Rapid detection and quantification of aminoglycoside phosphorylation products using direct-infusion high-resolution and ultra-high-performance liquid chromatography/mass spectrometry.

Rationale: Worldwide efforts are underway to determine the extent of antimicrobial resistance (AMR). In 2015, the World Health Organization (WHO) founded the Global Antimicrobial Surveillance System (GLASS) focusing on surveillance and dissemination of data. In addition, the WHO advocates method development focused on rapid determination and close to real-time monitoring of antibiotic usage and its effectiveness. Rapid determination of aminoglycoside modification by O-phosphorylation, the most prevalent mechanism of clinical resistance, was performed using direct flow and liquid chromatography/mass spectrometry (LC/MS).

Methods: A strain of Escherichia coli carrying a plasmid encoding an aminoglycoside modification enzyme (O-phosphotransferase) was incubated with kanamycin, an aminoglycoside. The antibiotic and its modified form were observed using direct flow and LC/MS. Direct flow high-resolution mass spectrometry (HRMS) using a Thermo Fisher Q-Exactive hybrid quadrupole-orbitrap mass spectrometer was employed for quantitative analysis and structural elucidation. Liquid chromatography coupled with the AB Sciex QTRAP 6500+ was also used for quantitative analysis.

Results: Detection of phosphorylated kanamycin was achieved in less than 4 h of incubation. Calibration curves for modified kanamycin from 2.5-250 and 10-200 μg mL  μg mL were obtained for LC/MS and direct injection high-resolution experiments, respectively. The high-resolution measurements were employed for conformation and structural elucidation of the novel precursor and product ion biomarkers with high mass accuracy (≤7 ppm). These results confirm previous in vitro O-phosphotransferase metabolite measurements.

Conclusions: A new analytical method capable of determination and quantification of the most common form of aminoglycoside resistance (via phosphorylation) was developed requiring short incubation times for a positive confirmation 100-fold lower than the minimum inhibitory concentration (MIC). High-resolution data simultaneously revealed quantitative abilities and provided numerous novel product ions confirming placement of the phosphate group on kanamycin.