Ultrasensitive HPLC determination of tetracycline antibiotics and their 4‐epimer derivatives based on dual effect of methanesulfonic acid

In reversed-phase liquid chromatography, tetracycline antibiotics yield broad and asymmetrical peaks, as a result of their ionic interaction with the anionic free silanol groups and metal ion present in the silica-based stationary phases (commonly derivatized with C18 groups). These frequently encountered difficulties were absolutely eliminated based on dual effect when methanesulfonic acid was employed as an eluent additive. The study revealed that the performance of methanesulfonic acid to minimize the "silanol effect" is mainly explained by both direct neutralization of the anionic silanol sites and electrostatic attraction with analytes. Based on these dual action mechanisms, an ultrasensitive method has been successfully developed for the simultaneous determination of tetracycline antibiotics and their derivatives (minocycline, oxytetracycline, tetracycline, chlortetracycline, metacycline, doxycycline, 4-epitetracycline, and 4-epichlortetracycline) in bovine milk with convenient ultraviolet detection within 15 min. Under the optimal conditions, the calibration curves showed good linearity (r(2) > 0.999) for all analytes in the range of 1 approximately 200 ng/mL with the instrument limits of detection as low as 0.3 ng/mL. The study sheds new light on suitable additives to analyze basic compounds with the advantage of good compatibility with MS detection.