Free serum concentrations of antibiotics determined by ultrafiltration: extensive evaluation of experimental variables
Creators: name: Lier, Constantin
name: Dejaco, Alexander
name: Kratzer, Alexander
name: Kees, Martin G
name: Kees, Frieder
name: Dorn, Christoph
Publisher: name: Taylor & Francis
Year: 2024
Subjects: subject: Biophysics
subject: Biochemistry
subject: Cell Biology
subject: Physiology
subject: FOS: Biological sciences
subject: Biotechnology
subject: Environmental Sciences not elsewhere classified
subject: Chemical Sciences not elsewhere classified
subject: Cancer

description: <b>Aim:</b> To assess the impact of experimental conditions on free serum concentrations as determined by ultrafiltration and HPLC-DAD analysis in a wide range of antibiotics. <b>Materials &amp; methods:</b> Relative centrifugation force (RCF), temperature, pH and buffer were varied and the results compared with the standard protocol (phosphate buffer pH 7.4, 37°C, 1000 × g). <b>Results:</b> Generally, at 10,000 × g the unbound fraction (<i>f</i><sub>u</sub>) decreased with increasing molecular weight, and was lower at 22°C. In unbuffered serum, the <i>f</i><sub>u</sub> of flucloxacillin or valproic acid was increased, that of basic or amphoteric drugs considerably decreased. Comparable results were obtained using phosphate or HEPES buffer except for drugs which form metal chelate complexes. <b>Conclusion:</b> Maintaining a physiological pH is more important than strictly maintaining body temperature. The determination of free serum concentrations of antibiotics is of particular importance, as all PK/PD indices should be referenced to the free concentrations. While ultrafiltration is the most popular approach to determine the free concentrations in the clinical setting, there exists no standard protocol. The proposed standard protocol includes buffering of serum to the physiological pH 7.4 and subsequent centrifugation at body temperature and a relative centrifugal force (RCF) of 1000 × g. The influence of RCF, temperature, pH control and buffer type were evaluated for 26 antibiotics, 2 antimycotics, as well as for paracetamol and valproic acid. The protein concentration in ultrafiltrate was measured in 525 (65%) samples using the Bradford assay. The protein concentration was below 0.1% of the normal serum protein concentration in 94.7% of the samples, and still below 1% in the remaining samples. None of the investigated 30 drugs adsorbed significantly to the Vivafree™ Hydrosart<sup>®</sup> regenerated cellulose membrane. At room temperature, the unbound fraction (<i>f</i><sub>u</sub>) was unchanged or lower than at 37°C except for clindamycin which showed a higher <i>f</i><sub>u</sub>. Ultrafiltration at 10000 × g resulted in a molecular weight-dependent decrease of <i>f</i><sub>u</sub>, with a maximum relative decrease of 24% for vancomycin. The increase of pH in unbuffered serum resulted in increased <i>f</i><sub>u</sub> of acidic drugs such as flucloxacillin or valproic acid, and in decreased <i>f</i><sub>u</sub> of basic or amphoteric drugs (clindamycin, fluoroquinolones, tigecycline). Buffering with HEPES proved to be equivalent to phosphate and preferable for drugs which form stable chelate complexes. Maintaining a physiological pH is more important than strictly maintaining body temperature.