RESUMEN
Copanlisib is a dual PI3K-δ inhibitor, used in follicular lymphoma treatment. In this research, we report a validated LC-MS/MS method for quantifying copanlisib from a mouse dried blood spot (DBS). We validated the method in line with the guidelines of the US Food and Drug Administration. The liquid-liquid extraction technique was used to extract copanlisib from the DBS discs. We used an Atlantis dC18 column and isocratic mobile phase for the chromatographic separation of copanlisib and the internal standard (idelalisib). The flow was 0.90 ml/min. Under the optimized chromatographic conditions, the retentions of copanlisib and the internal standard were 0.98 and 0.93 min, respectively. Each injection total run time was 2.50 min. The MS/MS ion transitions monitored were m/z 481.31 â 128.00 and 416.10 â 176.10 for copanlisib and the internal standard (IS) idelalisib, respectively. We have used a broad calibration range (1.01-4,797 ng/ml) with a determination coefficient (r2 ) of 0.997. All of the evaluated parameters met the acceptance criteria. Hematocrit did not influence the DBS copanlisib concentrations. We have used the validated method to derive the intravenous pharmacokinetic parameters by quantifying copanlisib in mouse plasma.
Asunto(s)
Quinazolinas , Espectrometría de Masas en Tándem , Ratones , Animales , Cromatografía Liquida/métodos , Espectrometría de Masas en Tándem/métodos , Pirimidinas , Inhibidores de las Quinasa Fosfoinosítidos-3 , Reproducibilidad de los Resultados , Pruebas con Sangre Seca/métodosRESUMEN
Paxalisib is a pan-PI3K and mTOR inhibitor, currently entering into Phase II clinical trials as a potential drug to treat glioblastoma patients. We report the development and validation of a high-performance liquid chromatography (HPLC) method for the quantitation of paxalisib in mouse plasma as per the US Food and Drug Administration regulatory guidelines. From the mouse plasma, paxalisib and the internal standard (IS; filgotinib) were extracted using ethyl acetate as an extraction solvent. The chromatographic separation of paxalisib and the IS was accomplished on a Symmetry C18 (250 × 4.6 mm, 5.0 µm) column maintained at 40°C using 10 mm ammonium formate and acetonitrile in gradient conditions at a 0.8 ml/min flow-rate. The injection volume was 20 µl. The elution was monitored using a photo-diode array detector set at λmax 280 nm. Paxalisib and the IS eluted at 6.5 and 5.9 min, respectively with a total run time of 10 min. The calibration curve was linear over the range of 111-4,989 ng/ml. Inter- and intraday precision and accuracy, stability studies, dilution integrity and incurred sample reanalysis were investigated and the results met the acceptance criteria. The validated HPLC method was extended to assess the pharmacokinetic parameters of paxalisib in mice.
Asunto(s)
Fosfatidilinositol 3-Quinasas , Inhibidores de Proteínas Quinasas , Ratones , Animales , Cromatografía Líquida de Alta Presión/métodos , Reproducibilidad de los Resultados , Inhibidores de Proteínas Quinasas/farmacocinética , Inhibidores mTOR , Serina-Treonina Quinasas TORRESUMEN
In this study, we report the development and validation of an LC-tandem mass spectrometry method for the simultaneous quantitation of bendamustine and copanlisib in mouse plasma as per the US FDA regulatory guidelines. The sample processing involves extraction of bendamustine and copanlisib along with internal standard (IS; warfarin) from 50 µL mouse plasma using a liquid-liquid extraction method. The chromatographic separation of bendamustine, copanlisib and the IS was achieved on an Atlantis dC18 column using an isocratic mobile phase (5 mM ammonium acetate:methanol, 20:80 v/v). Bendamustine, copanlisib and the IS eluted at 0.88, 1.39 and 0.74 min, respectively, with a total run time of 2.5 min. The calibration curve ranged from 3.99-2996 and 4.33-3248 ng/mL for bendamustine and copanlisib, respectively. Inter- and intra-day precision and accuracy, stability in processed samples and upon storage, dilution integrity and incurred sample reanalysis were investigated for both the analytes. The intra- and inter-day precisions were in the ranges of 2.01%-5.05% and 2.74%-6.13% and 1.98%-7.64 and 8.62%-9.04% for bendamustine and copanlisib, respectively. Stability studies showed that both analytes were stable on bench top for 6 h, in auto-sampler for 24 and at -80°C for 30 days. The validated method was successfully applied to a pharmacokinetic study in mice.
