We've recently started using a single quad LC/MS system (Shimadzu LC/MS-2020, for what it's worth) for mouse plasma AA analysis (the 20 common EAA and NEAA). Pre-column derivatization seems to be ideal for this purpose, as we've gotten greatly increased sensitivity when using the Phenomenex EZfaast kit, but ideally I'd like to be free from kit prison for my AA analysis. When looking online, I've found a variety of AA derivatization protocols but not one that stands out as the standard. Is there any standard/common derivatization protocol for AA for use with LC/MS, or is it really as variable as it seems?
I'm quite new to using LC/MS, so any and all advice is appreciated.
EDIT: I am using ESI-MS, didn't even consider the remember importance of that until Karen mentioned it. Thank you both for the answers, they're extremely helpful!
Dear Pszczolkowski,
I think attached papers are useful for your request.
Best wishes, Homayoon.
Hello, if you have LC/MS with electrospray ionisation ESI so for more sensitive analysis, derivatization is used and next to commercially available derivatization reagents such as:
1. dansyl chloride (DNS),
2. 9-fluorenylmethyl chloroformate (FMOC-Cl),
3. diethyl ethoxymethylenemalonate (DEEMM),
But if you looking for low LOD so new derivatization reagents are designed specially for LC–ESI-MS, like p-N,N,N-trimethylammonioanilyl N′-hydroxysuccinimidyl carbamate iodide (TAHS) which provides very low limits of detection.
Hope this info will help you.
Good luck!
I highly recommended 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) reagent. This will react with both primary and secondary amines in few seconds at pH 9.0, however heating at 55 C for 15 minutes is recommended for complete reaction. SPE is not required and the by-products do not interfere with the detection of the amino acids. You can achieve the separation of all physiological amino acids in 10-30 minutes depending on the column you use with sub-pmol to fmol sensitivity. MeCN and Water are the solvents and also you can use use buffers (< 5mM).
I must sadly and repeatedly say that HPLC-MS is not the quantitative method at all. I have forced to ask the Shimadzu Co. when I have been employed to The University of Tokyo, Yayoi-machi, Bunkyo-ku, Japan in 2012. The answer has been obtained from the kind engineer that "MS is not the quantitative detector at all" (my unpublished information from an kind engineer of Shimadzu Co., 1-Banchi, Nishinokyo-Kuwabara-machi, Nakagyo-ku, Kyoto, Japan, via a telephone).
I do not understand the term "gold standard" at all. Determination of amino aids or their derivative such as PTH amino acids can only be able to be performed in the condition of high recovery rate. Therefore, I have presented quantitative analyses of protein and D-biotin by measuring recovery rate with HPLC-photometric detection (please see files; Lysozyme by RP-HPLC and Netherlands biotin).
By the way, I have previously presented the PTH-amino acid determination method by RP-HPLC-photometric detection (please see the file; PTH Roma RP-HPLC).
Dear Pszczolkowski,
I think attached papers are useful for your request.
Best wishes, Homayoon.
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