Introduction

Hydroxyl groups can be reacted with acetic anhydride to form an acetate derivative (ethyl ester) that is suitable for injection into the GC/MS. Pyridine acts as a catalyst. The reaction is rapid and simple to perform, the reagents and byproducts are all volatile so no sample extraction is necessary, and the derivatized compounds are stable. The primary drawback to this method is that only alcohol groups are derivatized.

Materials

• acetic anhydride (should be stored in freezer)
• anhydrous pyridine
• methanol, DCM (for rinsing)
• heating block (70C)

Warnings

• Pyridine is a strong carcinogen and should be handled carefully. It is volatile, so there is risk of both contact and inhalation. Always work in the hood and wear gloves.
• Capped vials have occasionally burst while being heated. Keep the heating block in the hood with the sash lowered while samples are being heated.

Procedure

1. Transfer sample to a GC vial in an appropriate solvent (DCM, ether). Samples should be carefully dried prior to this step. Add any internal standard.
2. Add 20 uL acetic anhydride and 20 uL pyridine (note A) to the sample. This amount is sufficient for a sample containing <100ug of derivatizable material in ~100uL of solvent. If your sample is bigger, you will need to scale up the amount of reagent being added, or take a smaller aliquot of your sample for the reaction.
3. Cap the vial tightly and heat at 70C for ~20 minutes. The heating step is simply to ensure that the reaction goes to completion, so the the exact time and temperature are not critical. You should not need to heat samples for any longer than 30 minutes.
4. Let the sample cool to room temperature then inject on the GC/MS.
5. Clean all syringes that come in contact with the reagents thoroughly, first using methanol then DCM.

Notes

1. Pyridine is added as a catalyst for the reaction. Be sure to use anhydrous pyridine, as the normal stuff will contain lots of water and will cause a poor reaction yield.
2. If you are trying to derivatize hydroxyl groups on hydroxy-fatty acids, you need to methylate the carboxyl groups first. Otherwise the acid catalyst used in the methylation reaction will hydrolyze the acetate-hydroxyl ester.