6490 triple quadrupole

Crtc2^LKO and Crtc2^f/f tissues were obtained from Korea University. The weight of each tissue sample was measured; the tissues were pulverized with 500 μl of amino acid extraction buffer (acetonitrile: methanol: water, 40:40:20 v/v) using a centrifuge tube sample pestle. Standard branched-chain amino acid (BCAA) with heavy isotope labeling was spiked into each sample and incubated at −20 °C for 1 h. After incubation, samples were centrifuged at 16,000g for 10 min at 4 °C. The supernatants were collected, dried, and subjected to LC-MS/MS analysis using an Agilent Triple Quadrupole 6490. An XBridge BEH amide column was used for chromatographic separation with Sol A and Sol B (20 mM ammonium acetate, 20 mM ammonium hydroxide in 95:5 water: acetonitrile buffer at pH 9.45; 20 mM ammonium acetate, 20 mM ammonium hydroxide in acetonitrile at pH 9.45, respectively). A total of 30 min gradient with a flow rate of 0.25 ml/min was applied as follows: 85% of Sol B for 2 min, 15% to 20% of Sol A in 3 min, 20% to 25% of Sol A in 7 min, 25% to 30% of Sol A in 8 min, 30% to 50% of Sol A in 10 min, 50% to 75% of Sol A in 13 min, 75% to 100% of Sol A in 18 min, and 100% to 15% of Sol A in 24 min. The BCAAs transition was applied as follows: leucine light, 132.1/86; leucine heavy, 138.1/91.1; isoleucine, 132.1/86.1; isoleucine, 138.1/91; valine light, 118.1/76; and valine heavy, 123.1/72.

Samples were analyzed, using a previously described validated analysis method [16 (link)]. Analysis was performed with an UHPLC 1290 Infinity from Agilent Technologies (Waldbronn, Germany) and an Agilent Technologies 6490 Triple Quadrupole mass spectrometer, using a C18 column (ZORBAX Eclipse Plus C18 Rapid Resolution HD 2.1 × 100 mm 1.8-µ, Agilent) at 30 °C with 5.00 µL sample injection volume at a flow rate of 0.6 mL/min. Mobile phase A was 5 mM ammonium formate in water, mobile phase B consisted of 0.1% (v/v) formic acid in acetonitrile. Analytes were separated using the following elution: 65% mobile phase B for the first 4.5 min, changed to 80% B and increased to 80.9% B at 7.50 min, changed to 100% B till 9.50 min. The column was re-equilibrated with 65% B for 3.5 min. Total run time was 13 min. Analytes were measured using electrospray ionization (ESI) in positive and negative reaction mode with a gas temperature of 250 °C, gas flow of 15 L/min, nebulizer pressure of 20 psi, sheath gas heater temperature of 400 °C, sheath gas flow of 12 L/min, capillary voltage of 4000 V and a nozzle voltage of 1000 V. For evaluation of the data, the Agilent Mass Hunter software B 09.00 was used. Analytes were calibrated in blank human serum.