Zorbax eclipse plus rp uhplc column

All steps of solution and sample preparation, LC/MRM-MS parameters, are described in the article [3 (link)]. LC/MRM-MS analysis was performed with a Zorbax Eclipse Plus RP-UHPLC column on a 1290 Infinity UPLC system (all from Agilent Technologies) that was interfaced to a triple quadrupole mass spectrometer (Agilent 6490) via Agilent’s Jet Stream™ source, operated in the positive-ion ESI mode. Plasma tryptic digests were analyzed only once. The MRM data was visualized and examined with MassHunter Quantitative Analysis software (version B.07.00; Agilent). Quantitation was done using linear regression analysis, as described previously [8 (link)].

For details of the solution and sample preparation, the reader is referred to the Supplemental Information section. Fifteen-µL injections of the plasma tryptic digests were separated with a Zorbax Eclipse Plus RP-UHPLC column (2.1 × 150 mm, 1.8 µm particle diameter; on a 1290 Infinity UPLC system (all from Agilent Technologies). Peptide separations were achieved at a flow rate of 0.4 mL∕min over a 43 min run, using a multi-step LC gradient (3–90% mobile phase B; eluent compositions: A was 0.1% FA in 100% H₂O while B was 0.1% FA in 90% ACN), as described previously^{29 (link)}. The column and autosampler were maintained at 50 °C and 4 °C, respectively. A post-gradient equilibration time of 4 min was used after the analysis of each sample. All samples were processed individually.
The LC system was interfaced to a triple quadrupole mass spectrometer (Agilent 6490) via Agilent’s Jet Stream™ source, operated in the positive-ion ESI mode. The MRM acquisition parameters employed were identical to those reported previously^{29 (link)}. Note that the peptide parameters had previously been empirically optimized by direct infusion of the purified SIS peptides. In the quantitative analysis, the targets (852 total transitions for 142 peptides with 3 transitions/peptide form) were monitored during 800-ms cycles and 1-min detection windows.