Asunto(s)
Espectrometría de Masa por Ionización de Electrospray , Espectrometría de Masas en Tándem , Animales , Clorhidrato de Bendamustina , Cromatografía Líquida de Alta Presión/métodos , Cromatografía Liquida/métodos , Ratones , Pirimidinas , Quinazolinas , Reproducibilidad de los Resultados , Espectrometría de Masa por Ionización de Electrospray/métodos , Espectrometría de Masas en Tándem/métodosRESUMEN
Lumefantrine (LFN) is a chiral antimalarial drug. Enantioselective in vitro attributes and absolute oral pharmacokinetics for (-)-LFN and (+)-LFN have been characterized in mice. No stereoselectivity was seen with either of the enantiomers when compared with rac-LFN in the executed in vitro studies (solubility, metabolic stability, protein binding, permeability and blood partitioning). Post intravenous or oral administration of rac-LFN, the AUC0-∞ and MRT of (+)-LFN was higher over (-)-LFN, which is reflected in higher clearance value for (-)-LFN. Following (-)-LFN intravenous administration to mice, the key PK parameters were comparable to (-)-LFN from rac-LFN; however, post intravenous administration of (+)-LFN alone to mice, the AUC0-∞ was 1.3-fold higher than (+)-LFN from rac-LFN. Similarly, post oral administration of (-)-LFN to mice, both AUC0-∞ and Cmax were 1.3-fold higher than (-)-LFN from rac-LFN. On other hand, (+)-LFN showed 1.4-fold higher AUC0-∞ and 1.7-fold higher Cmax post oral administration over (+)-LFN from rac-LFN.
Asunto(s)
Antimaláricos/farmacocinética , Lumefantrina/farmacocinética , Administración Oral , Animales , Disponibilidad Biológica , Etanolaminas , Infusiones Intravenosas , Masculino , Ratones , Solubilidad , EstereoisomerismoRESUMEN
Immuno-oncology (IO) is an emerging option to treat cancer malignancies. In the last two years, IO has accounted for more than 90% of the new active drugs in various therapeutic indications of oncology drug development. Bioanalytical methods used for the quantitation of various IO small molecule drugs have been summarized in this review. The most commonly used are HPLC and LC-MS/MS methods. Determination of IO drugs from biological matrices involves drug extraction from the biological matrix, which is mostly achieved by simple protein precipitation, liquid-liquid extraction and solid-phase extraction. Subsequently, quantitation is usually achieved by LC-MS/MS, but HPLC-UV has also been employed. The bioanalytical methods reported for each drug are briefly discussed and tabulated for easy access. Our review indicates that LC-MS/MS is a versatile and reliable tool for the sensitive, rapid and robust quantitation of IO drugs.
