Permethylation “Standard Scale”


The purpose of the protocol is to perform permethylation of samples prior to mass spectrometry analysis.


The Preparation of the slurry NaOH/DMSO solution is made fresh every time.
Mortar, pestle, and glass tubes are washed with MilliQ water and dry beforehand.

Whenever possible, liquid reagents are handled with disposable glass pipettes.
Solvents are HPLC grade or higher.


  1. With a clean and dry mortar and pestle, grind 7 pellets of NaOH (Sigma-Aldrich, #S8045) in 3 ml of DMSO (Sigma-Aldrich, #D8418). Add 1 ml of this slurry solution to dry sample in a glass tube with a screw cap.
  2. Add 500 µl of Iodomethane (Sigma-Aldrich, #289566), close the lid tightly and shake the sample at RT for ~30 min. Caution: Pressure can build up within the tube, make sure it is fastened. The mixture should turn white and even become solid as it reaches completion.
  3. Slowly open the cap as gas pressure has built up. Add 1 ml of MilliQ water to stop the reaction, vortex the tube until all solid is dissolved.
  4. Add 1 ml of Chloroform and additional 3 ml of MilliQ water, vortex thoroughly the tube to mix both phases and centrifuge briefly to separate the chloroform and the water phases (~5000 rpm,
  5. Discard the aqueous top layer and repeat the wash 2 more times, adding 3 ml of MilliQ water.
  6. Dry the chloroform fraction with a SpeedVac (~20-30 min).
  7. Condition a C18 Sep-Pak (50 mg) column (Waters, #WAT054955) with Methanol, MilliQ water, Acetonitrile and MilliQ water. Resuspend the dry sample with 200 µl of 50% methanol and load onto the column. Wash the tube with 1 ml of 15% acetonitrile and load onto the column. Wash the column with 2 ml of 15% acetonitrile.
  8. Elute the column in a clean glass tube with 3 ml of 50% acetonitrile. Lyophilize the eluted fraction for mass spectrometry (MS) analysis.