Chromatography Forum

greeting
I'm from continental Europe, sorry for bad English. I have a problem with the determination of gamma hydroxy butyric acid in the blood. Is there any SPE phase which effectively binds the analyte? I tried the C18, extrelute (Merck) and Narc columns 1 and 2, but without success. I also had problems with liquid-liquid extraction, I can not find an appropriate solvent.

What solvents did you try? And, are you trying to put whole blood, plasma, or serum, or a fraction prepared from one of these on the SPE cartridge?

And, I have to be curious as to why you want to use an SPE step, there are published papers showing fairly simple preparation and analysis of GHB http://www.ncbi.nlm.nih.gov/pubmed/12829000 or in http://jat.oxfordjournals.org/content/25/7/576.full.pdf and no SPE is used (but there is fractionation to get rid of large molecules).

UCT has SPE methods for GHB, please check out the following links:

http://www.unitedchem.com/sitefiles/app ... action.pdf

http://www.unitedchem.com/sitefiles/app ... _Blood.pdf

LLE Procedure for GHB

1. Pipet 200 μL of each case sample
2. Add ISTD (GHB-D6)
3. Add 250 μL of 0.1N H2SO4
4. Add 6 mL Ethyl acetate
5. Vortex each sample for 30 seconds
6. Centrifuge at approximately 2500 rpm for 15 minutes
7. Transfer upper organic layer to new clean tube
8. Evaporate samples under nitrogen at 55°C
9. Add 60 μL acetonitrile to each sample
10. Add 30 μL BSTFA to each sample. Mix briefly
11. Heat samples at 70°C for 15 minutes
12. Transfer into GC vial and Inject onto GC/MS

What solvents did you try? And, are you trying to put whole blood, plasma, or serum, or a fraction prepared from one of these on the SPE cartridge?

I tried the whole blood:

I
1. deproteinization with methanol,
2. centrifugation, transfer organic layer
3. evaporation
4. 50 ul BSTFA + 50 ul IS (derivatized benzyl alcohol)
5. GC/MS, 1 ul inject

II
1. extraction with ethyl acetate (without deproteinization)
2,3,4,5 same as I

III
1. extraction with CH2Cl2:CH3OH (different ratios 10:90, 25:75, 50:50)
2,3,4,5 same as I

And, I have to be curious as to why you want to use an SPE step, there are published papers showing fairly simple preparation and analysis of GHB http://www.ncbi.nlm.nih.gov/pubmed/12829000 or in http://jat.oxfordjournals.org/content/25/7/576.full.pdf and no SPE is used (but there is fractionation to get rid of large molecules).

SPE step seems elegant. I'm trying to make it easier.

LLE Procedure for GHB
1. Pipet 200 μL of each case sample
2. Add ISTD (GHB-D6)
3. Add 250 μL of 0.1N H2SO4
4. Add 6 mL Ethyl acetate
5. Vortex each sample for 30 seconds
6. Centrifuge at approximately 2500 rpm for 15 minutes
7. Transfer upper organic layer to new clean tube
8. Evaporate samples under nitrogen at 55°C
9. Add 60 μL acetonitrile to each sample
10. Add 30 μL BSTFA to each sample. Mix briefly
11. Heat samples at 70°C for 15 minutes
12. Transfer into GC vial and Inject onto GC/MS

I may be wrong but GHB in acid environment will be converted to GBL?

Your first procedure looks somewhat similar to one of the published procedures noted above. However, you do not indicate how much methanol you use. You want to be sure there is enough so the analyte partitions adequately into the methanol.

Your internal standard - it is bet to get an internal standard into the extraction. And it should be as chemically similar to the analyte as possible. GHB-D6 is ideal. If you want to go for elegant, locate some 13C labeled GBH - it will give an exact coelution on the GC/MS. And that's where you open the chapter on isotope dilution quantification. (And the wallet for purchasing standards.)

I don't know exactly what you are putting 50 uL of in as the internal standard. If it is 50 uL neat Benzyl alcohol - TMS ester, you have way too much. If you have diluted the internal standard in a solvent, it needs to be aprotic (and dry).

On 0.1 N acid causing lactone formation: You have a lot of water present (mole for mole) which would push the equilibrium away from esterification and you handle the samples relatively quickly - so even if there is a strong driving force toward ring formation, it won't have time to do much. That's my expectation - and it is easily enough checked in the lab.

On shooting for elegant: Shoot for simple. Something that appears to be difficult or complex solved simply is elegant. Go for the increased number of steps if you have LOD issues or just need to burn up a budget.

I am preparing to adding 2.5 ml of methanol per 1 ml of blood. As for the internal standard I have no GHB-D6, in some brochures I found that using the derivatized benzyl alcohol. concentration of the internal standard was 0.052 ug/ml. Retention times and response are close GHB-TMS. My goal was to go down to the concentration of GHB to endogenous levels (0.01 ug/ml). I planned to go from 1 ug/ml and to descend to lower concentrations (SIM mode) but I got a very low reproducibility.
Can you tell me which is your limit of detection for GHB in the blood?