Some negative iRT values are expected. Typically you would assign the value 0 as the iRT score of your earliest peptide standard that you add to your sample, and 100 to the latest eluting peptide. However, there often will exist endogenous peptides whose retention time happens to be earlier than that first standard peptide and those peptides will have a negative iRT score.
If you would like, you can send us your Skyline document and we can see whether anything looks suspicious. In Skyline, you can use the menu item:
File > Share
to create a .zip file containing your Skyline document and supporting files including iRT database and extracted chromatograms.
If that .zip file is less than 50MB you can attach it to this support request.
Otherwise, you can upload it here: