RESUMEN

Appropriate substitution of acetonitrile (ACN) in mobile phases for reversed-phase liquid chromatography (RPLC) by low toxicity, ecologically friendly alternative solvents emerges as a greener approach in separation sciences. Ethyl lactate is considered as a green solvent in organic synthesis, industrial extraction processes and many other applicative fields. Its ability to substitute ACN in mobile phases for RPLC applications was barely investigated. The feasibility of such a replacement was tested for the separation of the mixture of 16 polycyclic aromatic hydrocarbons listed by the Environmental Protection Agency. The analytical approach was found to be achievable, with some compromises in terms of elution order, peak efficiency and UV detectability. Thermodynamic aspects of the chromatographic process were also comparatively assessed. Correlations between the elution order and some molecular descriptors were also discussed.

RESUMEN

AIM: Discrimination power evaluation of UV-Vis and (±) electrospray ionization/mass spectrometric techniques, (ESI-MS) individually considered or coupled as detectors to reversed phase liquid chromatography (RPLC) in the characterization of Ginkgo Biloba standardized extracts, is used in herbal medicines and/or dietary supplements with the help of Fuzzy hierarchical clustering (FHC). EXPERIMENTAL: Seventeen batches of Ginkgo Biloba commercially available standardized extracts from seven manufacturers were measured during experiments. All extracts were within the criteria of the official monograph dedicated to dried refined and quantified Ginkgo extracts, in the European Pharmacopoeia. UV-Vis and (±) ESI-MS spectra of the bulk standardized extracts in methanol were acquired. Additionally, an RPLC separation based on a simple gradient elution profile was applied to the standardized extracts. Detection was made through monitoring UV absorption at 220 nm wavelength or the total ion current (TIC) produced through (±) ESI-MS analysis. FHC was applied to raw, centered and scaled data sets, for evaluating the discrimination power of the method with respect to the origins of the extracts and to the batch to batch variability. RESULTS: The discrimination power increases with the increase of the intrinsic selectivity of the spectral technique being used: UV-Vis

Asunto(s)

Cromatografía de Fase Inversa/métodos , Lógica Difusa , Ginkgo biloba/química , Extractos Vegetales/normas , Espectrometría de Masa por Ionización de Electrospray/métodos , Espectrofotometría Ultravioleta/métodos , Análisis por Conglomerados

RESUMEN

BACKGROUND: Extreme efforts are made for the structural diversification of oximes used as AChE reactivators. Co-administration of different oximes should also be considered as a solution in therapy. Consequently, development of selective assays of oximes in biological matrices is of major importance. RESULTS: Three chromatographic separation mechanisms were evaluated: hydrophilic-interaction LC; mixed reversed-phase/cation exchange (DUET); and reversed-phase ion pairing based on per-fluorinated agents. MS was used to identify and quantify oximes. Alternative preparation of whole blood and plasma samples were used based on protein precipitation through addition of acetonitrile or ionic liquids. Quality characteristics of the proposed analytical approaches are discussed. CONCLUSION: The reversed-phase ion pairing based on per-fluorinated agents chromatographic separation mechanism and positive ESI-MS/MS detection produced the best results for the assay of bis-quaternary pyridinium oximes. LLOQ in the tenths of nanogram per milliliter range are achievable.

Asunto(s)

Reactivadores de la Colinesterasa/química , Cromatografía Liquida/métodos , Oximas/sangre , Oximas/química , Compuestos de Piridinio/sangre , Compuestos de Piridinio/química , Espectrometría de Masas en Tándem/métodos , Acetilcolinesterasa/química , Humanos

RESUMEN

Substitution of acetonitrile (ACN) as organic modifier in mobile phases for liquid chromatography by mixtures of propylene carbonate (PC) and ethanol (EtOH) may be considered a greener approach for pharmaceutical applications. Such a replacement is achievable without any major compromise in terms of elution order, chromatographic retention, efficiency and peak symmetry. This has been equally demonstrated for reverse phase (RP), ion pair formation (IP) and hydrophilic interaction liquid chromatography (HILIC) separation modes. The impact on the sensitivity induced by the replacement between these organic solvents is discussed for UV-vis and mass spectrometric detection. A comparison between Van Deemter plots obtained under elution conditions based on ACN and PC/EtOH is presented. The alternative elution modes were also compared in terms of thermodynamic parameters, such as standard enthalpy (ΔH°) and entropic contributions to the partition between the mobile and the stationary phases, for some model compounds. Van't Hoff plots demonstrated that differences between the thermodynamic parameters are minor when shifting from ACN/water to PC/EtOH/water elution on an octadecyl chemically modified silicagel stationary phase. As long as large volume injection (LVI) of diluents non-miscible with the mobile phase is a recently developed topic having a high potential of greening the sample preparation procedures through elimination of the solvent evaporation stage, this feature was also assessed in the case of ACN replacement by PC/EtOH.

