I promised to write the final protocol for the glutamate analysis when I had and now I am ready to say that I can determine the glutamate content in almost anything (at least I think so).
Pretreatment:
- If the sample is solid then I will dissolve it in MilliQ water, or a phosphate buffer (I need to remember nor to use an acetate buffer)
- If I can see some solids in it I centrifuge the sample (app. 10 min 14.000 rpm in an 1.5 mL eppendorf tube, or more if the particle don’t sediment)
Protein precipitation
Next step is to get rid of the proteins in the sample. They need to go as they could be denatured on the column and thereby block the HPLC column. As advised by a biochemist I do a TCA (trichloro acetic acid) precipitation
1.TCA solution:
Simply dissolve 50 g of TCA in 35 mL MilliQ water. I then keep this a 4 deg.
2. Precipitation
- To 500 myL of the sample I add 125 myL of the TCA solution. If there is a lot of protein in the sample, it immediately turn milky and one can see the denatured proteins as a white fluffy stuf.
- The sample then goes to the fridge (or cold room) for 5 to 10 min.
- The samples are the centrifuged for 10 min at 14.600 rpm (max speed of our centrifuge) and the at 4 deg.
-400 myL is them transferred to a new eppendorf for next step-
Removal of lipids
The lipis also have to be removed as they would stick pretty tightly to my HPLC column.
- To 400 myL of the protein free sample (in an 1.5 mL Eppendorf) I add 400 myL hexane and wortex it.
-The again 5 min in the centrifuge to separate the Hexane from the water
-300 myL is then transferred to a new 500 myL eppendorf . This is now ready for the analysis.
The glutamat anlysis
The method I am using is a general method for amino acid analysis based on the old Edman degradation, ie. reaction of the amino acid with phenylisothiocyanate (PITC). I use the method published by Chao-Yuh Yang and Felix I. Sepulveda in Journal of Chromatography, 346 (1985) 413-416. This method has been used for food stuff in this paper: Daniels DH, Joe FL Jr, Diachenko GW: “Determination of free glutamic acid in a variety of foods by high-performance liquid chromatography. Food Addit Contam. 1995 Jan-Feb;12(1):21-9. and probally in many other papers.
Derivatisation
This is a two step process. First a evaporation with an ammonia source present and then the reaction with the PITC.
The evaporation
Here we use a drying solution of triethylamine (TEA) Methanol and water. One part of each, usually 100myL of each. I make a new solution for every experiment.
- The sample (usually 3 myL of around .5mM or 30 myL of around 0.05 mM is used) and 30 myL of the drying solution is mixed in a glass vial.
- Then evaporate the sample to dryness on a Thermo Reacti-vap set at 60 deg and with a stream of nitrogen flowing over the sample. (Usually this takes around 20 min).
The actual derivatisation
The derivation solution is also mader fresh for each experiment and contains 100 myl TEA, 50 myL water, 50 myL PITC and 350 myL MeOH.
- to the dry sample 30 myL of the derivation solution is added. It then reacts for around 30 min at room temperature (Probably i could cut down this time, but It works and in 30 min I do something).
- The samples are again evaporated to dryness (or almost, it is difficult to get the TCA away) on the Reacti-vap.
- They are then dissolved in 300 myL of the buffer A for the HPLC analysis.
- Finally they are filtered through a 0.22myM filter syringe filter into the final vial for the HPLC auto sample.
When dissolving the sample in the last step I get some white crystals forming. Of what I don’t know, but that is primarily why I filter them.
HPLC
So now we are now ready for the HPLC. I have access to a Dionex Ultimate 3000 equipped with a auto sampler, 6 different columns and all what the is of extras. The column I use here is Acclaim 120 C-18 Reverse Phase.
The HPLC Buffers
-Buffer A: Is a sodium acetate Buffer (NaAc) with TEA and Acetonitrille (MeCN). In details for 5L: 57,25 g NaAc is dissolved in 3-4 L of MilliQ water. To this 2,5 mL of TEA is added and the ph is adjusted to 6.4. 300 ml of MeCN is added and the volume i made op to 5L.
-Buffer B: 600 mL of MeCN mixed with 400 mL og MilliQ water.
The HPLC gradient:
0-1.5 min 100% A
1.5-11.5 min Gradient to 88% A
11.5-22.5 min Gradient to 52% A
22.5-27.5 5% A
27.5-39 min 100% A
Adnd that is all, I need to do to determine glutamate content 
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