Targeted metabolomics analysis
SEPARATION
Targeted metabolomics analysis was performed to determine the Lac-Phe, La, and Phe levels in the collected plasma samples using a Vanquish Ultra High-performance Liquid Chromatography-Mass Spectrometry coupled with a Q Exactive operated in negative ion mode, as described by Hoene et al. (An ACQUITY UPLC HSS T3 column (pore size = 1.8 µm, length = 2.1 × 100 mm) (Waters, Milford, USA) was equipped to carry out the separation. The mobile phases comprised (A) 6.5 mM ammonium bicarbonate ((NHThe spray voltage was operated at 3.00 kV. Nitrogen sheath gas and nitrogen auxiliary gas were fixed at a flow rate of 45 and 10 AU, respectively, while capillary and aux gas heater temperatures were set at 300 °C and 350 °C, respectively. Parallel reaction monitoring was employed to achieve high-resolution of the Lac-Phe MS/MS spectra (m/z = 236.093) at 17,500 resolution and collision energy of 30 eV. The signal intensities were normalized by adding 0.8 µg/mL of internal standard d5-Phe (615870, Merck, Germany) in the extraction solvent.