Hi all,

Our lab is trying to look at the differences in metabolic profiles of control and HD (Huntington's disease) patients.

We have a few fundamental questions on how we should quantitate the endogenous metabolites using HPLC- ECD (Electrochemical detector- a non-specific detector like UV).

After reading some papers about the same question, I found that background subtraction and standard addition methods may not work for us, as they could be tedious or may not allow good sensitivity. However, I think the surrogate matrix approach could be our best bet because it may be more representative than quantitating from standards prepared in the solvent.

a) So, what can be a good surrogate matrix for human plasma? Is 5% BSA in PBS, a good one to quantitate metabolites in tryptophan and tyrosine pathways? I have read that in order to use a matrix as a surrogate matrix, recoveries must be similar in plasma and the surrogate matrix. In order to calculate the recoveries, should I make 3 sets of calibration curves, one in the solvent, one in plasma, and the other in 5% BSA in PBS? and calculate the recoveries in plasma and 5% BSA in PBS with respect to the calibration curve prepared in the solvent.

b) We also run a pooled sample (made from all patient samples) in every batch to check instrumentation effects. Even after preparing a calibration curve in a surrogate matrix, is it still necessary to normalize with the average pooled sample's value from all batches?

c) My plan is to have a batch with this template. The idea is to quantitate samples from the calibration curve made in the surrogate matrix and check for quality control using recoveries of spiked pool samples. Would you change this order?

Equilibration injections (2)

Calibration standards made in 5% BSA in PBS (low to high) (7)

Reagent Blank

Extraction Blank

Samples (10)

Pooled sample

Spiked pooled samples (3 levels and 3 respective controls with the same spike volume)

Wash

We were wondering if any of you had the same questions and if you could help us out? Please let me know if there are any articles or books that may help answer these questions.

Thank you very much for reading.

Vamsi