Bioanalytical method validation--a revisit with a decade of progress.
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Biological Assay↗
Biomedical subjects
C T Viswanathan
Publications and source records attributed to C T Viswanathan.
Effects of humidity and temperature on in vitro dissolution of carbamazepine tablets.
The rate and extent of dissolution of various approved marketed carbamazepine (CBZ) tablets exposed to 33, 52, 75, and 97% relative humidities at both room temperature and 40 degrees C, and saturated water vapor at room temperature were compared to fresh unstressed tablets. The dissolution data indicate that exposure of CBZ tablets to high humidity and temperature can have a profound effect on tablet disintegration and dissolution. The dissolution rates of some batches of CBZ products exposed to 97% humidity at 40 degrees C or saturated water vapor at room temperature were drastically reduced in only 6-7 days.
Carbamazepine↗
Pharmacokinetic evaluation of drug interactions with anti-HIV drugs, II: Effect of 2',3'-dideoxyinosine (ddI) on zidovudine kinetics in monkeys.
The pharmacokinetic basis of a drug interaction between zidovudine (AZT) and 2',3'-dideoxyinosine (ddI) was investigated in normal monkeys. Five animals received 20 mg/kg of AZT intragastrically in the absence and presence of ddI. ddI was administered intravenously to produce steady-state ddI plasma concentrations for 30 min. Plasma and urine samples were analyzed for AZT, its major glucuronide metabolite, GAZT, and ddI by high-performance liquid chromatography (HPLC). Resultant AZT and GAZT concentration data were analyzed by noncompartmental methods. Statistical analysis indicated no differences in AZT's apparent total clearance, apparent volume of distribution at steady-state, and elimination half-life due to ddI, however, the mean apparent total clearance decreased from 2.92 to 1.67 L/h/kg, and the mean apparent volume of distribution at steady-state decreased from 5.79 to 3.43 L/kg in the presence of ddI. Incomplete urine collections in most animals prevented conclusions from being made about ddI's effect on renal elimination parameters. Nonetheless, the urinary GAZT/AZT ratio, a parameter not influenced by incomplete urine collection, was significantly reduced in the presence of ddI. Although additional studies will be useful to characterize the full importance of the interaction, there is evidence to suggest that both renal and metabolic elimination of AZT and renal elimination of GAZT may be inhibited by ddI.
Animals↗
Pharmacokinetic evaluation of drug interactions with zidovudine. I: Probenecid and zidovudine in monkeys.
Pharmacokinetic evaluation of a drug interaction between zidovudine (AZT) and probenecid was conducted in monkeys. Six animals received 20 mg/kg of AZT as single intragastric (ig) and iv doses in the absence and presence of 50 mg/kg of probenecid administered ig. Plasma concentrations of AZT and its 5'-glucuronide metabolite (AZTG) were quantitated for 12 h by HPLC. Amounts of AZT and AZTG in urine were also measured, as were probenecid plasma concentrations. Non-compartmental methods were used to obtain pharmacokinetic parameters for AZT and AZTG. In the presence of probenecid, the total clearance of AZT decreased by 50%, renal clearance decreased, and elimination half-life increased. The volume of distribution at steady-state and systemic bioavailability of AZT were not significantly altered by probenecid. The areas under the plasma concentration-time curves and terminal half-lives of AZTG were increased, and renal clearances of AZTG were decreased. The alterations in AZT and AZTG pharmacokinetic parameters are consistent with inhibition of metabolism and renal tubular secretion by probenecid. Since AZT was administered by both oral and iv routes, clearance, volume of distribution, and bioavailability parameters were independently determined. Based on data reported for humans on the zidovudine-probenecid interaction, monkeys appear to be appropriate animal models for the evaluation of zidovudine drug interactions.
Animals↗
Pharmacokinetics of rifampin in children. II. Oral bioavailability.
