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

Authors: Perez Jr, Johnny; Chen, Chinyi

Submitted to: Rapid Communications in Mass Spectrometry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/11/2018
Publication Date: 9/4/2018
Citation: Perez Jr, J.J., Chen, C. 2018. Rapid detection and quantification of aminoglycoside phosphorylation products using direct infusion high resolution and ultra-high performance liquid chromatography-mass spectrometry. Rapid Communications in Mass Spectrometry. 32:1822-1828.

Interpretive Summary: Traditional microbiology antimicrobial determination techniques have been established for years, but require considerable time and resources to confirm resistance. Advances in mass spectrometry has made this technology capable of determining resistance in incredibly short periods of time (< 1 hour). Liquid chromatography, which separates molecules based on their interaction with a nonpolar (waxy) surface followed with “weighing” molecules in a mass spectrometer, was used to determine whether a bacterial sample was positive/negative for antibiotic resistance. In this paper, we use that unique ability to determine the most common form of resistance for the aminoglycoside family of antibiotics, the phosphorylation enzyme in several hours in comparison to conventional antibiotic susceptibility testing protocols, which required several days. This analytical technique can provide rapid antibiotic resistance determination presumably for all microorganisms expressing some form of the phosphotransferase enzyme.

Technical Abstract: 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, 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). Detection of phosphorylated kanamycin was achieved in less than four hours of incubation. Calibration curves for modified kanamycin from 2.5 – 250 and 10 – 200 µg mL-1 µg mL-1 were obtained for LC-MS and direct injection high resolution experiments, respectively. The high resolution measurements was 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.