title: Development and validation of a hydrophilic interaction liquid chromatography-tandem mass spectrometry method for the quantification of lipid-related extracellular metabolites in Saccharomyces cerevisiae.
abstract: 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 (150 mm × 4.6 mm, 5 μm) 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 0.5 mL/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 0.5-150 nM, with the lower limit of quantitation validated at 0.5 nM 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.
authors.affiliation: Department of Chemistry and Biochemistry, Duquesne University, Pittsburgh, PA, USA.
authors.forename: Tao
authors.initials: T
authors.lastname: Sun
authors.forename: Stephanie J
authors.initials: SJ
authors.lastname: Wetzel
authors.forename: Mitchell E
authors.initials: ME
authors.lastname: Johnson
authors.forename: Beth A
authors.initials: BA
authors.lastname: Surlow
authors.forename: Jana
authors.initials: J
authors.lastname: Patton-Vogt
mesh_terms: D002794:Choline / Q000032:analysis / Q000378:metabolism; D002853:Chromatography, Liquid / Q000379:methods*; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D017353:Gene Deletion; D020404:Glycerophospholipids / Q000032:analysis* / Q000378:metabolism; D057927:Hydrophobic and Hydrophilic Interactions; D007294:Inositol / Q000032:analysis / Q000378:metabolism; D016014:Linear Models; D050356:Lipid Metabolism; D059625:Liquid-Liquid Extraction; D055442:Metabolome; D055432:Metabolomics / Q000379:methods*; D010740:Phospholipases / Q000235:genetics / Q000378:metabolism; D015203:Reproducibility of Results; D012441:Saccharomyces cerevisiae / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012680:Sensitivity and Specificity; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D005656:Fungal Proteins; D020404:Glycerophospholipids; D007294:Inositol; D010740:Phospholipases; D002794:Choline