The absolute bioavailability of oral rifampin was determined in 20 pediatric patients. Intravenous doses of rifampin (mean 287 mg/m2) were compared with p.o. doses (mean 324 mg/m2). Serum concentrations of rifampin, 25-O-desacetylrifampicin, and 3-formylrifamycin SV were determined by high performance liquid chromatography. Following a 1/2-h intravenous infusion, serum rifampin concentrations declined in a monoexponential fashion. Pharmacokinetic analysis of the rifampin serum concentration data indicated that only 50 +/- 22% of a freshly prepared p.o. suspension was absorbed. The rifampin elimination half-life following i.v. administration (2.25 +/- 0.64 h) was not different from that observed following p.o. dose administration (2.61 +/- 1.35 h). Peak rifampin concentrations were significantly higher following i.v. administration when corrected to a 300 mg/m2 dose (27.4 vs. 9.1 micrograms/ml, respectively, p less than 0.0001) than after p.o. administration. The peak concentration following a p.o. dose occurred at 2.0 +/- 0.9 h. The ratio of desacetylrifampicin to rifampin areas under the curves were similar for i.v. and p.o. routes of administration (0.23 vs. 0.19), suggesting linear metabolism of rifampin to this metabolite. 3-formylrifamycin SV concentrations were lower than those of desacetylrifampicin and were detectable in less than half of the patients. The results of this study indicate the need for larger p.o. doses when serum concentrations similar to those obtained following intravenous doses are desired.
Administration, Oral↗
Diurnal oscillations of CSF valproate in monkey.
Valproate (VPA) was administered to four rhesus monkeys by constant-rate intravenous infusion for two weeks under controlled conditions. Plasma and CSF samples were collected for a period of 27 hours at 3-hour intervals during steady-state and post-infusion periods. The mean correlation coefficient between total plasma and CSF VPA concentrations was found to be 0.78 +/- 0.09. The CSF VPA levels reflected the changes in free VPA in plasma but the two were not equivalent. Diurnal fluctuations in CSF VPA concentration were similar to those found in plasma but the inter-animal variation was greater in CSF than in plasma.
Animals↗
Time-dependent kinetics viii: absence of diurnal oscillations in valproic acid disposition following single dose administration to rhesus monkey.
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Animals↗
Bioavailability of regular and controlled-release chlorpheniramine products.
The bioavailability of chlorpheniramine regular-release versus controlled-release products was compared using 15 human subjects. The dosage forms evaluated were an 8-mg barrier coated-bead capsule, an 8-mg repeat action tablet, two 4-mg tablets, and 4- and 8-mg syrups. Single doses of each product were administered orally in a 5-way crossover study, plasma samples were collected at specific time intervals, and chlorpheniramine levels assayed by HPLC. Pharmacokinetic analysis was based on a two-compartment open model. The average plasma elimination half-life of chlorpheniramine was calculated to be approximately 18.3 hr. The controlled-release products gave a higher Cmax than the 4-mg syrup, but less than two 4-mg tablets. The controlled-release products also extended the time necessary to attain peak drug levels compared to the 4- and 8-mg syrups. The area under the curve (AUC) data for the controlled-release products was not equivalent to equal amounts of the regular-release products. The study indicated that while the controlled-release chlorpheniramine products were successful in prolonging the time course of absorption, this was at the expense of incomplete bioavailability of the drug.
Adolescent↗
Diurnal oscillations in plasma protein binding of valproic acid.
In view of the observed variation of valproic acid (VPA) free fraction (fp) during a dosing interval and the competitive binding effect of free fatty acids (FFA) in vitro, this study was designed to address the existence of diurnal variations in the fp of VPA. Six subjects were hospitalized at 7 a.m. for 25 h, and plasma samples were collected every 2 h. The protocol was repeated in 4 of the 6 subjects one week later. In vitro binding of VPA (100 micrograms/ml) was determined by equilibrium dialysis (14C-VPA), and FFAs were assayed colorimetrically. Phenytoin (PHT) binding was also determined for comparison. VPA fp ranged from 8.10 +/- 1.16 to 9.63 +/- 1.54. Intrasubject variability was also measured by the ratio of maximum to minimum fp values (fp max/fp min) over 24 h: This ratio ranged from 1.30 to 1.68 (mean +/- %SD = 1.51 +/- 7.7%, n = 10). For PHT, fp ranged from 10.88 +/- 0.50 to 12.39 +/- 1.07, and fp max/fp min from 1.09 to 1.31 (1.17 +/- 5.1%, n = 10). The fp max was observed between 2 and 6 a.m. in 7 out of 10 cases for VPA and 5 out of 10 cases for PHT. FFA levels, although in the normal range, varied two- to fourfold within 24 h. A significant correlation was observed between mean FFA levels at each sampling time and the corresponding fp values for VPA (p less than 0.001), but not for PHT.
