NSF Project: Protocols

Preparation and Derivatization of polar metabolites

1. Polar metabolites were methoximated and trimethylsilylated to improve chromatographic behavior.
    
2. 120mL of 15mg/mL methoxyamine hydrochloride in pyridine was added, incubated at 50°C, and sonicated until crystallized metabolites went into solution.
    
3. 120mL MSTFA + 1% TMCS was subsequently added and the solution incubated for 30 min at 50°C.
    
4. 1.0 mL of the solution was injected at 15:1 split ratio onto a HP 6890 GC equipped with a 60M DB-5-MS column coupled to a HP 5973 MS.
    
5. The injection port and transfer arm were held at 280°C, and the oven ramped from 80°C (2 min) to 315°C (6 min) at 5°C/min.
    
6. The MS source was held at 250°C and the quadrupole at 150°C and scanned from 50-650.