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1.
Pharm Res ; 36(12): 164, 2019 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-31637544

RESUMO

PURPOSE: To describe a stepwise approach to evaluate the pH effect for a weakly basic drug by in vitro, in vivo and in silico techniques and identify a viable mitigation strategy that addresses the risk. METHODS: Clinical studies included assessment of the pH effect with famotidine. In vitro dissolution was evaluated in various biorelevant media and in a pH-shift test. PK studies in dogs were conducted under pentagastrin or famotidine pre-treatment and GastroPlus was employed to model human and dog PK data and simulate the performance in human. RESULTS: Clinical data indicated considerable pH dependent absorption of the drug when dosed in the presence of H2-antagonists. In vitro dissolution and in vivo dog data confirmed that the observed pH effect was due to reduced dissolution rate and lower solubility at increased gastric and intestinal pH. A salt form was identified to overcome the effect by providing fast dissolution and prolonged supersaturation. GastroPlus simulations predicted a mitigation of the pH effect by the salt. CONCLUSIONS: The drug exhibited a strong pH-effect in humans. The in vitro, in vivo and modeling approach provides a systematic workflow to evaluate the risk of a new drug and identify a strategy able to mitigate the risk.


Assuntos
Antiulcerosos/farmacocinética , Simulação por Computador , Composição de Medicamentos/métodos , Famotidina/farmacocinética , Absorção Intestinal , Modelos Biológicos , Administração Oral , Animais , Antiulcerosos/administração & dosagem , Disponibilidade Biológica , Cães , Famotidina/administração & dosagem , Feminino , Humanos , Concentração de Íons de Hidrogênio , Masculino
2.
Anal Chem ; 91(13): 8652-8659, 2019 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-31180205

RESUMO

Preparation of multisample external calibration curves and dilution of study samples are critical steps in bioanalytical sample processing for quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS) based bioanalysis of small-molecule compounds, biotherapeutics, and biomarkers, but they can be time-consuming and prone to error. It is highly desired to simplify or eliminate these two steps in order to improve the assay throughput and robustness. While multisample external calibration curve preparation using authentic matrices can be eliminated with a previously reported in-sample calibration curve (ISCC) approach using multiple isotopologue reaction monitoring (MIRM) of a stable isotopically labeled (SIL) analyte, dilution of study samples is still inevitable due to limited LC-MS/MS assay ranges. In this work, a one-sample multipoint external calibration curve and isotope sample dilution, both using MIRM of an analyte, for quantitative LC-MS/MS based bioanalysis are proposed and demonstrated. By spiking a known amount of an analyte into one blank authentic matrix sample, a one-sample multipoint external calibration curve in an authentic matrix can be established on the basis of the relationship between the calculated theoretical isotopic abundances (analyte concentration equivalents) and the MS/MS responses in the corresponding MIRM channels. This one-sample multipoint external calibration curve can be used in the same way as the traditional multisample external calibration curve for quantitative LC-MS/MS-based bioanalysis. As isotopic abundance in each MIRM channel can be calculated and measured accurately, isotope sample dilution can be achieved by simply monitoring one or a few of the MIRM channels of the analyte in addition to the most abundant MIRM channel for study samples. While the most abundant MIRM channel (isotopic abundance of 100%) is used for the quantitation of samples having concentrations within the assay calibration curve range, less abundant MIRM channels (isotopic abundance of IA%) can be used for the quantitation of samples having concentrations beyond the assay upper limit of quantitation (ULOQ), resulting in isotope dilution factors (IDF) of 100%/IA%. The approaches of one-sample multipoint external calibration curve and isotope sample dilution were evaluated and demonstrated in this work with an example of the quantitation of daclatasvir in human plasma extracted with liquid-liquid extraction. Using these approaches together with the MIRM-ISCC methodology, accurate and reliable LC-MS/MS bioanalysis can be achieved without the need of preparation of multisample external calibration curve and dilution of study samples.

