If peak retention times decrease quickly even when using a newly packed pre-column after 10-20 samples, the protein concentration may be too high. If you find that after preparing samples from tissue or blood, the protein concentration is too high, you can repeat the protein precipitation procedure. After spinning the sample and removing the supernatant, add another volume of 100% methanol equal to the new total volume. Thus, the final concentration will be 75% methanol. It may be necessary to dilute the sample with carrier solution before injecting in order to maintain good separation of the front peak from the nitrite peak.