Free serum concentrations of antibiotics determined by ultrafiltration: extensive evaluation of experimental variables

Abstract

Aim: 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. Materials & methods: 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). Results: Generally, at 10,000 × g the unbound fraction (f_u) decreased with increasing molecular weight, and was lower at 22°C. In unbuffered serum, the f_u 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. Conclusion: Maintaining a physiological pH is more important than strictly maintaining body temperature.

Article highlights

Background

• 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.

Protein leakage & non-specific binding

• 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^® regenerated cellulose membrane.

Impact of temperature or relative centrifugal force

• At room temperature, the unbound fraction (f_u) was unchanged or lower than at 37°C except for clindamycin which showed a higher f_u.

• Ultrafiltration at 10000 × g resulted in a molecular weight-dependent decrease of f_u, with a maximum relative decrease of 24% for vancomycin.

Impact of pH control & buffer

• The increase of pH in unbuffered serum resulted in increased f_u of acidic drugs such as flucloxacillin or valproic acid, and in decreased f_u 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.

Conclusion

• Maintaining a physiological pH is more important than strictly maintaining body temperature.

Keywords: :

• anti-infectives
• azole antimycotics
• beta-lactams
• HPLC-UV
• plasma protein binding
• therapeutic drug monitoring
• unbound fraction
• valproic acid

Supplemental material

Supplemental data for this article can be accessed at https://doi.org/10.1080/17576180.2024.2365526

Author contributions

C Lier: conception and design of the work; acquisition. analysis and interpretation of data for the work; drafting the work. A Dejaco: interpretation of data for the work; revising the work critically for important intellectual content. A Kratzer: design of the work, interpretation of data for the work; revising the work critically for important intellectual content. MG Kees: interpretation of data for the work; revising the work critically for important intellectual content. F Kees: conception and design of the work, acquisition. analysis and interpretation of data for the work; drafting the work. C Dorn: conception and design of the work, acquisition. analysis and interpretation of data for the work; drafting the work.

Financial disclosure

The authors have no financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Competing interests disclosure

The authors have no competing interests or relevant affiliations with any organization or entity with an interest in or conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending or royalties.

Writing disclosure

No writing assistance was utilized in the production of this manuscript.