RESUMEN
BACKGROUND: The inherent reproducibility of a bioanalytical approach is usually sustained through incurred sample reanalysis (ISR). Questions relating to the number of ISRs, the right moment for performing reanalysis, the way of performing an appropriate statistical refinement of experimental data and actions to be taken in the case of failure are frequently raised. RESULTS: Data resulting from ISR following a bioequivalence study for spironolactone formulations are discussed. Reanalysis of samples was carried out twice: immediately after the end of the study and after a period that overcame the long-term stability study achieved during method validation. The Bland-Altman approach was used to assess experimental results. ISR was successful over the short reanalysis period for both compounds. Data produced through reanalysis after the long-term period indicated a systematic positive bias for the metabolite canrenone (although results supported reproducibility). The results obtained for spironolactone were affected by a strong negative systematic bias and failed to support reproducibility. The explanation deals with the continuous conversion of spironolactone to canrenone in plasma samples. However, reproducibility of the method may be sustained by comparing original and repeated differences between concentration values in samples by means of a paired t-test, Wilcoxon sign rank-sum test and linear regression. CONCLUSIONS: Different statistical approaches for making data comparisons are discussed and may be successfully applied during reanalysis of samples from a bioequivalence study. Results of the evaluations may differ in accordance with the statistical procedure being applied, thus a definitive conclusion requires consideration of all specific experimental circumstances arising during production of the processed data.
Asunto(s)
Canrenona/sangre , Espironolactona/sangre , Femenino , Humanos , Masculino , Reproducibilidad de los Resultados , Espironolactona/metabolismo , Equivalencia TerapéuticaRESUMEN
Large volume injection of samples in strong diluents immiscible with the mobile phases used in reversed phase liquid chromatography (RPLC) has been recently introduced in practice. In the present work, the potential of the technique has been evaluated for bioanalytical applications. The process consists of the liquid-liquid extraction of indapamide from whole blood into 1-octanol, followed by the direct injection from the organic layer into the LC. Detection was made through negative electrospray ionization (ESI) and tandem mass spectrometry (MS(2)). The method was developed, validated, and successfully applied to a large number of samples in two bioequivalence studies designed for indapamide 1.5mg sustained release and 2.5mg immediate release pharmaceutical formulations. The performance of the analytical method is discussed based on data resulting from the validation procedure and the completion of the bioequivalence studies.
Asunto(s)
1-Octanol/química , Cromatografía Liquida/métodos , Monitoreo de Drogas/métodos , Indapamida/sangre , Cromatografía Liquida/instrumentación , Estudios Cruzados , Preparaciones de Acción Retardada , Relación Dosis-Respuesta a Droga , Diseño de Equipo , Humanos , Indapamida/administración & dosificación , Indicadores y Reactivos , Reproducibilidad de los Resultados , Solubilidad , Soluciones , Equivalencia TerapéuticaRESUMEN
BACKGROUND: The bioequivalence of two pharmaceutical formulations containing 10 mg quinapril was assessed by assaying the untransformed drug and its active metabolite quinaprilat from plasma samples. RESULTS: A gradient elution liquid chromatographic separation coupled to positive atmospheric pressure electrospray ionization and tandem mass spectrometry detection was used and validated. Sample preparation is simple and uses protein precipitation through addition of an acetonitrile:methanol (8:2 v/v) mixture. The method has a run time of 6.3 min. Carvedilol was used as an internal standard. The multiple reactions monitoring mode was used for both quantitation and structural confirmation of target compounds. Linear 1/x²-weighted regressions characterize detector response function up to concentrations of 1000 ng/ml for quinapril and 2000 ng/ml for quinaprilat. Low limits of quantitation of 5 ng/ml for quinapril and 10 ng/ml for quinaprilat were found. Intra- and inter-day variability of the results were found below 15%. Long- (-20°C/6 months) and short-term (25°C/48 h) stability of analytes in plasma, as well as freeze and thaw stability (six cycles) were demonstrated. CONCLUSION: The method was found to be selective, precise, accurate and robust when applied to a large number of unknown samples.
Asunto(s)
Carbazoles/sangre , Propanolaminas/sangre , Tetrahidroisoquinolinas/sangre , Adulto , Bioensayo , Calibración , Carbazoles/química , Carbazoles/farmacocinética , Carvedilol , Cromatografía Liquida , Estudios Cruzados , Evaluación de Medicamentos , Femenino , Humanos , Masculino , Propanolaminas/química , Propanolaminas/farmacocinética , Quinapril , Estándares de Referencia , Reproducibilidad de los Resultados , Extracción en Fase Sólida , Espectrometría de Masa por Ionización de Electrospray , Espectrometría de Masas en Tándem , Tetrahidroisoquinolinas/química , Tetrahidroisoquinolinas/farmacocinética , Tetrahidroisoquinolinas/farmacología , Equivalencia Terapéutica , Adulto JovenRESUMEN
A simple, high-throughput, highly selective and sensitive HPLC-FLD method for isolation and determination of furosemide and/or norfloxacin in human plasma samples following a simple organic solvent deproteinization step with acetonitrile as sample 'clean-up' procedure is reported. One of the two drug substances plays the internal standard role for the determination of the other. Separation of analyte and internal standard was achieved in less than 5.3 min (injection to injection) on a Chromolith Performance RP-18e column, using an aqueous component containing 0.015 mol/L sodium heptane-sulfonate and 0.2% triethylamine brought to pH = 2.5 with H(3)PO(4). The composition of the mobile phase was: acetonitrile-methanol-aqueous component = 70:15:15 (v/v/v) and the flow-rate was set up to 3 mL/min. The chromatographic method applied to the determination of furosemide relies on fluorescent detection parameters of 235 nm for the excitation wavelength, and 402 nm for the emission wavelength. In case of norfloxacin, the excitation wavelength is set up to 268 nm and the emission wavelength is set up to 445 nm. The overall method leads to quantitation limits of about 27 ng/mL for furosemide, and 19.5 ng/mL for norfloxacin, using an injection volume of 250 microL. The method was applied to the bioequivalence study of two furosemide-containing formulations.
Asunto(s)
Furosemida/sangre , Norfloxacino/sangre , Cromatografía Líquida de Alta Presión , Femenino , Fluorescencia , Humanos , Masculino , Estructura MolecularRESUMEN
An extraction-less sample preparation technique followed by a RPLC-UV method on sub-two microns particles packed short column were used for the assay of tenoxicam in plasma samples. Protein precipitation was made by means of trichloroacetic acid addition. Supernatant was injected to the chromatographic column without any further pH adjustment. The mobile phase consisted in a mixture of acetonitrile and aqueous 0.1% phosphoric acid, at 2 mL/min flow rate and gradient elution. The Zorbax SB-C18 column (50 mm length, 4.6 mm internal diameter and 1.8 microm particle size) was thermostated at 60 degrees C. The mobile phase gradient composition program allowed separation of tenoxicam and piroxicam (internal standard), column clean-up and re-equilibration within 4 min. UV detection was achieved at 368+/-10 nm. The method is characterized by a low limit of quantitation of 25 ng/mL for tenoxicam, with a linearity interval up to 5500 ng/mL. The use of a low volume detection cell and detector high frequency data acquisition rate produced high precision and accuracy through a whole bioequivalence study of tenoxicam in two commercially available tablet formulations, after a single oral administration dose. Full method validation is presented. The high throughput characteristic of the proposed method allowed full validation and bioanalytical study completion within a 96 h period.