Evaluation of an automated SPE method for samples derived from capillary microsampling

Experimental Samples were processed by dilution of spiked plasma with water containing the internal standard followed by online sample-preparation described below. Working-solutions and sample preparation: The analyte (MW 581) was dissolved in DMSO and diluted in ACN/ DMSO (50:50, v/v) to obtain working-solutions at concentrations twenty times higher than those of the final CAL and QC samples. Aliquots of the working-solutions were spiked into blank rat plasma and diluted with water containing the internal standard in a ratio of 1:10 (v/v). The samples were vortexed, centrifuged and transferred to the ITSP sample-preparation plate. Online-sample-preparation: ITSP (ITSP Solutions Inc.) cartridges were conditioned with 80µL MeOH and 80µL 0.2% aqueous formic acid before loading 10µL sample. After drying the cartridge with 80µL air, the ITSP cartridges were washed twice with 80µl 0.2% aqueous formic acid and dried again. The compounds were eluted using 80µL EtOH and 10µL of the eluate was injected onto the analytical column. Sample-preparation of each plasma sample was performed directly before the injection (see Figure 1 for the workflow and the results of the optimization process). Chromatography: 10 µL of the extracted samples were injected onto a Thermo Hypersil Gold (2.1 x 100 mm, 3 µm) analytical column. Eluent A consisted of 0.5% aqueous formic acid, eluent B of 0.5% formic acid in ACN and LC flow rate was 0.5 mL/min. The eluent composition was changed linearly from 70% to 5% A in 2.25 minutes. Eluent composition was kept at 5% B for one minute and then went back ballistically to starting conditions. Calibration range: 50.0 - 25’000 ng/mL (high-range, HR) and 5.00 - 2’500 ng/mL (low-range, LR). Instrumentation and MS-method: TSQ Quantum Ultra (Thermo Scientific), Rheos 2200 quarternary pump (Flux Instruments), HTX PAL autosampler (CTC Analytics). Data-Processing: Raw data were acquired with Xcalibur 2.0 software and processed with LCQuan 2.5 (Thermo Scientific). Validation parameters: Intra- and inter-assay accuracy and precision, linearity, and carry-over. Table 1. MS-parameter (SRM-assay). www.swissbioanalytics.com