The Analytical Unit provides sample preparation/lipid extraction, data normalization measurements (levels of inorganic phosphate/sample), and lipid analysis based on High-Performance Liquid Chromatography-tandem Mass Spectrometry (HPLC-MS/MS) or Supercritical Fluid Chromatography-tandem Mass Spectrometry (SFC-MS/MS) approaches.
The core provides both qualitative (compound identification) and quantitative mass spectral analysis of key lipids from different biological materials (cells, tissues, serum, blood) with assays to measure over 300 different sphingolipid and DAG components at a basic metabolomics level.
Quantitative analyses of sphingolipids, are based on eight-point calibration curves generated for each target analyte. The synthetic standards along with a set of internal standards are spiked into an artificial matrix, they are then subjected to an identical extraction procedure as the biological samples. These extracted standards are then analyzed by the HPLC/MS/MS or SFC/MS/MS system operating in positive multiple reaction-monitoring (MRM) mode employing a gradient elution. Plotting the analyte/internal standard peak area ratios against analyte concentrations generates the analyte specific calibration curves. Any lipid with no authentic standards is quantitated using the calibration curve of its closest counterpart.
Reference: “Sphingolipid levels were measured by high-performance liquid chromatography mass spectrometry (LC-MS/MS) methodology as previously described. Bielawski, J., et al. (2009) Chapter 22 Comprehensive Quantitative Analysis of Bioactive Sphingolipids by High-Performance Liquid Chromatography–Tandem Mass Spectrometry, In D. Armstrong (Ed.), Lipidomics: Vol1: Methods and Protocols (pp. 443-467) New York, NY: Humana Press.”
These instruments have available electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) probes for various applications.