Danilo César Galindo Bedor

Simultaneous determination of sulfamethoxazole and trimethoprim in biological fluids for high-throughput analysis: comparison of HPLC with ultraviolet and tandem mass spectrometric detection.

The comparison of two methods based on online solid phase extraction-liquid chromatography with UV (SPE-LC-UV) or mass spectrometry detection (SPE-LC-MS/MS) for the simultaneous quantification of sulfamethoxazole (SMZ) and trimethoprim (TMP) is presented. The methods were validated and proved to be accurate. The analysis of standard samples for SMZ at concentrations of 0.5, 1.5, 25 and 50microg/mL demonstrated a relative standard deviation of less than 6% for both methods (n=18), while TMP samples at concentrations of 0.05, 0.15, 1.5 and 5.0microg/mL were analyzed with R.S.D. of less than 4% (n=18). The method with mass spectrometric detection was approximately six times more sensitive than the method with ultraviolet detection. The total run time for the SPE-LC-MS/MS was 2.5min per sample as opposed to 18.0min for the SPE-LC-UV method. The method with MS detection in comparison with UV detection proved to be more rugged and was successfully applied to pharmacokinetics studies.

Physicochemical Characterization and Development of a Microemulsion System for Transdermal Use

A microemulsion (ME) carrier system containing diclofenac diethylamine (DDA) was developed and physicochemically characterized. In addition, a methodology for analytical determination of DDA by UV-Vis spectrophotometry was validated. The methodology used to produce the ME was simple and reliable, producing a stable ME system with a high DDA entrapment efficiency of 89.6 ± 14.19%. The ME system presents a mean droplet size, conductivity, pH, refractive index and viscosity of around 50 nm, 300 µS cm−1, 7.0, 1.399, and 110 mPa, respectively. Furthermore, the analytical method developed to evaluate the DDA content was validated and used to quantify DDA in the ME formulations.

Reversed phase HPLC determination of tamoxifen in dog plasma and its pharmaco-kinetics after a single oral dose administration

The analytical method developed to evaluate tamoxifen in dog plasma samples was precise, accurate, robust and linear in the range of 5–200 ng/mL. The limits of detection and quantification were 0.981 ng/mL and 2.97 ng/mL, respectively. Besides, the intra-day precision and accuracy variations were 8.78 and 10.16%, respectively. Tamoxifen concentrations were analyzed by combined reversed phase liquid chromatography and UV detection (λ=280 nm). The study was conducted using an open randomized 2-period crossover balanced design with a 1-week washout period between the doses. This simple, rapid and selective method is suitable for pharmacokinetic, bioavailability and bioequivalence studies.

Correlation between microstructure and bioequivalence in Anti-HIV Drug Efavirenz

Polymorphism and particle size distribution can impact the dissolution behaviour and, as a consequence, bioavailability and bioequivalence of poorly soluble drugs, such as Efavirenz (EFV). Nevertheless, these characteristics do not explain some failures occurring in in vitro assays and in in vivo studies. EFV belongs to Class II and the High Activity Antiretroviral Therapy (HAART) is considered the best choice in the treatment of adults and children. EFV is a drug that needs bioequivalence studies for generic compounds. In this work, six raw materials were analyzed and two of them were utilized with human volunteers (in vivo assays or bioequivalence). All the routine pharmaceutical controls of raw materials were approved; however, the reasons for the failure of the bioequivalence assay could not be explained with current knowledge. The aim of this work was to study microstructure, a solid-state property of current interest in the pharmaceutical area, in order to find an explanation for the dissolution and bioequivalence behaviour. The microstructure of EFV raw materials was studied by Whole Powder Pattern Modelling (WPPM) of X-ray powder diffraction data. Results for different EFV batches showed the biorelevance of the crystalline domain size, and a clear correlation with in vitro (dissolution tests) and in vivo assays (bioequivalence).

Relative bioavailability of two formulations of venlafaxine extended-release 75-mg capsules in healthy brazilian male volunteers: A single-dose, randomized-sequence, open-label, two-period crossover study in the fasting and fed states

