RESUMEN
Calibration data of LC-MS/MS rarely fit the pure least square regression model, especially for large concentration intervals. The response function of the MS instrument is corrected by weighted regression models or logarithms. The choice of a response linearization method is based on results produced through back-interpolation of experimental data and/or evaluation of correlation coefficients. Two bioequivalence studies carried out for pharmaceutical formulations containing metformin gave us the opportunity to appreciate the impact of the MS response linearization method (logarithm and 1/x weighted linear regression) on method quality characteristics. The sample preparation was based on protein precipitation with acetonitrile. Chromatographic separation was achieved on a Zorbax CN column (mobile phase acetonitrile and aqueous 10 m m ammonium acetate solution, pH 3.5). Tandem MS detection was performed on a triple quadrupole spectrometer equipped with an electrospray source, operated under positive-ion mode. The method was validated and used for evaluation of the bioequivalence of formulations containing 500 and 1000 mg metformin. The 500 mg metformin study used logarithms for linearization of the detector response, while the 1000 mg metformin study was based on 1/x linear weighted regression. Data resulting from validations and studies completion were compared with evaluate the impact of the response linearization on the method quality characteristics.
Asunto(s)
Metformina/sangre , Espectrometría de Masas en Tándem/métodos , Cromatografía Liquida , Estudios Cruzados , Humanos , Modelos Lineales , Metformina/farmacocinética , Modelos Estadísticos , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Espectrometría de Masas en Tándem/normas , Equivalencia TerapéuticaRESUMEN
The assay of diltiazem (DLTZ) and its active metabolites desacetyldiltiazem (DAcD) and desmethyldiltiazem (DMeD) in plasma samples was achieved by means of an HPLC/(ESI)MS(2) method. The diastereoisomer of diltiazem, namely {(2R,3S)-5-[2-(dimethylamino)ethyl]-2-(4-methoxyphenyl)-4-oxo-2,3,4,5-tetrahydro-1,5-benzothiazepin-3-yl acetate} was used as internal standard (IS). Sample preparation was based on protein precipitation by means of organic solvent addition (acetonitrile). The isocratic elution based on a reversed-phase mechanism allows good separation of the analytes within 15 min. Atmospheric pressure electrospray ionization was used. All analytes were monitored in the MS(2)-MRM mode. A fragmentation schema is proposed for the target compounds. As the method was designed for bioequivalence purposes, a full validation procedure was considered. On validation, nonlinear calibrations were observed. Consequently, concentration intervals requiring nonlinear calibrations are discussed. Low limits of quantification in the 0.6-1 ng/mL concentration range were obtained. The analytical method was successfully applied to a single dose (120 mg), open-label, randomized, two-period, two-sequence, crossover bioequivalence study of two commercially available solid oral dosage pharmaceutical formulations (tablets).
Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Diltiazem/sangre , Espectrometría de Masa por Ionización de Electrospray/métodos , Adulto , Calibración , Estudios Cruzados , Diltiazem/farmacocinética , Femenino , Humanos , Masculino , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Equivalencia TerapéuticaRESUMEN
Separation of metformin and glibenclamide was achieved within a single chromatographic run on a Zorbax CN column, under isocratic conditions, using acetonitrile and aqueous component (0.01 moles/L ammonium acetate adjusted at pH 3.5 with acetic acid) in volumetric ratio 1/1. Plasma sample preparation is based on protein precipitation by means of organic solvent addition. 1,3,5-Triazine-2,4,6-triamine (IS1) was used as internal standard for metformin, while gliquidone (IS2) played the same role for glibenclamide. Detection was performed with an ion trap mass analyzer, using atmospheric pressure chemical ionization (APCI). A single MS stage was used for detection of metformin and IS1, by extracting ion chromatograms corresponding to molecular ions. MS/MS detection in the SRM mode was used for glibenclamide (m/z transition from 494 to 369 Da) and IS2 (m/z transition from 528 to 403 Da). The method produces linear responses up to 2000 ng/mL for metformin and 400 ng/mL for glibenclamide, respectively. Low limits of quantification were found in the 40 ng/mL range for metformin and at the 4 ng/mL level for glibenclamide. Precision was characterized by relative standard deviations (RSD%) below 9%. The analytical method was successfully applied to a single dose, open-label, randomized, two-period, two-sequence, crossover bioequivalence study of two commercially available anti-diabetic combinations containing 400 mg metformin and 2.5 mg of glibenclamide per coated tablet.
