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Benazepril hydrochloride (�� 98%, HPLC) and ethyl acetate (100% p.a.) were obtained from Sigma-Aldrich (Seelze, Germany). Methanol (HiPerSolv Epigenetics inhibitor Chromanorm HPLC grade), water (super gradient grade), and acetone (AnalaR Normapur) were purchased from VWR (Germany). Alternative supplier of methanol (HPLC grade) was Fisher Scientific (Loughborough, United Kingdom). Formic acid (98�C100% p.a.) was delivered by AppliChem (Gatersleben, Germany). Ammonium formate (99%, HPLC grade) was obtained from Fluka (Seelze, Germany). Blank human serum was provided by employees of the Institute of Clinical Pharmacy and Pharmacotherapy (D��sseldorf, Germany). Oasis 96-well plates (30 and 10?mg) and XBridge BEH C18 3.5?��m columns (3.0?mm �� 150?mm) were obtained from Waters (Eschborn, Germany). 2.2. Preparation of Standard and Quality Control Stock solutions of enalapril, enalaprilat, and benazepril (internal standard) were prepared at 0.10?mg/mL in methanol. These stock solutions were diluted with water to obtain working solutions with 10?��g/mL enalapril and enalaprilat as well as 166?ng/mL benazepril. For the calibration curve, blank serum was spiked with the analytes of interest and serially diluted. The final calibration range of the mass spectrometry was 0.2�C200?ng/mL enalapril and 0.18�C180?ng/mL enalaprilat. Quality control (QC) samples R428 concentration were independently prepared at four concentration levels over the whole calibration range (LLOQ, low, medium, and ULOQ). 2.3. Sample Preparation, Extraction, and Scale-Up Based on preliminary investigations on the degree of sample dilution prior to solid-phase extraction a dilution ratio of 1?:?23 using water led to a robust method with high recovery rates for all analytes of interest. Solid-phase extraction was chosen for the extraction process of the biological fluid owing to the superior purification performance if compared to protein precipitation or liquid-liquid extraction. Based on the compound properties of enalapril and enalaprilat, strong mixed-mode ion exchangers were chosen as sorbent material for the purification. Both a cation exchanger that interacts with the carboxylic acid groups and an anion exchanger that binds with the amino E-64 group of all above-mentioned substances were evaluated on their applicability (Oasis MCX and MAX material). The SPE protocol included a conditioning step utilizing methanol to enable optimal wetting of the cavernous sorbent material. For the subsequent equilibration step, different pH values and acids in aqueous solutions were tested to warrant for best interaction conditions prior the aqueous sample was loaded into the cavity. Namely, 2% formic acid (v/v), 4% phosphoric acid (v/v), 0.2?N hydrochloric acid, and pure water were evaluated.