I want to detect TCA cycle intermediates, pyruvate, glyoxylic acid with GC MS, I have MTBSTFA, after derivatization, pyruvate, oxaloacete and glyoxylic acid cannot be detected, does anyone know how to quantify those metabolites with one method? I want to use GC MS bot LC MS
Hello,
Please read this Book Chapter by Professor Oliver Fiehn which has "all details" provided for 1 D GC-MS-based metabolomics where most of the TCA cycle intermediates (5-6 of them!), pyruvate, glyoxylate and other organic acids are easily quantifiable. This is one method, using one GC-MS instrument and one column (usually DB-5MS, HP-5MS, Rtx-5MS etc.) for 45-55 minutes. Simple ! : ) You need not develop any other methods for these basic/ generic compounds.
The Book Chapter is Metabolomics by Gas Chromatography–Mass Spectrometry: Combined Targeted and Untargeted Profiling: http://onlinelibrary.wiley.com/doi/10.1002/0471142727.mb3004s114/abstract;jsessionid=A22EBA30008C8506B27687EBACCE8A28.f02t02?userIsAuthenticated=false&deniedAccessCustomisedMessage=
You can read mor about GC-MS applications of metabolomics at his laboratory's website: http://fiehnlab.ucdavis.edu/staff/kind/metabolomics/structure-elucidation
And yes, he suggests the use of MSTFA and works well for all of us; however, you may use MTBSTFA as well- and may get similar results. Better stick to expert advice than anything else.
Hope it helps.
Thanks,
Biswa
thank you, sir, but I didn't find their result conclude TCA cycle intermediate, they have only pyruvate. And I have MSTFA and I have tried to derivatize with MSTFA but it didn't work.
Succinate, citrate, ketoglutarate, malate are there! What else you need? Did you refer the Chapter link I sent?
Try following the exact protocol, column it will work.
Can you send me your column,instrument, and GC-MS protocol and extraction and derivatization protocol by Email to [email protected] and then I can check for problems!
Hope it helps.
I think you may follow the extraction and derivatization protocol described by Roessner et. al., 2006 (Plant Physiol 142:1087–1101). I used this protocol to detect TCA cycle intermediates.
http://www.plantphysiol.org/content/plantphysiol/142/3/1087.full.pdf
Methoxyamine in pyrdine at 37C for 2h and then derivatize with MSTFA with TMCS, right? I will try this mwthod, thank you.
hi Xiaofang,
Could you please let me know it that method working? Are you able to extract pyruvic acid and glyoxylic acid now?
I need to do that as well.
Best regards,
Thao
I am sorry I didn't detect pyruvic acid and glyoxylic acid untill now. I tried many times with MSTFA, BSTFA, MTBSTFA and other methods suggested by other people.
I detect pyruvate and other TCA metabolites on a day to day basis in biological samples using MTBSTFA and/or chloroformates. You said that you cannot detect pyurvate post derivatisation with aforementioned compouds. One of the possible reason could be incomplete or no oximation of these a-keto acids and therefore, multiple products and cyclisation coould have occured and hence you were not able to detect them. Use anhydrous methoximine HCL and anhydrous pyridine and make fresh solutions although 2% - 4% solution can be stored in -20 for a month.
Alpesh Thakker have you ever tried MSTFA? I have MTBSTFA but haven't used it as I have a large amount of the MSTFA. I dilute my reagent 50% with pyridine. I too was having problems with pyridine methoximation. I think it was the anhydrous pyridine going bad that caused the reaction to fail.
Xiaofang Yuan
Hello,
Have you detected glyoxylic acid yet? If so, could you please help with the method.
Best regards
Maria
Maria Dolores Lola Camalle sorry, I didn't detect glyoxylic acid successfully untill now.
NMR is very commonly used for those intermediates. If you are using LC, is it HILIC LC?
Hi all, I could detect pyruvate and glyoxylic acid using GC-MS.
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