We want to quantify organic acids related to redox metabolism in NDUFS4 KO mice, heart liver and brain tissue. First, we are establishing whether the selected organic acids do indeed derivatize with MTBSTFA.
We made up standards for the first organic acids and amino acid namely: Oxaloacetic acid, Aspartic acid and 2-ketoglutaric acid.
These standards where made up to be 200 ppm.
50 µL's of each metabolite were dried under a nitrogen stream, thereafter the dried samples were derivatized with MTBSTFA (containing 1% TBDMCS) and left to incubate at 120 °C for 30 minutes.
After derivatization, the total volume of each sample was inserted into a GC-vial insert and analyzed on a GC-TOFMS system.
The GC-TOFMS system utilizes electron impact (EI) ionization as ionization technique at standard -70 eV.
After subsequent analysis we inspected the chromatogram and mass spectrum of each sample analyzed and found that these organic acids do indeed derivatize with MTBSTFA however, the [M-57] ion is not abundant at all. Furthermore, the mass spectrum showed an abundant [M-15] ion, characteristic to BSTFA derivatization.
How is it possible that the samples analyzed do not give an abundant [M-57] ion when MTBSTFA was used as derivatization agent? Furthermore, how is it possible that the [M-15] ion surpasses the [M-57] ion in terms of intensity?
For quite some time BSTFA and TMCS was most commonly used on this particular platform, could there be a possibility that these two derivatization reagents remain in the system causing some kind of interference with MTBSTFA.
Thank you for reading this far.
T. Jooste
- Badges
- Science topic
- Similar topics
- Computer Science
- Program