Azithromycin extraction from municipal wastewater and quantitation using liquid chromatography/mass spectrometry

Azithromycin is a broad spectrum antimicrobial agent that is approved in the United States for use in humans. Due to azithromycin's low rate of metabolism it is likely to be found in wastewater treatment plants, where its broad spectrum of antimicrobial activity could lead to development of resistance in bacteria. A liquid-liquid extraction using K2CO3 and methyl-t-butyl ether (MTBE) was used to extract azithromycin from 10 ml aliquots of wastewater. Liquid chromatography was performed using a Luna C18(2) (30 mm × 2.0 mm) column with a mass spectrometer friendly mobile phase containing 50:24:2:24 acetonitrile, methanol, tetrahydrofuran, and 0.04 M ammonium hydroxide. The mass spectrometer used an electrospray source with positive ionization and an ion trap detector. A linear standard curve from 5 to 200 pg/ml was validated and used to quantitate azithromycin in wastewater.