Multivariate analysis of capillary electrophoresis separation conditions for Z-E isomers of clomiphene.

Plackett-Burman (P-B) experimental design has been used to optimize the factors affecting separation of Z and E isomers of clomiphene (zuclomiphene and enclomiphene respectively) using capillary electrophoresis. The P-B design was used to simultaneously investigate the following five factors: buffer ionic strength, buffer pH, heptakis (2,3,6-tri-o-methyl) beta-cyclodextrin (TMCD) concentration, methanol concentration and injection time, each at three levels. In addition to these, a dummy variable was added to estimate the variability of the system. Effects on resolution and analysis time were calculated. Based on the information gained from the P-B design, the following set of conditions was chosen: 100 mM phosphate buffer pH 2.3, 5 mM TMCD, 5% methanol, and 1.7 s hydrodynamic injection time. These conditions gave well-resolved peaks for zuclomiphene and enclomiphene.

Normal phase LC-MS determination of retinoic acid degradation products.

The degradation products formed when 13-cis retinoic acid (13-cis RA) and all-trans RA were exposed to fluorescent light and air were investigated. These retinoids are known to undergo Z-E isomerization (due to the existence of four unsaturated double bonds) and oxidation when exposed to light and air. Analysis by LC was carried out on a 25 cm x 4.6 mm Zorbax Rx-SIL (5 microns) with a mobile phase (1.4 ml min-1) of heptane-THF-acetic acid (96.5:3.5:0.015) and an in-line UV (365 nm) detector. The LC eluate was coupled through a Vestec universal interface to a Finnigan 4023 mass spectrometer. EI-mass spectra were obtained at 77 eV from m/z 200 to 350 with multiplier voltage of 1200 V. Solid samples of 13-cis RA and all-trans RA exposed to light and air and also solutions of these retinoids in the mobile phase exposed to the same conditions were used for the analysis. Tentative identities of the degradation products from the mass spectra suggest the isomerization of the retinoids (Z-E isomerism) and the formation of the 5,6-epoxides of these isomers. Identities of the 5,6-epoxides were confirmed with chromatographic and mass spectral data from synthetic samples of the epoxides. Isomerization occurred more readily in solution than in the solid form and the 13-cis RA isomer oxidized more readily than the all-trans isomer.

Separation of 13-cis and all-trans retinoic acid and their photodegradation products using capillary zone electrophoresis and micellar electrokinetic chromatography (MEC).

Two retinoic acid isomers; 13-cis retinoic acid and all-trans retinoic acid and their photodegradation products were resolved with capillary electrophoresis (CE) (UV detector, 345 nm) using three different mobile phases: method 1--an acetonitrile modified borate buffer (pH 8.5); method 2--borate buffer (pH 8.5) modified with acetonitrile and alpha-cyclodextrin; and method 3--borate buffer (pH 8.5) modified with SDS (MEC). Concentration of acetonitrile in the buffer was varied from 10 to 50% in method 1 and resolutions of 0-1.9 were obtained for the two retinoic acid isomers. Similarly in method 2, concentration of alpha-cyclodextrin in the buffer (with 10% acetonitrile) was varied from 0 to 40 mM, giving resolutions of 0-3.8. In method 3, concentration of SDS in the buffer was varied from 5 to 60 mM resulting in resolutions of 1.3-4.1. Optimum separation conditions for the three methods were applied to the separation of photodegradation products of the two retinoids after exposure to fluorescent light for 36 h. A buffer modified with 45% acetonitrile and the same buffer modified with 10 mM SDS gave incompletely resolved electropherograms with a 72 cm x 50 microns capillary (50 cm to the detector). A buffer containing 20 mM alpha-cyclodextrin 10% acetonitrile gave completely resolved peaks for each isomer. The buffer containing 10 mM SDS gave completely resolved peaks for the photodegradation products when a 122 cm x 50 microns capillary (100 cm to detector) was used.

Association of beta-agonists with corresponding beta 2- and beta 1-adrenergic pentapeptide sequences.