Asunto(s)

Etanol/química , Tecnología Química Verde , Preparaciones Farmacéuticas/análisis , Propano/análogos & derivados , Solventes/química , Acetonitrilos/química , Acetonitrilos/toxicidad , Química Farmacéutica , Cromatografía Líquida de Alta Presión , Etanol/toxicidad , Estudios de Factibilidad , Límite de Detección , Preparaciones Farmacéuticas/química , Propano/química , Propano/toxicidad , Solubilidad , Solventes/toxicidad , Espectrometría de Masa por Ionización de Electrospray , Espectrofotometría , Espectrometría de Masas en Tándem , Termodinámica

RESUMEN

Dimethyl sulfate (DMS) is frequently used in pharmaceutical manufacturing processes as an alkylating agent. Trace levels of DMS in drug substances should be carefully monitored since the compound can become an impurity which is genotoxic in nature. Derivatization of DMS with dibenzazepine leads to formation of the N-methyl derivative, which can be retained on a reversed phase column and subsequently separated from other potential impurities. Such derivatization occurs relatively slowly. However, it can be substantially speed up if ionic liquids are used as reaction media. In this paper we report the use of 1-butyl-1-methylpyrrolidinium bis(trifluoromethylsulfonyl)imide (IL1) and 1-butyl-4-methylpyridinium tetrafluoroborate (IL2) as reaction media for the derivatization of DMS with dibenzazepine. It was determined that the stoichiometry between the substrate and DMS may be 1:1 or 2:1, in relation with the nature of the reaction media. An (+)ESI-MS/MS approach was used for quantitation of the derivatized product. Alternatively, DMS derivatization may be carried out with pyridine in acetonitrile (ACN). The N-methylpyridinium derivative was separated by hydrophilic interaction liquid chromatography (HILIC) and detected through (+)ESI-MS (in the SIM mode). In both cases, a limit of quantitation (LOQ) of 0.05 µg/ml DMS was achievable, with a linearity range up to 10 µg/ml. Both analytical alternatives were applied to assay DMS in 4-(2-methoxyethyl)phenol, which is used as a starting material in the synthesis of metoprolol.

Asunto(s)

Alquilantes/análisis , Mutágenos/análisis , Ésteres del Ácido Sulfúrico/análisis , Acetonitrilos/química , Alquilantes/química , Métodos Analíticos de la Preparación de la Muestra , Boratos/química , Química Farmacéutica , Cromatografía Líquida de Alta Presión , Cromatografía de Fase Inversa , Dibenzazepinas/química , Contaminación de Medicamentos/prevención & control , Interacciones Hidrofóbicas e Hidrofílicas , Imidas/química , Indicadores y Reactivos/química , Líquidos Iónicos/química , Cinética , Límite de Detección , Mutágenos/química , Piridinas/química , Compuestos de Piridinio/química , Pirrolidinas/química , Espectrometría de Masa por Ionización de Electrospray , Ésteres del Ácido Sulfúrico/química , Espectrometría de Masas en Tándem

RESUMEN

Green bioanalytical approaches are oriented toward minimization or elimination of hazardous chemicals associated to bioanalytical applications. LC/MS-MS assay of enalapril and enalaprilat in human plasma was achieved by elimination of acetonitrile from both sample preparation and chromatographic separation stages. Protein precipitation (PP) by acetonitrile addition was replaced by liquid-liquid extraction (LLE) in 1-octanol followed by direct large volume injection of the organic layer in the chromatographic column operated under reversed phase (RP) separation mechanism. At the mean time, acetonitrile used as organic modifier in the mobile phase was successfully replaced by a mixture of propylene carbonate/ethanol (7/3, v/v). Three analytical alternatives ((I) acetonitrile PP+acetonitrile based chromatographic elution; (II) 1-octanol LLE+acetonitrile based chromatographic elution; (III) 1-octanol LLE+propylene carbonate/ethanol based chromatographic elution) were validated and the quality characteristics were compared. Comparison between these alternative analytical approaches was also based on results obtained on incurred samples taken during a bioequivalence study, through application of the Bland-Altman procedure.

