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

Instrument manufacturers try to convince us that mass spec 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. 5 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 (e.g. 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 un-naturally 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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7) If you see the signal at 359.2 inspect the shape of the mass peaks. If they look unusually tailed or fronted or show inadequate resolution try re-tuning using the advanced parameters of your instrument. If this doesn’t resolve the problem, it’s time to vent and clean.

8) If the mass looks accurate, the mass peaks appear to be the correct shape, but it’s just that sensitivity is low then it’s time to clean the source.

9) Cleaning the source. If you’re going to all the trouble of venting the mass spec, don’t do half a job of cleaning the source. Take an extra 15mins to strip the instrument back as far as your manufacturers recommend – this should be almost back to the mass analyser i.e. the complete ion source and focusing lenses. Don’t skimp on the cleaning – take your time do it fully and correctly. Cut any corners here and it just means you’ll have to repeat the next day.

If you’re not comfortable doing this then it’s time you learned. Either read the manual, ask your service engineer to demonstrate it or get booked onto a training course. If you operate an LC-MS you need to be comfortable and competent at stripping and cleaning the source.

10) If you’ve done all this and the problem persists, then next step is have a chat with your service engineer. Many mass specs have software diagnostics that can allow an engineer to diagnose a problem over the telephone. Always try and chat to the engineer before booking them to visit – it may be something simple that you can fix yourself.


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