6495b mass spectrometer

The SIS peptide mixture was solubilized in 220 μl of 30% ACN/0.1% FA, transferred to a 15-mL Falcon tube, and then diluted to 10× LLOQ per μL with 0.1% FA. A 45-μL aliquot of plasma digest was transferred into a well of an Eppendorf LoBind skirted PCR plate and spiked with 45 μl of the SIS peptide mixture. For each standard curve point and each QC sample, 55 μl of BSA surrogate matrix digest (143 μg/ml) was spiked with 55 μl of the SIS peptide mixture, as well as 55 μl of a level-specific light peptide mixture at a ratio of 1:1:1 (v/v/v). Plasma samples were then concentrated by solid-phase extraction (SPE) using an Oasis HLB μElution plate. Briefly, the SPE plate was conditioned with 600 μl of MeOH, equilibrated with 600 μl of 0.1% aqueous FA, followed by sample loading. The wells were washed three times with 600 μl of H₂O, and the bound peptides were eluted with 55 μl of 70% ACN/0.1% FA. After the SPE step, the eluates were evaporated using a speed vacuum concentrator and were then stored at −80 °C. Plasma samples, standards, and QC samples were then resolubilized and analyzed on an Agilent 6495B mass spectrometer.