1290 infinity uhplc 6495b triple quadrupole mass spectrometer

Aliquots (25 μl) of each thawed yeast pellet on ice were homogenized in 175 μl of 50:50 water/acetonitrile with 0.3% FA at 4°C using tough microorganism lysing tubes in a Precellys homogenizer (Bertin Corp., Rockville, MD). Wash and postwash medium samples were aliquoted and prepared without dilution. The input and prewash medium samples were diluted 1000× in deionized water before sample preparation. Then, d₃-acetate and d₃-lactate (internal standard) were derivatized in 50 μl of aliquots of yeast homogenate and medium with O-benzylhydroxylamine through a coupling reaction with 1-ethyl-3-(-3-dimethylaminopropyl) carbodiimide at room temperature for 10 min. This mixture was extracted with 600 μl of ethylacetate with 50 μl of added deionized water followed by vortexing and centrifuging at 21,000g at 4°C. Aliquots of ethylacetate (50 μl) were dried under nitrogen at 30°C and reconstituted in 500 μl of 50% methanol in a 96-well plate. Derivatized d₃-acetate was quantitated by multiple reaction monitoring (d₃-acetate, m/z 169.1 to 91.1; d₃-lactate, m/z 199.1 to 91.1) with standard calibration curves on an Agilent 1290 Infinity UHPLC/6495B triple quadrupole mass spectrometer.