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- Posts: 5
- Joined: Mon Jun 25, 2018 10:36 pm
LC Settings: phase A=H2O, 0.1% formic acid; phase B=ACN, 0.1% formic acid; flow rate .3ml/min; column=CS-C18 (100x2.1) or EP-C18 guard column; 2-98% B on a scouting gradient. Analytes of interest= bile acids.
Starting with guard column and analytical column installed. Ran individual bile acid standards and was getting multiple peaks for each in MRM. Ran no injection blanks and found a reproducible RT peak for all transitions with precursor 391.3. Took off analytical column (left guard column on) and ran no injection blanks in MS2 scan and 391.3 peak was still there with 265 as a main large peak in the TIC. Took off guard column and installed a union and this produced a beautiful blank TIC <.5x10^2. Took off union and installed analytical column only, and got again a reproducible RT for 391.3 peak and 265 on the TIC. Tried changing Mobile phase B to 50:50 IPA:ACN with 0.1%formic and again got the same 391.3 peak at the same RT!!! with 265 as the main on the TIC. Changed to all new lots of original mobile phase in very clean glassware and this did not change anything. Today finally installed an EC-C18 analytical column (lightly used but naive to bile acids) and there is no 391.3 peak on extracted chromatogram from MS2 scan! I don’t know why this worked, but maybe ACN/formic and negative ion mode and CS-C18s don’t play nice together?
A few notes:
1. All runs after the standard injections were no injection blanks— therefore, this problem couldn’t be due to contamination from the needle injection (I.e., from the wrong septa, not enough wash cycles, etc.)
2. The abundance of 391.3 peak would start out high on the first run of the day and then decrease to a point where abundance would sort of plateau. And then increase again on the first injection the next day.
3. All components of mobile phase were HPLC or greater quality. When I remade new lots, every component was a new lot and the new mobile phase was all LCMS quality.
Hope this helps keep another poor soul from repeating this!