To develop the method for UHPLC-HRMS to determine the antibacterial potential of a central American medicinal plant

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The development of antibiotic resistance by microbials has long been acknowledged. The major challenge worldwide is to develop novel, natural, and potent antibiotics against the multidrug resistant bacteria. In this study, our aim was to develop the method for a highly sensitive instrument, ultra-high performance liquid chromatograph-high resolution mass spectrometer (UHPLC-HRMS), to evaluate the antibacterial property of a natural product. Aechmea magdalenae (Andre) Andre ex Baker, a plant belonging to the family Bromeliaceae, a native of Central America was used in this study. Based on the available literature, it was hypothesized that Aechmea magdalenae has antibacterial activity. In addition, the profiling done on A. magdalenae using gas chromatography-mass spectrometry (GC-MS) also revealed the presence of medicinally important chemical compounds, such as acetic acid. Minimum inhibitory concentration (MIC) of dried Aechmea plant extract was determined for the first time using 96-well plate assay, followed by determination of antibacterial potential using LC-MS. The reason being that other dried methanolic plant extracts, such as Vismia macrophylla, lined up for antibacterial testing have dark extracts, for which determining the antibacterial potential and reading the results with the naked eye would be challenging. To overcome the situation of dark plant extracts, a generalized novel LC-MS method was developed that was used for the plant A. magdalenae, and would be used further for other plants. A blue indicator called resazurin was added to the wells; resazurin, upon incubation with the living cells, got reduced to resorufin (which was pink), while it remained blue with bacterial growth inhibition. The mass difference created due to reduction of resazurin to resorufin was detected by using LTQ Orbitrap Discovery in positive ion mode to determine the antibacterial activity of the plant extract. The sample preparation for LC-MS assay included centrifugation of the samples taken from 96-well plate, followed by filtration of the supernatant, before exposing them to C-18 column. The results obtained from full scan LC-MS spectrum consistently demonstrated the presence of resorufin from wells with bacterial growth, and resazurin from wells with inhibition through peaks of relevant masses.

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