Development of analytical methods for some penicillins in bovine milk by ion-paired chromatography and confirmation by thermospray mass spectrometry

Analytical methods for the determination of cloxacillin, ampicillin/hetacillin, and amoxicillin in bovine milk were developed. The methods involved ultrafiltration of milk diluted with methanol, acetonitrile, and water on a 10,000-dalton cut-off filter. Separation of penicillins from other milk components was accomplished by ion-paired chromatography using a microbore column. The penicillins were detected using ultraviolet photodiode array (UV-PDA) detection and confirmed by thermospray liquid chromatography-mass spectrometry (LC-MS). The thermospray spectra of these compounds exhibited M + H]+ and M + Na]+ ions along with several fragment ions. The limits of detection for these antibiotics were estimated to be 50 to 100 ppb for LC with UV-PDA detection and 100-200 ppb for thermospray LC-MS detection.