Dear Jeannie Devereaux ,

You can analyze both types of analytes from the same sample. You can adopt the MPLEx method (biphasic) which gives high peptide enrichment besides metabolite extraction. If you are only interested in looking at tryptophan metabolism, then a targeted approach would be better.

Best of luck.

Some tryptophan metabolites - tryptophan, kynurenine, kynurenic acid retain well in C18 column and for sample prep - MeOH or ACN solid liquid extraction works well for brain tissues. I set up a method for tryptophan and kynurenine can be found here : Article Tryptophan Metabolism Contributes to Radiation-Induced Immun...

Thank you so much for you help

Hi,

For targeted identification or profiling of inflammatory cytokines in biological matrices, immunologic based assays like ELISpot or cytokine bead array would be more effective than chromatographic approaches.

The links below are useful for the subject.

Article Cytokine quantitation: Technologies and applications

Article A laboratory test based on determination of cytokine profile...

Although, if you are trying to observe quantitative differentiation at only a limited number cytokines, LC-UV systems may do the job. But the sample preparation steps should be able to make cytokines very specific, isolated and enriched for the LC detection. Otherwise, you can produce false-positive or false-negative results due to the simultaneous monitoring of too many interferences or sensivity problems.

On the other hand, you can easily establish the aminoacid analysis at HPLC or LC-MS even at GC-MS systems. At sample pretreatment protocols, you need to derivatize the Tryptophan with the chromophore moiety carrying reagents like OPA, or alkyl chloroformates after isolation from tissue and just before the injections. If you are investigating the total Tryptophan quantities at your samples, acidic/alkaline hydrolysis step is required prior to sample preparation since the free and bound amino acid content will be variable in tissue matrices.

Tryptophan derivates can be monitored by means of its UV, Fluorescence, and/or mass responses following these typical sample pretreatments...

Good Luck,

Emir...

Jeannie Devereaux,

I extracted neurotransmitters including serotonin from the zebrafish brain and estimated their levels of expressions using ESI-LCMS/MS. I used MeOH, ultrapure water and formic acid (or HCl) to extract the neurotransmitters. The results were encouraging except for acetylcholine (detected but too low to be quantified). If you keen, give a try. You can contact me if you need more details.

Best of luck!