ORIGINAL ARTICLE Measuring Unbound Versus Total Vancomycin Concentrations in Serum and Plasma: Methodological Issues and Relevance Veronique Stove, PharmD, PhD,* Louise Coene, PharmD,* Mieke Carlier, PharmD,† Jan J. De Waele, MD, PhD,‡ Tom Fiers, MD,* and Alain G. Verstraete, MD, PhD*† Background: Studies on the unbound fraction (f u ) of vancomycin report highly variable results. Great controversy also exists about the correlation between unbound and total vancomycin concentrations. As differences in (pre-)analytic techniques may explain these findings, we investigated the impact of the procedure used to isolate unbound vancomycin in serum/plasma on f u and the correlation between total and unbound concentrations. Methods: Patient samples (n = 39) were analyzed for total and unbound vancomycin concentrations after ultrafiltration (UF, Centrifree at 48C and 378C) or equilibrium dialysis (ED, using a Fast Micro- Equilibrium Dialyzer at 378C) on an Architect i2000SR. To investigate correlations with potential binding proteins, total protein, albumin, alpha-1-acid glycoprotein, and IgA concentrations were also measured. Results: The median f u after ED was 72.5% [interquartile range (IQR), 68.7%–75.0%]. Ultrafiltration at 48C and 378C resulted in a median f u of 51.6% (IQR, 48.6%–54.8%) and 75.2% (IQR, 69.3%–78.6%), respec- tively, with no significant difference between unbound vancomycin con- centrations after ED and UF at 378C(P = 0.13). Unbound concentrations obtained through ED and UF correlated linearly (48C: r = 0.9457; 378C: r = 0.9478; both P , 0.0001). Linear mixed-model regression showed that total vancomycin as such was the predominant determinant for the unbound concentration, allowing a reliable prediction (mean bias 6 SD, 5.0% 6 7.6%). The studied protein concentrations were of no added value in predicting the unbound concentration. Conclusions: Vancomycin f u after UF at 48C was on average 30.6% lower than that after UF at 378C, demonstrating the impor- tance of temperature during UF. Ultrafiltration at 378C resulted in unbound vancomycin concentrations equivalent with ED. As the unbound concentration could be reliably predicted based on total vancomycin concentrations as such, measurement of unbound van- comycin concentrations has little added value over measurements of total vancomycin concentrations. Key Words: vancomycin, unbound concentrations, ultrafiltration, protein binding, equilibrium dialysis (Ther Drug Monit 2015;37:180–187) INTRODUCTION Protein binding is an important determinant in the pharmacokinetic and pharmacodynamic behavior of a drug. The unbound or free drug fraction (f u ) is considered as the biologically active fraction, available for passive diffusion and binding at the site of action. 1,2 Changes in protein binding are of clinical importance in some patient groups, such as the critically ill, where hypoproteinemia is frequently seen. This phenomenon is particularly important for highly protein-bound drugs, where altered protein binding will lead to a significant change in the unbound concentration. In case of an increased unbound concentration, this will result in a more extensive distribution and clearance of the drug, the latter being clinically relevant in drugs with a high clearance rate. The effect of these pharmacokinetic changes has more relevance for drugs, like antibacterials, where dosing is difficult to correlate with the clinical effect. 1,2 Here, if therapeutic drug monitoring is avail- able, total antibiotic concentrations will be routinely monitored in serum/plasma. Although the determination of the unbound drug concentration is considered to be a better tool to optimize antibiotic therapy, this is still a rarely performed analysis. 1,2 A possible explanation why unbound drug concentrations are not frequently analyzed might be found in the (pre-)analytical challenges. 1,2 In the analysis of unbound drug concentrations, separating the unbound fraction of a drug is probably the most difficult step, and several methods have been used. 3 Equilibrium dialysis (ED) and ultrafiltration (UF) are the 2 most widely used in vitro methods, each having their own advantages and disad- vantages. In ED, 2 chambers are separated by a semipermeable membrane. These chambers are filled with serum/plasma and a buffer, respectively. This method is considered as the gold standard but has the limitation that it needs a long time to reach equilibrium. 3 Although nonspecific adsorption of drugs to the dialysis membrane has been described, this can be solved by measuring the concentration in both the buffer and plasma cham- ber. 4 The alternative method, UF, is fast and easy to perform but has the disadvantage of potential adsorption to the UF filter. Furthermore, in both methods, variables like pH 5 and tempera- ture 5,6 may influence protein binding. 4 Unfortunately, in practice, temperatures for preincubation, handling, and centrifugation of Received for publication February 7, 2014; accepted July 17, 2014. From the *Department of Laboratory Medicine, Ghent University Hospital; †Department of Clinical Chemistry, Microbiology and Immunology, Ghent University; and ‡Department of Critical Care Medicine, Ghent University Hospital, Belgium. Supported by the Research Foundation Flanders (M.C.). The authors declare no conflict of interest. Correspondence: Veronique Stove, PharmD, PhD, Department of Laboratory Medicine, Ghent University Hospital, De Pintelaan 185 Building 2P8, Ghent 9000, Belgium (e-mail: veronique.stove@UGent.be). Copyright © 2014 Wolters Kluwer Health, Inc. All rights reserved. 180 Ther Drug Monit  Volume 37, Number 2, April 2015 Copyright © 201 Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited. 5