Nexera x2 uhplc system

Extraction of 2HG from lysed cell pellets and urine samples was performed using methanol protein precipitation (containing deuterated 2HG internal standard) followed by derivatization of the enantiomers using Diacetyl-L-tartaric anhydride (DATAN). LC-MS/MS analysis was performed using a Shimadzu Nexera X2 UHPLC system coupled to a Sciex Triple Quad 6500 Mass Spectrometer. Derivatized enantiomers of 2HG and internal standard were separated on a Waters Acquity UPLC HSS T3, 100 × 2.1 mm, 1.8 μm column using a 125 mg/L Ammonium Formate (aq) pH3.6/Acetonitrile gradient.

Approximately 3 mg of hiPSCs were hydrolyzed with 6 M HCl for 24 h at 110 °C. Then, the resulting amino acids were quantified with hydrophilic interaction chromatography coupled tandem mass spectrometry (HILIC-MS/MS) using a Shimadzu Nexera X2 UHPLC system connected to a SCIEX QTRAP 6500+ triple quadrupole-linear ion trap mass spectrometer, equipped with an IonDrive™ Turbo V electrospray ionization source, as described previously [34 (link)]. Positive ion mode was used for all amino acids except cysteic acid, which was analyzed in negative mode. The sample was injected into an Infinity Lab Poroshell 120 Z-HILIC column (2.7 μm, 100 × 2.1 mm; Agilent Technologies, Santa Clara, CA, USA), and amino acids were eluted with a gradient of ammonium formate in water (A) and acetonitrile:water (90:10), pH 3.0, at a final concentration of 20 mM ammonium formate (B) with a constant flow of 0.25 mL/min, followed by 50% B over 6 min, then 100% B over 30 s, followed by 6.5 min to re-equilibrate the column. The electrospray ionization source parameters were as follows: ion spray voltage: 4.5 kV (ESI+ and ESI−); ion source temperature: 400 °C; source gas: 1:45; source gas: 2:40; and curtain gas: 35.