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- Posts: 6
- Joined: Tue Dec 10, 2024 1:32 am
- Location: usa
I'm having a difficult time with a method to quantify lyophilized peptides.
The proposed method is to determine purity and to quantify.
I run a 35 minute gradient with water/acn each 0.1% TFA. starts at 95% h20, end at 95% ACN.
For purity the method is working well, but my quantifications always seem off.
To quantify I ran a 7 point curve at (ppm) 50, 75,100,125,150,175,200
When I run a check standard using my standard im spot on. The r2 is .998
However anytime I run a sample with a known value Im off by 10-20% or greater. Label claim says 10mg/ml and according to the curve im at 12-14mg.
The calculation I uses is to get the value as a percent is
(instrument ppm)(dilution factor)(diluent volume)/1000
diluent volume is the water I put in the peptide vial, the dilution factor is the dilution from the peptide vial to the autosampler vial.
To prep the sample I simply add I ml of hplc water to the lyophilized vial, dilute to the ppm range of the curve and inject.
When this didnt work I did a 2 point curve injecting 1ul of a 5mg/ml standard, 4.9mg/ml and then injecting my sample. For that run I was within 5% of what the label claim was. Im a bit perplexed though why a 2 point curve would be working better than a 5+point curve.
I have just found the below reference on quantifying peptides, but I dont 100% under stand what they are doing.
It looks like they are taking the peak area of the sample multiplied by the mg of the reference sample multiplied by 50 ml which I assume is the dilution factor for the sample, they divide that by the average peak area of the std. this is what i dont understand, what exactly are they averaging? is it multiple injections of the same standard? is the 100ml what they are diluting the 5mg reference std in? Why would the sample be diluted to 100ppm and the std diluted to 50 ppm?
5mg % Label Claim = [(PeakAreaofSPl)(mgRS)(RSPurity)(50ml) / (AvgPeakAreaofStd)(100ml)(5mg)] x100
I have been running other estd curves on this instrument for analytes like nicotine in a glycerol matrix, caffeine in coffee, etc and have not had any issues. Its just the lyophilized peptides that I cant seem to get an accurate result with .
The proposed method is to determine purity and to quantify.
I run a 35 minute gradient with water/acn each 0.1% TFA. starts at 95% h20, end at 95% ACN.
For purity the method is working well, but my quantifications always seem off.
To quantify I ran a 7 point curve at (ppm) 50, 75,100,125,150,175,200
When I run a check standard using my standard im spot on. The r2 is .998
However anytime I run a sample with a known value Im off by 10-20% or greater. Label claim says 10mg/ml and according to the curve im at 12-14mg.
The calculation I uses is to get the value as a percent is
(instrument ppm)(dilution factor)(diluent volume)/1000
diluent volume is the water I put in the peptide vial, the dilution factor is the dilution from the peptide vial to the autosampler vial.
To prep the sample I simply add I ml of hplc water to the lyophilized vial, dilute to the ppm range of the curve and inject.
When this didnt work I did a 2 point curve injecting 1ul of a 5mg/ml standard, 4.9mg/ml and then injecting my sample. For that run I was within 5% of what the label claim was. Im a bit perplexed though why a 2 point curve would be working better than a 5+point curve.
I have just found the below reference on quantifying peptides, but I dont 100% under stand what they are doing.
It looks like they are taking the peak area of the sample multiplied by the mg of the reference sample multiplied by 50 ml which I assume is the dilution factor for the sample, they divide that by the average peak area of the std. this is what i dont understand, what exactly are they averaging? is it multiple injections of the same standard? is the 100ml what they are diluting the 5mg reference std in? Why would the sample be diluted to 100ppm and the std diluted to 50 ppm?
5mg % Label Claim = [(PeakAreaofSPl)(mgRS)(RSPurity)(50ml) / (AvgPeakAreaofStd)(100ml)(5mg)] x100
I have been running other estd curves on this instrument for analytes like nicotine in a glycerol matrix, caffeine in coffee, etc and have not had any issues. Its just the lyophilized peptides that I cant seem to get an accurate result with .