Synthesized beta 1- and beta 2-pentapeptide sequences corresponding to published adrenoceptor transmembrane activation site subtypes were investigated in vitro for selectivity in association for drug ligands of known selectivity. Both nuclear magnetic resonance spectroscopy and molecular mechanics demonstrated that structural differences among the corresponding pentapeptide activation-site sequences can explain agonist selectivity. Results suggest the agonists bind across the activation site loop on the second transmembrane alpha-helix by dipole/dipole interactions between a ligand and the peptide. Since electrostatic interactions within the membrane may determine the rate of intercellular ion flux, agonist association across the activation site sequence could thereby decrease electrostatic resistance to positive ion flux into the cell. Interactions between the peptides and the ligands may provide insight into the structures and mechanisms involved in association of ligands for the identical sequences on the beta-adrenoreceptors.

Nuclear magnetic resonance (NMR) spectroscopic investigation of interaction energies of ephedrine stereoisomers in noncrystalline solids and its correlation with thermodynamic data.

Equations relating the interaction energies of each of the binary mixtures of ephedrine from linear combinations of the energies of the individual isomers are presented. The interaction energies in the noncrystalline solid mixtures measured from NMR chemical shift data using cross-polarization magic angle spinning nuclear magnetic resonance 13C cross-polarization magic angle spinning nuclear magnetic resonance (13CP/MAS NMR) spectroscopy correlate strongly with interaction energy from thermodynamic data. The summation of changes in relative frequencies for structurally equivalent carbons is used as a measure of differences in electron shielding on mixing. The relative direction of polarization of individual stereoisomers is found to affect association in noncrystalline binary mixtures of solids. NMR chemical shift data of solids may be useful in confirming spectroscopically the interactions of stereoisomers observed thermodynamically.

Effect of dietary fat content on the bioavailability of a sustained release quinidine gluconate tablet.

Quinidine gluconate 324 mg sustained release tablets (Quinaglute) was administered as a single dose to 15 healthy male subjects following an overnight fast, immediately following a high fat (HF) breakfast or immediately following a low fat (LF) breakfast. Serum samples were obtained over a 48 h period and analyzed for quinidine content using a high performance liquid chromatographic assay. Under the conditions of the study, both the rate and extent of quinidine bioavailability was significantly affected by food. The extent of bioavailability was statistically significantly greater (p less than 0.05) following both the HF and LF meals as compared to that in the fasted state. Rate of bioavailability was significantly enhanced following the LF meal as compared to that of the other two treatment groups. Although peak concentrations were greater and time to peak concentrations somewhat later following the HF meal versus those under fasting conditions, these differences were not statistically significant. In addition, the characteristics of the serum concentration-time profile (as defined by the number, magnitude, and time of occurrence of the multiple absorption maxima) was unique for each of the three treatment groups. Possible mechanisms underlying these results are explored.

Chromatographic separation and photoelectrochemical detection of sodium nitroprusside and its degradative and metabolic products.

Ion-pair chromatography has been used for the separation of nitroprusside ion and its photochemical hydrolytic and metabolic products. Organic modifier and pH were adjusted for maximum separation of the ions. Methanol was selected as the organic modifier in a pH range 5-8 and tetrabutylammonium perchlorate was used as the ion-pairing reagent. Ions were detected with a photoelectrochemical detector as described by Krull. A modification of this procedure was used to detect nitroprusside ion in spiked serum samples.

Bioequivalency and dose proportionality of three tableted promethazine products.

Data from a five-way crossover study in human subjects using four talented promethazine products and a promethazine solution are presented. All products were administered as a single oral dose. The five objectives of the study were to investigate bioequivalency, to estimate dose proportionality at two dose levels, to establish validity of a reference production solution for future bioequivalency studies, to estimate intersubject variation, and to compare bioavailability/tablet dissolution data. Blood samples were collected at given intervals over a 24-hour period and analysed for promethazine using an HPLC technique. Pharmacokinetic parameters were calculated using standard procedures and a two-way analysis of variance (ANOVAR) was used to assess whether the differences were statistically significant. The AUC0----infinity data from the ANOVAR analysis showed that the 50 mg innovator and generic products and the 50 mg solution were not significantly different. However, the innovator product had a significantly lower Cmax and longer tmax than the solution. The generic product did not differ significantly from the solution. Promethazine was found to exhibit linear dose proportionality in the range and product studied. Intersubject variation was high for all parameters (23 to 63 per cent) and the in vivo and in vitro data showed a positive relationship.

