Development and validation of a liquid chromatography-tandem mass spectrometry method for the determination of ε-acetamidocaproic acid in rat plasma

A simple and rapid liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the quantification of e-acetamidocaproic acid (AACA), the primary metabolite of zinc acexamate (ZAC), in rat plasma by using normetanephrine as an internal standard. Sample preparation involved protein precipitation using methanol. Separation was achieved on a Gemini-NX C₁₈ column (150 mm × 2.0 mm, i.d., 3 μm particle size) using a mixture of 0.1% formic acid-water: acetonitrile (80: 20, v/v) as the mobile phase at a flow rate of 200 μl/min. Quantification was performed on a triple quadrupole mass spectrometer employing electrospray ionization and operating in multiple reaction monitoring (MRM) and positive ion mode. The total chromatographic run time was 4.0 min, and the calibration curves of AACA were linear over the concentration range of 20̃5000 ng/ml in rat plasma. The coefficient of variation and relative error at four QC levels were ranged from 1.0% to 5.8% and from -8.4% to 6.6%, respectively. The present method was successfully applied for estimating the pharmacokinetic parameters of AACA following intravenous or oral administration of ZAC to rats.