Measuring Unbound Versus Total Vancomycin Concentrations in Serum and Plasma: Methodological Issues and Relevance

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 4 degrees C and 37 degrees C) or equilibrium dialysis (ED, using a Fast MicroEquilibrium Dialyzer at 37 degrees C) 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 4 degrees C and 378C resulted in a median f(u) of 51.6% (IQR, 48.6%-54.8%) and 75.2% (IQR, 69.3%-78.6%), respectively, with no significant difference between unbound vancomycin concentrations after ED and UF at 378C (P = 0.13). Unbound concentrations obtained through ED and UF correlated linearly (4 degrees C: 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 +/- SD, 5.0% +/- 7.6%). The studied protein concentrations were of no added value in predicting the unbound concentration. Conclusions: Vancomycin f(u) after UF at 4 degrees C was on average 30.6% lower than that after UF at 37 degrees C, demonstrating the importance of temperature during UF. Ultrafiltration at 37 degrees C 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 vancomycin concentrations has little added value over measurements of total vancomycin concentrations.