Rotary pursuit, a measure of human performance, and plasma concentrations of promethazine.

Promethazine in doses of 50 mg has demonstrated detrimental effects upon the performance of visual tasks. The purpose of the study was to examine the relationship between the blood concentration levels of promethazine and two human performance tasks. Fifteen paid healthy male volunteers completed a randomized five-way crossover design which included a 25 mg and 50 mg dose of the innovator dosage form, a 50 mg dose of a generic dosage form, a 50 mg solution dosage form, and a placebo. Serial blood samples were obtained in addition to performance measures of rotary pursuit and a simple force choice reaction time. Analysis of the forced choice reaction depicted a mild relationship with the blood concentration levels of promethazine. However, the measures of rotary pursuit, a more sensitive determinant of human motor performance, proved to be more related to both the promethazine blood concentration and the inherent learning which was confounded in the experiment. The degree of impaired pursuit performance and reaction time differences could be defined in terms of a linear relationship to the promethazine concentration.

Stable-isotope methodology in the bioavailability study of 17 alpha-methyltestosterone using gas chromatography-mass spectrometry.

The application of a stable-isotope coadministration technique for estimating the relative bioavailability of 17 alpha-methyltestosterone is described. Eight healthy male subjects were administered orally a single 10-mg 17 alpha-methyltestosterone tablet together with a 10-mg 17 alpha-methyltestosterone-d3 solution. The serum concentrations of 17 alpha-methyltestosterone and 17 alpha-methyltestosterone-d3 were determined by gas chromatography-mass spectrometry with selected ion monitoring using 17 alpha-methyltestosterone-d6 as an internal standard. The extent of absorption from the tablet formulation was comparable to that from the oral solution. The stable-isotope methodology was compared with the conventional cross-over method for evaluating the bioavailability of 17 alpha-methyltestosterone.

In vitro and in vivo bioequivalence of commercial prednisone tablets.

The purpose of the study was to examine the bioequivalence of five commercially available oral prednisone products. The in vivo study utilized 18 healthy males, each of whom was administered 20 mg of prednisone as a reference solution or as a tablet in a 6-week, six-way crossover design. Blood was collected and serum was assayed, using an HPLC procedure specific for prednisone and prednisolone. Mean pharmacokinetic parameters (t 1/2, ke, Cmax, tmax, and AUC) were determined. ANOVA was performed on the prednisone and prednisolone data (F-test, p less than 0.05) as well as Duncan's multiple range analysis. Dissolution tests were also performed on each of the five products in order to test the relationship between dissolution and bioequivalence among prednisone products. The in vitro study consisted of a standard USP dissolution test which included tablets from the same lots as the tablets used in the in vivo study. The data showed no statistical difference in any of the pharmacokinetic parameters among tableted products, subjects, or dosing periods in the study. There was also no statistical difference in the dissolution study among the five commercial tablet forms.

An in vivo single- and multiple-dose study of several marketed brands of conventional and controlled-release theophylline.

In a single-dose study, 18 healthy adult males consumed each of six dosage forms of theophylline. A conventional-release tablet, a syrup, and four competing brands of controlled-release theophylline were studied. Serial serum samples were obtained and analyzed via high pressure liquid chromatography (HPLC). After achieving steady state, 15 healthy adult males consumed each of five dosage forms of theophylline in a multiple-dose study. Serial blood samples were obtained between 0 and 72 hours and subjected to analysis with HPLC. The results indicated that the controlled-release products were not bioequivalent, although they achieved longer time-to-peak values than did the immediate-release syrup and the conventional-release tablet. A single sustained-release product was uniquely different on most pharmacokinetic parameters when compared with the remaining three controlled-release products. In general, the dosage form variation exceeded the individual subject variation on the single-dose study, but the opposite was true for the multiple-dose study.

