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Ultrasensitive Quantification of Drug-metabolizing Enzymes and Transporters in Small Sample Volume by Microflow LC-MS/MS.

Authors
  • Ahire, Deepak Suresh1
  • Basit, Abdul1
  • Karasu, Matthew1
  • Prasad, Bhagwat2
  • 1 Department of Pharmaceutical Sciences, Washington State University, 12 E Spokane Falls Blvd, Spokane, WA 99202, USA.
  • 2 Department of Pharmaceutical Sciences, Washington State University, 12 E Spokane Falls Blvd, Spokane, WA 99202, USA. Electronic address: [email protected]
Type
Published Article
Journal
Journal of Pharmaceutical Sciences
Publisher
Elsevier
Publication Date
Jul 01, 2021
Volume
110
Issue
7
Pages
2833–2840
Identifiers
DOI: 10.1016/j.xphs.2021.03.020
PMID: 33785352
Source
Medline
Keywords
Language
English
License
Unknown

Abstract

Protein abundance data of drug-metabolizing enzymes and transporters (DMETs) are broadly applicable to the characterization of in vitro and in vivo models, in vitro to in vivo extrapolation (IVIVE), and interindividual variability prediction. However, the emerging need of DMET quantification in small sample volumes such as organ-on a chip effluent, organoids, and biopsies requires ultrasensitive protein quantification methods. We present an ultrasensitive method that relies on an optimized sample preparation approach involving acetone precipitation coupled with a microflow-based liquid chromatography-tandem mass spectrometry (µLC-MS/MS) for the DMET quantification using limited sample volume or protein concentration, i.e., liver tissues (1-100 mg), hepatocyte counts (~4000 to 1 million cells), and microsomal protein concentration (0.01-1 mg/ml). The method was applied to quantify DMETs in differential tissue S9 fractions (liver, intestine, kidney, lung, and heart) and cryopreserved human intestinal mucosa (i.e., CHIM). The method successfully quantified >75% of the target DMETs in the trypsin digests of 1 mg tissue homogenate, 15,000 hepatocytes, and 0.06 mg/ml microsomal protein concentration. The precision of DMET quantification measured as the coefficient of variation across different tissue weights, cell counts, or microsomal protein concentration was within 30%. The method confirmed significant extrahepatic abundance of non-cytochrome P450 enzymes such as dihydropyridine dehydrogenase (DPYD), epoxide hydrolases (EPXs), arylacetamide deacetylase (AADAC), paraoxonases (PONs), and glutathione S-transferases (GSTs). The ultrasensitive method developed here is applicable to characterize emerging miniaturized in vitro models and small volume biopsies. In addition, the differential tissue abundance data of the understudied DMETs will be important for physiologically-based pharmacokinetic (PBPK) modeling of drugs. Copyright © 2021 American Pharmacists Association. Published by Elsevier Inc. All rights reserved.

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