3.
Anal Chem ; 91(13): 8443-8452, 2019 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-31247719

RESUMO

We report a novel immunocapture (IC)-LC-MS/MS methodology to directly measure real time in vivo receptor occupancy (RO) for a covalent binding drug in blood lysate. A small molecule quencher was added immediately after sample collection to convert the free receptor to a quencher-bound receptor (QB-R) which was measured with the drug-bound receptor (DB-R) simultaneously by LC-MS/MS after immunocapture enrichment, followed by trypsin digestion. Addition of the quencher is necessary to prevent the free receptor from ex vivo binding with the drug. The real time RO was calculated based on the concentrations of DB-R and the free receptor (which is now QB-R) that were obtained from each sample. This strategy has been successfully applied to the measurement of the RO for Bruton's tyrosine kinase (BTK) in the blood lysate of monkeys after dosing with branebrutinib (BMS-986195), a covalent BTK inhibitor being evaluated to treat rheumatoid arthritis. A custom-made quencher, which is more reactive to BTK than branebrutinib, was added in excess amount to bind with all available free BTK to form quencher-bound BTK (QB-BTK) during blood sample collection. To measure a wide range of % BTK RO, including those of <5% or >95%, the required LLOQ at 0.125 nM for QB-BTK and 0.250 nM for drug-bound BTK (DB-BTK) in blood lysate were successfully achieved by using this IC-LC-MS/MS strategy. This proof-of-concept assay demonstrated its suitability with high throughput for real time in vivo BTK RO measurement as a pharmacodynamic (PD) biomarker for clinical drug development.

4.
Bioanalysis ; 11(8): 785-795, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30994008

RESUMO

In recent years, hybrid ligand-binding assays (LBAs)/LC-MS assays have been increasingly used for quantitation of protein biomarkers in biological matrices. However, unlike in LBAs where the importance of critical reagent screening and characterization is well understood and widely reported, benefits of well-characterized hybrid LC-MS assay reagents are frequently underestimated. Two groups of analyte-specific reagents, binding reagents and assay calibrators, are considered the critical reagents for biomarker assays. In this article, we summarize the similarities and differences of critical reagents used in LBAs and hybrid LC-MS assays, overview the benefits and approaches of critical reagent screening, characterization, antibody conjugation and discuss bioanalytical considerations in hybrid LC-MS assay development for robust measurements of protein biomarkers in biological matrices.


Assuntos
Biomarcadores/metabolismo , Cromatografia Líquida/métodos , Indicadores e Reagentes/química , Proteínas/metabolismo , Espectrometria de Massas em Tandem/métodos , Humanos
5.
J Med Chem ; 62(7): 3228-3250, 2019 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-30893553

RESUMO

Bruton's tyrosine kinase (BTK), a non-receptor tyrosine kinase, is a member of the Tec family of kinases and is essential for B cell receptor (BCR) mediated signaling. BTK also plays a critical role in the downstream signaling pathways for the Fcγ receptor in monocytes, the Fcε receptor in granulocytes, and the RANK receptor in osteoclasts. As a result, pharmacological inhibition of BTK is anticipated to provide an effective strategy for the clinical treatment of autoimmune diseases such as rheumatoid arthritis and lupus. This article will outline the evolution of our strategy to identify a covalent, irreversible inhibitor of BTK that has the intrinsic potency, selectivity, and pharmacokinetic properties necessary to provide a rapid rate of inactivation systemically following a very low dose. With excellent in vivo efficacy and a very desirable tolerability profile, 5a (branebrutinib, BMS-986195) has advanced into clinical studies.

6.
Anal Chem ; 91(3): 2536-2543, 2019 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-30615432

RESUMO

A novel methodology of in-sample calibration curves (ISCC) using multiple isotopologue reaction monitoring (MIRM) of multiple naturally occurring isotopologue transitions of a stable isotopically labeled (SIL) analyte for instant liquid chromatography-tandem mass spectrometry (LC-MS/MS) bioanalysis of biomarkers, biotherapeutics, and small-molecule compounds is proposed and demonstrated for the first time. The theoretical isotopic abundances of the SIL analyte in its MIRM channels can be accurately calculated based on the isotopic distributions of its daughter ion and neutral loss. The isotopic abundances in these MIRM channels can also be accurately measured with a triple quadrupole mass spectrometer. By spiking a known amount of a SIL analyte into each study sample, an ISCC can be established based on the relationship between the calculated theoretical isotopic abundances (analyte concentration equivalents) in the selected MIRM channels of the SIL analyte and the measured MS/MS peak areas in the corresponding MIRM channels in each individual study sample. The analyte concentration of each study sample can then be calculated individually with the ISCC instantly without using an external calibration curve. The MIRM-ISCC-LC-MS/MS methodology was evaluated and demonstrated in this work with the examples of quantitation of a protein biomarker in human and monkey serum processed with immunocapture and trypsin digestion; three surrogate peptides in trypsin-digested human colon tissue homogenates; and a small-molecule drug in human and rat plasma extracted with liquid-liquid extraction. The potential applications of the MIRM-ISCC-LC-MS/MS methodology in quantitative proteomics, clinical laboratories, and other areas are also discussed in this paper. Without the need for using external calibration curves, this novel MIRM-ISCC-LC-MS/MS methodology can provide accurate and reliable bioanalysis in many potential applications, especially for cases where authentic matrices for external calibration curves are not available.