Asunto(s)
Antineoplásicos Inmunológicos/análisis , Antineoplásicos Inmunológicos/aislamiento & purificación , Cromatografía Liquida , Espectrometría de Masas en Tándem , Animales , Antineoplásicos Inmunológicos/uso terapéutico , Cromatografía Líquida de Alta Presión , Humanos , Extracción Líquido-Líquido , Ratones , Neoplasias/tratamiento farmacológico , Extracción en Fase SólidaRESUMEN
AMG 510 is the first-in-class KRASG12C inhibitor, currently in phase 2 clinical trials as an orphan drug to treat non-small cell lung cancer patients. We developed and validated a sensitive, selective, and high-throughput HPLC-MS/MS method for the quantitation of AMG 510 in mouse plasma per the regulatory guideline of the US Food and Drug and Administration. AMG 510 and the IS (MRTX-1257) were extracted from mouse plasma using tert-butyl methyl ether and chromatographed using an isocratic mobile phase (0.2% formic acid:acetonitrile; 25:75, v/v) at a flow rate of 0.65 mL/min on an Atlantis dC18 column. AMG 510 and the IS eluted at ~0.95 and 0.73 min, respectively. AMG 510 and the IS were detected by positive electrospray ionization in multiple reaction monitoring using transition pair (Q1 â Q3) m/z 561.1 â 134.1 and m/z 566.5 â 98.2, respectively. Excellent linearity was achieved in the concentration range of 1.08-5040 ng/mL (r > 0.0996). No matrix effect and carryover were observed. Intra- and inter-day accuracies and precisions were within the acceptance range. AMG 510 was demonstrated to be stable under the tested storage conditions. This novel method has been applied to a pharmacokinetic study in mice.
Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Piperazinas/sangre , Piperazinas/farmacocinética , Piridinas/sangre , Piridinas/farmacocinética , Pirimidinas/sangre , Pirimidinas/farmacocinética , Espectrometría de Masas en Tándem/métodos , Animales , Modelos Lineales , Masculino , Ratones , Ratones Endogámicos BALB C , Piperazinas/química , Proteínas Proto-Oncogénicas p21(ras)/antagonistas & inhibidores , Piridinas/química , Pirimidinas/química , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
Phosphatidylinositol 3-kinase (PI3K) inhibitors are a novel class of anticancer drugs that are approved to treat various malignancies. We report the development and validation of a HPLC method for the simultaneous quantitation of three PI3K inhibitors, namely copanlisib, duvelisib and idelalisib, in rat plasma as per the regulatory guidelines of the United States Food and Drug Administration. The method involves extraction of copanlisib, duvelisib and idelalisib along with an internal standard (IS; filgotinib) from rat plasma (100 µL) using a liquid-liquid extraction process. The chromatographic separation of the analytes was achieved using step-wise gradient elution on a Hypersil Gold C18 column. The UV detection wavelength was set at λmax = 280 nm. Copanlisib, duvelisib, idelalisib and the IS eluted at 7.16, 12.6, 11.9 and 9.86 min, respectively, with a total run time of 15 min. The calibration curve ranged from 50 to 5000 ng/mL for all the analytes. Inter- and intra-day precision and accuracy, stability studies, dilution integrity and incurred sample reanalysis were investigated for all three analytes, and the results met the acceptance criteria. The validated HPLC method was successfully applied to a pharmacokinetic study in rats.
Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Extracción Líquido-Líquido/métodos , Inhibidores de las Quinasa Fosfoinosítidos-3/sangre , Inhibidores de las Quinasa Fosfoinosítidos-3/farmacocinética , Animales , Antineoplásicos/sangre , Antineoplásicos/química , Antineoplásicos/farmacocinética , Isoquinolinas/sangre , Isoquinolinas/química , Isoquinolinas/farmacocinética , Modelos Lineales , Masculino , Inhibidores de las Quinasa Fosfoinosítidos-3/química , Purinas/sangre , Purinas/química , Purinas/farmacocinética , Pirimidinas/sangre , Pirimidinas/química , Pirimidinas/farmacocinética , Quinazolinas/sangre , Quinazolinas/química , Quinazolinas/farmacocinética , Quinazolinonas/sangre , Quinazolinonas/química , Quinazolinonas/farmacocinética , Ratas , Ratas Sprague-Dawley , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
Aurora B plays critical role in the process of chromosome condensation and chromosome orientation during the regulation of mitosis. The overexpression of Aurora B has been observed in several tumor types. As a part of our ongoing effort to develop Aurora B inhibitors, herein, we described the design, synthesis and evaluation of phenyl/pyridine diazepine analogs. The diazepane aniline pyrimidine (4a) was identified as an initial hit (Aurora B IC50 6.9 µM). Molecular modeling guided SAR optimization lead to the identification of 8-fluorobenzodiazepine (6c) with single digit nM potency (Aurora B IC50 8 nM). In the antiproliferation assay 6c showed activity across the cell lines with IC50 of 0.57, 0.42, and 0.69 µM for MCF-7, MDA-MB 231, and SkoV3 respectively. In the in vivo PK profile. 6c has shown higher bioavailability (73%) along with good exposure (AUC of 1360 ng.h/mL).
