The AAPS Journal

, 21:5 | Cite as

Effect of the Plasticizer DEHP in Blood Collection Bags on Human Plasma Fraction Unbound Determination for Alpha-1-Acid Glycoprotein (AAG) Binding Drugs

  • Nicholas Ingram
  • Christopher Dishinger
  • Jennifer Wood
  • J. Matthew Hutzler
  • Sherri Smith
  • Michael HuskinEmail author
Research Article


Fraction unbound (fu) is a critical drug distribution parameter commonly utilized for modeling efficacious dosage and safety margin predictions. An over-estimation of fu for 13 chemically diverse small molecule drugs primarily bound to alpha-1-acid glycoprotein (AAG) in human plasma was discovered when in vitro results from our screening lab were compared to literature values. Di-(2-ethylhexyl) phthalate (DEHP), a plasticizer known to be used in the manufacture of blood collection bags, was extracted from plasma obtained through three common techniques that allowed contact with DEHP, and drug fu values in plasma from each collection method were estimated using the HTDialysis protein binding methodology. Additionally, fu of test compounds in plasma spiked with varying concentrations of DEHP (0–800 μM) was determined, and DEHP extractions were performed from plasma stored in Terumo bags over 7 days. Blood stored in Terumo bags, blood collected in Terumo bags, but immediately transferred to conical vials, and vacutainer-collected blood yielded DEHP concentrations of 300–1000 μM, 1–10 μM, and 0.1–2 μM, respectively. This finding corresponded with the fu of tested drugs in DEHP-spiked plasma increasing between 2- and 5-fold. Additionally, DEHP was discovered to leach from the Terumo bag, with concentrations increasing 10-fold over a 7-day test period. In summary, the presence of DEHP in commercially available blood collection bags confounds in vitro fu estimation for drugs that bind primarily to AAG. It is recommended that vacutainer-collected human plasma, which contains negligible DEHP, be used for the most accurate estimation of fu in human plasma.


alpha-1-acid glycoprotein equilibrium dialysis plasma protein binding plasticizer di-(2-ethylhexyl) phthalate (DEHP) 



The authors would like to thank Will Plentl and team at ZenBio, Inc. for collecting and storing donated plasma under our specified experimental conditions, and Sabrina Giordano at BioreclamationIVT for information regarding plasma storage conditions.


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Copyright information

© American Association of Pharmaceutical Scientists 2018

Authors and Affiliations

  • Nicholas Ingram
    • 1
  • Christopher Dishinger
    • 1
  • Jennifer Wood
    • 1
  • J. Matthew Hutzler
    • 1
  • Sherri Smith
    • 2
  • Michael Huskin
    • 1
    Email author
  1. 1.Q2 Solutions, Bioanalytical and ADME LabsIndianapolisUSA
  2. 2.H3 Biomedicine, Inc.CambridgeUSA

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