7.
J Pharm Biomed Anal ; 165: 198-206, 2019 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-30553110

RESUMO

Stable isotope labeled (SIL) compounds have been commonly used as internal standards (IS) to ensure the accuracy and quality of liquid chromatography-mass spectrometry (LC-MS) bioanalytical assays. Recently, the application of SIL drugs and LC-MS assays to microdose absolute bioavailability (BA) studies has gained increasing attention. This approach can provide significant cost and time saving, and higher data quality compared to the accelerator mass spectrometry (AMS)-based method, since it avoids the use of radioactive drug, high-cost AMS instrumentation and complex measurement processes. It also eliminates potential metabolite interference with AMS-based assay. However, one major challenge in the application of this approach is the potential interference between the unlabeled drug, the microdose SIL drug, and the SIL-IS during LC-MS analysis. Here we report a convenient and cost-effective strategy to overcome the interference by monitoring the isotopic ion (instead of the commonly used monoisotopic ion) of the interfered compound in MS analysis. For the BMS-986205 absolute BA case study presented, significant interference was observed from the microdose IV drug [13C7,15N]-BMS-986205 to its SIL-IS, [13C7,15N, D3]-BMS-986205, since the difference of nominal molecular mass between the two compounds is only 3 mu, and there is a Cl atom in the molecules. By applying this strategy (monitoring the 37Cl ion for the analysis of the IS), a 90-fold reduction of interference was achieved, which allowed the use of a synthetically accessible SIL compound and enabled the fast progress of the absolute BA study. This strategy minimizes the number of stable isotope labels used for avoiding interference, which greatly reduces the difficulty in synthesizing the SIL compounds and generates significant time and cost savings. In addition, this strategy can also be used to reduce the MS response of the analyte, therefore, avoiding the detector saturation issue of LC-MS/MS assay for high concentration BMS-986205. A LC-MS/MS assay utilizing this strategy was successfully developed for the simultaneous analysis of BMS-986205 and [13C7, 15N]-BMS-986205 in dog plasma using [13C7,15N, D3]-BMS-986205 as the IS. The assay was successfully applied to a microdose absolute BA study of BMS-986205 in dogs. The assay was also validated in human plasma and used to support a human absolute BA study. The same strategy can also be applied to other compounds, including those not containing Cl or other elements with abundant isotopes, or other applications (e.g. selection of internal standard), and the applications were presented.


Assuntos
Acetamidas/análise , Cromatografia Líquida/métodos , Inibidores Enzimáticos/análise , Quinolinas/análise , Espectrometria de Massas em Tandem/métodos , Acetamidas/administração & dosagem , Acetamidas/farmacocinética , Animais , Disponibilidade Biológica , Cromatografia Líquida/economia , Análise Custo-Benefício , Cães , Inibidores Enzimáticos/administração & dosagem , Inibidores Enzimáticos/farmacocinética , Humanos , Marcação por Isótopo , Quinolinas/administração & dosagem , Quinolinas/farmacocinética , Espectrometria de Massas em Tandem/economia
8.
Bioanalysis ; 10(18): 1473-1485, 2018 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-30215261

RESUMO

AIM: A robust LC-MS/MS assay was developed to quantify endogenous 1, 14-tetradecanedioic acid (TDA) and 1, 16-hexadecanedioic acid (HDA) in human plasma as potential biomarkers for evaluating drug-drug interactions mediated by the hepatic drug transporters, organic anion-transporting polypeptides. RESULTS: This assay was validated using fit-for-purpose approach over standard curve range of 2.5-1000 nM for TDA and HDA using analyte-free charcoal-stripped human plasma as the surrogate matrix. Chromatographic separation condition was successfully optimized to separate TDA from an interference peak while maintaining both analytes in neutral forms to minimize carryover issue. CONCLUSION: The described assay is currently applied to a clinical study for evaluating TDA/HDA as potential substitute biomarkers for drug-drug interaction studies.


