I have been looking for articles about the quantification of free amino acids in cell lysate by LC-MS/MS. The method should not incorporate a complex sample preparation, and a trap and elute approach would be very interesting to be used. Google scholar as well as web of science always directs me to methods for peptide analysis. Has anyone a good idea?
You might try to use HILIC (hydrophilic interaction chromatography). Regarding the expected high concentrations in cell lysate you might probably try to "dilute and shoot".
Attached you find a publication of mine as an example. Hope this helps...
Reverse phase HPLC, aqueous methanolic extraction followed by MRM quantification does well for us too.
http://www.phenomenex.com/Products/AminoAcidDetail/EZfaast?returnURL=/Search
look at this site to see if it is useful for you.
Thanks @ Narong! Well, the EZ-Fast Kit ist not really an option for this application. We even have it, but I did not give a thought about it, honestly. One reason is that the sample volume for this application is less than 1 microliter. Depending on the concetration we may dilute the sample, however we don't want to derivatise anyway since the amino acids are only a model for other metabolites in the cell lysate.
But thanks anyway!
In most cases you'll find articles with the derivatization step in order to improve the sensitivity of the method. There are not many articles for free AA detection in biological fluids but try this one:
http://www.ncbi.nlm.nih.gov/pubmed/20038084
LC-MS/MS is by far the best instrument for AA determination either with or without the derivatizattion step.
Currently I work with the EZ:faast kit that was mentioned previousely, which serves very well for my application in AA detection in plants using LC-MS/MS. 100 microliter is ideal volume for me, no saturation is observed, but you may always dilute the sample. I tried to detect AA without derivatization and it gave satisfactory results when at least 2 transitions were chosen. Plant matrix is quite complex, so even though derivatization is not indispensable when using LC-MS/MS, we stuck to it, with good results.
If you have some questions, please do not hesitate to contact me and I'll try to help.
I don't derivatise so far! As I mentioned I would like to avoid that, but if I can't find a different solution I may have to adopt such a kit. However I don't want to limit my approach for only amino acids, we may want to search for other metabolites also. The total volume of the sample is below 1 Microliter. Dilution up to 5 Microliter is possible (maybe more if we are more sensitive), But the concentrations are not very high anyway (less than 1 ng/mL). Currently we are running a amino acid method by direct injection without derivatisation out of dried blood spots. However this works fine, but now I would like to improve this setting by using a trap column in order to analyze the cell lysate. Using the trap would also allow me to dilute the sample more and to pre-concnetrate it on the first column prior to separation. I think the Zic.-HILIC is a good appoach for that.
You can find different methods in Alterman, M. A., & Hunziker, P. (2012). Amino Acid Analysis: Methods and Protocols Methods in Molecular Biology, v828, that you can to adapt to your needs, but I don't know any method using trap column.
It seems you have three way to go: HILIC, reverse phase and Carbon. Zic-HILIC is probably one of the best HILIC colums. The two other phases require the use of ion pairing agents that in some cases can contaminate the MS. In case of using HILIC the amino acids (if you want an important retention) should be injected in high percentage of organic solvent, so you would have to dilute your sample or evaporate and the reconstitute in organic solvent with possible solubility problems.
Just a thought: ion exchange with volatile buffers using perhaps a low-capacity ion chromatography column so buffer concentrations can be low. A reversed phase trap should be very effective.
Christian, Waters provides a HPLC and UPLC-MS based AA analysis kit: AccQTag(TM). It looks good to me, but I have never tried. You can contact Dr. Claudio Pasqual ([email protected]), who certainly can give you some details. In addition, ABSciex also provides a kit, aTRAQ (TM) , LC-MS/MS based. Maybe you can explore this as well and let me know how good it is. ;-).
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