Determination of oxysterols in cancer stem cells using on-line automated filtration and filter-flush solid phase extraction liquid chromatography tandem mass spectrometry

Abstract

A robust on-line automatic filtration and filter-flush solid phase extraction liquid chromatography tandem mass spectrometry (AFFL-SPE-LC- MS/MS) method for determination of 25hydroxycholesterol (25HC), 24S hydroxycholesterol (24SHC) and 22S hydroxycholesterol (22SHC) in cell samples has been developed. Only one sample transfer (from sample tube to LC vial) was needed for the entire process of cell lysis, derivatization and determination of the analytes.

In general, cell lysate samples (200-400 µL) were mixed with internal standard and evaporated into dryness. The samples were resolved in 2-propanol and phosphate buffer (pH 7) followed by an oxidation with cholesterol oxidase. Subsequently, methanol and acetic acid were added to the samples and the samples were derivatized with Girard T reagent. Aliquots of 100 µL were injected directly on to the AFFL-SPE-LC-MS/MS system.

The method was validated and showed good linearity (R2>0.99 in the concentration range 0.3- 33 nM). The limit of detection was 0.06 nM (2.5 pg injected on column) and the limit of quantification was found to be 0.3 nM. Repeatability was satisfying at three concentration levels, low (0.3 nM), medium (2.1 nM) and high (33 nM), where the relative standard deviation (RSD) between replicates (n=6) were 15-19 %, 5-13 % and 5-6 %, respectively. RSD between days (n=5) were 6-16 %, 9-12 % and 5-9 %, respectively.

The method was applied for analysis of different cell lines (NIH3T3, SHh-L2, SUFU-/-, HEK293, HCT15 and HCT116). The between cell flask concentration variation was low (RSD=5-35 %, except for 24SHC in Shh-LII (RSD= 65%)) implying method robustness. The method was also applied successfully for a cell subpopulation of 330 000 cells.