Assuntos
Análise Química do Sangue/métodos , Transportadores de Ânions Orgânicos/metabolismo , Ácidos Palmíticos/sangue , Espectrometria de Massas em Tandem , Métodos Analíticos de Preparação de Amostras , Biomarcadores/sangue , Calibragem , Cromatografia Líquida , Humanos , Limite de Detecção , Modelos Lineares
9.
Bioanalysis ; 10(13): 987-995, 2018 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-29972311

RESUMO

In recent years, immunocapture enrichment coupled with LC-MS technology has seen more applications for the measurement of low abundant protein therapeutics and biomarkers in biological matrices. In this article, several critical considerations for the application of immunocapture enrichment to LC-MS bioanalysis of protein therapeutics and biomarkers, including reagent selection, reagent characterization, designing of capture format, etc. are discussed. All these considerations are critical in developing reliable and robust bioanalytical assays with high assay specificity and sensitivity. Successful examples using the immunocapture LC-MS approach in the quantification of biotherapeutic and low abundant protein biomarkers will also be discussed.


Assuntos
Proteínas/análise , Biomarcadores/análise , Cromatografia Líquida , Humanos , Espectrometria de Massas , Proteínas/imunologia
10.
Bioanalysis ; 9(2): 183-192, 2017 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-27960545

RESUMO

AIM: It is challenging to develop a multiple reaction monitoring (MRM) method for some disulfide-bonded peptides with inefficient collision-induced dissociation fragmentation. This study describes a new methodology using differential mobility spectrometry (DMS) combined with multiple ion monitoring (MIM) to enhance bioanalytical sensitivity for sunflower trypsin inhibitor. RESULTS: By combining DMS with MIM to monitor the intact precursor ion in Q1 and Q3 MS analyzers, a lower limit of quantitation at 0.125 ng/ml was achieved to quantify sunflower trypsin inhibitor in rat plasma, representing a 40-fold sensitivity improvement over MIM without DMS. CONCLUSION: DMS coupled with MIM method provides triple quadrupole MS users an effective means to overcome challenges in analyzing disulfide-bonded peptides or other analytes that do not have useful collision-induced dissociation fragment ions for MRM analysis.


Assuntos
Técnicas de Química Analítica/métodos , Dissulfetos/química , Espectrometria de Massas , Peptídeos Cíclicos/sangue , Animais , Cromatografia Líquida de Alta Pressão , Íons/química , Limite de Detecção , Ratos
11.
Bioanalysis ; 8(23): 2429-2443, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-27855510

RESUMO

AIM: A UHPLC-MS/MS assay was developed to quantify urinary dehydroepiandrosterone (DHEA), 7ß-hydroxy-DHEA, cortisone and 6ß-hydroxycortisone as potential biomarkers to predict CYP3A activity. RESULTS: A sensitive assay at LLOQ of 0.500 ng/ml with good accuracy and precision was developed for the four analytes in human urine. This UHPLC-MS/MS assay was optimized by eliminating nonspecific loss of the analytes in urine, ensuring complete hydrolysis of the conjugates to unconjugated forms and use of the product ions of [M+H-H2O]+ for multiple reaction monitoring detection of DHEA and 7ß-hydroxy-DHEA. CONCLUSION: This assay was successfully applied to a pilot clinical study. It is also suitable for future drug-drug interaction studies to continue evaluating the potential of these steroids as biomarkers for CYP3A inhibition and induction.


Assuntos
Biomarcadores/urina , Cortisona/urina , Citocromo P-450 CYP3A/metabolismo , Desidroepiandrosterona/urina , Espectrometria de Massas em Tandem , Urinálise/métodos , Cromatografia Líquida de Alta Pressão/normas , Cortisona/metabolismo , Cortisona/normas , Citocromo P-450 CYP3A/química , Desidroepiandrosterona/metabolismo , Desidroepiandrosterona/normas , Interações de Medicamentos , Humanos , Hidroxilação , Limite de Detecção , Extração Líquido-Líquido , Controle de Qualidade , Espectrometria de Massas em Tandem/normas , Urinálise/instrumentação
12.
Anal Chim Acta ; 934: 170-9, 2016 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-27506357

