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Anal Chim Acta. 2012 Mar 2;717:67-76. doi: 10.1016/j.aca.2011.12.005. Epub 2011 Dec 24.

Development of a multiplex UPLC-MRM MS method for quantification of human membrane transport proteins OATP1B1, OATP1B3 and OATP2B1 in in vitro systems and tissues.

Author information

1
Pfizer Global Research and Development, Andover/Cambridge Laboratories, Andover, MA 01810, USA. chengjie.ji@pfizer.com

Abstract

OATP1B1, OATP1B3 and OATP2B1 are important members of the organic anion transporting polypeptides (OATP) family and are implicated in the hepatic disposition of endobiotics and xenobiotics. Quantitating the expression levels of human OATP1B1, OATP1B3 and OATP2B1 in in vitro systems and tissue samples could significantly improve attempts to scale up in vitro data and result in more effective in vitro-in vivo correlation of transporter-mediated effects on drug disposition, such as hepatic clearance. In the present study, a quantification method was developed, characterized, and implemented for simultaneous determination of human OATP1B1, OATP1B3 and OATP2B1 in HEK cells transfected with OATP-expressing plasmid vectors (SLCO1B1, SLCO1B3, and SLCO2B1, respectively), human hepatocytes, human brain capillary endothelial cells, and humanized mouse liver tissue using UPLC-MRM MS. Purified membrane protein standards prepared and characterized as previously reported (Protein Expr. Purif. 2008, 57, 163-71) were first used as standards for absolute quantification of the expression levels of the three human OATP membrane proteins. The specificity of the optimized MRM transitions were characterized by analyzing the tryptic digests of the membrane protein fraction of wild type HEK cells and control mouse liver tissue using the herein reported UPLC-MRM MS method. The linearity of the calibration curve spanned from 0.2 μg mL(-1) (0.040 μg mg(-1)) to 20 μg mL(-1) (4.0 μg mg(-1)), with accuracy (% RE) within 15% at all concentrations examined for all three OATPs of interest in this study. The intra- and inter-day assay accuracy (% RE) and coefficient of variations (% CV) of triplicates are all within 15% for all levels of quality control samples prepared by mixing the membrane fraction of control mouse liver tissue with the required amount of purified human OATP1B1, OATP1B3 and OATP2B1.

PMID:
22304817
DOI:
10.1016/j.aca.2011.12.005
[Indexed for MEDLINE]

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