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Elution was assessed by acidified methanol for MAX sorbent material and ammonium in methanol for MCX sorbent material. The kind of acid (or base), its concentration, and the corresponding elution volumes were investigated to obtain all analytes within one fraction E-64 by reducing the coeluted residual matrix to a minimum. To meet current demands in sample throughput within a clinical study, the scale-up to offline positive pressure extraction was conducted. The switch to 96-well format came along with a changeover from vacuum extraction to positive pressure extraction. SPE-formats with higher amounts of SPE cavities per plate were commercially not available. For the critical steps of sample load, washing, and sample elution, the positive pressure was kept between 1 and 2?psi to warrant an intensive interaction between analyte and sorbent material as well as reproducible flow rate. Figure 1 illustrates the corresponding scale-up. Figure 1 Scale-up process and optimization steps to establish a high-throughput approach of all bioanalytical assays utilizing solid-phase extraction. On the left side the development approach of sample preparation and purification is illustrated (a). The solid-phase R428 cost ... 2.4. Chromatographic and Mass Spectrometric Conditions The utilized modular HPLC system (Shimadzu Deutschland GmbH, Duisburg, Germany) consisted of a controller SCL-10Avp, two separate pumps LC-10ADvp, three-channel online degasser DGU-20A3 prominence, autosampler SIL-10ADvp, and a column oven (L-2300, VWR/Hitachi). For the separation of enalapril and enalaprilat an XBridge BEH C18 3.5?��m column (3.0?mm �� 150?mm) was used. After injection of 10?��L sample solution (methanol/water 40?:?60, v/v) the samples were separated under gradient conditions within 6-minute run time utilizing a methanol/water mixture (40?:?60, v/v) buffered with formic acid (1%, v/v) and ammonium formate (2?mM). The applied gradient started with 40% of methanol and increased stepwise after 0.5 minute to 60% and after 1 minute to 80%. Between 1 and 4 minutes the amount of methanol was continuously JQ1 increased to 95% and reduced at 4.5 minutes to 40% of methanol again. It stayed at this level till the end of the run time. The flow rate was 0.4?mL/min and the column temperature was maintained at 50��C which resulted in a moderate back pressure of 125 bar. Triple-quadrupole tandem mass spectrometric detection was performed on an Applied Biosystems SCIEX API 2000 (Applied Biosystems/MDS SCIEX, Concord, Canada) with an electrospray ionization (ESI) interface running in positive ionization mode. The device screened the transitions channels 377.2 to 234.2 m/z (enalapril), 349.1 to 206.1 m/z (enalaprilat), and 425.3 to 351.2?m/z (benazepril) in multiple reaction monitoring (MRM) mode. All dwell times were set to 250?ms. 2.5.