RESUMO

Dried saliva spot (DSS) sampling is a non-invasive sample collection technique for bioanalysis that can be potentially implemented at the patient's home. A UHPLC-MS/MS assay was developed using detergent-assisted sample extraction to quantify BMS-927711, a drug candidate in development for the treatment of migraines, in human DSS. By implementing DSS sampling at the patients' home, the bioanalytical sample collection for pharmacokinetic evaluation can be done at the time of the acute migraine attack without the need for clinical visits. DSS samples were prepared by spotting 15 µL of liquid saliva onto regular Whatman FTA™ DMPK-C cards and verified with a UV lamp (at λ 254 nm or 365 nm) during DSS punching. The 4-mm DSS punches in a 96-well plate were sonicated with 200 µL of [(13)C2, D4]-BMS-927711 internal standard (IS) solution in 20/80 MeOH/water for 10 min, followed by sonication with 50 µL of 100 mM NH4OAc with 1.0% Triton-X-100 (as detergent) prior to liquid-liquid extraction with 600 µL EtOAc/Hexane (90:10). UHPLC-MS/MS was performed with an Aquity(®) UPLC BEH C18 Column (2.1 × 50 mm, 1.7 µm) on a Triple Quad™ 5500 mass spectrometer. The assay was linear with a concentration range from 2.00 to 1000 ng mL(-1) for BMS-927711 in human saliva. The intra- and inter-assay precision was within 8.8% CV, and the accuracy was within ±6.7% Dev of the nominal concentration values. This UHPLC-MS/MS assay has been successfully applied to determine the drug's pharmacokinetics within a clinical study. For the first time, we observed BMS-927711 exposure in human DSS, confirming the suitability of this sampling technique for migraine patients to use at home. Detergent-assisted extraction with Triton-X-100 could be very useful in DSS or other dried matrix spot (DMS) assays to overcome low or inconsistent analyte recovery issues.


Assuntos
Detergentes/química , Piperidinas/análise , Piridinas/análise , Saliva/química , Cromatografia Líquida de Alta Pressão , Humanos , Extração Líquido-Líquido , Espectrometria de Massas em Tandem
13.
J Pharmacol Exp Ther ; 358(1): 138-50, 2016 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-27189973

RESUMO

The pharmacokinetics, pharmacodynamics, safety, and tolerability of BMS-932481, a γ-secretase modulator (GSM), were tested in healthy young and elderly volunteers after single and multiple doses. BMS-932481 was orally absorbed, showed dose proportionality after a single dose administration, and had approximately 3-fold accumulation after multiple dosing. High-fat/caloric meals doubled the Cmax and area under the curve and prolonged Tmax by 1.5 hours. Consistent with the preclinical pharmacology of GSMs, BMS-932481 decreased cerebrospinal fluid (CSF) Aß39, Aß40, and Aß42 while increasing Aß37 and Aß38, thereby providing evidence of γ-secretase enzyme modulation rather than inhibition. In plasma, reductions in Aß40 and Aß42 were observed with no change in total Aß; in CSF, modest decreases in total Aß were observed at higher dose levels. Increases in liver enzymes were observed at exposures associated with greater than 70% CSF Aß42 lowering after multiple dosing. Although further development was halted due to an insufficient safety margin to test the hypothesis for efficacy of Aß lowering in Alzheimer's disease, this study demonstrates that γ-secretase modulation is achievable in healthy human volunteers and supports further efforts to discover well tolerated GSMs for testing in Alzheimer's disease and other indications.


Assuntos
Secretases da Proteína Precursora do Amiloide/metabolismo , Peptídeos beta-Amiloides , Compostos de Anilina/farmacologia , Compostos de Anilina/farmacocinética , Anti-Inflamatórios não Esteroides/farmacologia , Anti-Inflamatórios não Esteroides/farmacocinética , Pirimidinas/farmacologia , Pirimidinas/farmacocinética , Adolescente , Adulto , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/enzimologia , Peptídeos beta-Amiloides/sangue , Peptídeos beta-Amiloides/líquido cefalorraquidiano , Compostos de Anilina/efeitos adversos , Compostos de Anilina/química , Anti-Inflamatórios não Esteroides/efeitos adversos , Anti-Inflamatórios não Esteroides/química , Área Sob a Curva , Cromatografia Líquida , Relação Dose-Resposta a Droga , Método Duplo-Cego , Feminino , Voluntários Saudáveis , Humanos , Limite de Detecção , Masculino , Espectrometria de Massas , Pessoa de Meia-Idade , Pirimidinas/efeitos adversos , Pirimidinas/química , Adulto Jovem
14.
Anal Chim Acta ; 916: 42-51, 2016 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-27016437

