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Routine therapeutic drug monitoring (TDM) reports only total vancomycin (VAN) concentrations, although protein binding varies and it is generally accepted that only free VAN is active. The aims of this study were to examine the correlation between free and total VAN concentrations in order to estimate whether free VAN levels can be predicted based on its total concentration. A high-performance liquid chromatography (HPLC) method was set up and validated (against routine laboratory immunoassays) for measurement of free [ultrafiltration (Centrifree(R)); cut-off 30 kDa] and total [solid-phase extraction (Oasis(R) MCX cartridge)] VAN in serum. Samples (n = 65) from patients (n = 15) treated by continuous infusion were analysed. There was a wide variation in free to total VAN ratios [range 12-100%; mean 63.6+/-25.8%, with 59 values falling outside the 95% confidence interval (57.3-69.9%); median 70.2%]. The correlation between free and total VAN was poor (R-2 = 0.55). Artefacts such as pH variation of sera could be excluded. Both intrapatient and interpatient variabilities were large and no correlation could be made with patients’ clinical conditions. Total VAN concentration is not predictive of free VAN concentration, suggesting that actual determination of free VAN might be recommended as an improved method of TDM. (C) 2009 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.