quinidine had inhibitory effects on the C. echinulata metabolism of phenazopyridine, suppressing formation of 4′-hydroxyphenazopyridine and its sulfate monoester. Key words: Cunninghamella echinulata, biotransformation, GLC analysis of phenazopyridine, HPLC analysis of phenazopyridine, phenazopyridine, sulfate conjugate.

Diltiazem (DTZ) is a calcium antagonist widely used in the treatment of angina and related heart diseases. It is extensively metabolized into a host of metabolites, some of which have potent pharmacological activities. Previous work has shown that DTZ and its major metabolite N-desmethyl-DTZ (MA) were unstable and readily decomposed to deacetyl-DTZ (M1) and deacetyl N-desmethyl-DTZ (M2), respectively. This report describes the stability of DTZ and its metabolites in plasma samples stored at -20 and -70 degrees C for different periods up to 12 weeks. The results indicate that in those samples obtained from volunteers who received DTZ, no deterioration of DTZ or MA occurred up to 8 weeks, but considerable deterioration of DTZ to M1 and MA to M2 (p less than 0.01) occurred after 12 weeks. However, in samples prepared by adding DTZ and its metabolites to outdated plasma (spiked plasma), deterioration of DTZ occurred after 4-6 weeks of storage, but there were no concomitant increases in concentrations of M1 or M2. Thus, it appears that decomposition of DTZ and MA was affected by the nature of the plasma materials, but the reason for the differences in analyte stability observed between volunteers' and spiked plasma is not known. Also, it appeared that DTZ and its metabolites in plasma samples stored at -70 degrees C may be more stable than those at -20 degrees C, although further studies are required to substantiate this observation. On the basis of these results, plasma samples obtained from patients or volunteers receiving DTZ should be analyzed within 8 weeks when the samples are stored frozen at -20 degrees C.

Delta-9-tetrahydrocannabinol (Δ-9-THC) is the main psychoactive ingredient of cannabis (marijuana). The present review focuses on the pharmacokinetics of THC, but also includes known information for cannabinol and cannabidiol, as well as the synthetic marketed cannabinoids, dronabinol (synthetic THC) and nabilone. The variability of THC in plant material (0.3% to 30%) leads to variability in tissue THC levels from smoking, which is, in itself, a highly individual process. THC bioavailability averages 30%. With a 3.55% THC cigarette, a peak plasma level of 152±86.3 ng/mL occured approximately 10 min after inhalation. Oral THC, on the other hand, is only 4% to 12% bioavailable and absorption is highly variable. THC is eliminated from plasma in a multiphasic manner, with low amounts detectable for over one week after dosing. A major active 11-hydroxy metabolite is formed after both inhalation and oral dosing (20% and 100% of parent, respectively). THC is widely distributed, particularly to fatty tissues, but less than 1% of an administered dose reaches the brain, while the spleen and body fat are long-term storage sites. The elimination of THC and its many metabolites (from all routes) occurs via the feces and urine. Metabolites persist in the urine and feces for severalweeks. Nabilone is well absorbed and the pharmacokinetics, although variable, appear to be linear from oral doses of 1 mg to 4 mg (these doses show a plasma elimination half-life of approximately 2 h). As with THC, there is a high first-pass effect, and the feces to urine ratio of excretion is similar to other cannabinoids. Pharmacokineticpharmacodynamic modelling with plasma THC versus cardiac and psychotropic effects show that after equilibrium is reached, the intensity of effect is proportional to the plasma THC profile. Clinical trials have found that nabilone produces less tachycardia and less euphoria than THC for a similar antiemetic response.

Pharmacokinetic and bioavailability parameters of propranolol were estimated in 10 healthy adult subjects after single oral doses of two commercial tablet formulations of propranolol hydrochloride (2 X 40 mg). Plasma concentrations of propranolol were determined by a high-performance liquid-chromatographic (HPLC) assay. Peak plasma concentrations of propranolol glucuronide were 6-8 times those of the corresponding peak propranolol plasma concentrations. The mean resident time (MRT) of propranolol and of propranolol glucuronide was determined for each subject for both formulations. The MRT of the parent drug was found to be longer than the MRT of the glucuronide metabolite for each of the subjects examined. Statistical moment analysis indicated that this phenomenon is attributable to extensive presystemic glucuronidation of the parent drug.

