RESUMEN
The synthetic peptide drug taspoglutide, developed for treatment of diabetes, must be quantified at low pg/mL levels in biological samples. This manuscript describes the improvement of a previous method, featuring orthogonal hydrophilic interaction to reversed-phase chromatography column switching and tandem mass spectrometric detection. Signal-to-noise ratio was enhanced and isobaric interferences were reduced by ultra-performance separation using a basic mobile phase in 'wrong-way-round' ionization mode and monitoring a selective fragment ion. Tedious solid-phase extraction cleanup was abandoned in favor of simple protein precipitation. Urine required the addition of surfactants to prevent adsorptive drug loss. Dissociation of complexes with possibly formed anti-drug antibodies was achieved with formic acid. Lower limits of quantitation (LLOQ) were 4 pg/mL in human plasma and 10 pg/mL in urine using a 250 µL sample, and an LLOQ of 50 pg/mL was obtained in animal plasma using 50 µL. Precision, accuracy and incurred samples reproducibility fulfilled regulatory requirements. Simultaneous determination of unlabeled and stable isotope labeled taspoglutide, interesting for clearance studies in which both compounds are co-administered, was realized using a structural analog as internal standard. The described method offered excellent sensitivity with low sample consumption, reasonable throughput, moderate costs and high robustness for routine analysis.
Asunto(s)
Cromatografía Liquida/métodos , Péptidos/sangre , Péptidos/orina , Espectrometría de Masa por Ionización de Electrospray/métodos , Estabilidad de Medicamentos , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Péptidos/química , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
BACKGROUND: Studies evaluating the CNS penetration of a novel tyrosine kinase inhibitor, entrectinib, proved challenging, particularly due to discrepancies across earlier experiments regarding P-glycoprotein (P-gp) interaction and brain distribution. To address this question, we used a novel "apical efflux ratio" (AP-ER) model to assess P-gp interaction with entrectinib, crizotinib, and larotrectinib, and compared their brain-penetration properties. METHODS: AP-ER was designed to calculate P-gp interaction with the 3 drugs in vitro using P-gp-overexpressing cells. Brain penetration was studied in rat plasma, brain, and cerebrospinal fluid (CSF) samples after intravenous drug infusion. Unbound brain concentrations were estimated through kinetic lipid membrane binding assays and ex vivo experiments, while the antitumor activity of entrectinib was evaluated in a clinically relevant setting using an intracranial tumor mouse model. RESULTS: Entrectinib showed lower AP-ER (1.1-1.15) than crizotinib and larotrectinib (≥2.8). Despite not reaching steady-state brain exposures in rats after 6 hours, entrectinib presented a more favorable CSF-to-unbound concentration in plasma (CSF/Cu,p) ratio (>0.2) than crizotinib and larotrectinib at steady state (both: CSF/Cu,p ~0.03). In vivo experiments validated the AP-ER approach. Entrectinib treatment resulted in strong tumor inhibition and full survival benefit in the intracranial tumor model at clinically relevant systemic exposures. CONCLUSIONS: Entrectinib, unlike crizotinib and larotrectinib, is a weak P-gp substrate that can sustain CNS exposure based on our novel in vitro and in vivo experiments. This is consistent with the observed preclinical and clinical efficacy of entrectinib in neurotrophic tropomyosin receptor kinase (NTRK) and ROS1 fusion-positive CNS tumors and secondary CNS metastases.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP , Proteínas Tirosina Quinasas , Subfamilia B de Transportador de Casetes de Unión a ATP , Animales , Benzamidas , Diferenciación Celular , Indazoles , Ratones , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas , RatasRESUMEN
A highly sensitive liquid chromatographic method with online solid-phase extraction (SPE) and tandem mass spectrometric detection was developed for the quantification of the synthetic peptide drug taspoglutide in human and animal plasma. Sample preparation consisted of simple protein precipitation or automated off-line SPE using mixed mode cation exchange material, optionally preceded by cleavage of potential antidrug antibody complexes. Excellent selectivity was obtained by a novel orthogonal column switching approach, employing hydrophilic interaction liquid chromatography (HILIC) for online SPE followed by chromatographic separation on a 300 A pore size C18 column. The use of a stable isotope labeled drug (C-terminal arginine containing six (13)C and four (15)N atoms) as internal standard improved the quality and ruggedness of the method. The lower limits of quantitation (LLOQ) in human and animal plasma were 10.0 and 50.0 pg/mL, respectively, extracting only 250 or 100 microL sample aliquots. Precisions and accuracies were below 15% CV and between 89% and 114%, respectively. The method was successfully employed to analyze samples from pharmacokinetic studies.