Asunto(s)
Antineoplásicos/farmacología , Aurora Quinasa B/antagonistas & inhibidores , Azepinas/farmacología , Descubrimiento de Drogas , Inhibidores de Proteínas Quinasas/farmacología , Animales , Antineoplásicos/síntesis química , Antineoplásicos/química , Aurora Quinasa B/metabolismo , Azepinas/síntesis química , Azepinas/química , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Ratones , Estructura Molecular , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/química , Relación Estructura-ActividadRESUMEN
Levodropropizine (LDP) is a non-opioid anti-tussive. The stereoselective pharmacokinetics and tissue distribution (TD) of LDP vs. dextrodropropizine (DDP) have been characterized after oral and intravenous (IV) administration of LDP and rac-dropropozine in rats.Oral/IV doses of 50/5.0 mg/kg and 25/2.5 rac-dropropizine and LDP were employed. TD study focused on tissues such as liver, lung and kidney. Blood samples were collected for pharmacokinetic and TD evaluation. Validated methods were used to quantitate LDP, DDP and rac-dropropizine.No stereoselectivity in pharmacokinetics was observed between LDP vs. DDP following rac-dropropizine. However, LDP pharmacokinetics after LDP administration (oral/IV) appeared to be different compared to LDP derived from rac-dropropizine.TD data were similar between the two enantiomers regardless of oral/IV rac-dropropizine administration. When LDP alone was administered, levels were comparable to those derived for LDP from rac-dropropizine after oral/IV. However, in the lung and kidney tissues, the exposure after oral dosing was higher for LDP alone as compared to LDP from rac-dropropizine.In summary, complete characterization of stereoselective pharmacokinetics and TD of rac-dropropizine has been reported after oral/IV routes. It was evident that the presence of DDP, increased the plasma/tissue exposure of LDP which was evident after oral rac-dropropizine dosing.
Asunto(s)
Antitusígenos/farmacocinética , Glicoles de Propileno/farmacocinética , Administración Intravenosa , Administración Oral , Animales , Masculino , Ratas , Ratas Sprague-Dawley , EstereoisomerismoRESUMEN
Cefuroxime is one of the widely used antibiotics. The objective of this study was to determine pharmacokinetics and disposition in various ocular tissues following topical (TOP), intracameral (IC) and intravitreal (IVT) administration of cefuroxime to rabbits.Following TOP, IC and IVT dosing plasma and various ocular tissues (aqueous humor (AH), vitreous humor (VH), conjunctiva, trabecular mesh (TM), lens and retina-choroid (RC)) were collected and analyzed to understand the disposition of cefuroxime. Postintravenous administration plasma samples were collected to determine the systemic pharmacokinetics.Post-TOP dosing cefuroxime concentrations were observed only in conjunctiva up to 48 h. IC administration showed cefuroxime concentrations in AH up to 8 h; in conjunctiva, TM and plasma, the concentration lasted up to 4 h and in RC and VH till 1 h. IVT administration of cefuroxime showed concentrations in all ocular tissues (up to 8 h) and lasted up to 48 h except in conjunctiva and RC.There was evidence that the mechanism(s) of cefuroxime entry into the eye by via IVT, IC and TOP routes is clearly different. The present ocular tissue data may aid clinicians for considering appropriate choice in the treatment of post-operative ocular complications due to bacterial infections including endophthalmitis.
