We are running an assay to simultaneously determine vitamin A, E, and carotenoids in human serum. Now we experienced problems with significant decrease in peak areas of most compounds. After discontinuing the analysis for a period of time, we found there were significant decreases in peak areas of most compounds: first carotenes and then retinol and tocopherols. All the changes seemed to be suddenly dropped to next step (I mean it was not gradual decrease), and then kept at consist level. We have checked the entire HPLC system, such as flow rate, flow cell, guard column, tubing, injection volume, mobile phase, lamp, and replaced with a new column. But we still could not figure out the problems, and the sizes of peak areas are still significant small than before. I am wondering some people could share their experience with us. I appreciate it. I can be reached directly at tjiang@jhsph.edu.

Regards,