A sensitive and specific high‐performance liquid chromatographic procedure for the determination of chlorpropamide or tolbutamide in plasma in the presence of their metabolites is described. The ether extract of acidified plasma is redissolved in the mobile phase, 17% acetonitrile in 0.05 M aqueous ammonium formate, and chromatographed on a reverse‐phase column on a high‐performance liquid chromatograph fitted with a UV absorbance detector. Quantitation of plasma samples containing less than 0.5 μ g/ml of chlorpropamide and 5 μ g/ml of tolbutamide is reported, using these drugs as mutual internal standards. The retention times of the metabolites are such that they do not interfere in the procedure. The assay method was tested in a human volunteer with both drugs and found suitable for single‐dose pharmacokinetic studies.

A rapid, sensitive, accurate method for determination of quinidine in plasma has been developed using ion-pair extraction and high-performance liquid chromatography. The method, which is capable of distinguishing between quinidine and dihydroquinidine, involves acidification of plasma with perchloric acid, extraction with methyl isobutyl ketone and chromatography of the carbonate-washed extract on a silica gel column with a mobile phase of methylene chloride—hexane—methanol—perchloric acid (60:35:5.5:0.1) followed by fluorometric detection. The procedure is sensitive to below 50 ng/ml (coefficient of variation 6.6%) and compares favourably with a standard spectrofluorometric method when tested with plasma from volunteer subjects.

A novel method for the quantitative estimation of warfarin in plasma is described. Plasma containing warfarin to which a known amount of phenylbutazone is added as internal standard is acidified and extracted with ethylene dichloride. The drug and the internal standard are then back-extracted into alkali which, in turn, is acidified and reextracted with ethylene dichloride. The organic extract, after washing with phosphate buffer (pH 7.2), is evaporated and the evaporated extract is reacted with an ethereal solution of diazomethane (100 μl). The reacted mixture is evaporated and then dissolved in 25 μl of carbon disulfide. Ali-quots (2–3 μl are injected into a gas chromatograph equipped with a flame-ionization detector. The methyl derivatives of warfarin and the internal standard give sharp, well-separated, symmetrical peaks. The method is of sufficient sensitivity to determine plasma levels in humans after single doses (20 mg) of warfarin (sensitivity of 0.25 μg/ml).

A GLC method for the quantitative estimation of phenprocoumon from plasma is described. Plasma containing phenprocoumon, to which a known amount of phenytoin is added as the internal standard, is acidified and extracted with ethylene dichloride. The drug and the internal standard are then back-extracted into alkali, which is acidified and reextracted with ethylene dichloride. The organic extract is evaporated, and the evaporated residue is mixed with 50 mul of trimethylanilinium hydroxide in methanol. Aliquots (1-2 mul) are injected into a gas chromatography equipped with a flame-ionization detector in which the injection port is held at 325 degrees. The methyl derivatives of phenprocoumon and the internal standard give sharp, well-separated, symmetrical peaks. The method is of sufficient sensitivity to determine 0.125 mug/ml of the drug in plasma with a coefficient of variation of 7%.

High performance liquid chromatography and mass spectrometry were used to isolate and identify theophylline, theobromine and 1,7-dimethylxanthine (paraxanthine) in plasma of human volunteers following administration of 300 mg caffeine to methylxanthine-free volunteers. Plasma from these subjects was extracted and the dimethylxanthines were separated from each other and caffeine by high performance liquid chromatography. The effluents at the chromatographic peaks corresponding to the dimethylxanthine metabolites were collected, rechromatographed in a second system and the dried residues were subjected to mass spectrometry. By comparison of their retention times and mass spectra with those of authentic compounds, theophylline, theobromine and paraxanthine were positively identified as metabolic products of caffeine.

Two betamethasone tablet formulations, and betamethasone phosphate solution were compared in a plasma level study. The tablet formulations (A and B) and a solution of betamethasone phosphate (C) were administered in single 2 mg doses to nine volunteers according to a three times repeated Latin square design. Plasma samples were obtained over 72 h following each dose and plasma was analysed for betamethasone by radioimmunoassay. Pharmacokinetic evaluation of the data, obtained according to the one-compartment open model, indicated that there were no significant differences between the extent of absorption, and the first-order elimination rate constants. As would be expected, the solution (C) gave a faster absorption rate than tablets A and B.

