Abstract
Aim
Antibiotic concentration at the infected site is a relevant information to gain knowledge about deep-seated infections. The combination of antibiotic therapy and debridement is often indicated to treat these infections. At University Hospital Basel the most frequently administered antibiotic before debridement is amoxicillin in combination with clavulanic acid. Amoxicillin is a fragile beta-lactam antibiotic that brings multiple challenges for its quantification.
As for many sample materials only little material is available, the aim of this work was to establish a sensitive and reliable quantification method for amoxicillin that only requires a small sample mass. We did not quantify clavulanic acid as we focused on the drug with antibiotic action.
Method
Usually discarded sample material during debridement was collected and directly frozen. The thawed tissues were prepared using simple protein precipitation and manual homogenization with micro pestles followed by a matrix cleanup with online solid-phase extraction. Separation was performed by HPLC followed by heated electrospray ionization and tandem mass spectrometry.
Results
During method development, amoxicillin showed partial formation of a covalent methanol adduct when performing protein precipitation. Furthermore, multiple in-source products of amoxicillin during ionization could be observed. Adding an aqueous buffer to the samples before protein precipitation and summing up the signals of amoxicillin and its in-source acetonitrile-sodium-adduct led to successful method validation for a calibration range of 1–51 mg/kg using 10 mg of each tissue sample. The imprecision was < 8% over the entire concentration range and the bias was ≤ 10 %. The quantitative matrix effect was < 6 % in six different tissue samples.
Until now we measured amoxicillin in samples from nine patients with prosthetic joint infection, bursitis, or an abscess who obtained amoxicillin between 5 hours and 15 minutes before sampling and found concentrations between 1.4 and 35 mg/kg.
Conclusions
With this method, we developed a fast, simple, and sensitive quantification assay for amoxicillin in tissue samples with little material that can now be applied to different study samples.
