Hi everyone,
I'm doing metabolomics of placental tissue in my lab. I added 1ml of 80% MeOH along with 2 isotopic internal standards namely, phenylalanine and palmitic acid to my sample after grinding it in liquid nitrogen, followed by sonication. My concern is, I could not detect the internal standard at the LC-MS level.
If someone can help me out with this or suggest me some other protocol, it would be a huge help.
Thanks in advance!!
P.S.: I used 10 different serial conc. from 0.25-7.5uM.
Hi
You should add IS to the sample before starting to purify it. The internal standard is therefore "internal" because, along with the sample, it goes through all the processes (purification evaporation etc etc) that the sample undergoes up to reconstitution prior to analysis.
See FDA document with nice examples how IS works:
Evaluation of Internal Standard Responses During Chromatographic Bioanalysis: Questions and Answers Guidance for Industry
https://www.fda.gov/media/130451/download
Best regards
Tomasz
There are some reasons:
- your IS are more solube in fatty tissue than MeOH --> lost IS during extraction
- Suppression effect from matrices
Suggestion:
- Add IS just before injection, if you find that the signal of IS are closely the same in standard with mobile phase --> losing IS during extraction
- Add cleanup step during extraction to remove fatty substances - -> remove matrix effects.
Thanks to both of you, Tomasz Grabowski & Bui Van Hoi , for answering. Can you suggest me that which method is better for metabolite extraction for untargeted metabolomics, single phase or biphasic!!!
Hi
Im not expert in metabolomics so I can advice you in such specific case. Maybe one advice for you, Your IS should be similar by phys/chem prperties to your analytes. Only then internal standarisation make sense, and only then your IS will behaves like your analyte. This is why for example in case of drugs analysis in samples like blood or serum best Internal standard is labeled analyte. Then you can be sure that analytes and IS will responce in the same way for your purification steps and ionisation methods etc. It makes life easier if it can be implemented.
Best regards
Tomasz
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