Asunto(s)
Cromatografía Liquida/métodos , Gliburida/sangre , Hipoglucemiantes/sangre , Espectrometría de Masas/métodos , Metformina/sangre , Adulto , Femenino , Gliburida/farmacocinética , Humanos , Hipoglucemiantes/farmacocinética , Masculino , Metformina/farmacocinética , Sensibilidad y Especificidad , Equivalencia TerapéuticaRESUMEN
Bioequivalence data for two pharmaceutical formulations (solid oral dosage forms) containing carvedilol is presented for both racemic and enantiomers of the active substance. This was achieved by on-line coupling of two liquid chromatographic separations followed by fluorescence detection. The first LC dimension was used for a fast separation of racemic carvedilol from propranolol (IS) and the endogenous matrix, by means of a reversed phase mechanism. The peak of racemic carvedilol was on-line transferred to the second enantioselective LC dimension, based on a reversed phase separation on cellulose tris(3,5-dimethyl-phenylcarbamate) stationary phase. Both stages were monitored over a single run by means of a fluorescence detector operated at an excitation wavelength of 285 nm and an emission wavelength of 355 nm. Automated shortcutting of the racemic carvedilol peak to the chiral column and simultaneous detection over the two LC dimensions have been obtained by using an experimental set-up based on two six-port rotative switching valves. Linearity was demonstrated on the interval 2-150 ng/mL for racemic carvedilol and on 1-75 ng/mL intervals for enantiomers. LLOQ fits between 0.7 and 1.4 ng/mL. Recoveries of the target compounds are 87+/-4 and 81+/-4% for the IS. Precision ranged from 0.6 to 2.5% and the mean accuracy obtained on quality control samples (measured as % bias) over the whole study falls between -0.8 and 6.3%.
Asunto(s)
Antagonistas Adrenérgicos beta/sangre , Carbazoles/sangre , Cromatografía Liquida/métodos , Propanolaminas/sangre , Adolescente , Antagonistas Adrenérgicos beta/farmacocinética , Adulto , Calibración , Carbazoles/farmacocinética , Carvedilol , Humanos , Masculino , Persona de Mediana Edad , Preparaciones Farmacéuticas/química , Propanolaminas/farmacocinética , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Estereoisomerismo , Equivalencia TerapéuticaRESUMEN
A simple and fast method intended for large-scale bioequivalence studies for the determination of glibenclamide in plasma samples is presented. The chromatographic separation was achieved on a monolithic octadecyl chemically modified silicagel column and a mobile phase containing 42% aqueous 0.1% HCOOH solution (v/v) and 58% acetonitrile, at a flow rate of 1 mL/min, in isocratic conditions. Preparation of plasma samples was based on protein precipitation with acetonitrile. Gliquidone was used as internal standard. The target analytes were transferred into an ion trap mass analyzer via an atmospheric pressure chemical ionization interface. The precursor ions with mass 494 a.m.u. for glibenclamide and 528 a.m.u. for gliquidone were isolated, while in the second MS stage product ions 369 a.m.u. and 403 a.m.u., respectively, were monitored. The analytical process was characterized by a low limit of quantitation of 1.5 ng/mL. The mean recovery for glibenclamide was 98.1+/-2.8% over a concentration interval ranging from 1 to 500 ng/mL. Intra-day and inter-day precision calculated over 2-400 ng/mL concentration interval ranged from 15.4% to 3.4%. Inter-sequence accuracy expressed as % bias from theoretical concentration values over the concentration interval of 10-400 ng/mL fall within -13.9% and +14.6%. The method was applied for evaluation of the bioequivalence between two formulations containing 3.5mg glibenclamide per dose.
