Pharmaceutical Research

, Volume 24, Issue 12, pp 2281–2296 | Cite as

Multichannel Liquid Chromatography–Tandem Mass Spectrometry Cocktail Method for Comprehensive Substrate Characterization of Multidrug Resistance-Associated Protein 4 Transporter

  • Yasuo Uchida
  • Junichi Kamiie
  • Sumio Ohtsuki
  • Tetsuya Terasaki
Research Paper



To develop a comprehensive substrate-screening method for the ATP-binding cassette (ABC) transporter, and identify new substrates for multidrug resistance-associated protein 4 (MRP4/ABCC4).


Human MRP4-expressing membrane vesicles were incubated with a mixture of 50 compounds, including methotrexate, a known MRP4 substrate. The amounts transported were simultaneously determined by liquid chromatography–tandem mass spectrometry.


From 49 compounds, 12 were identified as substrate candidates for MRP4 in the first screening. The second screening was performed involving the uptake of mixture using single quadrupole multichannel mode, and the third screening was performed involving the uptake of individual compounds using multiple reaction monitoring multichannel mode. As a result, eight substrate candidates were additionally identified. Subsequently, in the fourth step, osmotic pressure-dependent transport was demonstrated for 18 compounds (cefmetazole, piperacillin, rebamipide, tetracycline, ampicillin, benzylpenicillin, bumetanide, cephalosporin C, enalapril, pipemidic acid, furosemide, ceftazidime, pravastatin, hydrochlorothiazide, sulbactam, baclofen, bezafibrate and alacepril) among the 20 substrate candidates, thereby confirming them as MRP4 substrates. By contrast, the uptakes of meloxicam and nateglinide did not depend on osmolarity, indicating that these compounds were not substrates, but bound to MRP4.


The new comprehensive substrate-screening method for ABC transporters allowed the identification of 18 new substrates for MRP4.

Key words

ATP-binding cassette transporter human multidrug resistance-associated protein 4 liquid chromatography–tandem mass spectrometry substrate screening vesicle uptake study 



ATP-binding cassette


angiotensin-converting enzyme


blood–brain barrier


blood–cerebrospinal fluid barrier




central nervous system


dehydroepiandrosterone sulfate


electrospray ionization


liquid chromatography–mass spectrometry


liquid chromatography–tandem mass spectrometry


multiple reaction monitoring


multidrug resistance-associated protein




membrane uptake-index difference


organic anion transporter


single quadrupole



We thank H. Yabuuchi (GenoMembrane, Yokohama, Japan) for kindly supplying the human MRP4-expressing membrane vesicles and the control vesicles. We also thank N. Funayama for secretarial assistance. This study was supported, in part, by a Grant-in-Aid for Scientific Research on Priority Area 17081002 from The Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan, and by a 21st Century Center of Excellence (COE) Program Grant from the Japan Society for the Promotion of Science. This study was also supported, in part, by the Industrial Technology Research Grant Program from New Energy and the Industrial Technology Development Organization (NEDO) of Japan.


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Copyright information

© Springer Science+Business Media, LLC 2007

Authors and Affiliations

  • Yasuo Uchida
    • 1
  • Junichi Kamiie
    • 1
    • 2
  • Sumio Ohtsuki
    • 1
    • 2
  • Tetsuya Terasaki
    • 1
    • 2
  1. 1.Division of Membrane Transport and Drug Targeting, Graduate School of Pharmaceutical SciencesTohoku UniversitySendaiJapan
  2. 2.SORSTJapan Science and Technology Agency (JST)KawaguchiJapan

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