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1.
Bioanalysis ; 9(3): 251-264, 2017 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-28097886

RESUMO

AIM: Immobilized metal ion affinity chromatography is widely employed for purifying polyhistidine-tagged recombinant proteins from cell lysates. The technique can be applied for quantification of therapeutic proteins in biological matrices by LC-MS/MS. RESULTS: A protein reagent-free workflow was developed for quantifying polyhistidine-tagged proteins by LC-MS/MS. The workflow includes target protein enrichment by immobilized metal ion affinity chromatography, on-bead trypsin digestion and quantification of signature peptides by LC-MS/MS. It was applied to quantify a 6×His-tagged protein in a mouse pharmacokinetic study with assay sensitivity of 10.0 ng/ml and linearity up to 10,000 ng/ml. CONCLUSION: The protein reagent-free workflow developed herein can overcome reagent limitation and serve as a viable approach for quantifying polyhistidine-tagged therapeutic proteins to support discovery pharmacokinetic and pharmacodynamic studies.


Assuntos
Cromatografia de Afinidade/métodos , Cromatografia Líquida/métodos , Histidina/química , Fragmentos de Peptídeos/análise , Proteínas Recombinantes/farmacocinética , Espectrometria de Massas em Tandem/métodos , Animais , Metais/química , Camundongos , Camundongos Nus , Ratos , Distribuição Tecidual
2.
J Pharm Biomed Anal ; 56(4): 778-84, 2011 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-21840665

RESUMO

A simple, robust, and sensitive liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was developed for the measurement of endogenous adenine in mouse, rat, cynomolgus monkey, and human plasma. A "surrogate analyte" strategy was adopted by employing [(13)C(U)]-adenine as the surrogate analyte. The plasma samples were processed by protein precipitation, and the extracted supernatant samples were subjected directly to LC-MS/MS analysis. The analysis was carried out in the negative ion detection mode using selected-reaction monitoring (SRM). The method achieved a lower limit of quantification (LLOQ) of 5.0nM with a signal-to-noise ratio of 10. The intra- and inter-day assay coefficients of variation (CV) were ≤6.67% in rat plasma, and the mean recoveries and matrix effects across species and at various concentrations ranged from 88.8% to 104.2% and 86.0% to 110.8%, respectively. Using this methodology, the endogenous concentration of adenine in plasma of four species was found to range from 8.7nM in human to 93.1nM in cynomolgus monkey plasma. The assay was further applied to both an adenine pharmacokinetic study and a pivotal pharmacodynamic study evaluating the plasma concentration of adenine after a dose of 5'-deoxy-5'-methylthioadenosine (MTA).


Assuntos
Adenina/sangue , Adenina/farmacocinética , Cromatografia Líquida/métodos , Espectrometria de Massas em Tandem/métodos , Adenina/química , Adenina/metabolismo , Animais , Gatos , Haplorrinos , Humanos , Modelos Lineares , Camundongos , Ratos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Razão Sinal-Ruído
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