Determination of penicillin G, ampicillin, amoxicillin, cloxacillin and cephapirin by high-performance liquid chromatography—electrospray mass spectrometry

Abstract

This report contributes to a preliminary investigation of high-performance liquid chromatographic (HPLC)—mass spectrometric (MS) methods for confirming β-lactam antibiotic residues in bovine milk. Initial work for each antibiotic evaluated the collisional activated dissociation (CAD) spectra that could be generated between the capillary and skimmer in the electrospray (ESP) interface. The drugs show various characteristic fragmentation, mostly within the β-lactam ring and the amide group. Response for a particular compound in a given solvent can vary drastically. Usually, the more organic component in the solvent, the higher the ESP response. In many cases use of acetonitrile also results in slightly better ion currents than for methanol when comparing equal percentages of either organic solvent in water. The ESP response of most of the tested antibiotics can be enhanced by the addition of formic acid or acetic acid to the mobile phase methanol—water (1:1). In general, the negative ion spectra are lower in intensity, exhibiting an [M  H] − ion and producing less fragmentation at higher CAD voltages as compared to positive ion spectra. An isocratic reversed-phase HPLC method for the separation of a mixture of five common β-lactam antibiotics was developed using acetic acid as a mobile phase additive and optimized for detection with a new ESP HPLC-MS interface. A post-column split ratio of 70:1 for the eluent from a 150 × 2 mm I.D. column was chosen to provide the required lower flow-rate (approximately 4 μ1/min). The limit of detection for the simultaneous determination of these antibiotics was estimated to be 100 ppb. Electrospray HPLC-MS could be used to confirm these antibiotics for quantities down to about 100 pg entering the mass spectrometer. Multiresidue analysis with microbore HPLC-ESP-MS has the advantage that no post-column splitting of the eluent is required and all of the analyte (on-column injected) will be transferred into the ESP interface. Preliminary work showed good mass spectrometric sensitivity down to the level of regulatory interest, but chromatographic separation efficiency must be improved.