Preparation and analysis of non-polar metabolites

1. Nonpolar metabolites were hydrolyzed and trimethylsilylated.

2. Hydrolysis was performed with 0.5 mL of 1.25 M HCl in methanol and 0.8 mL chloroform at 50°C for five hours.

3. Following hydrolysis, the solvents and HCl were evaporated under nitrogen.

4. The sample was then resuspended in 80mL pyridine and 20mL MSTFA + 1% TMCS and incubated for 1hr at 50°C.

5. One mL of the sample was injected at 1:1 split rationas described for polar samples, with the exception that the oven temperature was held at 315°C for 11 minutes.