Adolescent↗
Plasma protein binding interaction between valproic and salicylic acids in rhesus monkeys.
The effects of three levels of salicylic acid on the steady-state plasma concentrations of free and total valproic acid were examined in catheterized rhesus monkeys. Valproate was infused intravenously for a total of 41 hr, and salicylate was added after the first 8 hr. The three salicylate infusions were randomly assigned to each monkey. Valproate free fraction was determined by equilibrium dialysis. Statistically significant increases in valproate free fraction and total body clearance were observed after addition of salicylic acid. The increase in valproate clearance was positively correlated with the molar ratio of salicylate to valproate steady-state plasma concentrations. There was no significant change in valproate free concentration after salicylate treatment. The proposed mechanism of this in vivo interaction includes plasma protein binding displacement with no change in valproate intrinsic clearance.
Animals↗
An extensive exploration of the rat brain for sites mediating prostaglandin-induced hyperthermia.
Experiments designed to ascertain the site or sites within the brain where prostaglandins act to evoke hyperthermia were carried out. At an ambient temperature of 20 degrees C, microinjections of PGE1 (50-100 ng) or saline vehicle were made in restrained rats. The 272 sites tested formed a matrix encompassing much of the subcortical tissue rostral to the medulla. The sensitivity of each site to PGE1 was evaluated on the basis of the dose required to elicit a criterion level of effect and on the intensity of the effect evoked by a fixed dose. Injections of PGE1 into a sensitive site typically produced a rapidly developing, short lasting, monophasic rise in body temperature. Saline injected at these sites produced no initial hyperthermia. Almost all of the active sites were located within the anterior hypothalamic/preoptic region (AH/PO). The sites of greatest reactivity were found in the ventral aspect of the tissue lying between the anterior commissure and the optic chiasm. These data suggest that the AH/PO region is an important and probably the only supramedullary site of PGE1 action in the rat brain.
Animals↗
Interference by p-hydroxyphenobarbital in the 125I-radioimmunoassay of serum and urinary phenobarbital.
A radioimmunoassay for barbiturates is shown to be equally sensitive to phenobarbital and its major urinary metabolite, p-hydroxyphenobarbital, in serum and urine. Interference by the metabolite can be essentially eliminated by selectively extracting phenobarbital into chloroform. The extraction efficiency of the method for phenobarbital was 93+/-2% (SD) over the concentration range studied. Although cross reactivity between barbiturates and their metabolites may be less important in determining cases of barbiturates abuse or overdose, it may be extremely important if data on serum or urine are required for accurate estimates of drug disposition, or in establishing dose/response relationships.
Cross Reactions↗
Effect of central cholinergic blockade on the hyperthermia evoked by prostaglandin E1 injected into the rostral hypothalamus of the rat.
The possibility of a cholinergic involvement in the hyperthermic action produced by the injection of prostaglandin E1 (PGE1) into the anterior hypothalamic/preoptic region of the rat brain was examined. Intracerebral injection of either atropine or mecamylamine prior to the injection of PGE1 failed to attenuate the PGE1-induced hyperthermia. Both atropine and mecamylamine by themselves produced a rise in colonic temperature. It thus seems unlikely that PGE1 evokes hyperthermia in the rat by releasing endogenous acetylcholine at muscarinic or nicotinic synapses in the rostral hypothalamus. The possibility that PGE1 acts by inhibiting the release of acetylcholine within this region requires additional investigation.
Animals↗
High performance liquid chromatographic analysis of six conjugated and unconjugated estrogens in serum.
