2.5 Chromatography and Mass Spectrometry
Chromatography and mass spectrometry instrumentation were the same as described previously in “Assessment of Renal Injury”. 2 µL of sample was injected from each vial. Sample injection order was randomized, and the quality control pooled sample was injected after every 6 sample injections throughout the run. Chromatographic separation was achieved using a Waters ACQUITY UPLC HSS T3 column (1.8 µm particle size, 2.1 mm x 100 mm) maintained at 45°C. The mobile phase consisted of A) water + 0.1% formic acid and B) ACN + 0.1% formic acid. Mobile phase flow rate was set to 0.45 mL min-1 and the following mobile phase conditions were used: 0–2 mins, 1–60% B; 2–6 mins 60–85% B; 6–8 mins 85–99% B; 8–10 mins 99–1% B. Features were measured in both positive and negative electrospray ionization modes with the following mass spectrometer parameters: capillary voltage, 2kV; cone voltage, 40V; source temperature, 150°C; desolvation gas flow and temperature, 1200L h-1 and 600°C; cone gas flow, 50L h-1. Data was acquired in centroid, using the MSE method in resolution mode. The MSE method generates both the precursor ion (function 1) and fragment ions (function 2) in one acquisition. The acquisition period was 11 minutes, with a 0.05 second scan time and a mass range of 50 – 1200 Da. Collision energy was set to 0 V for function 1 and was ramped from 15 – 50 V for function 2. Leucine-enkephalin (0.9 μM) was used as lockspray solution to ensure mass accuracy. The lockspray solution was infused at a flow rate of 10 μL mL-1. Lockmass was acquired at intervals of 10 seconds and averaged over 3 scans.