analysis of GHB on ion trap

[Stoo]

Hi all,

I'm in the middle of a research project to develop analytical methods for GHB (gamma-hydroxybutyrate) and BHB using LC/MS on the ion trap or triple quad.

First port of call, the ion trap. I've been trying for the past week to optimise the MS by direct infusion of GHB standards into the ion trap. The target ion is 105 (ESI+) but so far have failed to get anything much other than noise although peaks ranging from 104-107 have been seen. However, the largest peak is not stable and it fluctuates in this range when it is visible at all.

I've varied everything I can think of:
the concentration of GHB (1mg/L - 200mg/L)
the solvent (acidic water 1% HCOOH, and 10% HCOOH; acidic MeOH 0.1% HCOOH)
the voltages in the detector

I know GHB is a bit of a problem drug but if anyone has any advice as to how I might go about getting a clear spectrum I'd really appreciate it, as I'm tearing my hair out. My next step will be to use conc NH3 in water and use the ESI- mode but I'm really not hopeful. The lab would much prefer to use the ion trap over the triple quad so if it's possible I want to do that.

Thanks in advance for any replies.

[zokitano]

Try in ESI negative mode. If you're infusing the GHB solution into the ESI source by syringe pump, make sure to add at least 20% of methanol in your infusing solution, in order to aid the spray formation and solvent evaporation.
You could try dissolving your analyte in methanol / water mixture containing (5-10mM) ammonium bicarbonate or acetate, or even ammonia.
If your MS is well mass calibrated and functions properly you shouldn't have problems with detection of the deprotonated molecule [M-H]- of GHB.

Good luck!

[Stoo]

Thanks very much for that. I'll do that.

[Stoo]

Well, that didn't work. Used 1% NH3 in water:MeOH (80:20). Just got more of the same, just noise.

So far I've tried 0.1%, 1% and 10% HCOOH in water, and the above. Plus 0.1% HCOOH in water:MeOH (80:20).

Noise.

I am at a loss. The solutions I've used have been specified in the literature so I've no idea why I'm not seeing a peak. For info, I ran a 1mg/L standard of codeine and got a nice spectrum, so I'm convinced the instrument is working correctly, and it was tuned before I started running the standards

[Peter Apps]

GC-MS would probably be easier with a molecule this small.

Peter

[sepscientologist]

Peter Apps: Absolutely! I run 3 hydroxybuytric acid (the propyl ester) every day on my GCMS
and it works great. I would like to have a small analytical sample of GHB to see if it is in my
samples. Does the original poster know where I could get some? Not for personal consumption
of course!

[Stoo]

Hi

Yeah the lab currently uses GCMS for GHB but considers it very messy due to the derivitisation needed. The point of my project is to get something that's quick and simple, and there are a number of published methods for LCMS of GHB with simple extractions from blood. The GHB just won't show up on the mass spec. I've tried every variable I can think of, from the solvent to the parameters on the ion trap, but nothing has worked. I'm waiting to speak to someone at Agilent about it too.

seps,
there are a number of companies that supply standards, one of which is
http://www.cerilliant.com/ShopOnline/Pr ... x?text=ghb

[zokitano]

Hi Stoo,

I haven't personaly analyzed this compound with LC-MS, but i analyzed similar hydroxy-monocarboxylic acids which are chemically similar to GHB. Usually, they all give nice peaks of their deprotonated molecules when using negative ESI. I used ammonia solution or other ammonia LC-MS compatible salts together with methanol and/or ACN and always I was getting good signal and sensitivity. And the MS used was QqLIT.

At which m/z you're looking for your analyte? I don't have the information about your ion trap, but do you know (maybe is in the settings) what is the lowest m/z cut-off of your MS? Have you tried to change the scanning range and see if you're in the proper m/z window?
Sorry for the stupid questions, but i am really surprised that you cannot obtain any signal under LC-MS.

Regards

[zokitano]

I am at a loss. The solutions I've used have been specified in the literature so I've no idea why I'm not seeing a peak. For info, I ran a 1mg/L standard of codeine and got a nice spectrum, so I'm convinced the instrument is working correctly, and it was tuned before I started running the standards

I didn't understand, you tuned your MS with GHB or with some other compound? Have you tried to tune the MS voltages, ion optics parameters, etc. with a standard solution of your compound? The tuning settings for codeine might be inadequate for detecting the low molecular monoacid.

[Stoo]

Questions are fine with me

The first thing we did was clean the chamber and after that was done the MS was tuned using the direct infusion of tuning solution provided, which has a range of ions but m/z 922 was the major one we were looking for during tuning. Pretty standard practice I'd expect.

After running blanks through for a while we went straight into running a 1mg/L standard of GHB, as is standard practice in the lab, by ESI+ mode, looking for m/z 105. The scan range is 30-300. I think I tried acidic water and acidic MeOH. The transitions I'm looking for are 105 > 87 > 45 and shouldn't be a problem as far as the cut off is concerned.

The codeine was only run after I'd run quite a few GHB standards and got nothing. It was only to check the instrument was working ok and to let me see what a clear spectrum was supposed to look like. That worked fine.

I hope that's clearer now.

[zokitano]

Interesting article regarding the topic:

http://www.sciencedirect.com/science/ar ... 3806004762

that uses 3D ion trap and one-step derivatization. Detection of the n-butyl derivative at m/z 161 (positive ESI) and using m/z 161 -> 143 (qualifier) and m/z 161 -> 87 (quantifier) transitions.
The mobile phase and sample/std solvent was ACN:5 mM ammonium formate (1:1 for sample/std solvent).

And for the end the authors stated this: "As a fairly small molecule GHB is not suitable for a direct and specific determination by ion-trap mass spectrometry. Therefore, GHB was converted into its corresponding n-butyl ester."

Hope this helps

[lmh]

Your ion-trap clearly isn't the same as mine, because mine only goes down to m/z 50, but despite claiming to go to 50, it really doesn't give much signal in MS below 120 (although it's very good at giving MS2 spectra down to 50 from a suitably small precursor ion). The stated cut-off may not be the cut-off that's really happening.

Probably best not to tune to 922 if you want to work at 105. Try to tune to something nearer the correct mass.

Consider also whether the free acid of your analyte has a low boiling point. If it does, you may get a stronger signal by reducing temperaturers.

Good luck!

[Stoo]

success!

Following a suggestion from Agilent to use 10mM ammonium acetate in MeOH, I have a nice spectrum in ESI -ve mode.

Lower concentrations (c. 1mM) of that eluent increased the intensity so I'm off and running.

Thanks to all who posted