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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.

Objectives To examine the pharmacokinetic and pharmacodynamic interactions between quinidine and diltiazem because both drugs can inhibit drug metabolism. Methods Twelve fasting, healthy male volunteers (age, 24 ± 5 years; weight, 75 ± 10 kg) received a single oral dose of diltiazem (60 mg) or quinidine (200 mg), alone and on a background of the other drug, in a crossover study. Background treatment consisted of 100 mg quinidine twice a day or 90 mg sustained-release diltiazem twice a day for 2 days before the study day. Results Pretreatment with diltiazem significantly (p < 0.05) increased the area under the curve of quinidine from 7414 ± 1965 to 11,213 ± 2610 ng · hr/ml and increased its terminal elimination half-life (t1/2) from 6.8 ± 1.1 to 9.3 ± 1.5 hours. Its oral clearance was decreased from 0.39 ± 0.1 to 0.25 ± 0.1 L/hr/kg, whereas the maximal concentration was not significantly affected. Diltiazem disposition was not significantly affected by pretreatment with quinidine. Diltiazem pretreatment increased QTc and PR intervals and decreased heart rate and diastolic blood pressure. No significant pharmacodynamic differences were shown for diltiazem alone versus quinidine pretreatment. Conclusion Diltiazem significantly decreased the clearance and increased the t1/22 of quinidine, but quinidine did not alter the kinetics of diltiazem with the dose used. No significant pharmacodynamic interaction was shown for the combination that would not be predicted from individual drug administration. Clinical Pharmacology & Therapeutics (1996) 60, 255–264; doi

Pharmacokinetic and pharmacodynamic interactions of alcohol and nifedipine were assessed in 10 healthy human volunteers. Doses of 20 mg (2 × 10-mg capsules) of nifedipine were administered with either 150 ml of orange juice or 75 ml of alcohol (94%) in 75 ml of orange juice according to a crossover randomized design. Plasma nifedipine levels were monitored for 16 hr after each dosing, along with pulse rate and blood pressure. The relative bioavailability of nifedipine, measured as AUC, was increased by 54% (533 vs 346 ng · hr/ml) after the dose of alcohol (P < 0.0002). However, there were no significant differences between treatments in Cmax,tmax, or t1/2. Although there was no difference in the systolic and diastolic blood pressure and pulse rate between the two treatment groups, the time to reach peak heart rate was significantly faster in the group treated with alcohol (1.4 vs 2.2 hr). This study shows that ethanol increases the bioavailability of nifedipine and decreases the time for onset of increased heart rate.

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.

Cyclosporine in whole blood samples from renal and cardiac transplant recipients was measured by high-performance liquid chromatography (HPLC) and by CYCLO-Trac SP specific radioimmunoassay (RIA). The fresh samples assayed by RIA were remeasured in a second laboratory after storage at -20 degrees C for two to six months and good interlaboratory agreement was obtained on the 59 samples assayed (y = 0.926x + 14.8 micrograms/L, r = 0.982). The calibration curve for RIA was not influenced by fresh whole blood, hemolyzed whole blood or serum matrix from normal volunteers. However, comparison of the RIA results from patient samples with those from an HPLC procedure showed that RIA values averaged about double those measured by HPLC. The difference is attributable to differences between the blood matrix of transplant and nontransplant subjects, rather than exclusively to the apparent nonspecific nature of the antibody.

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.

To examine the specificity of the Incstar Cyclo-Trac SP RIA kit, individual blood samples from 28 cardiac allograft patients on cyclosporine A (CsA) therapy were extracted and chromatographed by HPLC. Initially, eluates from a pool of the above samples were collected at regular intervals and measured by RIA to locate possible cross-reacting compounds. Unknown cross-reacting materials were detected in a fraction (UNK) that was collected before elution of CsA. For each patient's sample, fraction UNK and the fraction containing CsA were then collected and measured by RIA. In 9 of 28 samples, cross-reactivity was detected in fraction UNK; range 11 to 36%, mean 22 +/- 7.5%. Cross-reactivity was not apparent in fraction UNK of CsA-free blood samples from normal volunteers.

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.