Asunto(s)
Cefuroxima/farmacocinética , Animales , Cefuroxima/administración & dosificación , Humanos , Inyecciones Intraoculares , Conejos , Distribución TisularRESUMEN
Quantitation of drugs used for the treatment of chronic lymphocytic leukemia in various biological matrices during both pre-clinical and clinical developments is very important, often in routine therapeutic drug monitoring. The first developed methods for quantitation were traditionally done on LC in combination with either UV or fluorescence detection. However, the emergence of LC with mass spectrometry in tandem in early 1990s has revolutionized the quantitation as it has provided better sensitivity and selectivity within a shorter run time; therefore it has become the choice of method for the analysis of various drugs. In this article, an overview of various bioanalytical methods (HPLC or LC-MS/MS) for the quantification of drugs for the treatment of chronic lymphocytic leukemia, along with applicability of these methods, is given.
Asunto(s)
Antineoplásicos , Cromatografía Liquida , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Antineoplásicos/análisis , Antineoplásicos/farmacocinética , Antineoplásicos/uso terapéutico , Monitoreo de Drogas , Humanos , Espectrometría de Masas en TándemRESUMEN
Different options on performing incurred sample reanalysis (ISR) on dried blood spot (DBS) cards were investigated using drugs belonging to various therapeutic areas: (a) darolutamide (to treat prostate cancer) and (b) filgotinib (to treat rheumatoid arthritis). The proposed novel methodology included the generation of half-DBS and quarter-DBS discs after initial blood collection using the full-DBS discs. Accordingly, blood collection via DBS was performed in male BALB/c mice following intravenous and oral dosing of darolutamide; in male Sprague Dawley rats following intravenous and oral dosing of filgotinib. The ISR data generated from the full-DBS disc, half-DBS disc and quarter-DBS disc were compared for the assessment of the proposed methodology. Quantification of darolutamide and filgotinib was accomplished using liquid chromatography-electrospray ionization/tandem mass spectrometry methods. Darolutamide and filgotinib ISR samples, which were collected and prepared using full-, half- and quarter-DBS discs, met the acceptance criteria for ISR analysis. In conclusion, this is the first report showing a viable tool for the performance of ISR on DBS cards. The use of quarter- or half-DBS discs would aid in not only ISR but also in long-term storage experiments of analytes because it would avoid the need for additional blood sampling in patients.
Asunto(s)
Pruebas con Sangre Seca/métodos , Pirazoles/sangre , Piridinas/sangre , Triazoles/sangre , Animales , Cromatografía Liquida/métodos , Límite de Detección , Modelos Lineales , Masculino , Ratones , Ratones Endogámicos BALB C , Ratas , Ratas Sprague-Dawley , Reproducibilidad de los Resultados , Espectrometría de Masas en Tándem/métodosRESUMEN
In this paper, we present the incurred sample reanalysis (ISR) data for cefuroxime in various ocular tissues of rabbits. Based on the cefuroxime concentration vs. time profile in various ocular tissues, three chosen time points enabled ISR assessment. Cefuroxime was quantitated in the ocular tissues using a published liquid chromatography-electrospray ionization/tandem mass spectrometry method operated under the multiple reaction-monitoring mode in positive ion mode. Regardless of the ocular tissue, the linearity range was 12.7-2760 ng/ml with a correlation coefficient (r2 ) of ≥0.996. All of the ISR samples representing various ocular tissues met the acceptance criteria. To the best of our knowledge, this is the first report showing the ISR of ocular tissues in any species.
