Chromatography Forum

Hello,

I'm trying to set up a method for the determination of Clenbuterol by GC/MS in urine samples. The main problem so far is a good derivatization method in aqueous phase. Anyone with experience who could lend me a hand?

Thanks.

J

Why not extract into organic solvent and then derivatize? You are going to transfer to organic solvent before running on the GC, right? Most derivatization reagents will react with water.

Do a Google Search for "analysis of Clenbuterol"

Fourth hit was "Gas Chromatography-mass spectrometric analysis of clenbuterol from urine".

http://cat.inist.fr/?aModele=afficheN&cpsidt=1673449

Now that wasn't difficult was it?

Do a Google Search for "analysis of Clenbuterol"

Fourth hit was "Gas Chromatography-mass spectrometric analysis of clenbuterol from urine".

http://cat.inist.fr/?aModele=afficheN&cpsidt=1673449

Now that wasn't difficult was it?

Thank you, but if I had access to those papers I wouldn't ask here.

Hi Jerole,

I wonder also why you would derivatize in aquos phase. I`ve done beta agonists (not only clenbuterol) out of urine, liver, water and retina some times ago. We extracted, after hydrolysis with glucuronidase/sulfatase, with CS-DAU SPE columns. The organic solution was blown to dryness with nitrogen at 40°C an redissolved in acetonitrile/hexadimethylsilazane (1:1) and tempered at 70°C for 90 min.
This solution has been analyzed with GC-MS in PCI mode. Taget mass for clenbuterol was 349 Qualifiers 333, 259 and 351.

Some of my collegues are using MSTFA or BSTFA for derivatization at 75°C / 90 min.
By the way, all reactions are trimethylsilylations which are very sensitive to water
If you are in need I can try to scan the method (whole SOP for urine) and mail to you. But it may take a few days becaus our QS system is audited at the moment.

Best regards
Stephan

Hi jerole

The summary to the article provided by "ods-at-pacific" should be enough to get you started even though you do not have full acess to the article.

Like others and the author of the article I have done similiar liquid/liquid extractions of similar compunds using a gentle ~pH 12 buffert and an organic solvent like toluene or the solvents mentioned in the article.

Once extracted you can gently dry and proceed with dervatization.

Good Luck

Thanks for the replies.

I am used to work with organometallic compounds that can be easily derivatized in aqueous solutions and was hoping it can be applied in this case too. But it seems that it doesn't work in this case. I will try the LLE and derivatization of the dried extract you proposed.

Jerole

I've been analysing clenbuterol in urine (and liver, and retina and hair) using GC/MS for about 10 years. As everybody has said, first you need the molecule in organic solvent after (usually) SPE and then it has to be derivatized to do GC/MS. For that I don´t recomend to use MSTFA, BSTFA and the like since they result in a trimethylsylil derivative that break completely in the ion source in EI conditions. So that useful ions in terms of intensity have low m/z values (base peak is 72 if I recollet rigthly) and ions with reasonable m/z have a very low intensities. I use a derivatitation using CMDCS and diethylamine in n-Hx at 60ºC. The derivative is a cyclic dimethylsylil that is very stable in the EI source: the M+ ion is easily detected at m/z 346. I can give you more information if you are interested.

Jerole

I've been analysing clenbuterol in urine (and liver, and retina and hair) using GC/MS for about 10 years. As everybody has said, first you need the molecule in organic solvent after (usually) SPE and then it has to be derivatized to do GC/MS. For that I don´t recomend to use MSTFA, BSTFA and the like since they result in a trimethylsylil derivative that break completely in the ion source in EI conditions. So that useful ions in terms of intensity have low m/z values (base peak is 72 if I recollet rigthly) and ions with reasonable m/z have a very low intensities. I use a derivatitation using CMDCS and diethylamine in n-Hx at 60ºC. The derivative is a cyclic dimethylsylil that is very stable in the EI source: the M+ ion is easily detected at m/z 346. I can give you more information if you are interested.

Yes, I have noticed. The base peak is 86, no molecular ion with EI and the other fragment abundances at high masses are weigh too low and can be hardly used for quantification or even identification.
I would be very grateful if you can send me more information. (jeroleATgmail.com)

Hi Jerole! I could not reach you in the email address in your last post .

Anyway, here’s the complete protocol for the derivatitation :

Evaporate your extract to dryness under nitrogen sparging in a vial. (I use 3 ml derivatitation vials from Alltech)

Mix 0.320 ml of CMDMCS (Aldrich 22,618-1), 0.2 ml of diethylamine and 4 ml of n-hexane in a stoppered tube. Mix gently. Warning: a white precipitate appears, it is normal. Centrifuge at about 1900 g to pellet the precipitate. Add 0.1 ml of the supernatant to the dry extract, stopper and hold at 60ºC for 40 minutes. Cool to room temperature and evaporate the remaining solvent under nitrogen. Redisolve in toluene ( I use 50 ul ). Note: usually a white residue remains after the last evaporation; if so, just centrifuge the vial after adding the toluene at the same speed for 2 minutes and use the supernatant for GC/MS.

The diagnostic ions are 346 (M+), 331, 289, 187 and 128.

The original reference for the derivatitation reaction is: Dumasia M.C., Houghthon, E.; J. Chromatogr. B, 564, 503, 1991.

This reaction works fine for clenbuterol, cimaterol and other b-agonist with no OH moieties (excepting the OH in C2 of the aliphatic sidechain common to all b-agonist compounds). If you need to analyze other b-agonist with additional OHs you’ll have to use MSTFA, BSTFA

Hope it helps. Good luck

Thanks a lot.
I will try this derivatization next week and let you know how it worked.

Carras, I've been testing your derivatization method this week, but without any success yet. It seems like the CMDMCS is very labile, do you work under an inert atmosphere? Can you tell me if the derivatization mixture is stable for a longer period once prepared and centrifuged?
Thanks in advance.

With the GC/MS methods, what LOQ and LODs are you attaining?

I have developed a method for Clenbuterol in Blood and Vitreous fluid by UPLC/MS/MS with an LOQ of 0.1 ng/mL and a LOD of 0.05 ng/mL.

LODs are not the problem here, but implementing Council Directive 96/23/EC concerning the performance of analytical methods and the interpretation of results. For the confirmation of substances listed in Group A of Annex I of Directive 96/23/EC, a minimum of 4 identification points shall be required. Using GC-MS (EI) each Ion gives you 1 identification point, that means that you need 4 masses from 4 ions(fragments).

The usual derivatization methods produce mainly a base peak at 87 (quite low mass range, not ideal) and a few ions of higher m/z, but with very low abundances, wich are too low to be used as identification points. Therefore, these derivatization methods are not suitable to meet the Directive requirements.

Thus, we are looking for an alternative derivatization method that produces fragments with higher abundances, else we have to go to MS/MS which is not cheap

Hi, Jerole! The derivatitation mixture must be used inmediately. I don't use inert atmosphere.
Two thougths: I am assuming that you've tried the procedure with a standard, if that's the case be sure that you have evaporated completely the solvent you've pipeted into the derivatitation vial.
The TIC of a derivated standard in full scan (or even in SIM) will show several peaks. Use the diagnostic ions to locate the peak.
In case it helps, I redisolve in 50 ul of toluene and inject 2 ul (splitless) and use SIM for detection. In these conditions the derivatitation of 10ng of clembuterol should give a nice peak.