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In particular for high-throughput analytics��as it is useful in clinical study approaches��the LLE is not the first choice. Consequently, SPE was selected owing to its superior purification properties and flexibilities in extraction protocols to cope with diversity of analytes, purifications solvents, and biological fluids. The selected drug combination (calculated log?P values of enalapril: 2.5; enalaprilat: ?0.9; benazepril: 3.5) carried the risk of improper binding to the sorbent due to Coulomb repulsion, losing hydrophilic analytes during too extensive and not well-balanced washing steps or by incomplete recovery of the more lipophilic compounds from the sorbent material during elution. All this may be attributed to low recovery or bad reproducibility which in turn narrows the calibration range, because especially lower concentrations might Selleckchem JQ1 not conform to international guideline requirements. Available sorbent phases characterized by different interaction possibilities (van der Waals forces, ionic interaction, etc.) and different amounts of sorbent per cavity and the high flexibility in the SPE protocol on how intensive the purification of sample needs to be conducted represent some of many useful selleck inhibitor tools to overcome those drawbacks. First extraction attempts were undertaken by utilizing Oasis MCX. This polymeric material is characterized by a strong cation exchanger (on sulfonic acid base) binding the carboxylic acids groups of the analytes of interest. However, purified samples showed a split peak for the selected transition of enalaprilat if determined by HPLC-MS/MS. The corresponding chromatograms revealed a peak occurring at an earlier retention time plus a second peak at the expected retention time of the compound (Figure 3). Figure 3 Determined split peak in serum with the transition E-64 349.1 �� 206.1 m/z during method development. The split peak was measured on several HPLC columns after SPE purification by Oasis MCX. In grey, the ion count of a low enalaprilat concentration ... The peak area and intensity of the first peak did not alter with different enalaprilat concentrations per sample and accounted therefore most likely for a residual serum matrix component. It was not possible to erase the peak neither by thought-out SPE nor by any number of different LC gradients or by different HPLC columns featured with opposed chromatographic properties (Atlantis T3, XBridge, and XSelect). The checks for contamination in mobile phase, autosampler, or solutions for SPE were additionally negative. At the lowest concentration level of the calibration curve, the first peak and the one of enalaprilat were comparable in shape, intensity, and peak area. This phenomenon carried the risk of less robustness if automated integration of the chromatogram is preferred. After also scanning for the second most intense transition of enalaprilat (349.1 �� 303.1 m/z), clarity was brought to question as the first peak did not belong to enalaprilat.