BACKGROUND The oral antidepressant venlafaxine hydrochloride is a selective serotonin-norepinephrine reuptake inhibitor. OBJECTIVE The aim of this study was to evaluate the bioequivalence of a new generic formulation of venlafaxine extended-release 75-mg capsules (test) and the available branded formulation (reference) to comply with regulatory criteria for marketing of the test product in Brazil. METHODS This single-dose, randomized-sequence, open-label, 2-period crossover study was conducted in healthy male volunteers and consisted of separate fast- ing and fed phases. A single oral dose of the test or reference formulation was followed by a 7-day washout period, after which subjects received the alternative formulation. There was a 3-month interval between the fasting and fed portions of the study. There was no standardization of race because of the difficulty of achieving standardization in the Brazilian population. Blood samples were collected before dosing and at 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, 8, 10, 12, 24, 36, and 48 hours after dosing. Venlafaxine concentrations were determined using an HPLC-MS/MS method. The formulations were considered bioequivalent if the 90% CIs of the geometric mean ratios (test:reference) for C(max) and AUC(0-t) were within the regulatory range of 80% to 125%. Adverse events were monitored through-out the study based on vital signs, laboratory tests, interviews, and spontaneous patient reports. RESULTS Forty-eight subjects were enrolled in both phases of the study; all 48 subjects completed the fasting phase, and 1 subject withdrew during the fed phase. The mean (SD) age of participants in the fasting and fed phases was 24.96 (5.5) and 24.90 (4.7) years, respectively; their mean weight was 69.65 (9.6) and 71.00 (10.6) kg and their mean height was 172.0 (6.9) and 173.0 (6.6) cm. Under fasting conditions, the arithmetic mean venlafaxine C(max) was 35.705 (23.946) ng/mL for the test formulation and 34.470 (20.639) ng/mL for the reference formulation, with a geometric mean ratio of 1.04. The arithmetic mean AUC(0-t) for the respective formulations was 562.015 (481.875) and 508.509 (439.456) ng · h/mL, with a geometric mean ratio of 1.11. The arithmetic mean T(max) was 6.188 (1.560) and 5.885 (1.648) hours. Under fed conditions, the arithmetic mean venlafaxine C(max) was 42.892 (24.348) ng/mL for the test formulation and 46.275 (23.011) ng/mL for the reference formulation, with a geometric mean ratio of 0.93. The arithmetic mean AUC(0-t) for the respective formulations was 737.218 (603.998) and 682.124 (524.713) ng · h/mL, with a geometric mean ratio of 1.08. The arithmetic mean T(max) was 6.787 (1.769) and 5.957 (1.661) hours. There were no significant increases in venlafaxine C(max), AUC(0-t), or T(max) for either formulation in the fed phase compared with the fasting phase. In both the fasting and fed portions of the study, the 90% CIs for the ratio (test:reference) of log-transformed C(max) (fasting: 93.24-105.93; fed: 84.67-97.85) and AUC(0-t) (fasting: 102.90-116.71; fed: 98.19-114.41) were within the acceptance range for bioequivalence. The most common adverse events (≥ 5% of subjects) in the fasting phase were nausea (46%), diarrhea (29%), headache (29%), vomiting (15%), and colic (6%); the most common adverse events in the fed phase were nausea (15%), headache (13%), and dizziness (9%). CONCLUSION In this single-dose study in healthy fasting and fed volunteers, the test formulation of venlafaxine extended-release 75-mg capsules met Brazilian regulatory criteria for bioequivalence to the reference formulation.

A sensitive and robust lc-ms/ms method with monolithic column and electrospray ionization for the quantitation of efavirenz in human plasma: application to a bioequivalence study

An LC-MS/MS method has been developed for the determination of efavirenz (EFZ) in human plasma using hydrochlorothiazide as internal standard (I.S.). An ESI negative mode with multiple reaction-monitoring was used monitoring the transitions m/z 313.88→69.24 (EFZ) and 296.02→204.76 (I.S.). Samples were extracted using liquid-liquid extraction. The total run time was 2.0 min. The separation was achieved with HPLC-RP using a monolithic column. The assay was linear in the concentration range of 100 - 5000 ng mL-1. The mean recovery was 83%. Intra- and inter-day precision were < 9.5% and < 8.9%, respectively and accuracy was in the range ± 8.33%. The method was successfully applied to a bioequivalence study.

Determination of Ampicillin in Human Plasma by Solid-phase Extraction -Liquid Chromatography -Tandem Mass Spectrometry (SPE-LC-MS/MS) and its Use in Bioequivalence Studies

A simple, fast, sensitive and selective solid-phase extraction-liquid chromatography-tandem mass spectrometry (SPE-LC-MS/MS) method for the quantitative analysis of ampicillin (CAS 69-53-4) in human plasma was developed using amoxicillin as internal standard, and sample extraction by solid-phase extraction (SPE). Extracts were separated by reversed-phase C18 with aqueous mobile phase (acetonitrile, 80:20, v/v) with 0.1% formic acid. The method was validated and successfully applied in a bioequivalence study of capsules 500 mg of ampicillin. Using a short running time of 2.5 min, the lower limit of quantification (LLOQ) for obtained ampicillin was 0.1 microg/ml for a plasma sample of 250 microl and a recovery of 94.38% +/- 4.05. Bioequivalence between the products was determined by calculating 90% confidence intervals (CI) for the ratio of Cmax, AUC0-t and AUC0-inf values for the test and reference products, which were within the 0.80-1.25 interval proposed by FDA and EMEA. It is concluded that the two formulations are bioequivalent in their rate and extent of absorption, and thus, may be used interchangeably.

