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Determination of enalapril and enalaprilat in small human serum quantities for pediatric trials by HPLC–tandem mass spectrometry


Stephanie Läer, Department of Clinical Pharmacy and Pharmacotherapy, Heinrich-Heine-Universität Düsseldorf, Building 26.22.02, Room 22–24, Universitätsstrasse 1, 40225 Düsseldorf, Germany. E-mail:


The angiotensin converting enzyme-inhibitor enalapril is the prodrug of enalaprilat and used in the treatment pediatric hypertension and chronic heart failure. Pharmacokinetic data are lacking to provide adequate dosing and for pediatric pharmacotherapeutical trials it is imperative to minimize sample volume. Therefore an HPLC-tandem mass spectrometry (MS) method for the determination of enalapril and enalaprilat in 100 μL of human serum was developed and validated with benazepril as internal standard (IS). After solid-phase extraction, chromatography was performed on a Luna® RP-C18(2) column with methanol–water–formic acid (65:35:1, v/v/v) as mobile phase and a flow rate of 0.4 mL/min. The MS was set to positive-mode electrospray ionization and multiple reaction monitoring, analyzing the m/z transitions channels 377.3 → 234.2, 349.3 → 206.1 and 425.3 → 351.2 for enalapril, enalaprilat and IS. Calibration curves were linear in the range of 1.61–206 ng/mL (enalapril) and 1.84–236 ng/mL (enalaprilat) with coefficients of determination >0.99. Relative standard deviations of intra- and inter-run precisions were below 7% and relative errors were below 6 ± 7% for both analytes. Also stabilities were acceptable for both analytes. As an application example, concentrations of enalapril and enalaprilat in serum after oral administration of 20 mg enalapril maleat in a healthy volunteer were determined. Copyright © 2011 John Wiley & Sons, Ltd.