Asunto(s)
Gliburida/sangre , Hipoglucemiantes/sangre , Espectrometría de Masas/métodos , Presión Atmosférica , Gliburida/farmacocinética , Humanos , Hipoglucemiantes/farmacocinética , Estándares de Referencia , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Equivalencia TerapéuticaRESUMEN
The bioequivalence of two formulations of 15 mg tablets of meloxicam (CAS 71125-38-7), Meloxicam (LaborMed Pharma) and a commercially available preparation as reference in 24 healthy male and female Caucasian volunteers was assessed based on a validated non-extractive HPLC-DAD method. The sample preparation procedure was based on protein precipitation with a mixture of methanol/acetonitrile, trifluoacetic acid and sodium sulfate solution. Piroxicam (CAS 36322-90-4) was used as internal standard. The stepwise gradient elution profile of the chromatographic method allows injection of a high volume of the sample (500 microL) with the focusing of both analytes in a Chromolith Performance RP-18e column. The mobile phase constituents are methanol and aqueous 20 mmol/L Na2HPO4 buffer solution brought to pH = 6 with H3PO4. A flow-rate of 2 mL/min achieves a complete chromatographic run (including column equilibration) within 12 min. UV detection at 356 nm allowed a quantitation limit around 30 ng/mL. Bioequivalence study was based on an open-label, randomized, two-period, two-sequence, single dose, crossover design with a 2-week wash-out period between consecutive oral administrations. The main pharmacokinetic parameters (C(max), t(max), t 1/2, AUD and AUC(0-infinity)) were considered as evaluation criteria for the bioequivalence of the test drug against the reference.
Asunto(s)
Antiinflamatorios no Esteroideos/sangre , Tiazinas/sangre , Tiazoles/sangre , Adolescente , Adulto , Antiinflamatorios no Esteroideos/farmacocinética , Área Bajo la Curva , Calibración , Cromatografía Líquida de Alta Presión , Estudios Cruzados , Femenino , Semivida , Humanos , Masculino , Meloxicam , Estándares de Referencia , Equivalencia Terapéutica , Tiazinas/farmacocinética , Tiazoles/farmacocinéticaRESUMEN
Trimetazidine and internal standard [1-(2,4,5-trimethoxybenzyl)piperazine] were isolated from plasma by protein precipitation with trifluoroacetic acid. The neutralized supernatant was separated on a C(8) column with methanol-aqueous 0.11% triethylamine adjusted to pH 3.3 with formic acid (1:4, v/v) at a flow rate of 0.85 mL/min. The separation was achieved within 8 min and the column ef fluent was transferred into an ion trap analyzer via an atmospheric pressure chemical ionization interface. The mass analyzer was used in the selected reaction monitoring mode, to enhance detection selectivity. The method was fully validated with a quantitation limit for trimetazidine of 1.5 ng/mL. The method was successfully applied to assess bioequivalence of two immediate and two modified commercially available pharmaceutical formulations containing 20 and 35 mg of trimetazidine, respectively.
Asunto(s)
Cromatografía Liquida/métodos , Espectrometría de Masas/métodos , Trimetazidina/sangre , Trimetazidina/farmacocinética , Presión Atmosférica , Preparaciones de Acción Retardada/farmacocinética , Estabilidad de Medicamentos , Femenino , Humanos , Masculino , Reproducibilidad de los Resultados , Equivalencia TerapéuticaRESUMEN
Indapamide and internal standard (5-chloro-2-methoxy-N-[2-(4-sulphamoylphenyl)ethyl]benzamide) were isolated from plasma by a single step liquid-liquid extraction in t-butyl methyl ether. The chromatographic separation was achieved on a reversed-phase C(18) monolithic column with a mobile phase consisting in a methanol/aqueous 0.1% formic acid mixture and a flow rate of 0.8 ml/min, in isocratic conditions, within 11 min. Target compounds were transferred in an ion trap analyzer via an atmospheric pressure electrospray interface (AP-ESI). The mass analyzer was used in a selected reaction monitoring (SRM) mode, in order to enhance on detection selectivity. Whole method produces quantitation limit for indapamide of 1 ng/ml. Method was successfully applied to assess bioequivalence of two sustained release marketed pharmaceutical formulations of indapamide 1.5 mg coated tablets, carried-out in a single/multiple doses, randomized design.