Asunto(s)

Cromatografía Liquida/métodos , Enalapril/sangre , Enalaprilato/sangre , Tecnología Química Verde/métodos , Espectrometría de Masas en Tándem/métodos , 1-Octanol/química , Acetonitrilos/química , Estudios Cruzados , Enalapril/química , Enalapril/aislamiento & purificación , Enalaprilato/química , Enalaprilato/aislamiento & purificación , Etanol/química , Humanos , Modelos Lineales , Extracción Líquido-Líquido , Propano/análogos & derivados , Propano/química , Ensayos Clínicos Controlados Aleatorios como Asunto , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Equivalencia Terapéutica , Agua/química

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 Terapéutica

RESUMEN

BACKGROUND: Liquid-liquid extraction of target compounds from biological matrices followed by the injection of a large volume from the organic layer into the chromatographic column operated under reversed-phase (RP) conditions would successfully combine the selectivity and the straightforward character of the procedure in order to enhance sensitivity, compared with the usual approach of involving solvent evaporation and residue re-dissolution. Large-volume injection of samples in diluents that are not miscible with the mobile phase was recently introduced in chromatographic practice. The risk of random errors produced during the manipulation of samples is also substantially reduced. RESULTS: A bioanalytical method designed for the bioequivalence of fenspiride containing pharmaceutical formulations was based on a sample preparation procedure involving extraction of the target analyte and the internal standard (trimetazidine) from alkalinized plasma samples in 1-octanol. A volume of 75 µl from the octanol layer was directly injected on a Zorbax SB C18 Rapid Resolution, 50 mm length × 4.6 mm internal diameter × 1.8 µm particle size column, with the RP separation being carried out under gradient elution conditions. Detection was made through positive ESI and MS/MS. Aspects related to method development and validation are discussed. CONCLUSIONS: The bioanalytical method was successfully applied to assess bioequivalence of a modified release pharmaceutical formulation containing 80 mg fenspiride hydrochloride during two different studies carried out as single-dose administration under fasting and fed conditions (four arms), and multiple doses administration, respectively. The quality attributes assigned to the bioanalytical method, as resulting from its application to the bioequivalence studies, are highlighted and fully demonstrate that sample preparation based on large-volume injection of immiscible diluents has an increased potential for application in bioanalysis.

Asunto(s)

Cromatografía Líquida de Alta Presión/métodos , Espectrometría de Masas/métodos , Compuestos de Espiro/análisis , Compuestos de Espiro/farmacocinética , Cromatografía Líquida de Alta Presión/instrumentación , Espectrometría de Masas/instrumentación , Reproducibilidad de los Resultados , Equivalencia Terapéutica

RESUMEN

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 Terapéutica

RESUMEN

A new sensitive HPLC/MS/MS method for simultaneous determination of furosemide, spironolactone and canrenone in human plasma samples is presented. Electrospray ionization source (ESI) has been used. The tandem MS detection was performed under MRM conditions, in the negative ion mode for furosemide and indapamide (internal standard 1) and in the positive ion mode for spironolactone, canrenone and nitrazepam (internal standard 2). A simple plasma protein precipitation with acetonitrile was used as sample preparation technique. The chromatographic separation was carried out under the reversed phase mechanism, on a 250 mm length column packed with octadecyl modified silicagel and thermostated at 35 degrees C. The column was operated under isocratic conditions (3:7 aqueous 0.1% formic acid and methanol, v/v) at a flow rate of 0.8 mL/min. Quantitation intervals of 20-1600 ng/mL for furosemide and 2-100 ng/mL for spironolactone and canrenone have been concluded through validation. Precision and accuracy were situated within the accepted thresholds (maximum 15% relative standard deviation and +/-15% percentage bias). The most sensitive aspects relating to the analytical method development and validation were highlighted and critically assessed in order to reach an objective opinion about the real performances and inherent applicability of the method in bioanalysis.

