How to identify gammahydroxybutyric acid (GHB) via GC-MS using HFBA (heptafluorobutyric anhydride) chemical derivatization-a preprint

New Modified-HFBA (Heptafluorobutyric Anhydride) chemical derivatization protocol for identifying gamma-hydroxybutyric acid (GHB).? This method will allow for the identification of GHB v gamma-butyrolactone (GBL).? Another compound of interest is the 1,4-BD (butanediol).

Typically, GHB is converted to GBL by the thermal action of the inlet in the gas chromatograph-mass spectrometer (GC-MS).? Using the HFBA method, the attachment of the HFBA fraction protects the degradation of the inlet and it will readily identify the GHB-HFBA mass spectrum whereas the GBL will not be affected.? The HFBA molecule will be attached to the primary hydroxyl group on the GHB compound.? The whole process takes about five minutes.

Materials:

1.????? Saturated sodium bicarbonate solution (prepared by adding 20 grams sodium bicarbonate/100 mL of distilled water).

2.????? A small bottle of HFBA reagent (5 mL size bottle)

3.????? A bottle of mass spectral grade hexane

4.????? 10 x 75 mm test tubes.

5.????? 5 uL automatic pipettor

6.????? 500 uL automatic pipettor

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Protocol and Procedure:

1.????? Place a small amount of sample into a 10 x 75 mm test tube (1-10 mg)

2.????? Using an automatic pipettor, inject 5 uL of HFBA into the sample.? Dispose of the tip.

3.????? Vortex briefly about (10 seconds)

4.????? Add 500 uL of hexane to the sample mixture and vortex for 15 seconds.

5.????? Immediately after vortexing, add 500 uL of the saturated sodium bicarbonate to the same test tube.

6.????? Vortex for approximately 20-30 seconds.

7.????? Transfer top layer to a glass insert in a 2 mL mass spec vial.

8.????? Run-on GC-MS

GC-MS considerations

To analyze thermally sensitive compounds such as GBL, GHB, and 1,4-BD a few changes must be done to assist in the identification and analysis of these compounds.? There are three things to consider.? Gammabutyrolactone is an early eluter so the setting for the GC solvent delay must be set below 1 minute or in these sample runs the program it to have no solvent delay as I will refer to in the next comment.? The solvent of choice in this analysis is hexane.? Some of the newer GC-MS and columns will demonstrate impurity peaks around 1 minute to 1.5 minutes.? If you incorporate a solvent delay of 1.5 minutes you may miss the peak for GBL. Consider lowering the column temperature to start at 80o C or lower.? Finally, this is important, set the MS range on the GC-MS down to 30.? There is an ion for 1,4-BD at 31 m/z.

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Results:

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GC-Total Ion Chromatogram.

GC-MS of GHB chemical derivatized by heptafluorobutyric anhydride (HFBA)

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The structure of GHB and the resulting structure of GHB-HFBA

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GHB

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GHB-HFBA fragment, a 255 which correspond to the MS

GHB-HFBA fragment, a 226 ion on the MS

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GHB-HFBA producing two fragments,? a 283 ion and a 17 ion which below becomes a 213 ion.

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GHB-HFBA resulting ion from the fragmentation of a 17 ion and the HFBA compound.

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Conclusion
        

This may be a simple method for the confirmation of GHB v GBL        

I am sorry that you can't see the attachments, but if you email me I will attached the whole article. My email address is [email protected]. This is another tidbit and that is to add hexane first into your sample before adding the HFBA. Allow it to sit, after you add the HFBA for 5 minutes before adding saturated NaHCO(3). Because I don't have the premium Linked-In account I think I am limited to what I write. You can follow me or contact me on Researchgate.org.