Sensitive and rapid radlolmmunoassay (RIA) procedures for the determination of plasma concentrations of psychotroplc drugs--chlorpromazlne, Imipramlne, desipramlne, amltrlptyline, and nortrlptyllne--are These procedures employing 20 to 200FI of plasma can determine subnanogram concentrations of these drugs and are shown to be suitable for therapeutic monitoring of these drugs In patients. For the antidepressant drugs, the RIA procedures were found to compare favorably with gas chromatographic procedures employing alkali flame ionization detectors. The RIA procedure for chlorpromazine measures r as well as Its active metabolite, 7-hydroxychlorpromazlne. Cross-reactivity profiles for the antiserum of each drug were obtained, and In each case the antiserum of the drug dlstlnguished the drug from its inactive metabolites and most of the other psychotropir drugs tested.

This study examined the effect of changes in plasma concentrations of total protein, albumin, α1-acid glycoprotein, and free fatty acids occurring after heart operations on the protein binding of chemically basic drugs. Plasma protein and free fatty acid concentrations were measured simultaneously with in vitro determinations of the protein binding of lidocaine, quinidine, and propranolol: immediately before operation, immediately on weaning from cardiopulmonary bypass, on arrival in the recovery room, and 12, 24, 72, and 120 hours postoperatively. Initial decreases in the concentrations of all proteins were followed by a rise in a,-acid glycoprotein to 254% of baseline at 72 to 120 hours. The free fractions of drug were initially increased to 168% of baseline for lidocaine, 206% for quinidine, and 200% for propranolol and fell progressively with time, reaching sustained troughs of 65% for lidocaine, 50% for quinidine, and 57% for propranolol at 72 to 120 hours. Regression analysis indicated a major influence of changing α1-acid glycoprotein concentrations on free fractions of all three drugs, with a smaller effect of albumin that reached statistical significance only for lidocaine. There were no significant perioperative changes in plasma concentrations of free fatty acids when the in vitro effects of heparin were controlled. In conclusion, sequential changes in plasma protein concentrations after cardiac operations predictably alter the protein binding of lidocaine, quinidine, and propranolol and should be considered when interpreting total plasma drug concentrations.

The bioavailability of three isoniazid formulations was assessed using a procedure specific for the free drug. Nine human volunteers, all slow acetylators, were each given 4 × 100 mg of isoniazid of three different tablet formulations at weekly intervals; the plasma drug levels were measured at different times during the first 24 hr. No significant differences ( p > 0.05) were detected among the three products as to relative bioavailability, peak plasma concentrations, C max , and the time of C max , t max -Analysis of variance of the pharmacokinetic parameters obtained according to a one-compartment open model did not demonstrate any significant formulation or time effect but revealed a significant intersubject variation in all parameters involved.

The purpose of this study was to dissociate the authentic and artifactual effect of in vivo heparin on drug protein binding using protamine as an inhibitor of ex vivo lipolysis. A mixture of ethylenediamine tetra-acetic acid (EDTA, 5 mg ml-1) and protamine in the concentration range of 0 to 7.5 mg ml-1 was added to blood samples from 23 cardiac catheterized patients before (control) and 10 min after 3000 IU of intravenous heparin. In control samples, protamine does not interfere with the protein binding of lidocaine (L), quinidine (Q) or propranolol (P) when plasma pH is readjusted to 7.4. In the absence of protamine, heparin induced a significant increase in the free fraction by 40, 130, and 30 per cent for L, Q, and P, respectively (p less than 0.001), while free fatty acid (FFA) levels increased 2 to 6 fold. When protamine was present, the heparin-induced elevation in free fraction was significantly lower for L (16 per cent) and Q (77 per cent) but not for P; FFA levels were decreased at all protamine concentrations. Residual increases in free fraction and FFA levels compared to control values may represent the true in vivo effect of heparin at the peak activity of lipoprotein lipases. For L and Q, variations in free fraction were strongly associated with variations in FFA, but for P, no significant correlation was observed (r = 0.492). These results indicate that variations in free fraction of L and Q caused by heparin are, to a large extent, artifactual but may be prevented by use of protamine in collection tubes (5 to 7.5 mg ml-1). For P, the increase in free fraction was not mediated by variations of FFA indicating that another mechanism must be involved.