Asunto(s)

Canrenona/sangre , Cromatografía Líquida de Alta Presión/métodos , Furosemida/sangre , Espectrometría de Masa por Ionización de Electrospray/métodos , Espironolactona/sangre , Espectrometría de Masas en Tándem/métodos , Humanos , Estándares de Referencia , Reproducibilidad de los Resultados

RESUMEN

Analytical aspects related to the assay of pentoxifylline (PTX), lisofylline (M1) and carboxypropyl dimethylxanthine (M5) metabolites are discussed through comparison of two alternative analytical methods based on liquid chromatography separation and atmospheric pressure electrospray ionization tandem mass spectrometry detection. One method is based on a 'pure' reversed-phase liquid chromatography mechanism, while the second one uses the additional polar interactions with embedded amide spacers linking octadecyl moieties to the silicagel surface (C-18 Aqua stationary phase). In both cases, elution is isocratic. Both methods are equally selective and allows separation of unknowns (four species associated to PTX, two species associated to M1) detected through specific mass transitions of the parent compounds and owning respective structural confirmation. Plasma concentration-time patterns of these compounds follow typical metabolic profiles. It has been advanced that in-vivo formation of conjugates of PTX and M1 is possible, such compounds being cleaved back to the parent ones within the ion source. The first method was associated with a sample preparation procedure based on plasma protein precipitation by strong organic acid addition. The second method used protein precipitation by addition of a water miscible organic solvent. Both analytical methods were fully validated and used to assess bioequivalence between a prolonged release generic formulation and the reference product, under multidose and single dose approaches.

Asunto(s)

Cromatografía Liquida/métodos , Pentoxifilina/sangre , Pentoxifilina/metabolismo , Espectrometría de Masas en Tándem/métodos , Humanos

RESUMEN

A sensitive method for determination of free captopril as monobromobimane derivative in plasma samples is discussed. The internal standard (IS) was 5-methoxy-1H-benzimidazole-2-thiol. Derivatization with monobromobimane immediately after blood collection and plasma preparation prevents oxidation of captopril to the corresponding disulfide compound and enhances the ionization yield. Consequently, derivatization enhances sample stability and detection sensitivity. Addition of the internal standard was made immediately after plasma preparation. The internal standard was also derivatized by monobromobimane, as it contains a thiol functional group. Preparation of plasma samples containing captopril and IS derivatives was based upon protein precipitation through addition of acetonitrile, in a volumetric ratio 1:2. The reversed-phase liquid chromatographic separation was achieved on a rapid resolution cartridge Zorbax SB-C(18), monitored through positive electrospray ionization and tandem MS detection using the multiple-reaction monitoring mode. Transitions were 408-362 amu for the captopril derivative and 371-260 amu for the internal standard derivative. The kinetics of captopril oxidation to the corresponding disulfide compound in plasma matrix was also studied using the proposed method. A linear log-log calibration was obtained over the concentration interval 2.5-750 ng/mL. A low limit of quantitation in the 2.5 ng/mL range was obtained. The analytical method was fully validated and successfully applied in a three-way, three-period, single-dose (50 mg), block-randomized bioequivalence study for two pharmaceutical formulations (captopril LPH 25 and 50 mg) against the comparator Capoten 50 mg.

Asunto(s)

Compuestos Bicíclicos con Puentes/química , Captopril/sangre , Cromatografía Liquida/métodos , Espectrometría de Masa por Ionización de Electrospray/métodos , Adolescente , Adulto , Análisis de Varianza , Área Bajo la Curva , Captopril/química , Captopril/farmacocinética , Estudios Cruzados , Estabilidad de Medicamentos , Femenino , Humanos , Concentración de Iones de Hidrógeno , Cinética , Masculino , Persona de Mediana Edad , Dinámicas no Lineales , Oxidación-Reducción , Reproducibilidad de los Resultados , Equivalencia Terapéutica , Adulto Joven

RESUMEN

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 Joven

RESUMEN

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 Terapéutica

RESUMEN

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 Terapéutica

RESUMEN

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 Terapéutica

RESUMEN

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 Terapéutica

RESUMEN

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/farmacocinética

RESUMEN

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 Terapéutica

RESUMEN

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.

Asunto(s)

Antihipertensivos/sangre , Antihipertensivos/farmacocinética , Indapamida/sangre , Indapamida/farmacocinética , Espectrometría de Masa por Ionización de Electrospray/métodos , Preparaciones de Acción Retardada/análisis , Estabilidad de Medicamentos , Indapamida/administración & dosificación , Equivalencia Terapéutica