Absolute protein quantification of clinically relevant cytochrome P450 enzymes and UDP-glucuronosyltransferases by mass spectrometry-based targeted proteomics.
J Pharm Biomed Anal
; 100: 393-401, 2014 Nov.
Article
en En
| MEDLINE
| ID: mdl-25218440
ABSTRACT
Cytochrome P450 (CYP) enzymes and UDP-glucuronosyltransferases (UGT) are major determinants in the pharmacokinetics of most drugs on the market. To investigate their impact on intestinal and hepatic drug metabolism, we developed and validated quantification methods for nine CYP (CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4 and CYP3A5) and four UGT enzymes (UGT1A1, UGT1A3, UGT2B7 and UGT2B15) that have been shown to be of clinical relevance in human drug metabolism. Protein quantification was performed by targeted proteomics using liquid chromatography with tandem mass spectrometry (LC-MS/MS)-based determination of enzyme specific peptides after tryptic digestion using in each case stable isotope labelled peptides as internal standard. The chromatography of the respective peptides was performed with gradient elution using a reversed phase (C18) column (Ascentis(®) Express Peptide ES-C18, 100mm×2.1mm, 2.7µm) and 0.1% formic acid (FA) as well as acetonitrile with 0.1% FA as mobile phases at a flow rate of 300µl/min. The MS/MS detection of all peptides was done simultaneously with a scheduled multiple reaction monitoring (MRM) method in the positive mode by monitoring in each case three mass transitions per proteospecific peptide and the internal standard. The assays were validated according to current bioanalytical guidelines with respect to specificity, linearity (0.25-50nM), within-day and between-day accuracy and precision, digestion efficiency as well as stability. Finally, the developed method was successfully applied to determine the CYP and UGT protein amount in human liver and intestinal microsomes. The method was shown to possess sufficient specificity, sensitivity, accuracy, precision and stability to quantify clinically relevant human CYP and UGT enzymes.
Palabras clave
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Glucuronosiltransferasa
/
Espectrometría de Masa por Ionización de Electrospray
/
Sistema Enzimático del Citocromo P-450
/
Proteómica
/
Espectrometría de Masas en Tándem
/
Cromatografía de Fase Inversa
/
Yeyuno
/
Hígado
Tipo de estudio:
Guideline
Límite:
Adult
/
Aged
/
Female
/
Humans
/
Male
/
Middle aged
Idioma:
En
Revista:
J Pharm Biomed Anal
Año:
2014
Tipo del documento:
Article
País de afiliación:
Alemania