For polyamine extraction, snap-frozen tissues were pulverized on dry ice using mortar and pestle and subjected to 5% trichloroacetic acid (TCA) extraction using 20–25 mg pulverized tissue or 20 µl whole blood and a final extract volume of 750 µl. Quantitative HPLC–MS/MS-based determination of spermidine, putrescine, ornithine and spermine was performed as described before by employing stable-isotope labeled internal standards26 (link).
Samples were incubated on ice (1 h) and centrifuged at 10,000 g at 4 °C (10 min). 150 µl supernatant was mixed with 800 µl double distilled water, 125 µl sodium carbonate buffer (1 M, pH 9) and 25 µl isobutyl chloroformate, then incubated at 35 °C (15 min). The solution was centrifuged at 10,000 g (1 min) and the resulting supernatant was measured in the Ultimate 3000 HPLC system (Thermo Fisher Scientific, USA) coupled to a triple-quadrupole mass spectrometer, a Quantum TSQ Ultra AM controlled by Xcalibur Software version 4.0 (both Thermo Fisher Scientific, USA).
Samples were incubated on ice (1 h) and centrifuged at 10,000 g at 4 °C (10 min). 150 µl supernatant was mixed with 800 µl double distilled water, 125 µl sodium carbonate buffer (1 M, pH 9) and 25 µl isobutyl chloroformate, then incubated at 35 °C (15 min). The solution was centrifuged at 10,000 g (1 min) and the resulting supernatant was measured in the Ultimate 3000 HPLC system (Thermo Fisher Scientific, USA) coupled to a triple-quadrupole mass spectrometer, a Quantum TSQ Ultra AM controlled by Xcalibur Software version 4.0 (both Thermo Fisher Scientific, USA).
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