RESUMO

To quantify a therapeutic PEGylated protein in monkey serum as well as to monitor its potential in vivo instability and methionine oxidation, a novel ultra high performance liquid chromatography-high resolution mass spectrometric (UHPLC-HRMS) assay was developed using a surrogate disulfide-containing peptide, DCP(SS), and a confirmatory peptide, CP, a disulfide-free peptide. DCP(SS) was obtained by eliminating the step of reduction/alkylation before trypsin digestion. It contains an intact disulfide linkage between two peptide sequences that are essential for drug function but susceptible to potential in vivo cleavages. HRMS-based single ion monitoring (SIM) on a Q Exactive™ mass spectrometer was employed to improve assay specificity and sensitivity for DCP(SS) due to its poor fragmentation and low sensitivity with SRM detection. The assay has been validated for the protein drug in monkey serum using both surrogate peptides with excellent accuracy (within ±4.4%Dev) and precision (within 7.5%CV) with a lower limit of quantitation (LLOQ) at 10 ng mL(-1). The protein concentrations in monkey serum obtained from the DCP(SS)-based assay not only provided important pharmacokinetic parameters, but also confirmed in vivo stability of the peptide regions of interest by comparing drug concentrations with those obtained from the CP-based assay or from a ligand-binding assay (LBA). Furthermore, UHPLC-HRMS allowed simultaneous monitoring of the oxidized forms of both surrogate peptides to evaluate potential ex vivo/in vivo oxidation of one methionine present in each of both surrogate peptides. To the best of our knowledge, this is the first report of using a surrogate disulfide-containing peptide for LC-MS bioanalysis of a therapeutic protein.


Assuntos
Proteínas Sanguíneas/metabolismo , Cromatografia Líquida de Alta Pressão/métodos , Dissulfetos/química , Polietilenoglicóis/análise , Proteínas/uso terapêutico , Espectrometria de Massas em Tandem/métodos , Animais , Calibragem , Haplorrinos , Controle de Qualidade
15.
J Pharm Biomed Anal ; 119: 145-51, 2016 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-26683987

RESUMO

Asunaprevir (BMS-650032) is a selective hepatitis C virus (HCV) NS3 protease inhibitor with potent activity against HCV genotypes 1, 4, 5 and 6. It has been developed in conjunction with direct-acting antiviral agents, in interferon- and ribavirin-free regimen, to improve existing therapies for HCV infection. To support the pharmacokinetic analyses in asunaprevir clinical studies, we have developed and validated a highly sensitive and robust LC-MS/MS method to quantify asunaprevir in human EDTA plasma with an LLOQ of 0.05ng/mL, which was a 20-fold sensitivity improvement over a previously reported assay for asunaprevir. A deuterated labeled [D9]-asunaprevir was used as the internal standard (IS). The analyte and the IS were extracted using a semi-automated liquid-liquid extraction (LLE) at pH 7 with methyl-t-butyl ether (MTBE) in a 96-well plate containing 10µL of 10% CHAPS as the surfactant to prevent non-specific binding issue. Chromatographic separation was achieved on a Genesis C8 column (2.1×50mm, 4µm) with a gradient elution using 0.1% formic acid in water as mobile phase A and a mixture of methanol: acetone: formic acid (95:5:0.1; v/v/v) as the mobile phase B. Positive electrospray ionization was performed using multiple reaction monitoring (MRM) with transitions of m/z 748→648 for asunaprevir and m/z 757→649 for [D9]-asunaprevir,and a collision energy of 30 electron Volts (eV). The assay was validated over a standard curve range from 0.05 to 50ng/mL for asunaprevir in human plasma. The intra- and inter assay precisions were within 7.1% CV, and the % deviation was within 5.5% of their nominal concentrations. This assay has been successfully applied to multiple clinical studies with excellent assay ruggedness and reproducibility.


Assuntos
Antivirais/sangue , Cromatografia Líquida de Alta Pressão/métodos , Hepacivirus/enzimologia , Isoquinolinas/sangue , Sulfonamidas/sangue , Espectrometria de Massas em Tandem/métodos , Proteínas não Estruturais Virais/antagonistas & inibidores , Antivirais/química , Antivirais/farmacocinética , Calibragem , Humanos , Isoquinolinas/química , Isoquinolinas/farmacocinética , Limite de Detecção , Extração Líquido-Líquido , Estrutura Molecular , Padrões de Referência , Reprodutibilidade dos Testes , Sulfonamidas/química , Sulfonamidas/farmacocinética
16.
J Chromatogr A ; 1424: 27-36, 2015 Dec 11.
Artigo em Inglês | MEDLINE | ID: mdl-26525666

