José A. Morais

Simple high-performance liquid chromatographic assay for the determination of ciprofloxacin in human plasma with ultraviolet detection

A simple high-performance liquid chromatographic method is described for the quantitative analysis of ciprofloxacin in human plasma. Following protein precipitation from plasma by means of 6% perchloric acid, the upper layer which contains the analyte and the internal standard lomefloxacin, was analysed on a reverse phase column LiChrospher 60 RP-select B (5 microm) (EcoCART 125-3) with ultraviolet detection at 280 nm. The mobile phase was acetic acid 5%-methanol-acetonitrile (90:5:5, v/v/v). The assay was linear for ciprofloxacin over the concentration range of 0.050 to 6.00 microg ml(-1). The limit of quantification (LOQ) was 0.050 microg ml(-1). The method was successfully applied to a bioavailability study with five different ciprofloxacin formulations.

Determination of the antidepressant fluoxetine in human plasma by LC with UV detection

A selective and sensitive isocratic high-performance liquid chromatographic (HPLC) method was developed for the quantitative analysis of low concentrations of fluoxetine (FLX) in human plasma, with ultra-violet detection at 226 nm. A reversed-phase column, LiChrospher 60 RP-Select B (125 x 3 mm i.d., 5 microm) (Merck), was used to resolve FLX and diazepam (DZP) (internal standard) from endogenous matrix interferences. FLX was isolated from plasma by liquid-liquid extraction. Two identical HPLC systems were used, both validated under the same study conditions. Each chromatographic separation was completed in 30 min and the results showed a mean relative recovery of 101 and 99.3% and an overall precision (RSD%) of 4.78 and 6.09 for each HPLC system. The standard curve was linear for FLX concentrations over the range of 5.00-50.0 ng ml(-1) (R = 0.997 and 0.998). The limit of quantitation of FLX was 5 ng ml(-1) for both HPLC systems. The method described was applied to the analysis of plasma samples obtained from healthy subjects treated with one single oral dose of 40 mg of fluoxetine.

Comparative bioavailability of two immediate release tablets of enalapril/hydrochlorothiazide in healthy volunteers

SummaryA bioequivalence study of two oral formulations of 20/12.5 mg tablets of enalapril/hydrochlorothiazide was carried out in 20 healthy male volunteers according to a single dose, two-sequence, crossover randomized design. One washout period of nine days was observed between the two periods. Multiple samples were collected over 96 hours post-dosing. Bioavailability was evaluated on the basis of plasma concentrations of enalapril and its main active metabolite, enalaprilat and hydrochlorothiazide. Plasma samples were assayed for enalapril, enalaprilat and hydrochlorothiazide using a selective and sensitive high-performance liquid chromatography method with mass spectrometry detection (LC-MS).The pharmacokinetic parameter values of Cmax and tmax were obtained directly from plasma data, ke was estimated by log-linear regression, and AUC was calculated by trapezoidal rule. Different statistical tests were performed on the basis of untransformed and log-transformed data and the overall residual variance from ANOVA. Assuming the accepted tolerance intervals, a β-error of 20% and 90% confidence intervals (α=0.10), all the generally accepted tests (Schuirmann test and Wilcoxon-Tukey and Hauschke nonparametric tests) showed that the formulations can be considered as bioequivalent with respect to the extent of absorption, given by the AUC0−∞ and with respect to rate of absorption as assessed by Cmax and tmax.

Sensitive method for the determination of phenytoin in plasma, and phenytoin and 5-(4-hydroxyphenyl)-5-phenylhydantoin in urine by high-performance liquid chromatography

An isocratic high-performance liquid chromatographic method is described for the quantitative analysis of phenytoin (PHT) in plasma and both phenytoin and its main metabolite, 5-(4-hydroxyphenyl)-5-phenylhydantoin (p-HPPH) in urine. Following ethyl acetate (plasma) and Extrelut-1 (urine) extraction, samples are analysed by means of a reversed-phase column (Nova-Pak C18), using a mobile phase consisting of methanol-water-tetrahydrofuran (40:60:4, v/v/v) with UV detection at 230 nm. The chromatography is complete in 10 min and the results show good precision (RSD 1.23-4.49%) and sensitivity for a linear range of 0.4-4.0 micrograms ml-1 for PHT in plasma, 0.1-1.0 microgram ml-1 for PHT in urine and 0.1-1.2 microgram ml-1 for p-HPPH in urine. The results indicate the method to be suitable for pharmacokinetic studies.