Asunto(s)
Cefuroxima/análisis , Cromatografía Liquida/métodos , Ojo/química , Espectrometría de Masas en Tándem/métodos , Animales , Modelos Lineales , Conejos , Manejo de EspecímenesRESUMEN
Larotrectinib is a first-generation tropomyosin kinase inhibitor, approved for the treatment of solid tumors. In this paper, we present a validated dried blood spot (DBS) method for the quantitation of larotrectinib from mouse blood using HPLC-MS/MS, which was operated under multiple reaction monitoring mode. To the DBS disc cards, acidified methanol enriched with internal standard (IS; enasidenib) was added and extracted using tert-butyl methyl ether as an extraction solvent with sonication. Chromatographic separation of larotrectinib and the IS was achieved on an Atlantis dC18 column using 10 mm ammonium formate-acetonitrile (30:70, v/v) delivered at a flow-rate of 0.80 ml/min. Under these optimized conditions, the retention times of larotrectinib and the IS were ~0.93 and 1.37 min, respectively. The total run time was 2.50 min. Larotrectinib and the IS were analyzed using positive ion scan mode and parent-daughter mass to charge ion (m/z) transitions of 429.1 â 342.1 and 474.1 â 267.1, respectively, were used for the quantitation. The calibration range was 1.06-5,080 ng/ml. No matrix effect or carryover was observed. Hematocrit did not influence DBS larotrectinib concentrations. All of the validation parameters met the acceptance criteria. The applicability of the validated method was shown in a mouse pharmacokinetic study.
Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Pruebas con Sangre Seca/métodos , Pirazoles/sangre , Pirimidinas/sangre , Espectrometría de Masas en Tándem/métodos , Animales , Límite de Detección , Modelos Lineales , Masculino , Ratones , Pirazoles/química , Pirazoles/farmacocinética , Pirimidinas/química , Pirimidinas/farmacocinética , Reproducibilidad de los ResultadosRESUMEN
Filgotinib is a selective JAK1 (Janus kinase) inhibitor, filed in Japan for the treatment of rheumatoid arthritis. In this paper, we report a validated liquid chromatography coupled with tandem mass spectrometry for the quantification of filgotinib in rat plasma using tofacitinib as an internal standard (IS) as per the Food and Drug Administration regulatory guidelines. Filgotinib and the IS were extracted from rat plasma using ethyl acetate as an extraction solvent and chromatographed using an isocratic mobile phase (0.2% formic acid:acetonitrile; 20:80, v/v) at a flow rate of 0.9 mL/min on a Gemini C18 column. Filgotinib and the IS were eluted at ~1.31 and 0.89 min, respectively. The MS/MS ion transitions monitored were m/z 426.3 â 291.3 and m/z 313.2 â 149.2 for filgotinib and the IS, respectively. The calibration range was 0.78-1924 ng/mL. No matrix effect and carryover were observed. Intra- and inter-day accuracies and precisions were within the acceptance range. Filgotinib was stable for three freeze-thaw cycles: on bench-top up to 6 h, in an autosampler up to 21 h, and at -80°C for 1 month. This novel method has been applied to a pharmacokinetic study in rats.
Asunto(s)
Cromatografía Liquida/métodos , Piridinas/sangre , Piridinas/farmacocinética , Espectrometría de Masas en Tándem/métodos , Triazoles/sangre , Triazoles/farmacocinética , Animales , Estabilidad de Medicamentos , Modelos Lineales , Masculino , Piridinas/química , Ratas , Ratas Sprague-Dawley , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Triazoles/químicaRESUMEN
We developed and validated a simple, sensitive, selective, and reliable LC-MS/MS-ESI method for the direct quantitation of lumefantrine (LFN) enantiomers [(-)-LFN and (+)-LFN] in mice plasma as per regulatory guideline. LFN enantiomers and carbamazepine (internal standard) were extracted from mice plasma using Strata X SPE (solid-phase extraction) cartridges. Good resolution between enantiomers was achieved on a Chiralpak IA-3 column using an isocratic mobile phase (0.1% of diethyl amine in methanol), which was delivered at a flow rate of 0.8 mL/min. Detection and quantitation were performed using multiple reaction monitoring mode following the transitions m/z 530.27 â 512.30 and 237.00 â 194.00 for LFN enantiomers and the internal standard, respectively, in the positive-ionization mode. The proposed method provided accurate and reproducible results over the linearity range of 2.39-895 ng/mL for each enantiomer. The intra- and inter-day precisions were in the range of 1.03-6.14 and 6.36-8.70 and 2.03-4.88 and 5.82-11.5 for (-)-LFN and (+)-LFN, respectively. Both (-)-LFN and (+)-LFN were found to be stable under different stability conditions. The method was successfully used to delineate stereoselective pharmacokinetics of LFN enantiomers in mice after an oral administration of rac-LFN (20 mg/kg). The pharmacokinetic results indicated that the disposition of LFN enantiomers was stereoselective in mice.
Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Lumefantrina , Espectrometría de Masas en Tándem/métodos , Animales , Modelos Lineales , Lumefantrina/sangre , Lumefantrina/química , Lumefantrina/farmacocinética , Masculino , Ratones , Ratones Endogámicos BALB C , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , EstereoisomerismoRESUMEN
A selective, sensitive and rapid LC-MS/MS method has been developed and validated as per US Food and Drug Administration regulatory guidelines for the simultaneous quantitation of colchicine and febuxostat in rat plasma. Colchicine and febuxostat were extracted from the rat plasma using 10% tert-butyl methyl ether in ethyl acetate using colchicine-d6 as an internal standard (IS). The chromatographic separation of colchicine, febuxostat and the IS was achieved using a mobile phase comprising 5 mm ammonium formate and 0.025% formic acid in acetonitrile (20:80, v/v) in isocratic mode on an Eclipse XDB-C18 column. The injection volume and flow rate were 5.0 µl and 0.9 ml/min, respectively. Colchicine and febuxostat were detected by positive electrospray ionization in multiple reaction monitoring mode using transition pairs (Q1 â Q3) of m/z 400.10 â 358.10 and 317.05 â 261.00, respectively. The assay was linear in the ranges of 0.25-254 and 2.60-622 ng/ml for colchicine and febuxostat, respectively. The inter- and intra-day precision values were 0.58-13.0 and 1.03-4.88% for colchicine and febuxostat, respectively. No matrix or carryover effects were observed during the validation. Both analytes were stable on the bench-top, in the autosampler and in storage (freeze-thaw cycles and long-term storage at -80°C). A pharmacokinetic study in rats was performed to show the applicability of the validated method.
Asunto(s)
Colchicina/sangre , Colchicina/farmacocinética , Febuxostat/sangre , Febuxostat/farmacocinética , Animales , Cromatografía Líquida de Alta Presión/métodos , Colchicina/química , Febuxostat/química , Límite de Detección , Modelos Lineales , Masculino , Ratas , Ratas Sprague-Dawley , Reproducibilidad de los Resultados , Espectrometría de Masas en Tándem/métodosRESUMEN
Ceramides are known to be involved in various biological processes with their physiological levels elevated in various disease conditions such as diabetes, Alzheimer's, atherosclerosis. To facilitate the rapid screening of Cer d18:1/16:0, d18:1/24:0, d18:1/24:1, d18:1/18:0, d18:1/14:0, d18:1/20:0, and d18:1/22:0 inhibition in HepG2 cells, a RapidFire coupled to tandem mass spectrometry (RF-MS/MS) method has been developed. The RF platform provides an automated solid-phase extraction system that gave a throughput of 12.6 s per sample to an MS/MS system using electrospray ionization under the positive ion mode. Chromatographic separation of Cer d18:1/16:0, d18:1/24:0, d18:1/24:1, d18:1/18:0, d18:1/14:0, d18:1/20:0, and d18:1/22:0 was achieved using a ternary gradient on C8 type E cartridge. The MS/MS ion transitions monitored were 538.2 â 264.2, 650.7 â 264.2, 648.6 â 264.2, 566.4 â 264.2, 510.4 â 264.2, 594.4 â 264.2, 622.5 â 264.2, and 552.3 â 250.2 for Cer d18:1/16:0, d18:1/24:0, d18:1/24:1, d18:1/18:0, d18:1/14:0, d18:1/20:0, d18:1/22:0, and the internal standard (Cer d17:1/18:0), respectively. The RF-MS/MS methodology showed an excellent performance with an average Z' value of 0.5-0.7. This is the first report of an RF-MS/MS assay for screening of ceramides which is amenable for high-throughput screening.
