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Analysis of free drug fractions in human serum by ultrafast affinity extraction and two-dimensional affinity chromatography

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Abstract

Ultrafast affinity extraction and a two-dimensional high performance affinity chromatographic system were used to measure the free fractions for various drugs in serum and at typical therapeutic concentrations. Pooled samples of normal serum or serum from diabetic patients were utilized in this work. Several drug models (i.e., quinidine, diazepam, gliclazide, tolbutamide, and acetohexamide) were examined that represented a relatively wide range of therapeutic concentrations and affinities for human serum albumin (HSA). The two-dimensional system consisted of an HSA microcolumn for the extraction of a free drug fraction, followed by a larger HSA analytical column for the further separation and measurement of this fraction. Factors that were optimized in this method included the flow rates, column sizes, and column switching times that were employed. The final extraction times used for isolating the free drug fractions were 333–665 ms or less. The dissociation rate constants for several of the drugs with soluble HSA were measured during system optimization, giving results that agreed with reference values. In the final system, free drug fractions in the range of 0.7–9.5 % were measured and gave good agreement with values that were determined by ultrafiltration. Association equilibrium constants or global affinities were also estimated by this approach for the drugs with soluble HSA. The results for the two-dimensional system were obtained in 5–10 min or less and required only 1–5 μL of serum per injection. The same approach could be adapted for work with other drugs and proteins in clinical samples or for biomedical research.

General scheme for the analysis of free drug fractions in serum by ultrafast affinity chromatography and two-dimensional affinity chromatography. The circles represent the drug and the half-circles on the left are serum proteins that can undergo reversible binding with this drug. The rectangles represent (from left-to-right) a guard column, an affinity microcolumn, and an analytical affinity column that are used to isolate and measure the drug's free fraction in serum

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Acknowledgments

This work was supported by the National Institutes of Health under grants R01 GM044931 and R01 DK069629. M. Podariu was supported through a UCARE fellowship from the University of Nebraska-Lincoln. The concept of free fraction analysis by rapid affinity chromatography is the subject of U.S. Patent 6,720,193 (D.S. Hage and W.A. Clarke).

Conflict of interest

The authors declare that they have competing interests.

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Authors and Affiliations

  1. Department of Chemistry, University of Nebraska, Lincoln, NE, 68588, USA

    Xiwei Zheng, Maria Podariu, Ryan Matsuda & David S. Hage

Authors
  1. Xiwei Zheng
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  2. Maria Podariu
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  3. Ryan Matsuda
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  4. David S. Hage
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Correspondence to David S. Hage.

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Zheng, X., Podariu, M., Matsuda, R. et al. Analysis of free drug fractions in human serum by ultrafast affinity extraction and two-dimensional affinity chromatography. Anal Bioanal Chem 408, 131–140 (2016). https://doi.org/10.1007/s00216-015-9082-7

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  • DOI: https://doi.org/10.1007/s00216-015-9082-7

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