RESUMO

The current LC-MS/MS approach for bioanalysis of protein therapeutics requires generating peptides from protein molecules via trypsin digestion, followed by sensitive detection of these surrogate peptides by multiple reaction monitoring (MRM). However, the presence of huge amount of matrix-related interference peptides generated from trypsin digestion often causes substantial matrix effect or isobaric interferences during LC-MS/MS analysis. Solid phase extraction (SPE) exhibits great potential in sample extraction to overcome these challenges due to its characteristic features of high selectivity, reproducibility, cost-effectiveness and potential to be automated. Here, we report an effective SPE methodology for the bioanalysis of protein therapeutics involving post-pellet-digestion precipitation and SPE cleanup prior to UHPLC-MS/MS analysis. Specifically, proteins in serum samples were first precipitated with methanol to enrich the protein analyte in the pellet prior to trypsin digestion of the pellet (pellet-digestion). The trypsin digest was further processed by a post-pellet-digestion precipitation (second precipitation) to remove matrix-related clog-causing components prior to SPE on OASIS™ MAX (Mixed-Mode Anion-Exchange) SPE plate. This methodology successfully overcame SPE clogging issue and enabled extraction of 100µL of monkey serum on SPE with significant sensitivity improvement to achieve a lower limit of quantitation (LLOQ) of 50ng/mL for a human monoclonal antibody of the IgG4 subclass. This UHPLC-MS/MS assay was validated in a concentration range of 50-5000ng/mL with intra- and inter-assay precisions of within 9.6% CV, and assay accuracy of within ±2.9% Dev of their nominal concentrations. To our best knowledge, this is the pellet digestion with SPE method for LC-MS/MS bioanalysis of a monoclonal antibody for the first time to achieve a LLOQ in the low ng/mL concentration range.


Assuntos
Anticorpos Monoclonais/isolamento & purificação , Imunoglobulina G/isolamento & purificação , Animais , Anticorpos Monoclonais/sangue , Anticorpos Monoclonais/química , Cromatografia Líquida de Alta Pressão/métodos , Haplorrinos , Humanos , Imunoglobulina G/sangue , Imunoglobulina G/química , Peptídeos/química , Reprodutibilidade dos Testes , Soro , Extração em Fase Sólida/métodos , Espectrometria de Massas em Tandem/métodos , Tripsina/química
17.
Artigo em Inglês | MEDLINE | ID: mdl-25746754

RESUMO

Apixaban (Eliquis™) was developed by Bristol-Myers Squibb (BMS) and Pfizer to use as an antithrombotic/anticoagulant agent and has been recently approved for the prevention of stroke and systemic embolism in patients with nonvalvular atrial fibrillation. A clinical study of apixaban, sponsored by BMS and Pfizer, included a pilot exploratory portion to evaluate the potential for future drug concentration monitoring using dried blood spot (DBS) sample collection. For DBS sample collection, a fixed blood volume was dispensed onto a DBS card by either regular volumetric pipette (venous blood collection) or capillary dispenser (finger prick blood collection). A 96-well semi-automated liquid-liquid extraction sample preparation procedure was developed to provide clean extracts for UHPLC-MS/MS quantitation. Assays using both partial-spot center punch and whole spot punch were developed and validated. The linear dynamic ranges for all the analyses were from 0.5 to 500 ng/mL. The coefficient of determination (r(2)) values was >0.9944 for all the validation runs. For the center punch approach, the intra-assay precision (%CV) was within 4.4% and inter-assay precision was within 2.6%. The assay accuracy, expressed as %Dev., was within ± 5.4% of the nominal concentrations. One accuracy and precision run was performed using the whole spot approach, the intra-assay precision (%CV) was within 7.1% and the accuracy was within ± 8.0% of the nominal concentrations. In contrast to the center punch approach, the whole spot approach eliminated the effect of hematocrit and high lipids on the analysis of apixaban in human DBS when an accurate sample blood volume was collected on DBS cards.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Teste em Amostras de Sangue Seco/métodos , Pirazóis/sangue , Piridonas/sangue , Espectrometria de Massas em Tandem/métodos , Estabilidade de Medicamentos , Humanos , Limite de Detecção , Modelos Lineares , Extração Líquido-Líquido , Pirazóis/química , Piridonas/química , Reprodutibilidade dos Testes
18.
Artigo em Inglês | MEDLINE | ID: mdl-25464088

