My LC–MS isn’t behaving! Where do I start?

Instrument manufacturers try to convince us that mass spectrometry is just another detector. Most of us who work with LC–MS know that’s simply not the case - they can be maintenance intensive, unforgiving, and generate complex information. When they’re not working it can be difficult to work out exactly where the problem lies. Here’s some advice to point you in the right direction:

The first step is working out if the problem is related to  method/sample or if it’s an instrument related fault.

1) Establish a benchmarking method - a simple, quick method that you know works 100% reliably every time. Five replicate injections of a solution of prednisone onto a short C18 column are ideal. Generate a set of data when the instrument is working well and use this to refer to when it isn’t working. Switch to the benchmarking method when you get a problem - if the benchmark works then the problem is related to something with your method/samples and not a fault with the instrument.

If you find that the benchmark doesn’t work then the problem is with your system. Your system is made of two components: the HPLC bit and the MS bit. You need to work out which one of the two is at fault.

2) If the problem is related to retention times then it’s nothing to do with the mass spec. The mass spec gives information on the chromatogram AFTER it’s been generated by the HPLC. Any problems relating to chromatography such as wrong retention time, retention time drift and most peak shape problems are caused by the HPLC, so don’t waste your time troubleshooting the mass spec.

3) Some problems can be caused by a fault with either the HPLC or the mass spec (for example, poor repeatability, poor peak height). Prednisone is detected by both ms and UV detectors. Add a UV detector to the system and run the benchmarker. If results are poor on both UV and ms then it’s an HPLC fault.

If results are OK by UV and poor by mass spec then the problem is with the mass spec, so we’ll go through some basic steps in trying to figure out what’s wrong.

Rule here is to keep things simple and move on one step at a time.

4) Using the benchmark mobile phase perform a tee-ed infusion of prednisone in full scan +ve ion 350 – 370amu, using MCA or profile acquisition. Optimise the signal using normal tuning approach.

5) If you still have no signal at this stage, use a scan range of 50-500amu and look for any masses attributable to background/bleed/noise ions. If the whole baseline looks unnaturally flat, try cleaning the source.

6) If you see a signal for prednisolone, verify that this is optimal at c.359.2amu. If it isn’t, this indicates a mass accuracy problem so the ms should be recalibrated.

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