Highly automated nano-LC/MS-based approach for thousand cell-scale quantification of side chain-hydroxylated oxysterols

Underivatized oxysterols and nanoLC–ESI-MS: A mismatch

“…We therefore wanted to investigate whether the enhanced sensitivity of nanoLC-ESI-MS could compensate for the poor ionization of underivatized oxysterols, to allow sufficient detection of oxysterols in limited samples (e.g. million-scale cell numbers) in a shorter time than our present method [8]. This was partly inspired by the study of McDonald et al [10] who could monitor oxysterolssalt complexes (along with a number of other lipids) in biological samples using ESI and an AB Sciex API 5000 triple quadrupole mass spectrometer without derivatization.…”

“…derivatization with Girard reagents [6]. Such approaches can enable mass detection limits in pg-fg levels [6][7][8][9][10].…”

“…In metabolomics, the use of nanoLC is still very limited. However, nanoLC-ESI-MS can allow analysis of very small samples, and attomolar concentrations of 24S-OHC, 25-OHC and 27-OHC (derivatized with Girard T reagent) could be measured in just 10,000 pancreatic cancer cells, with excellent repeatability [8].…”

“…SPE clean-up (for removal of excess derivatization reagent) can be automated [8,15]. We therefore wanted to investigate whether the enhanced sensitivity of nanoLC-ESI-MS could compensate for the poor ionization of underivatized oxysterols, to allow sufficient detection of oxysterols in limited samples (e.g.…”

Quantitative Charge-Tags for Sterol and Oxysterol Analysis

“…Earlier studies (13)(14)(15)(16)(17) adopted an LC-MS approach to oxysterol analysis, but combined the approach with chemical derivatization to enhance analytical sensitivity. In the work described here, we introduce novel isotope labels into our derivatives to improve the imprecision of quantitative work.…”