Six estrogens-estrone, equilin, estradiol, sodium estrone sulphate, sodium equilin sulphate and sodium 17-alpha-dihydroequilin sulphate in dog serum were successfully separated and quantified by a high performance liquid chromatographic method developed in our laboratories. The mobile phase was optimized by studying the effects of an organic modifier (acetonitrile) and an ion pairing reagent (tetrabutylammonium hydroxide). The serum sample work-up procedure was designed to recover both conjugated and unconjugated estrogens. The optimum method involved acetonitrile to precipitate serum proteins/peptides and extract estrogens. Residues were reconstituted in 50% acetonitrile in water for injection. The detection limits for these six estrogens via UV detection ranged from 0.5 to 5 ng on-column with a signal-to-noise ratio (S/N) of 10 for a 20 microL injection and via fluorescence 0.1 ng on-column for 17-beta-estradiol. Validation data are included for all six estrogens.
Acetonitriles↗
Bioavailability of oral and intramuscular phenobarbital.
The absorption of phenobarbital was compared in healthy adult subjects after oral and intramuscular therapeutic doses. Serum levels of phenobarbital were determined for 21 days after dosing by means of radioimmunoassay. Serum levels were similar from both dosage routes, with peak levels occurring at 1-3 hours after dosing and then declining slowly with an elimination half-life of about 90 hours. The overall efficiency of phenobarbital absorption from intramuscular doses was approximately 80 per cent of that from equivalent oral doses. Except in cases where oral dosing is not appropriate, there is no clinical advantage in giving phenobarbital intramuscularly to adult patients.
Administration, Oral↗
Pharmacokinetics of phenobarbital following single and repeated doses.
Serum levels of phenobarbital, and also urinary excretion of phenobarbital and p-hydroxyphenobarbital, were examined after single and repeated oral doses of phenobarbital to three male subjects. Serum levels of phenobarbital at steady state were approximately ten times as high as those after a single dose. The overall elimination rate constant for loss of phenobarbital from serum, Kel, was significantly reduced after repeated doses, and Cmax infinity values calculated from single-dose data poorly predicted observed Cmax infinity values. Five-day urinary excretion of phenobarbital and p-hydroxyphenobarbital accounted for 16 and 21 per cent, respectively, of the initial dose. Due to extensive drug accumulation, 83 per cent of the final dose was excreted in five-day urine as phenobarbital and 85 per cent, as p-hydroxyphenobarbital. Comparison of plasma and renal clearances indicated that the rate of phenobarbital metabolism was reduced owing to repeated dosing, while the rate of urinary excretion of parent drug was unchanged.
Adult↗
Analytical methods validation: bioavailability, bioequivalence and pharmacokinetic studies. Conference report.
This is a summary report of the conference on Analytical Methods Validation: Bioavailability, Bioequivalence and Pharmacokinetic Studies. The conference was held from December 3 to 5, 1990 in the Washington, DC area and was sponsored by the American Association of Pharmaceutical Scientists, US Food and Drug Administration, Federation International Pharmaceutique, Health Protection Branch (Canada) and Association of Official Analytical Chemists. The purpose of the report is to represent our assessment of the major agreements and issues discussed at the conference. The report is also intended to provide guiding principles for validation of analytical methods employed in bioavailability, bioequivalence and pharmacokinetic studies in man and animals. The objectives of the conference were: 1. To reach a consensus on what should be required in analytical methods validation and the procedures to establish validation; 2. To determine processes of application of the validation procedures in the bioavailability, bioequivalence and pharmacokinetic studies; 3. To develop a report on analytical methods validation (which may be referred to in developing future formal guidelines). Acceptable standards for documenting and validating analytical methods with regard to processes, parameters or data treatments were discussed because of their importance in assessment of pharmacokinetic, bioavailability and bioequivalence studies. Other topics which were considered essential in the conduct of pharmacokinetic studies or in establishing bioequivalency criteria, including measurement of drug metabolites and stereoselective determinations, were also deliberated.
Biological Availability↗