A sensitive reverse-phase ultra performance liquid chromatography-tandem mass spectrometry method for the determination of tobramycin in Pseudomonas aeruginosa cell lysate

Abstract

Measurement of antibiotic accumulation in bacteria is essential for full understanding of the mechanisms of antimicrobial resistance but requires a highly sensitive analytical assay. A suitable ultra performance reverse phase liquid chromatographic tandem mass spectrometry (UPLC-MS/MS) method was developed in the electrospray negative ionisation mode for quantifying tobramycin in Pseudomonas aeruginosa cell extracts. P. aeruginosa cell lysate extracts were prepared and spiked with tobramycin and kanamycin (internal standard). Following a protein precipitation extraction procedure, the sample was applied to a reverse phase C18 column, equilibrated in 10 mM ammonium hydroxide at pH 11. Tobramycin and kanamycin were eluted using an acetonitrile gradient and detected in the electrospray negative ionisation mode. The retention times for kanamycin and tobramycin were 1.9 and 2.5 min, respectively and total run time of 10 min. The assay demonstrated linearity in the range of 0.02 – 1 µg mL⁻¹ (R = 0.9999), with limits of detection and quantitation at 1.42 ng mL⁻¹ and 10 ng mL⁻¹, respectively. The precision, expressed as the coefficient of variation, ranged from 1.4 % to 6.5 %, and the accuracy, expressed as bias, ranged from 0.4 % to 17.1 % (ICH, 1996). Using a simple protein precipitation method, the recoveries (%) of tobramycin from cell lysate supernatant for quality controls were 99.7–105.2 % and this result shows that the assay is quantitative. This validated analytical protocol will facilitate future studies aimed at determining the cellular uptake kinetics of tobramycin by P. aeruginosa under various controlled conditions and it could be used for other applications.