Background: Bile acid assay online SPE setup with 1290 and 1260 pumps and 1290 II multisampler on a 6470B. 3ul injection from vial inserts with multiwash using IPA for 10 seconds for seat and needle wash and then starting mobile phase for 10 seconds for seat and needle wash.
A new issue arose this week. Approximately 1 out of every 5-10 injections is "failing" and looking like it didn't inject the full amount of sample (very low peak areas for all compounds, but not as low as blanks). When rerunning the exact same line in the worklist file with a "2" added to the end of the data file name, sometimes the peak areas will return to normal. Specifically looking at a known amount of internal standards added to every sample. In all other samples, the peak areas for these compounds will be 30-35k. Then in the "failed" runs the peak areas will be anywhere from 50-2000 for these same compounds. But on reinjection from the same vial, will get 30k peak area.
troubleshooting steps so far:
- there is definitely enough liquid sample in the vials - 100ul in 250ul inserts and default offset
- peak areas are normal on reinjection (sometimes takes 2 times reinjecting)
- not likely something inherently wrong with the sample
- pressure curves are identical between "failed" runs and good runs
- likely not from sample debris blocking the needle
- no pattern in the reinjections - i.e., for one sample, first injection had peak areas of 2000, second had peak areas of 50, third had peak areas of 30,000.
- sample isnt concentrating from evaporation on the autosampler
- Tune is good - abundances in positive unit dropped by half from 1 month ago, but negative mode unit abundances are near same, and vast majority of compounds in this assay are in neg mode <500 m/z. Cleaned the source and examined nebulizer with magnifying tool- all good.
Any ideas on what else could be causing this? Last week, we were testing 1ul vs 3ul injections and the ratios looked spot on. So if there is suddenly a problem in the metering device, how does one test that?
Thank you for any suggestions!