Intrada organic acid column

The aqueous phase extract was analyzed by LC-MS/MS using a Shimadzu HPLC system (UFLC-XR; Shimadzu Corporation, Kyoto, Japan) connected to an AB Sciex triple quadrupole MS system (QTRAP 6500; AB Sciex, Foster City, USA). Amino acids and sugars (50 (link), 51 (link)) were separated on an Infinity Lab Poroshell 120 Z-HILIC column (2.7 μm, 100 × 2.1 mm; Agilent Technologies, Santa Clara, CA, USA), while sugar phosphates and organic acids (52 (link)) were separated on an Imtakt Intrada Organic Acid column (150 × 2 mm, 3 μm; Kyoto, Japan). Amino acids were detected in positive ion mode while sugars, sugar phosphates, and organic acids were detected in negative ion mode following previously described methods and MS parameters (50 (link)–52 (link)). For quantification of pool sizes and biomass composition, metabolite concentrations and recoveries were calculated on the basis of calibration curves of the individual standard and the internal standard ribitol, respectively.

The LC-MS system consisted of a Q-Trap 6500 (Sciex, Framingham, MA, USA) equipped with a Shimadzu LC-30AD HPLC system (Shimadzu Corporation, Kyoto, Japan). The stable isotope-labeled internal standards APDSTAG® Amino Acids Internal Standard Mixture Solution (FujiFilm-Wako, Osaka, Japan) and KIV-¹³C₅ (Cambridge Isotope Laboratories, Inc., Tewksbury, MA, USA) were added to the samples to facilitate quantification. For BCAA analysis, an Intrada Amino Acid column (100 × 3 mm, 3.0 μm; Imtakt, Kyoto, Japan) was used with an acetonitrile/100 mM ammonium formate/formic acid gradient of 85:15:0.1 to 0:100:0 (v/v/v) and a flow rate of 0.6 mL/min. For BCKA analysis, an Intrada Organic Acid column (150 mm × 2 mm, 3.0 μm; Imtakt) was used with an acetonitrile/water/100 mM ammonium formate/formic acid gradient of 10:90:0:0.1 to 10:0:90:0 (v/v/v/v) and a flow rate of 0.2 mL/min. For monitoring and quantifying BCAA and BCKA levels, a multiple reaction monitoring (MRM) method was developed with signature ion pairs Q1 (parent ion)/Q3 (characteristic fragment ion).