Melamine GC/MS

Discussions about GC-MS, LC-MS, LC-FTIR, and other "coupled" analytical techniques.

16 posts Page 1 of 2
Hi all I am investigating a melamine method via GC/MS. My company makes hydrolyzed vegetable proteins and buys protein and we want to test for melamine.

I don't have an LC/MS. I have a GC/MS and have read the apps about the older FDA method. Extract with water/ACN/diethylamine cent/filt, evap and silylate.

I am looking to replace the silylation with chlororformate derivatization either propyl or isobutyl. I just wish it were possible not to use diethlamine as it will have to be evaporated first or it will use up all the chloroformate. I understand the diethylamine breaks up complexes of melamine. Is there a nonamine alternative? I'm surprised noone has made a chloroformate paper on melamine as it works very well on amines.
I know most of the standards are shipped in dilute diethylamine which I figured was to stabilize it, so not sure how easy it would be to get around it. I never used diethylamine in my extraction, but it was present in the standards for certain.
The past is there to guide us into the future, not to dwell in.
I've read some older posts and the general consensus is injecting the silylation mix will mess up your column, liner and EI source quickly. Isobutyl Chloroformate should work. It is a classic bring your analyte from water soluble to organic phase so it is very clean and the excess reagent can be quenched with alkaline methanol or Sat NaHCO3. However with the diethlamine it will still have to be evaporated off first even though the derivatization does not need to be done under anhydrous conditions in fact most of the protocols are for water, acetonitrile, pyridine, methanol mix with extraction by chloroform or chloroform isooctane mix.

It works on nucleobases which have a similar structure so I am surprised that there are no literature reports.
https://jast-journal.springeropen.com/a ... 016-0090-9

I ordered the standard and materials so I will give it a try probably next week.
The normal way definitely does a number on the source. I ran a set about a year ago and every time I ran them I would lose 20-50% sensitivity. It didn't seem to hurt the column and I would get some sensitivity back cleaning the inlet but mostly I had to clean the source to get it all back. That was on my 7000QQQ, can't imagine what it would do to one with less vacuum.

Removing the diethylamine may be the reason nobody is doing it currently. I have been doing the EPA544 method for microcystins and you are evaporating a mixture of 20% water in Methanol and it takes forever when you can't exceed 60C.
The past is there to guide us into the future, not to dwell in.
I Have the same problem with my sugars method. everything is in water and I have to evaporate to dryness. I just keep adding methanol and MeCl2 and just give it time with nitrogen and heating to 50-60 deg C on my crude home made drying station.

I do a lot of chloroformate derivatization of amino acids and free fatty acids and it works well and does not mess up the instrument much. the paper I linked indicated ppb sensitivity on a single quad which is better than reported with the FDA method. I eventually need to buy a better evaporator.

I'll post how it goes next week when I get the materials.
So far I did about 7 different derivatizations conditions with methyl, ethyl, propyl, and isobutyl chloroformate and so far all failed. Inject and find nothing that could be the derivatization product of melamine (a tricarbamate). I tried everything from 0.1N NaOH to carbonate pH 9 to phosphate pH10 to various organic mixes (acetonitrile, my standard amino acid protocol, several publications...

I can't understand it. Chloroformates react with amines to produce carbamates so it should work.

I also found diethylamine is unnecessary. Dilute HCl solutions protonate the melamine and break up the H-bonded complexes.

Try a few more things then I'll try silylation. Agilent recommends a backflush method which since I have the quickswap I can do to save the EI source.
Could the triazine ring be interfering with the reaction.

https://en.wikipedia.org/wiki/Triazine


Since Melamine is actually an Amide, would that interfere with the reaction?

Also the complex that the diethylamine is used to prevent is this one

https://en.wikipedia.org/wiki/Melamine_cyanurate

If that is forming it might be difficult to break up.
The past is there to guide us into the future, not to dwell in.
yea that is what I was thinking chloroformates don't react with amides (actually asparagine gets converted to a nitrile by them). Guess that is why there is not publication.

Guess I will try the silylation with backflush as per agilent.

The USDA LC/MS method doesn't use diethylamine. They just add some 0.1N HCl which breaks up the complexes.
The method I used was based off the FDA one in combination with one from Restek. I think the Diethylamine was only in the standard I bought, not in the actual process.

Will you be doing the backflush with the half column method, where the Tee is in the middle of the column?
The past is there to guide us into the future, not to dwell in.
Not precisely. I don't have a t, The quickswap is like a purged union where a .1 mmID capillary goes through the transferline and the column ends at it with a steel ferrule. So I'd just use constant pressure and turn the pressure up to 40psi at the quickswap after acrylamide elutes to flush all the larger crap back into the inlet and out the split.
http://www.agilent.com/cs/library/appli ... 4071EN.pdf

I saw the Restek note. They reduce the silylating reagent ammount.

I may also try HMDS 1% TMCS. I have a ton of that around and it is cheaper than BSTFA. The derivatives aren't that low boiling where the reagent peaks are a concern.
MSCHemist wrote:
Not precisely. I don't have a t, The quickswap is like a purged union where a .1 mmID capillary goes through the transferline and the column ends at it with a steel ferrule. So I'd just use constant pressure and turn the pressure up to 40psi at the quickswap after melamine elutes to flush all the larger crap back into the inlet and out the split.
http://www.agilent.com/cs/library/appli ... 4071EN.pdf

I saw the Restek note. They reduce the silylating reagent ammount.

I may also try HMDS 1% TMCS. I have a ton of that around and it is cheaper than BSTFA. The derivatives aren't that low boiling where the reagent peaks are a concern.
There are also HPLC-UV methods out there, if that's an option for you.
Unfortunately I don't have an HPLC. That is why I do everything I can via GC/MS or FID.

Otherwise I looked at ELISA.
according to this Agilent app you can just extract the melamine with straight methanol though you might as well add the diethylamine it boils at 55deg. and you have to evaporate everything anyways. That should make it easier with the evap. 0.5g to 40ml is a big sample dilution that will impact sensitivity.

I never understand methods where they are going for sensitivity and dilute the heck out of the sample with solvent.

https://www.agilent.com/cs/library/slid ... 011309.pdf
Along these same lines I have a client looking for sub ppb levels of Glyphosate and I am finding several people are doing it by FOMC derivatives. Some are actually doing the reaction overnight in some of the older publications I have found. Do you think it should take that long? Agilent has a method for doing FOMC on Amino Acids in the autosampler vial automated, would they react that much faster than Glyphosate?
The past is there to guide us into the future, not to dwell in.
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