I am interested in obtaining metabolic profile of a serum sample (blood devoid of cells and clotting factors). I would like to run this sample on LC/MS on both positive and negative modes. My question is what kind of internal standards I can use in this experiment? As far as I have understood this, I can use any compound which is not present in serum. If I prepare a calibration curve from that compound, then I can use that compound as an internal standard. Also, if I do not include any internal standard and I normalize my serum samples based on say protein concentration per ml of serum. In that case, can I avoid using internal standards all together?
I think you can use a fixed amount of internal standard and compare the ratio of the internal standard to your compound of interest. The internal standard must be either a dueterated compound of the compound of interest, or another compound not found in serum with most similar properties with the compound of interest. You may not necessarily do a calibration curve for your internal standard.
Hi Vishesh, the two main reasons for using internal standards are 1) to correct for all procedural errors, such as losses due to extractions, absorption, dilutions etc, and 2) to correct for possible matrix effects on ionisation in the area of your peaks of interest. I'm sorry to say that the only way I know these two can be catered to is through the use of deuterated internal standards. Good luck
Two aspect should be considered:
internal std (istd) is generally act as two role:
1) the process of sample trearment, the recovery of istd should be similar to your target compound
2) the rresponses of istd and the target should be similar to eachother
Thanks a lot to everyone. So I now understand that choice of internal standard will depend upon target compound and as stated by most of you the standard should preferably be deuterated. I should have mentioned one more thing that I am interested in metabolomic analysis of sample. I really apologize for this. Metabolomics means study of all small/medium weight compounds present in the serum except for biomacromolecules (such as proteins). In that case I can not think of an internal standard and as far as I have understood the concept, an internal standard is essential for comparison LC/MS profiles from different samples.
Hi Clayton. i have an LC/MS equipment in central facility of our institute but unfortunately i don't know the make of system. I'll inquire about it and 'll let you know. also I am trying to understand what could be the best way to do the experiment. So again I apologize for this but I have not yet designed any experimental plan for this particular study.
HI Cristina. Thanks for the reply. I am reading your paper on similar problem and I'll come back to this forum in case I have any doubts.
Ok. its because some equipaments works with quantification by using label free samples. We work here this manner.
We use four types of quality control, pooled samples that is primarily used for batch corrections, synthetic samples with 40 or so chemical standards to measure drift in retention times, exact mass and intensity, NIST metabolomics standards for inter lab variability, and blanks. QC samples are run about every 10th sample so 40% of our analyses are for QC. When doing metabolic focused analysess, spiking with labeled standards can also be done.
If you are conducting metabolomic analysis are you using a data analysis software such as Simca p as this has built in internal quality control approach to analysing data for peaks of significance
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