Nexerax2 sil 30ac

RP-HPLC analysis was performed using a NexeraX2 SIL-30AC chromatograph and the LC Solution software (Shimadzu). Globin chains were separated by HPLC using a 250 mm × 4.6 mm, 3.6-μm Aeris Widepore column (Phenomenex). Samples were eluted with a gradient mixture of solution A (water/acetonitrile/trifluoroacetic acid, 95:5:0.1) and solution B (water/acetonitrile/trifluoroacetic acid, 5:95:0.1). The absorbance was measured at 220 nm.

A total of 10 µl human plasma was added into 990 µl 0.1% formic acid in methanol, and the sample solution was mixed using ThermoMixer C (Eppendorf) for 5 min at 4°C. After incubation on ice for 10 min, the sample solution was centrifuged at 15,000 × g for 10 min. The resultant supernatant was 2-fold diluted using 0.1% formic acid in methanol, and 300 µl diluted supernatant was applied into a LabTotal Vial (Shimadzu Corporation). The vial was inserted to the sample rack of the autosampler (Nexera X2 SIL-30AC; Shimadzu Corporation), and 3 µl sample was injected into the column for LC separation.
LC/ESI-MS was performed using the high-pressure LC installed LCMS-8060 (Shimadzu Corporation) system. To analyze the lipid components in human plasma, the LC/MS/MS Method Package for Phospholipid Profiling (Shimadzu Corporation) was used following the manufacturer's instructions. Kinetex C8 column (Kinetex C8, 150×2.1 mm i.d., 3.6-µm particle size; Phenomenex), mobile phase A (20 mM ammonium formate in water) and mobile phase B (acetonitrile: Isopropanol 1:1 v/v) were used for LC separation. The concentration of mobile phase B was programmed as 20% (0 min)-20% (1 min)-40% (2 min)-92.5% (25 min). The oven temperature was 45°C. Data processing and molecular identification/quantification were performed automatically by using the LabSolutions software (version 5.82 SP1; Shimadzu Corporation).