Asunto(s)
Cromatografía Liquida/métodos , Péptidos/sangre , Extracción en Fase Sólida/métodos , Espectrometría de Masas en Tándem/métodos , Secuencia de Aminoácidos , Animales , Humanos , Marcaje Isotópico , Datos de Secuencia Molecular , Péptidos/aislamiento & purificación , Péptidos/farmacocinéticaRESUMEN
An acylated peptide with MW ~4.5 kDa was measured in samples from pharmacokinetic, toxicology and clinical studies using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Lower limits of quantitation of 2 ng/mL and 50 pg/mL were achieved for animal and human plasma, respectively. Repeated drug administration may lead to anti-drug antibodies (ADA) which can inactivate the drug by formation of drug-ADA complexes. Hence, the LC-MS/MS assay incorporated cleavage of potential drug-ADA complexes to quantify the total plasma concentration. To obtain information on active drug levels, an assay that measures the free concentration or alternatively the ADA-unbound concentration would be needed. Ultrafiltration experiments through 100 kD cutoff membranes to remove Ig-bound peptide were not successful due to nonspecific binding. Extraction of Ig-bound drug using Protein A or G (bacterial cell wall proteins with high affinity to the Fc region of IgG) was suitable to distinguish between ADA-bound drug and [free+protein bound (not ADA-bound)] drug and correlated with findings from ELISA ADA measurement.
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Cromatografía Líquida de Alta Presión/métodos , Inmunoglobulina G/metabolismo , Péptidos/metabolismo , Preparaciones Farmacéuticas/metabolismo , Espectrometría de Masas en Tándem/métodos , Animales , Proteínas Bacterianas , Humanos , Inmunoglobulina G/sangre , Inmunoglobulina G/química , Límite de Detección , Macaca fascicularis , Péptidos/sangre , Péptidos/química , Péptidos/inmunología , Preparaciones Farmacéuticas/sangre , Preparaciones Farmacéuticas/química , Unión Proteica , Ratas , Reproducibilidad de los Resultados , Extracción en Fase Sólida , Proteína Estafilocócica A , UltrafiltraciónRESUMEN
BACKGROUND: Using dried blood spots (DBS) for quantitation of the antiviral drug oseltamivir (Tamiflu(®)), an ester prodrug, and its active metabolite oseltamivir carboxylate could provide ethical and logistic benefits. Hence, its feasibility was investigated using a previously developed column-switching LC-MS/MS method. RESULTS: Sensitivity, precision and accuracy in DBS were comparable to standard plasma assays. Chemically treated cards provided enhanced ex vivo stability of the ester prodrug in rodent blood. Online extraction was realized using the manual TLC-MS interface or the fully automated Sample Card and Prep system. Rat pharmacokinetic study data showed good correlation between plasma, liquid blood and DBS. CONCLUSION: From a bioanalytical perspective, DBS is potentially suited for Tamiflu analysis in animals and humans. Automation of the process by online DBS extraction promises workload reduction and throughput increase.
Asunto(s)
Análisis Químico de la Sangre/métodos , Recolección de Muestras de Sangre/métodos , Fraccionamiento Químico/métodos , Oseltamivir/análogos & derivados , Oseltamivir/sangre , Oseltamivir/aislamiento & purificación , Métodos Analíticos de la Preparación de la Muestra , Animales , Cromatografía Liquida , Cromatografía en Capa Delgada , Humanos , Masculino , Sistemas en Línea , Oseltamivir/farmacocinética , Ratas , Ratas Wistar , Reproducibilidad de los Resultados , Espectrometría de Masas en TándemRESUMEN
This manuscript describes the determination of Ganciclovir (GCV), active component of the antiviral drug Valcyte®, and its ester prodrug Valganciclovir (VGC) in human and rat plasma, using liquid chromatography coupled to tandem mass spectrometry. Protein precipitation with acetonitrile was followed by hydrophilic interaction liquid chromatography on a silica column with 4 min run time. After electrospray ionization, the compounds were detected in positive ion selected reaction monitoring (SRM) mode. The lower limits of quantification (LLOQ) were 16 ng/mL for GCV and 4 ng/mL for VGC in human and rat plasma. Inter-day and intra-day precisions and inaccuracies were below 15% and between 85 and 115%, respectively. Five-fold deuterated GCV and VGC were used as internal standards and compensated for any matrix effect. The method was successfully applied to samples from a rat pharmacokinetic study. The feasibility of blood analysis as dried blood spots (DBS) was investigated.
Asunto(s)
Cromatografía Liquida/métodos , Ganciclovir/análogos & derivados , Ganciclovir/sangre , Espectrometría de Masas en Tándem/métodos , Animales , Recolección de Muestras de Sangre , Estabilidad de Medicamentos , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Ratas , Ratas Sprague-Dawley , Ratas Wistar , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , ValganciclovirRESUMEN
BACKGROUND: A thin-layer chromatography-mass spectrometry interface was previously reported to extract compounds from dried blood spots online. Here, we integrated this interface into a column-switching LC-MS/MS system for online solid-phase extraction and addressed internal standard addition. RESULTS: A drug was analyzed in dried blood spots with a lower limit of quantitation of 50 pg/ml. The internal standard was added via an autosampler to the extraction solvent. The method featured online aqueous dilution of the dried blood spots extract with subsequent trapping and gradient analytical separation. Good precision, accuracy and recovery were obtained. CONCLUSION: The thin-layer chromatography-mass spectrometry interface provided high reproducibility and good extraction efficiency. Although manually operated, it significantly reduced sample preparation efforts. The combination with online solid-phase extraction allowed enrichment of larger sample/extract volumes and efficient clean-up.