On-line Coupling of Three-Dimensional Liq- uid Chromatography Based Sample Prepar- ation with Reversed-Phase Liquid Chromato- graphy for the Determination of Enkephalin Derivatives as Model Compounds in Biofluids

Abstract

In bioanalysis sample preparation frequently is the most time consuming and laborious step. As a result, automated and/or on-line sample preparation techniques are preferred. Here, an automated 3D-sample preparation procedure is described for the determination of enkephalin derivatives in spiked human plasma. The sample preparation procedure consists of an initial treatment based on the exclusion principle. The required selectivity is obtained in a subsequent step by combining a strong cation-exchanger with a reversed-phase column. Detection is performed by using UV absorbance at 200 nm or by using electrospray-ionization mass spectrometry. Human plasma samples, which are only five-fold diluted and acidified to 5% v/v acetic acid, can be injected into the system without any problems. As a result, off-line manual labor-intensive sample preparation steps, which generally form the bottleneck in bioanalysis, are reduced to a less than a one-minute liquid-handling process. The developed 3D-sample preparation RP–LC procedure shows good repeatability in the retention time (<0.5% RSD) and peak area response (<1.3% RSD) for the determination of enkephalin derivatives in spiked human plasma samples. Limits of detection of the procedure were 200 nmol·L−1 using UV detection and 10 nmol·L−1 using mass spectrometric detection.