Serum C4 Quantification by LC-MS/MS

Serum C4 measurement was performed by LC-MS-MS. C4 was extracted using a salting-out method as previously described [24 (link), 25 (link)]. Serum samples were prepared alongside of authentic C4 standards. Briefly, 100 μL of serum was diluted with 200 μL of distilled water, to which 5 ng of d7-C4 (used as internal standard) and 500 μL of acetonitrile were added. Then, 100 mg of ammonium sulfate was added, tubes vortexed for 1 minute and centrifuged at 2,000 g at 4°C for 5 minutes. The supernatant acetonitrile phase was collected and dried under nitrogen at 35°C. The residues were reconstituted with 200 μL methanol, vortexed for 1 minute, incubated for 10 minutes and centrifuged at 20,000 g for 5 minutes. Clear supernatants were transferred to 250 μL inserts for LC-MS-MS analysis. The LC-MS-MS system consisted of an Agilent UHPLC 1290 LC system coupled to an ABSciex QTRAP 5500 in positive ESI MRM mode. Chromatographic separation was performed using a XB-C18 kinetex column (50 x 3.0 mm, 2.6 μm; Phenomenex) operated at a flow rate of 800 μL/minute and eluted isocratically with a mobile phase consisting of acetonitrile/water (98/2, v/v) with 0.1% trifluoroacetic acid. Quantitation of C4 was achieved by comparing the deuterium-to-protium ratio of the samples with a standard using the Analyst 1.6 software.