The development and validation of a method for quantifying olanzapine in human plasma by liquid chromatography tandem mass spectrometry and its application in a pharmacokinetic study.

1. A rapid method using liquid chromatography tandem mass spectrometry for the quantification of olanzapine (OLZ) in human plasma was developed and validated. Venlafaxine was used as the internal standard (IS), and the samples were extracted from 400-μL human plasma with methyl tert-butyl ether for liquid-liquid extraction. 2. Chromatography was performed using an ACE C18, 125 × 4.6-mm i.d., 5-μm column. The mobile phase consisted of water with 0.1% formic acid for solvent A and acetonitrile with 0.1% formic acid for solvent B (50 : 50 v/v) in isocratic mode. The flow rate was 1.2 mL/min. The retention times for OLZ and the IS were 0.78 and 1.04 min, respectively. Tandem mass spectrometry operating in positive electrospray ionization mode with multiple reaction monitoring was used to detect OLZ and the IS (m/z: 313.1 > 256.1 and 278.1 > 260.2, respectively). 3. No significant matrix effects were observed on OLZ and the IS retention times, and the mean recovery of OLZ was 90.08%. The assay was linear in the concentration range of 1-20 ng/mL (R(2) = 0.9976). The intra- and inter-day precision were < 11.60% and the accuracy was < 1.66%. 4. This validated method was successfully applied to a pharmacokinetic study in which 10-mg OLZ tablets were administered to healthy volunteers and their plasma OLZ levels were monitored over time. The tests showed that the OLZ test and reference drug (Zyprexa(®)) were bioequivalent, as 90% of the confidence intervals were within the 80-125% interval proposed by regulatory agencies.A rapid method using liquid chromatography tandem mass spectrometry for the quantification of olanzapine (OLZ) in human plasma was developed and validated. Venlafaxine was used as the internal standard (IS), and the samples were extracted from 400‐μL human plasma with methyl tert‐butyl ether for liquid–liquid extraction. Chromatography was performed using an ACE C18, 125 × 4.6‐mm i.d., 5‐μm column. The mobile phase consisted of water with 0.1% formic acid for solvent A and acetonitrile with 0.1% formic acid for solvent B (50 : 50 v/v) in isocratic mode. The flow rate was 1.2 mL/min. The retention times for OLZ and the IS were 0.78 and 1.04 min, respectively. Tandem mass spectrometry operating in positive electrospray ionization mode with multiple reaction monitoring was used to detect OLZ and the IS (m/z: 313.1 > 256.1 and 278.1 > 260.2, respectively). No significant matrix effects were observed on OLZ and the IS retention times, and the mean recovery of OLZ was 90.08%. The assay was linear in the concentration range of 1–20 ng/mL (R2 = 0.9976). The intra‐ and inter‐day precision were < 11.60% and the accuracy was < 1.66%. This validated method was successfully applied to a pharmacokinetic study in which 10‐mg OLZ tablets were administered to healthy volunteers and their plasma OLZ levels were monitored over time. The tests showed that the OLZ test and reference drug (Zyprexa®) were bioequivalent, as 90% of the confidence intervals were within the 80–125% interval proposed by regulatory agencies.

Determination of indinavir in human plasma and its use in pharmacokinetic study

We report the development and validation of a new sensitive, accurate and precise HPLC method with ultraviolet detection for the determination of indinavir sulfate (IND) in human plasma and its application to a bioequivalence study of a new generic formulation. The extraction of IND from plasma samples was achieved by using liquid-liquid extraction with a mean recovery of 73.9%. The lower limit of quantification was 0.05 µg/mL. Bioequivalence between the products was determined by calculating 90% confidence intervals (CI) for the ratio of Cmax, AUC0-t and AUC0-inf values for the test and reference products, within the 0.80-1.25 interval proposed by ANVISA and FDA. Therefore the medications are bioequivalent and inter-exchangeable.

Biopharmaceutical Characterization of Nebulized Antimicrobial Agents in Rats: 5. Oseltamivir Carboxylate

ABSTRACT The aim of this study was to determine the biopharmaceutical characteristics of oseltamivir carboxylate (OC) after pulmonary delivery. After OC bolus and intratracheal nebulization (NEB) in rats, blood was collected and bronchoalveolar lavages (BALs) were performed. Epithelial lining fluid (ELF) concentrations were estimated from BAL fluid. The area under the curve (AUC) ratio for ELF to plasma was 842 times higher after NEB than after intravenous (i.v.) administration, indicating that OC nebulization offers a biopharmaceutical advantage over i.v. administration.