ARS | Publication request: Reversed phase and aqueous normal phase retention in multiclass LC-MS analysis of antibiotics

Submitted to: American Laboratory
Publication Type: Trade Journal
Publication Acceptance Date: April 1, 2008
Publication Date: August 1, 2008
Citation: Mastovska, K., Lightfield, A.R. 2008. Reversed phase and aqueous normal phase retention in multiclass LC-MS analysis of antibiotics. American Laboratory.

Technical Abstract: In creating LC-MS/MS methodology for the quantification and confirmation of veterinary drugs, the analytical chemist is challenged to include as many drug classes within a single analysis. For LC-MS/MS analysis of antibiotics, such as beta-lactams, sulfonamides, fluoroquinolones, tetracyclines, and macrolides, reversed phase chromatography with a suitable C18 column can be used to meet this objective. However, the inclusion of a widely used antibiotic class of aminoglycosides into this methodology presents a problem due to their high polarity, thus practically no retention on C18 columns unless an ion-pairing reagent is added to the mobile phase. In our study, we employed a hydride-based silica bonded column (Cogent Bidentate C18) that can provide reversed phase, normal phase or aqueous normal phase retention mechanisms depending on the mobile phase composition and analyte properties. We optimized reversed phase and aqueous normal phase conditions for the retention of the 6 classes of antibiotics using one mobile phase system (A: 0.1% formic acid in water and B: 0.1% formic acid in acetonitrile). Unfortunately, tetracyclines produced very broad, unfocused peaks; probably due to their strong interactions with the stationary phase. Therefore, we decided to design a system employing switching valves and 2 analytical columns (a Cogent Bidentate C18 and a traditional C18 column) that enabled alternate analyses in aqueous normal and reversed phase modes using a single LC-MS/MS and mobile phase system for a time- and cost-efficient screening of the 6 most important antibiotics classes.