Asunto(s)
Ceramidas/química , Ensayos Analíticos de Alto Rendimiento/métodos , Espectrometría de Masas en Tándem/métodos , Ceramidas/aislamiento & purificación , Células Hep G2 , Humanos , Extracción en Fase SólidaRESUMEN
Objective: The objective of the current research was to prepare self-micro-emulsifying drug delivery systems (SMEDDS) for BCS class II drug, nilotinib to enhance its oral bioavailability.Methodology: Different types of excipients like oil, surfactant, and co-surfactant were evaluated for drug solubility. Among the screened excipients, Capryol 90, Transcutol HP, and Tween 80 were selected as oil, co-surfactant, and surfactant, respectively, to construct a ternary phase diagram to identify a homogenous mixture. Characterization performed for the prepared SMEDDS for its particle size/droplet size, emulsification time, phase separation, droplet morphology, in vitro drug release, and oral bioavailability.Results: Prepared SMEDDS showed the highest of 87% drug release in in vitro drug release experiment. SMEDDS drug release was superior over suspension formulation, which could be attributed to oil/surfactant ratios and particle size of the SMEDDS. The acquired pharmacokinetic parameters indicate that twofold increase in systemic exposure of SMEDDS compared with nilotinib suspension formulation. A similar twofold increase in relative oral bioavailability was also observed when compared SMEDDS formulation with suspension formulation. Delayed Tmax (time to reach peak plasma concentrations) was observed with SMEDDS over suspension formulation, which was evident by slow rate of absorption of nilotinib from SMEDDS.Conclusion: This research demonstrated that SMEDDS could be an effective approach to improve solubility and oral bioavailability for the BCS class II poorly soluble nilotinib.
Asunto(s)
Antineoplásicos/administración & dosificación , Sistemas de Liberación de Medicamentos , Excipientes/química , Pirimidinas/administración & dosificación , Administración Oral , Animales , Antineoplásicos/química , Antineoplásicos/farmacocinética , Disponibilidad Biológica , Química Farmacéutica , Liberación de Fármacos , Emulsiones , Masculino , Tamaño de la Partícula , Pirimidinas/química , Pirimidinas/farmacocinética , Ratas , Ratas Sprague-Dawley , Solubilidad , Tensoactivos/químicaRESUMEN
A sensitive, selective and rapid LC-ESI-MS/MS method has been developed and validated for the quantification of copanlisib in mouse plasma using enasidenib as an internal standard (IS) as per regulatory guideline. Copanlisib and the IS were extracted from mouse plasma using ethyl acetate as an extraction solvent and chromatographed using an isocratic mobile phase (0.2% formic acid-acetonitrile; 25:75, v/v) on a HyPURITY C18 column. Copanlisib and the IS eluted at ~0.95 and 2.00 min, respectively. The MS/MS ion transitions monitored were m/z 481.1 â 360.1 and m/z 474.0 â 456.0 for copanlisib and the IS, respectively. The calibration range was 3.59-3588 ng/mL. The intra- and inter-batch accuracy and precision (RE and RSD) across quality controls met the acceptance criteria. Stability studies showed that copanlisib was stable in mouse plasma for one month. This novel method has been applied to a pharmacokinetic study in mice.