RESUMO

BMS-791325 is a hepatitis C virus (HCV) non-structural protein 5B (NS5B) RNA polymerase inhibitor that is being developed for the treatment of HCV infection. A rugged and accurate LC-MS/MS method was developed and validated for the quantitation of BMS-791325 and its metabolite, BMS-794712, in rat and dog plasma. This method utilized stable-isotope labeled [D6]-BMS-791325 and [13CD3]-BMS-794712 as internal standards. The samples were extracted using liquid-liquid extraction with n-butyl-chloride. Chromatographic separation was achieved with gradient elution on a Waters Atlantis dC18 analytical column (2.1mm×50mm, 3.0µm). Analytes and their stable isotope labeled internal standards were detected by positive ion electrospray tandem mass spectrometry. The standard curves, which ranged from 5.00 to 2000ng/mL for BMS-791325 and from 1.00 to 400ng/mL for BMS-794712, were fitted to a 1/x2 weighted linear regression model. For both species, the intra-assay precision was within ±4.3% CV, the inter-assay precision was within ±6.2% CV, and the assay accuracy was within ±10.8% of the nominal values for BMS-791325 and BMS-794712. The validated method was successfully applied to support pre-clinical toxicokinetic studies.

19.
Bioanalysis ; 6(18): 2441-59, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25384595

RESUMO

Robotic liquid handlers (RLHs) have been widely used in automated sample preparation for liquid chromatography-tandem mass spectrometry (LC-MS/MS) bioanalysis. Automated sample preparation for regulated bioanalysis offers significantly higher assay efficiency, better data quality and potential bioanalytical cost-savings. For RLHs that are used for regulated bioanalysis, there are additional requirements, including 21 CFR Part 11 compliance, software validation, system qualification, calibration verification and proper maintenance. This article reviews recent advances in automated sample preparation for regulated bioanalysis in the last 5 years. Specifically, it covers the following aspects: regulated bioanalysis requirements, recent advances in automation hardware and software development, sample extraction workflow simplification, strategies towards fully automated sample extraction, and best practices in automated sample preparation for regulated bioanalysis.


Assuntos
Métodos Analíticos de Preparação de Amostras/métodos , Cromatografia Líquida/métodos , Espectrometria de Massas em Tandem/métodos , Animais , Automação , Fracionamento Químico , Humanos , Software
20.
J Pharm Biomed Anal ; 89: 240-50, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24316424

RESUMO

An UHPLC-MS/MS method was developed and validated to quantify BMS-927711, a drug candidate to treat migraine, in rat dried blood spots (DBS). The DBS samples were extracted using an improved liquid-liquid extraction (LLE) strategy involving in the sonication of DBS punches in 20% MeOH aqueous solution containing the internal standard, [(13)C2, D4]-BMS-927711, and then with a 100mM NH4OAc buffer solution, followed by an automated LLE with EtOAc-hexane (70:30, v/v). The presence of 20% MeOH as an organic modifier in the elution solution significantly improved the analyte elution efficiency and assay performance. A novel inter-well volume replacement dilution workflow was introduced for DBS sample dilution before LLE step. This was a simple two-step process, firstly a small portion of the DBS blank solution was discarded, and then the same volume of a concentrated DBS sample solution was spiked into the leftover blank solution to achieve a desired dilution. Chromatographic separation was achieved on an Acuity UPLC(®) BEH C18 column (2.1mm×50mm, 1.7µm) and the analyte was detected by selected reaction monitoring (SRM) with positive electrospray ionization on an AB Sciex Triple Quad 5500 mass spectrometer. The standard curve was linear from 5.00 to 5000ng/mL with assay precision ≤4.9% CV, and assay accuracy within ±3.1%Dev of the nominal values. Accurate sample dilution was achieved by using inter-well volume replacement with a precision of ≤4.2% CV and an accuracy of ±3.3% for dilution QC at 50,000ng/mL with 100-fold dilution (n=18). This robust UHPLC-MS/MS assay has been successfully applied to the non-clinical studies in rats. By using inter-well volume replacement workflow, accurate dilution was demonstrated using only one DBS blank sample for a typical dilution of <50-fold, and using only two blank DBS samples for a dilution of up to 625-fold. Moreover, this new workflow makes it easier to automate DBS sample dilution.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Teste em Amostras de Sangue Seco/métodos , Piperidinas/química , Piridinas/química , Animais , Técnicas de Diluição do Indicador , Limite de Detecção , Extração Líquido-Líquido/métodos , Ratos , Reprodutibilidade dos Testes , Espectrometria de Massas em Tandem/métodos , Fluxo de Trabalho
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