Liquid chromatography of codeine in plasma with fluorescence detection.

This analytical method for easier determination of codeine in human plasma is based on "high-performance" liquid chromatography for separation and the natural fluorescence of codeine for detection. Codeine is extracted from alkalinized plasma with a mixture of hexane and dichloromethane, and the extract is further purified and chromatographed. The method can be used for routine assay of codeine at the concentrations of 10 micrograms/L or greater in human plasma. As little as 4 micrograms/L can be detected. Coefficients of variation for the assay of codeine in the concentration range of 10 to 100 micrograms/L were 2.2-7.4% (n = 6). We used this method to establish a concentration/time profile for plasma from a human volunteer after a 60-mg oral dose of codeine sulfate.

Determination of oxfendazole in cow milk by reversed-phase high-performance liquid chromatography.

A specific and sensitive high-performance liquid chromatographic method for the analysis of oxfendazole in cow milk is described. Oxfendazole was extracted from milk using a mixture of acetone and chloroform under alkaline conditions. The solvents were evaporated, and the oily residue was purified by hexane-acetonitrile partition and acid-base extraction. The residue obtained after cleanup was redissolved in methanol for chromatographic analysis. Chromatography was performed on a reversed-phase column with acetonitrile-water as the mobile phase. As low as 0.005 microgram of oxfendazole/g can be measured by this method using 50 g of milk. The method was applied to measure oxfendazole in the milk of a cow given an oral 5-mg/kg dose.

Radioimmunoassay of flunisolide in human plasma.

A simple radioimmunoassay was developed for the measurement of flunisolide in human plasma or serum. Plasma extraction was not required. Antiserums were produced in rabbits by immunization against the flunisolide 21-hemisuccinate-bovine serum albumin conjugate. Cross-reactivities were determined for cortisol and a major metabolite of flunisolide and were 0.02 and 0.06%, respectively. Assay sensitivity is in the 100--200-pg/ml range. Accuracy studies gave regression lines of y = 1.06x, r = 1.00, for a 0.1-ml plasma aliquot and y = 0.99x, r = 0.99, for a 0.2-ml plasma aliquot. The accuracy of the method was estimated to be at least +/- 15%. The method was used to determine plasma concentration-time profiles in human subjects after the administration of a 1.0-mg iv dose.

High-performance liquid chromatographic measurement of cloprednol in human plasma.

A rapid and specific high-performance liquid chromatographic method for the quantitative determination of cloprednol in human plasma is described. Samples were extracted using methylene chloride-ether (40:60) and then purified further by solvent and pH partitioning techniques. Cloprednol was analyzed using normal-phase chromatography and UV detection at 254 nm. The final recovery after losses during the cleanup procedure for cloprednol from human plasma was 80.8%. The lowest concentration that could be measured with confidence was 8 ng/ml.

Disposition of flunisolide in the rat, mouse, dog, rhesus monkey, and cynomolgus monkey.

Flunisolide (6 alpha-fluoro-11 beta, 16 alpha, 17 alpha, 21-tetrahydroxypregna-1,4-diene-3,20-dione 16,17-acetonide) administered as a single iv or oral dose to rats, mice, dogs, rhesus monkeys, and cynomolgus monkeys had a plasma t 1/2 of 1-3.5 hr and was eliminated mainly via the bile. After iv administration of 14C-labeled flunisolide, radioactivity was widely distributed into tissues and organs. The apparent volume of distribution of flunisolide in these five species was 3.0-8.0 liters/kg. A major metabolite isolated from rhesus monkey urine was shown to be 6 beta, 11 beta, 16 alpha, 17 alpha, 21-pentahydroxypregna-1,4-diene-3,20-dione 16,17-acetonide (6 beta-OH metabolite). Free 6 beta-OH metabolite was a major radioactive entity detected in urine of all species given radiolabeled flunisolide, whereas flunisolide conjugated with glucuronic acid and/or sulfate was a major metabolite detected in the bile of rats, dogs, and cynomolgus monkeys. Following the oral administration of radiolabeled flunisolide, radioactivity was rapidly and efficiently absorbed in all species, but in the rhesus and cynomolgus monkeys most of the plasma radioactivity was due to the 6 beta-OH metabolite and to water-soluble conjugates, suggesting extensive first-pass metabolism of flunisolide.

Radioimmunoassay of oxfendazole in bovine, equine, or canine plasma or serum.

A simple radioimmunoassay was developed for the determination of oxfendazole in plasma. Oxfendazole N-1(3)-valerate was coupled to polylysine via a carbodiimide reaction, and antiserum was developed in rabbits after inoculation with oxfendazole--polylysine conjugate. The assay was developed so that oxfendazole could be measured directly in a 0.1-ml aliquot of diluted or undiluted plasma. With the developed procedure, 200 pg of oxfendazole/ml of plasma can be determined quantitatively. Cross-reactivity was determined for closely related compounds and metabolites. The method was used to determine plasma concentration--time profiles in dogs and calves.

GLC determination of atenolol and beta-blocking agents in biological fluids.

