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

• Published in: Journal of chromatography. B, Analytical technologies in the biomedical and life sciences Analytical technologies in the biomedical and life sciences, 2012-05, Vol.897, p.1-9
• Main Authors: Sun, Tao, Wetzel, Stephanie J., Johnson, Mitchell E., Surlow, Beth A., Patton-Vogt, Jana
• Format: Article
• Language: English
• Subjects: acetonitrile; ammonium acetate; choline; Choline - analysis; Choline - metabolism; Chromatography, Liquid - methods; Fungal Proteins - genetics; Fungal Proteins - metabolism; Gene Deletion; glycerol; Glycerophospholipid metabolites; Glycerophospholipids - analysis; Glycerophospholipids - metabolism; HILIC–MS/MS; hydrophilicity; Hydrophobic and Hydrophilic Interactions; Inositol - analysis; Inositol - metabolism; ionization; Linear Models; Lipid Metabolism; liquid chromatography; Liquid-Liquid Extraction; Metabolic footprinting; metabolites; Metabolome; Metabolomics; Metabolomics - methods; monitoring; myo-inositol; Phospholipases - genetics; Phospholipases - metabolism; quality control; Reproducibility of Results; Saccharomyces cerevisiae; Saccharomyces cerevisiae - chemistry; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - metabolism; Sensitivity and Specificity; spectrometers; storage quality; tandem mass spectrometry; Tandem Mass Spectrometry - methods
• ISSN: 1570-0232; 1873-376X
• DOI: 10.1016/j.jchromb.2012.03.034

► A new analytical method to determine extracellular levels of lipid-related metabolites. ► Five metabolites are separated in 20min with lower limit of quantification at 0.5nM. ► The developed method is successfully applied to monitor the extracellular levels of metabolites in S. cerevisiae. 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.