An improved GLC procedure was developed for the simultaneous determination of phenytoin and its metabolite, 5‐( p ‐hydroxyphenyl)‐5‐phenylhydantoin, in plasma and urine following enzyme hydrolysis. After extraction, the drug, the metabolite, and the internal standard, 5‐( p ‐methylphenyl)‐5‐phenylhydantoin, are measured by GLC with flame‐ionization detection as their respective methyl derivatives following flash‐heater methylation with tri‐methylanilinium hydroxide. The drug and metabolite give well‐resolved symmetrical peaks on a phenyl methyl silicone column, and the method has a sensitivity of 150 ng/ml of phenytoin and 125 ng/ml of the metabolite. GLC–mass spectral evidence is presented for the formation and intact determination of methyl derivatives of the drug, its metabolite, and the internal standard.

A rapid, selective and reproducible high-performance liquid chromatographic assay with electrochemical detection was developed for the determination of nalbuphine in human plasma. The method involves extraction with chloroform-isopropanol at pH 9.4, back-extraction into dilute phosphoric acid and reversed-phase chromatography on a microBondapak phenyl column. The recovery of nalbuphine and naltrexone (internal standard) was greater than 90%. Calibration curves were linear over a concentration range of 3-36 ng/ml with coefficients of variation, within-day or between-day, not exceeding 8% at any level. Although the limit of detection was 0.3 ng/ml based on a signal-to-noise ratio of 3, the reliable limit of quantitation was 1 ng/ml (coefficient of variation 12%) using 1 ml of plasma. The dual-electrode detector was operated in the screening mode of oxidation (electrode 1, 0.3 V and electrode 2, 0.6 V), providing a greater specificity and reducing background noise. This procedure was applied to a large number of clinical samples in an intravenous dose-range pharmacokinetic study in patients.

The nitromethane-Hyamine method for assay of nitrofurantoin in whole blood has been improved: smaller volumes of blood (0.8 ml) are required, the sensitivity is greater (0.2 pg/mI), and the solution to be read is more stable than in the original method. The analysis detects 70 to 72% of 1 to 4 Ag of nitrofurantoin added per milliliter of whole blood. Blood concen-trations after the usual therapeutic peroral dose (100 mg) of nitrofurantoin are reported.

A quantitative method for the gamma-oxo metabolite of phenylbutazone from plasma is described. The procedure involved an ethylene dichloride extraction of acidified plasma to which an internal standard, acenocoumarol, had been added. The extracted gamma-oxo metabolite and the internal standard were methylated and analyzed by GLC. Determination of 0.25 microgram of gamma-oxo metabolite/ml with a relative standard deviation of 6.5% was accomplished.

A GLC method for qualitative and quantitative determination of 3,4-methylenedioxyamphetamine in biological fluids is described. After extraction of the drug and the internal standard, 4-methoxyamphetamine, from plasma and urine, the solvent is evaporated and the residue is mixed with 20 mul of freshly distilled ether. Aliquots (1-2 mul) then are injected into the gas chromatograph. Both the drug and the internal standard give well-separated symmetrical peaks. Flame-ionization detection allows concentrations of 0.125 mug of 3,4-methylenedioxyamphetamine from plasma to be determined with a precision of 3.16%. The method is applicable to the estimation of 4-methoxyamphetamine, employing 3,4-methylenedioxyamphetamine as the internal standard. For identification purposes, various derivatives such as Schiff bases, isothiocyanates, trifluoroacetates, and heptafluorobutyrates of both compounds were formed following extraction from urine and their GLC behavior was investigated. Derivative formation was confirmed by GLC-mass spectrometry. Mass spectral data for these derivatives are presented.

A recently described GLC method for the determination of phenylbutazone and its major metabolite, oxyphenbuta- zone [1 -phenyl-2- (p -hydroxyphenyl) -3,5-dioxo-4-n -butylpyrazoli- dine], in plasma was extended to the estimation of the second major metabolite, γ-hydroxyphenylbutazone [l,2-diphenyl-3,5-dioxo-4-(3-hydroxybutyl)pyrazolidine]. The method is sensitive to 1.0 μg/ml.