Blood levels following multiple oral dosing of chlorpheniramine conventional and controlled release preparations.

An examination of steady-state performance of chlorpheniramine conventional versus controlled release products was conducted using 15 male subjects in a 3-way crossover study with a 2-week washout period between studies. The study was designed to determine if chlorpheniramine formulations provide consistent pharmacokinetic performance between individual units upon going from single dose to multiple dose therapy. In addition, the validity of predicting steady-state levels for these kinds of products using only single oral dose data was examined. The dosage forms evaluated were a conventional 4 mg tablet, and 8 mg barrier coated-bead capsule, and an 8 mg repeat action tablet. Multiple doses of each product were orally administered to each subject for 6 days prior to the study day to achieve steady-state levels and on the actual study day. Serum samples were collected at specific time intervals on the study day, and chlorpheniramine levels assayed by HPLC. Pharmacokinetic analysis was based on a two-compartment open model. The mean plasma elimination half-lives of the various dosage forms were in the range 24.5-25.4 h. There was no rapid release of drug from the controlled release products nor did they have drug release problems during the dosing interval. Good agreement was obtained between predicted average drug concentration at steady-state and drug concentration actually present for all the formulations studied. Based upon comparative examination of AUC, Cmax, and fraction of dose absorbed data, the controlled release products administered every 12 h were comparable in performance to a conventional release tablet administered every 6 h. Since the half-life of chlorpheniramine is approximately 1 day, therapeutic management may possibly be gained with dosing the patient once daily with a controlled release product or twice daily with a conventional tablet.

Pharmacokinetics and bioavailability of hydromorphone following intravenous and oral administration to human subjects.

In a relatively small pilot study, the half-life of elimination of hydromorphone in six subjects was 2.64 +/- 0.88 hours and the drug had a high volume of distribution, 1.22 l./kg. In addition, the drug was rapidly but incompletely absorbed after oral administration. An equation to predict the plasma concentration of hydromorphone on oral administration was developed from the data of these six subjects.

Radioimmunoassay of hydromorphone and hydrocodone in human plasma.

A radioimmunoassay for hydromorphone in human plasma was developed using a commercially available morphine-6-antiserum and tritiated dihydromorphine. In the assay, free and bound drug are separated using dextran-coated charcoal. The method quantitates hydromorphone in the 2.5-20-ng/ml range with minimum sensitivity at 1.0 ng/ml. Within-run precision for hydromorphone as shown by the standard error of the estimate of the linear regression equation is +/- 1.01 ng/ml. Between-run precision data for hydromorphone at the 2,5-, 10-, and 20-ng/ml levels gave percent relative standard deviations of 22.35, 10.96, and 8.55%, respectively. A plasma concentration-time curve from a subject administered a single oral dose of hydromorphone demonstrates the usefulness of the assay in monitoring drug levels in a bioavailability study. The method also is applicable to the analysis of hydrocodone in human plasma in the 10-80-ng/ml range with minimum sensitivity at 3.0 ng/ml. Within-run precision for hydrocodone as shown by the standard error of the estimate of the linear regression equation is +/- 1.06 ng/ml. Between-run precision data at the 15-, 30, and 60-ng/ml levels gave percent relative standard deviations of 12.48, 7.67, and 6.03%, respectively.

Plasma levels of clobazam after 10-, 20-, and 40-mg tablet doses in healthy subjects.

It is evident that substantial intersubject and intrasubject varition in the bioavailability of clobazam exists following ingestion of 10, 20 and 40 mg doses in these 12 volunteers. Peak concentrations and area under the plasma level-time curve were directly proportional to the dose of clobazam and the mean plasma half-life of clobazam was about 18 hours regardless of dose administered. The t1/2 value was less than that previously reported, as the current results allow differentiation of parent drug from metabolites. This 18 hr t1/2 compares favorably with the half-life of other benzodiazepines.