Centrifree ym 30

This method was performed in the Department of Laboratory Medicine at the National Institutes of Health, Bethesda, MD. The updated method was similar to our previous method [20 (link),21 (link)], with minor modifications. Human serum/plasma samples were thawed at room temperature. 400 µL of human serum/plasma was placed in a 30 kDa ultrafiltration device (Centrifree YM-30, Millipore) and centrifuged in an Eppendorf temperature controlled centrifuge with a fixed angle rotor at 2700 rpm and a temperature of 37 °C for 30 min. To a 1.5 mL conical plastic Eppendorf centrifuge tube, 150 µL of ultrafiltrate was added and mixed with 450 µL methanol containing internal standards, deuterium-labeled T4 (T4-d₅) and carbon-labeled T3 (T3-¹³C₆), for deproteinization. The tube was capped, vortex mixed vigorously for 30 s, and centrifuged for 10 min at 13,000 rpm. After centrifugation, 500 µL of supernatant was diluted with 600 µL of distilled de-ionized water and 400 µL aliquot was injected onto a Phenomenex Kinetex 2.6 µm C18 column (75 × 2.1 mm). The procedure involved an online extraction step followed by activation of a built-in valco switching valve and subsequent sample introduction into the mass spectrometer. After washing, the switching valve was activated and the analyte compounds were eluted from the column with a water/methanol gradient into the MS/MS system (see Table 1a).