An electron-capture GLC procedure for determination of plasma ephedrine concentrations is described. The procedure is capable of determining 2 ng/ml of ephedrine and is adequate for following profiles after 25-mg single doses. Pentane extraction of the drug and the internal standard and formation of the N-pentafluorobenzoyl derivatives were followed by GLC. Plasma ephedrine concentrations following a 24-mg dose of ephedrine hydrochloride to a human volunteer are presented. Formation of N-trifluoroacetyl, N-pentafluoropropionyl, N-heptafluorobutryl, and N-pentafluorobenzoyl derivatives and their GLC-mass spectrometric identification are discussed together with comparative electron-capture sensitivities of these derivatives toward a nickel-63 detector. The detection of the N-pentafluorobenzoyl derivative of ephedrine is at least 100-fold greater in sensitivity than detection of the N-trifluoroacetyl derivative.

Sensitive specific methods are described for the determination of phenylbutazone and its metabolite oxyphenbutazone from the same plasma sample. The sample, to which an internal standard 5-(4-hydroxyphenyl)-5-phenylhydantoin (Standard II) is added, is first extracted with ether to remove interfering substances and then with n-heptane under acidic conditions to separate phenylbutazone, which is determined on a gas chromatograph by flash methylation (310°) with trimethylanilinium hydroxide using 1-(o-chlorophenyl)-1-(p-chlorophenyl)-2,2,2-trichloroethane as the external standard (Standard I). The aqueous acidic residue from which the phenylbutazone has been selectively removed is shaken again with ether to extract the oxyphenbutazone, which is analyzed with a different GLC system with flash methylation (310°) against Standard II. The methods are of sufficient sensitivity to determine plasma levels in humans after a 200-mg dose of phenylbutazone (phenylbutazone, 1 μg/ml; oxyphenbutazone, 0.5 μ/gml).

A sensitive and specific method for the estimation of theophylline-7-acetic acid in plasma by high performance liquid chromatography is described. Acidified plasma is extracted with chloroform-n-butanol (85: 15), back extracted with phosphate buffer (0.5 mmol-1, pH 6.5), and finally the acidified aqueous phase extracted again with chloroform-butanol. After evaporation of the extract the residue is redissolved in the chromatographic mobile phase (hexane-dichloromethane-ethanol-acetic acid, 10 : 86 : 3 : 1) and chromatographed on silica gel. The method was used to follow the plasma theophylline-7-acetic acid concentrations in two volunteers after single oral doses of 1.0 g of the drug. The results confirm previous reports, based on urine data and on plasma data following intravenous administration, that the drug is poorly absorbed (apparent bioavailability of 1 and 2 per cent), rapidly eliminated (half-life of 1.0 and 2.4 h) and not converted to theophylline.

A high-performance liquid chromatographic procedure is described for the determination of disopyramide and its mono-N-dealkyl metabolite which offers simplicity of extraction with excellent selectivity, sensitivity and reproducibility. The drug and metabolite, following basic diethyl ether extraction and back-extraction with acetic acid, are injected into a reversed-phase high-performance liquid chromatographic column and the absorbance of the eluate measured at 254 nm. Detectability limits of 0.05 μg/ml were obtained with both compounds, and studies of the reproducibility, precision, recovery, stability during storage and effect of time in separating plasma from erythrocytes are described. Applications of this high-performance liquid chromatographic procedure to plasma samples from patients on disopyramide therapy and to plasma and urine from a healthy dog administered single doses are reported.

A catechol metabolite, 5-(3,4-dihydroxyphenyl)-5-phenylhydantoin, and a methylated catechol metabolite, 5-(3-methoxy-4-hydroxyphenyl)-5-phenylhydantoin, were identified as urinary metabolites in humans, monkeys, and dogs following the administration of phenytoin. These metabolites were separated from each other and from other known metabolites of phenytoin as n-butyl derivatives by GLC and positively identified by combined GLC-mass spectrometry.