Bioequivalence evaluation of three different oral formulations of ciprofloxacin in healthy volunteers

SummaryTwo bioequivalence studies were performed in twenty four healthy male volunteers with the objective of comparing the bioavailability of three different oral formulations of ciprofloxacin as immediate release tablets 250, 500 and 750 mg (test formulations) with a reference formulation at 500 and 750 mg strengths forms.In study 1, the subjects were enrolled in a single-dose, open-label, 3-period, crossover randomised study, designed to compare the bioavailability of two test formulations of ciprofloxacin (A and B) as 250 and 500 mg tablets, compared to the reference formulation (C), as 500 mg tablets.In study 2, the same 24-subjects were included in a single-dose, open-label, 2-period, crossover randomised study, designed to compare the bioavailability of one test formulation of ciprofloxacin (A) as compared to the reference formulation (B), both products as 750 mg tablets. In both studies multiple blood samples were collected over 24 hours post-dosing. One washout period of six days was observed between the periods. Plasma was harvested and assayed for ciprofloxacin using a selective and sensitive high-performance liquid chromatography (HPLC) method with UV detection.The pharmacokinetic parameter values of Cmax and tmax were obtained directly from plasma data, ke was estimated by log-linear regression, and AUC was calculated by trapezoidal rule. Different statistical tests were performed on the basis of untransformed and log-transformed data and the overall residual variance from ANOVA. Assuming the accepted tolerance intervals, a β-error of 20% and 90% confidence intervals (α=0.10) of all the generally accepted tests (Westlake, Schuirmann test and Wilcoxon-Tukey nonparametric tests) showed that the formulations can be considered as bioequivalent with respect to the extent of absorption, given by the AUC0-∞ and with respect to rate of absorption as assessed by Cmax and tmax.

Comparative bioavailability of two immediate release tablets of cisapride in healthy volunteers

SummaryRelative bioavailability of cisapride was investigated after oral administration of a test versus a reference formulation of immediate release tablets of cisapride, both with 10 mg per unit. The study was conducted in a two-way cross-over design, as a single dose open-label randomised trial. The two formulations were administered in two treatment days, separated by a washout period of 6 days, in fasted subjects who received one single oral dose of 20 mg of one study medication of cisapride as two 10 mg tables. Multiple samples were collected over 24 h post-dosing. Plasma samples were assayed for cisapride using a selective and sensitive high-performance liquid chromatography (HPLC) method with UV detection. The pharmacokinetic parameter values (mean±RSD%) of cisapride as the test formulation were: AUC0−∞=329±20.9 ng.h/ml, Cmax=52.8±22.6 ng/ml, tmax=1.26±22.0 h and t1/2=4.08 \+-15 h. Following administration of the reference formulation the values obtained for the same parameters were: AUC0\t-\t8=317\+-19.2 ng.h/ml, Cmax=49.2\+-21.3 ng/ml, tmax=1.38\+-30.1 h and t1/2=4.52\+-24.8 h. These results show that the two cisapride formulations can be considered as bioequivalent, with respect to the above mentioned parameters.

A comparative bioavailability study to estimate the influence of an antacid on droxicam pharmacokinetics

SummaryThe influence of an antacid on droxicam pharmacokinetics was investigated in 12 healthy male volunteers. A two way cross-over study was performed after the oral administration of 20 mg of droxicam (Ombolan capsules) and droxicam together with an antacid (Mucal powder). The plasma concentrations of piroxicam, the active moiety of droxicam, were determined by a high-performance liquid chromatographic method. The pharmacokinetic parameter values (mean ± RSD) of piroxicam after administration of droxicam alone were: AUC0→∞=125.5 ± 25.1 μg.h/l. Cmax=2.08 ± 19.9 μg/l, tmax=7.08±36.8 h and t1/2=46.3 ± 27.0 h. Following administration of droxicam together with the antacid the values obtained for the same parameters were: AUC0→∞=135.1 ± 24.1 μg.h/l, Cmax=1.85 ± 23.9 μg/l, tmax=8.17 ± 34.9 h and t1/2=52.0 ± 22.4 h. These results indicate that concurrent administration of the antacid does not significantly change the bioavailability and pharmacokinetics of droxicam.

Absorption of intra-bronchial diprophylline-methodology and preliminary results

SummaryWith the objective of measuring alveolo-capillary permeability, a diprophylline solution (molecular weight 254 D) was instilled as a bolus. Instillation was performed through distallized impacted fiber bronchoscope and 3 normal volunteers and 3 patients with pulmonary fibrosis were evaluated. Blood samples were collected at regular intervals with subsequent pharmacokinetic study, mainly aimed at determining absorption kinetics. This technique enabled us to distinguish the two groups of individuals which had different absorption rates. In controls, kinetics apparently followed a simple, one compartment model for absorption, which was slower and occurred to a lesser extent Patients with a pulmonary fibrosis had a faster and higher degree of absorption, with a larger plasma Cmmax. In the latter group two-compartment kinetics for absorption was found. We conclude that absorption kinetic parameters can disclose data on structural integrity of epithelia and possible lesions of the lung interstitium.