Dear all,

I am working on small peptides project. We have two peptides with molecular weights around 6000. These two peptides were soluble in water.

Objective is to validate the bioanalytical method in human plasma using LC-MS/MS.

Stock solution prepared in water and secondary dilutions made in 50-50 water-acetonitrile. Internal standard is also a very similar peptide.

Both analytes and IS were estimated in multiple charge state.

Precision and accuracy of the method is very good with recovery of peptide-I is 80% and peptide-II is 114%. This was tested at 3 different concentrations corresponding to low, mid and high QC level.

1. Question is ‘why did I get 114% recovery, what would be the probable reason?

Note: Extraction set and post extraction spiked set are at equal concentration.

Matrix factor: obtained by comparing analyte over IS response from post extraction spiked set with neat solution standards.

Peptide-I: MF is 0.5 and Peptide-II is 0.7

2. What could be the reason to get this result? Though MF is similar across the range of QCs, why I am not getting close to 1?

If I have to assume this as matrix effect, my PA batches are going well. In this case, can I use the method as is or I have to fix the matrix effect issue before I move further?

Appreciate your kind in puts.

Thank you\Raja