(LCMS) LC-MS and LC-MS/MS Common Troubleshooting Measures

This article details a workflow of instrument areas to be checked when troubleshooting low sensitivity or signal-to-noise, with steps below each to expand the testing and troubleshooting procedure. The LCMS Troubleshooting Guide is also linked at the bottom of this article for additional troubleshooting steps and fixes.

1. Check the method file loaded - Is this correct for the assay? Is the MS acquisition windowed or acquiring throughout the run?
   • Check MS acquisition parameters are correct against previously acquired data, research, method packages, or literature, whatever appropriate.
   • Check m/z and transitions against the Compound Table within the Data Processing Parameters, and correct where appropriate. 
   • Open up all MS acquisition windows to the full runtime. If peaks have moved, check the mobile phases and column in step 3.
   • Redownload the correct method file and ensure the system is equilibrated in terms of backpressure, flow, oven temperature and source parameters.
2. Check the instrument parameters and status - Are all LC, MS, and source parameters as expected? Are all source parameters correct, such as gas flows, temperatures, and voltages? Are there any issues with pressure, such as inconsistent readings, pressure looping, pressure too high or too low?
   • Change each source parameter individually and observe if changing.
   • Check pressure readings from current data to past data - if pressure is high, try flushing with 100% organic/strong solvent at a high flow suitable to the pressure rating of the column for thirty minutes to remove any heavily retained compounds, checking the pressure after. If pressure is low, there may be a leak.
3. Check the column, mobile phases, and mobile phase lines - Is the correct column being used, and (if applicable) is the column selection valve in the correct position? Are the mobile phases correct? Are the mobile phase lines full or contain any air bubbles?
   • Swap between previous and freshly prepared mobile phases, if possible. If performance returns with old mobile phases, check solvents and additives individually.
   • Swap guard cartridges or precolumn filters to cartridges/filters from a new batch/lot, and/or swap to a previously used clean column or new column, if available.
   • Large air bubbles in mobile phase lines may result in air entering the pumps if not fully degassed. If air bubbles are in the pump, the pressure will 'loop' (increase slowly a few bar, then drop suddenly and repeat). To remove, purge the lines and pumps with IPA, followed by water, then back to mobile phases.
   • Large air bubbles in rinse solvent lines may mean the needle is not fully aspirating. To remove, place all lines in IPA and purge by pressing 'Purge' on the front of the autosampler, or in the LabSolutions software.
4. Check all connections, tubings and valves - Are all the connections properly tightened, with the correct fitting and matching ferrule, where required? Are all valves, such as waste divert, online trap or SPE, in the correct position?
   • Ensure all valves are set to bypass additional system components and flow is going to the MS. If there are additional pumps for online trapping, stop the flow and see if the MS spray is still present. If not, this and related valves need correcting.
   • Ensure all fittings are correctly made (e.g. the ferrule is not at the tip of the PEEK or stainless steel tubing, not overtightened or locked to the tubing, and is not warped in shape or visibly damaged) and leak free. Increase the flow rate of aqueous solvent (water) if working with low flows to make this more apparent, and reduce the column oven temperature if high above room temperature (40 degrees or more).
5. Check the injection made - Is this the correct vial/well, tray, and injection volume? Does the vial/well have suitable volume within? Is the needle stroke depth deep enough? Has the vial been weighed pre- and post-injection to ensure liquid is being taken?
   • Use a fresh vial, extract or tray, or reference material at a concentration where a response exceeds signal-to-noise requirements.
   • Check the needle depth is set appropriately. Default settings for 1mL, 1.5mL, 4mL, 10mL vials and microtubes is 47.0mm. Default settings for MTP/DWP 96 and MTP/DWP 384 is 48.0mm.
   • Fill a vial/well with LCMS grade water, weigh, inject 10uL, weight, record the difference, and repeat 3-5 times. 10uL water is equivalent to 10mg.
6. Check the MS spray - Is the capillary protruding 1mm or less and still visible? Is the spray cone visible? Is it consistently flowing without any sputters or interruptions? If it is not visible and the pressure allows, try increasing the flow rate?
   • Replace the capillary with a spare, if available. Ensure the tip can be seen, but no longer than 1mm.
   • Flush the capillary manually with 50:50 v/v water:methanol, 1% formic acid, to clean and see if any blockages are dislodged.
7. Check the chemistry of the compound - If this is a new method, check literature to see if the compound is suitable for the current LC and MS method. If the wrong columns or mobile phases are being used, it may be the compound is totally unretained, or too strongly retained. Additionally, it may require a different ion source type or additives to effectively ionise for detection.

If this has not resolved the issue, contact [e-mail] for additional support.

For continued troubleshooting, more guidance is available in our LCMS Troubleshooting Guide, linked below.