Quantifying Trehalose Uptake by GC-MS

Trehalose uptake quantification by gas chromatography and mass spectrometry was performed precisely as reported19 (link). ¹³C₁₂ trehalose (Omicron Biochemical, South Bend IN) was used as a sample extraction and derivatization internal standard. 5 nmol internal standard were added to the samples which were then extracted into 200 μl isopropanol:CH₃CN:H₂O. Samples were centrifuged and the supernatant was taken to dryness under nitrogen. Samples were derivatized using 100 ul N-Methyl-N-(Trimethysilyl) trifluoroacetamide (MSTFA): 10% pyridine in CH₃CN (1:3) at room temperature overnight. Derivatized samples were analyzed using an Agilent 7890 A gas chromatograph interfaced to an Agilent 5975 C mass spectrometer. The GC column was a HP-5MS (30 m, 0.25 mm internal diameter, 0.25 um film coating, P.J. Cobert, St. Louis, MO). A linear temperature gradient was used. The initial temperature (80 °C) was held for 2 min and increased to 300 °C at 10 °C/min. The temperature was held at 300 °C for 2 min. Samples were run by electron ionization (EI) mode and the source temperature, electron energy and emission current were 200 °C, 70 eV and 300 μA, respectively. The injector and transfer line temperatures were 250 °C. The ions monitored were m/z 361 and 13-Trehalose at m/z 367.