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 gas-liquid chromatographic procedure employing an alkali flame ionization detector (GLC-AFID) for the quantitation of imipramine and its major active metabolite, desipramine, in plasma was developed. This method validated a previously reported radioimmunoassay for imipramine and desipramine. The GLC-AFID method was also compared with a GLC-mass spectrometric procedure which employed selected ion monitoring (GLC/MS-SIM) in which specific ions for imipramine and desipramine were monitored. The two methods using gas chromatographic separation were shown to be suitable for therapeutic monitoring of these drugs in patients. The direct immunoassay, which measures the total imipramine plus desipramine, is very useful when large numbers of samples are to be analysed rapidly and it compares favorably with the other two analytical procedures.

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.

Dissolution studies of eight different lots of acetaminophen tablets were investigated by means of three simple dissolution procedures. None of the methods provided complete correlation with physiological availability of acetaminophen when estimated from blood or urine profiles. Two lots of tablets, which were several years old, released the drug more slowly in vitro than more recently purchased lots of the same brands. All of the recently manufactured products required less than 15 min. for 50% dissolution; this time appears to be a reasonable limit for tablet formulations of acetaminophen.

A radioimmunoassay for chlorpromazine in plasma is described. The antiserum was obtained by immunizing rabbits with a conjugate of bovine serum albumin and N-(2-carboxyethyl)desmethylchlorpromazine. It is specific for chlorpromazine and its minor active metabolite, N-desmethylchlorpromazine. Other known active or inactive chlorpromazine metabolites and other psychotropic drugs tested do not cross react with the antiserum. Less than 34 pg of the drug can be detected in 200 muL of plasma. As many as 100 samples can be processed in a day by one technician. Concentrations of chlorpromazine can be measured in 200-muL samples of plasma collected as late as 48 h after a single oral 25-mg dose.

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.

For the development of radioimmunoassay procedures for chlorpromazine and its active metabolites, three chlorpromazine haptens, 7-(or 8-)(3-carboxypropionyl)chlorpromazine, N -(3-carboxypropionyl)desmethylchlorpromazine, and N -(2-carboxyethyl)desmethylchlorpromazine, were synthesized and characterized by GLC–mass spectrometry, PMR spectrometry, and IR spectrophotometry. Each hapten was coupled to bovine serum albumin, and the number of hapten residues per mole of bovine serum albumin was calculated by UV spectrophotometric methods. Antibodies to each hapten–protein conjugate were obtained in rabbits, and titers of the antiserums were checked by evaluating their binding characteristics to tritiated chlorpromazine.

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).

1. Metabolism of the anorectic agent, fenfluramine, was studied in man to detect phenolic and/or alcoholic metabolites.2. Two new metabolites identified as 1-(m-trifluoromethylphenyl)-2-propanol and 1-(m-trifluoromethylphenyl)-1,2-propanediol, were detected in human urine by g.l.c. and g.l.c.-mass spectrometry.

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.

For the development of radioimmunoassay procedures for chlorpromazine and its active metabolites, three chlorpromazine haptens, 7-(or 8-)(3-carboxypropionyl)chlorpromazine, N -(3-carboxypropionyl)desmethylchlorpromazine, and N -(2-carboxyethyl)desmethylchlorpromazine, were synthesized and characterized by GLC–mass spectrometry, PMR spectrometry, and IR spectrophotometry. Each hapten was coupled to bovine serum albumin, and the number of hapten residues per mole of bovine serum albumin was calculated by UV spectrophotometric methods. Antibodies to each hapten–protein conjugate were obtained in rabbits, and titers of the antiserums were checked by evaluating their binding characteristics to tritiated chlorpromazine.

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.

A modification of the erythrocyte partitioning method for the rapid estimation of plasma-free drug fractions (fu) is described and applied to five basic drugs. In the procedure, which uses readily available clinical laboratory equipment, fu is calculated from measurements of drug partitioning between plasma and erythrocytes, and between buffer and erythrocytes. Results obtained are compared with those from equilibrium dialysis or ultrafiltration techniques for amitriptyline, imipramine, quinidine, lidocaine, and propranolol. For each drug, the mean value of fu obtained with the erythrocyte partitioning procedure was not found to be significantly different from that determined by one of the two other classical techniques. The erythrocyte partitioning method lead to reproducible (mean C.V. = 6.25) and precise values of fu when compared to the other methods; its clinical application to lidocaine gave results which agreed with those obtained by equilibrium dialysis.

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.

Thin-layer chromatographic systems and spray reagents are described for the rapid differentiation of 10 penicillins. Comparative behavior of 18 other antibiotics under these conditions is presented together with other supporting data.

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.