Quantification of trehalose and glucose by LC-MS/MS

Chromatographic separation was performed on an ACQUITY BEH Amide column (100 mm × 2.1 mm, 1.7 mm particles, Waters) in combination with a VanGuard precolumn (5 mm × 2.1 mm, 1.7 mm particles) using an Acquity UPLC H-Class System (Waters). Elution was performed at 30 °C under isocratic conditions (0.3 mL/min, 70% acetonitrile and 30% 10 mM ammonium bicarbonate, pH 10.0). The mass spectrometric analysis was performed using a Xevo TQD triple quadrupole mass spectrometer (Waters) coupled with an electro-spray ionization source in the negative ion mode. The MRM transitions of m/z 341.2 >119 and m/z 182.1 >88.9 were used to quantify trehalose and mannitol-¹³C, respectively. Analytical conditions were optimized using standards solution. Sample concentrations were calculated from the standard curve obtained from serial dilution of each standard. The amounts of trehalose were normalized to the levels of mannitol-1-¹³C and further normalized to the levels at time 0 to determine the relative hydrolysis rate in each mutant. TPP activity in trehalose synthesis is Mg²⁺ dependent, whereas the Treh activity is not affected by the addition of EDTA (data not shown). Therefore, we assumed that there was no de novo production of trehalose during the incubation period. For the quantification of glucose in the haemolymph, the MRM transition of m/z 179.1 >89.0 was used to detect glucose under conditions identical to those described above.