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Development and validation of a hydrophilic interaction liquid chromatography–tandem mass spectrometry method for the quantification of lipid-related extracellular metabolites in Saccharomyces cerevisiae

Sun, Tao, Wetzel, Stephanie J., Johnson, Mitchell E., Surlow, Beth A., Patton-Vogt, Jana
Journal of chromatography 2012 v.897 pp. 1-9
Saccharomyces cerevisiae, acetonitrile, ammonium acetate, choline, glycerol, hydrophilicity, ionization, liquid chromatography, metabolites, monitoring, myo-inositol, pH, quality control, spectrometers, storage quality, tandem mass spectrometry
A highly sensitive hydrophilic interaction liquid chromatography–tandem mass spectrometry (HILIC–MS/MS) method was developed and validated for the quantification of glycerophosphoinositol (GroPIns), glycerophosphocholine (GroPCho), glycerol 3-phosphate (GroP), inositol, and choline in the extracellular medium of Saccharomyces cerevisiae. The media samples were pretreated with a single two-phase liquid extraction. Chromatographic separation was achieved on a Waters Xbridge HILIC (150mm×4.6mm, 5μm) column under isocratic conditions using a mobile phase composed of acetonitrile/water, 70:30 (v/v) with 10mM ammonium acetate (pH adjusted to 4.5) at a flow-rate of 0.5mL/min. Using a triple quadrupole tandem mass spectrometer, samples were detected in multiple reaction monitoring (MRM) mode via an electrospray ionization (ESI) source. The calibration curves were linear (r²≥0.995) over the range of 0.5–150nM, with the lower limit of quantitation validated at 0.5nM for all analytes. The intra- and inter-day precision (calculated by coefficient of variation, CV%) ranged from 1.24 to 5.88% and 2.46 to 9.77%, respectively, and intra- and inter-day accuracy (calculated by relative error, RE%) was between −8.42 to 8.22% and −9.35 to 6.62%, respectively, at all quality control levels. The extracellular metabolites were stable throughout various storage stability studies. The fully validated method was successfully applied to determine the extracellular levels of phospholipid-related metabolites in S. cerevisiae.