Chromatography Forum

Hello Everybody,

I´ve been recently injecting quite a lot of standards with a GC-QqQ system and I´m facing some interesting issues with amino acid standards; when I start working with amino acids I get a nice peak(s) from the first injection but then nothing from the second one, even if I´m just re-injecting the first sample. If I inject something different, such as fatty acids, for while I again get a nice peak(s) from the first injection but nothing from the following injection. Peaks in the first injection are nice and sharp, no fronting or tailing and no detector saturation. Injector is performing as it should be as I have no problems with other classes of chemicals. I´m using Rxi-5Sil MS column. For derivatization I´m using methoxyamine and MSTFA.

Any ideas on what I might be doing wrong?

So far I have tried to:
- change liner (RESTEK deactivated)
- change septa
- vary sample concentration
- use deactivated vials
- different split value

Cheers,
Otto

I do not understand what happens for fatty acids: do you mean that you have the same proble; with them, OR that fatty acids work fine and when you inject afterwards amino acids you have the problem>

That's exactly what happened to me. I had clean inner and outer ion sources, this restored performance. After several injections, again. Finally I switch to the Waters accq chemistry for AA analysis

I meant that fatty acids are working just fine. No problems with them, just with amino acids.

I hope that I will get this working without any kits...

I never experienced anything like this, but to me it seems a problem regarding something tha you are injecting with your analytes and accumulates in the injector. To start, why do you use methoxyamine with aminoacids? It should be used only to derivatize molevules with a keto group (e.g. carbohydrates)

I have much better results with amino acids (except arginine can't do it) with ethyl chloroformate and a 1701 column. Silylation hasn't worked well for me on most of the analytes I've tried it on (acrylamide, mononucleotides).

I should have remembered to mention that methoxyamine in combination with MSTFA is used as standard derivatization for plasma samples in our lab and I was prepping standard samples with the same protocol. However, it is hard to figure out how could methoxyamine cause this issue but I`ll give it a try and test if this problem still appears if I only do silylation.

I tested with two amino acid standards with MSTFA only and it seems to be working now! I did three repeated injections from both samples without loosing any signal. I also tried adding methoxyamine in pyridine to the same vial and lost signal after it. I just wonder what is the reason behind this problem? I´m just adding methoxyamine in pyridine and after it I´m losing amino acid signal.

Methoxylamine is for aldehydes and ketones such as sugars or ketoacids. Why is is used for amino acids?

After a lot of troubleshooting it all came back to my needle wash solvents. I was first washing the needle with hex and after it with MeOH After changing to EtAC it seems to be working again!

Thanks for all!

That makes sense. silylation reagents and their derivatives get hydrolyzed by water or any protic solvent (alcohols). Ethyl acetate, acetone, or acetonitrile would be a good choice for a rince solvent.