HPLC of Peptides
Posted: Wed Mar 23, 2005 7:49 pm
Hi!
Development and validation of HPLC-UV methods for peptides is new to me. I am finding a couple of difficult issues. First, I am having a problem with linear range of my peptide (incidently, about 3000 Da). At the low end of the of my linear range, say 10 - 75% of a 1.0 mg/mL target concentration, I lose linearity. The y-intercept of the slope is always above zero and usually outside the range of +/- 3% of the origin relative to 100%.
The response factor plot illustrates this very well with a downward trend of the data as the concentration decreases. Even when I base my linearity curve on a target concentration of 10 mg/mL, I see the same sort of behavior. I think this due to nonspecific binding of the peptide - glassware, stainless steel and peek tubing of the HPLC system, etc., but I am not sure. This makes determining a large linear range difficult and also makes determining LOQ very difficult as well.
Any ideas? Suggestions?
Second, I have been trying to determine response factor for several major degradation products. At first glance, it looks like the response factors are not the same and that a response factor ratio will need to be determined to accurately quantitate degradation/impurity peaks. I've been told that peptides don't behave like small molecules in this regard and not to establish response factor ratios. Sounds like bunk to me, but I want to hear some insights from analysts with more experience with peptides/proteins than I do. The degradants are all very similar - some only different by a sulfonated side chain on one of the amino acid residues, some by a cross-linking.
Any comments?
Many thanks.
Development and validation of HPLC-UV methods for peptides is new to me. I am finding a couple of difficult issues. First, I am having a problem with linear range of my peptide (incidently, about 3000 Da). At the low end of the of my linear range, say 10 - 75% of a 1.0 mg/mL target concentration, I lose linearity. The y-intercept of the slope is always above zero and usually outside the range of +/- 3% of the origin relative to 100%.
The response factor plot illustrates this very well with a downward trend of the data as the concentration decreases. Even when I base my linearity curve on a target concentration of 10 mg/mL, I see the same sort of behavior. I think this due to nonspecific binding of the peptide - glassware, stainless steel and peek tubing of the HPLC system, etc., but I am not sure. This makes determining a large linear range difficult and also makes determining LOQ very difficult as well.
Any ideas? Suggestions?
Second, I have been trying to determine response factor for several major degradation products. At first glance, it looks like the response factors are not the same and that a response factor ratio will need to be determined to accurately quantitate degradation/impurity peaks. I've been told that peptides don't behave like small molecules in this regard and not to establish response factor ratios. Sounds like bunk to me, but I want to hear some insights from analysts with more experience with peptides/proteins than I do. The degradants are all very similar - some only different by a sulfonated side chain on one of the amino acid residues, some by a cross-linking.
Any comments?
Many thanks.