A GLC procedure was developed for the quantitative estimation of intact chlorpropamide and tolbutamide concentrations in plasma; the drugs are used as mutual internal standards. After extraction of plasma containing the drug and internal standard with toluene, the dried residue is treated with ethereal diazomethane to form the methyl derivatives of tolbutamide and chlorpropamide. Aliquots of the ethereal solution are injected into a gas chromatograph equipped with a glass‐lined injection port and glass column packed with a phenyl methyl silicone fluid (OV‐25) on Chromosorb W, which facilitates the intact determination of the methyl derivatives of the drugs. The response to the flame‐ionization detector was linear over a range of 0.20–25 μg/ml, with a 0.05–μg/ml limit of detectability for both drugs. The method compares favorably with a recently developed high‐pressure liquid chromatographic procedure and is adequate for following blood level profiles of single doses of chlorpropamide (125 mg) and tolbutamide (250 mg). Mass spectral evidence showing that intact sulfonylureas are measured is presented.

The period of time after administration over which blood level measurements are required to obtain a reliable bioavailability comparison of two or more formulations of the same drug was considered by the analysis of bioavailability data taken from the literature. The drugs examined, selected to represent a range of absorption and elimination half-lives, were acetaminophen, aminosalicylic acid, chloramphenicol, chlordiazepoxide, digoxin, isoniazid, phenylbutazone, sulfamethizole, tetracycline, and warfarin. For most drugs, ratios of areas under the curve changed little between the end of the absorption period and the time when blood sampling was terminated. Reliable bioavailability comparisons among different brands of the drugs apparently could have been made by blood sampling over 24 hr or less.

1. The fungus C. bainieri, incubated for 7 days with methoxyphenamine alone or in combination with either sparteine or quinidine, gave N-desmethylmethoxyphenamine and its N-acetyl conjugate as major metabolites, while O-desmethylmethoxyphenamine, 5-hydroxymethoxyphenamine and 2-hydroxyamphetamine were produced in lesser amounts. In addition, 1-(2-hydroxyphenyl)-2-aminopropane, 1-hydroxy-1-(2-methoxyphenyl)-2-propanone, beta-hydroxymethoxyphenamine, and 1-(5-hydroxy-2-methoxyphenyl)-2-aminopropane were tentatively identified as minor components of the fungal biotransformation of methoxyphenamine. 2. As observed in mammalian systems, the addition of either sparteine or quinidine decreased the rate and extent of methoxyphenamine biotransformation. 3. C. bainieri may be a useful model for drug interaction studies.

The USP/NF dissolution test (Method I or rotating-basket method) was applied to different commercial tablet formulations of sulfisoxazole, sulfamethoxazole, and tolbutamide. It was found that the hydrochloric acid solution required for testing the sulfa drug tablets corroded the basket so that it could not be used after 40 hr. of exposure to the acid. Because the dissolution time of a sulfamethoxazole formulation in phosphate buffer (pH 7.2) was close to that obtained using compendial conditions, it is suggested that the official dissolution medium could be replaced, with advantage, by the phosphate buffer. The dissolution times of nine different sulfisoxazole formulations tested in a less acidic medium were not comparable in rank order and magnitude to those obtained with the compendial test. Dissolution times of eight different tolbutamide formulations in phosphate buffer (pH 7.6) were similar in rank order and magnitude to those obtained with the compendial test. It is suggested that this buffer replace the tromethamine [tris-(hydroxymethyl)aminomethane] buffer, currently official, which has a large temperature coefficient of pH.

Objective: To determine if therapeutic doses of naproxen affect the in vivo disposition of zidovudine. Methods: This was designed as a randomized, two-period, two-treatment, crossover study. The patients were 12 men infected with human immunodeficiency virus who had acquired immunodeficiency syndrome (AIDS) or AIDS-related complex. On two separate occasions 14 days apart, patients received either zidovudine alone (200 mg every 4 hours while awake) or zidovudine (200 mg every 4 hours while awake) and naproxen (500 mg every 12 hours for 4 days). On the morning of the fifth day, each patient received the final dose of each regimen and blood and urine were serially collected for 8 hours. Pharmacokinetic parameters (area under the serum concentration—time curve [AUC], maximum plasma concentration, terminal half-life, renal clearance, and urinary recovery) were assessed for zidovudine and its glucuronide metabolite. Main results: Naproxen had no significant effect ( 0.15, ANOVA) on the above pharmacokinetic parameters for both zidovudine and its metabolite. Although the power of the study to detect these small differences was