A rapid, sensitive, and specific method of analysis for atenolol is described. Metoprolol is used as the internal standard. Atenolol and metoprolol are extracted into 1-butanol--benzene. Interfering components present in palsma and urine, but not discolored saliva, are removed during an acid wash and reextraction into ether. Drug and internal standard are converted to the pentafluoropropionate derivatives, which are quantitated by GLC with electron-capture detection and characterized by chemical-ionization mass spectrometry. The method should be applicable to measurement of other beta-adrenergic blocking agents with similar structures.

Development of a procedure for the assay of an experimental steroid drug in dog plasma.

Studies of the estimation of 16alpha-cyano-3beta-cyclopentyloxypregn-5-en-20-one (an experimental drug) in dog plasma are described. Extraction using a salt/solvent pair (ammonium carbonate/ethyl acetate) is followed by a rapid chromatographic procedure employing Lipidex 5000, which affords a substantially purified fraction. After preparation of the t-butyldimethylsilyloxime, quantification of the drug is performed by selected ion monitoring. The [2H9]cyclopentyloxyl analogue is used as an internal standard. In a preliminary experiment, the advantages (in terms of both sensitivity and selectivity) of the use of an open tubular GLC column are demonstrated.

Quantitative determination of prednisone and prednisolone in human plasma using GLC and chemical-ionization mass spectrometry.

A method for the quantitative determination of prednisolone and prednisone in human plasma utilizing GLC and chemical-ionization mass spectrometry is described. Corticosteroids are extracted from plasma into ether, and the extract is purified either by passing through a magnesium silicate column or by solvent partitioning. Interference from endogenous hydrocortisone is removed by selective derivatization with Girard Reagent T. Following derivatization, prednisolone can be quantitatively separated from the water-soluble hydrocortisone derivative by simple solvent partitioning. The extracted prednisone and prednisolone are converted to their corresponding methoxyimino trimethylsilyl derivatives, and subjected to GLC-mass spectrometry. Prednisone and prednisolone plasma profiles following a 15-mg oral dose of prednisone in a human volunteer are presented. The method can measure prednisone and prednisolone in plasma at the nanogram per milliliter level.

Kinetics, salivary excretion of amphetamine isomers, and effect of urinary pH.

Amphetamine was administered to healthy subjects as the racemic mixture and as (+)- and (-)-isomers under conditions of urine acidification and alkalinization. Plasma and saliva concentration of each isomer was measured and the kinetics of the individual isomers were determined. (+)-amphetamine was eliminated more rapidly than the (-)-isomer. The difference in half-life between isomers was maximal under basic urinary pH conditions. Saliva amphetamine levels were higher than plasma levels and in the postabsorptive phase were predictably proportional to plasma drug levels.

Plasma glucagon suppression by phenformin in man.

In an attempt to elucidate the mechanism of action of phenformin, eleven juvenile-onset, insulin-requiring diabetic subjects underwent four different treatment regimens during standard breakfast tests. These four treatments were: control (no insulin or phenformin); insulin alone (15 U regular insulin administered subcutaneously one-half hour before breakfast); phenformin alone (50 mg of the timed-release capsule given twice daily for three days before the study and two and one-half hours before breakfast on the day of study); and phenformin plus insulin (in the amounts and at the times stated above). Phenformin was found to decrease postprandial hyperglycaemia significantly when compared with control values, and its addition to insulin further decreased the postprandial glucose rise below that found with insulin alone (p less than 0.005). These effects were associated with a reduction in early (30-min) postprandial hyperglucagonaemia (p less than 0.05). Triglyceride levels, gastrin secretion, growth hormone levels, and increments of alpha-amino nitrogen were not affected by phenformin. Thls, suppression of postprandial hyperglucagonaemia may be an additional mechanism in the reduction of postprandial hyperglycaemia after phenformin.

Serum phenformin concentrations in patients with phenformin-associated lactic acidosis.

Phenformin concentrations were measured in serum from seven patients with phenformin-associated lactic acidosis, and initial values ranging from 20 to 625 ng./ml. were obtained. Five of the seven patients had serum concentrations within the usual therapeutic range of up to 241 ng./ml. Serum phenformin concentrations were measured serially, and apparent half-lives of 5, 25, and 30 hours were obtained in three patients with serum creatinine concentrations of 1.7, 7.6, and 6.0 mg./dl., respectively. Although the half-life of phenformin was prolonged in azotemic patients, no correlation between serum creatinine concentration and serum phenformin could be demonstrated; furthermore, the severity of lactic acidosis as measured by arterial pH and lactate concentration did not correlate with the serum creatinine concentration.

Quantitative determination of enantiomeric compounds. I--Simultaneous measurement of the optical isomers of amphetamine in human plasma and saliva using chemical ionization mass spectrometry.

A sensitive and specific method for the quantitative determination of the optical isomers of amphetamine from human plasma and saliva is described. Amphetamine was extracted from basified plasma or saliva with hexane and then derivatized by reaction with N-pentafluorobenzoyl-S-(-)-prolyl-1-imidazolide. The reaction of the amphetamine enantiomers with this chiral reagent yields diasteriomers which are easily resolved by gas liquid chromatography. The resolved diasteriomers upon elution from the gas chromatograph were measured quantitatively by chemical ionization mass spectrometry. Plasma and saliva levels achieved following the oral administration of 10 mg of dl-, d- and l-amphetamine in man are reported.