Liquid chromatography-tandem mass spectrometry for the simultaneous quantitation of enmetazobactam and cefepime in human plasma.

A simple ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed and validated for the simultaneous analysis enmetazobactam (also known as AAI101) and cefepime in human plasma. Sample preparation was based on protein precipitation with acetonitrile. Separation was performed on Acquity BEH HILIC column (50 mm × 2.1 mm, 1.7 μm) with a mobile phase containing ammonium formate in water and acetonitrile. The analytes were analyzed with the corresponding isotopically labeled internal standards and were detected in multiple reactions monitoring (MRM) using API 5000 triple-quadrupole mass spectrometer with electrospray (ESI) source operating in positive ion mode. The calibration curves were linear over the selected ranges (r > 0.9970 for both analytes). The intra and inter-assay precision of the Quality Control samples showed CV ≤ 15% and the accuracy was within 85 and 115% in all cases for both compounds. The lower limit of quantification was 0.05 μg/mL for enmetazobactam and 0.5 μg/mL for cefepime.