HPLC/MS/MS quantitation with 2000 QTRAP: signal instability

[chern_es]

Dear colleagues,

Please, answer, can 2000 QTRAP's signal be stable in time? What should we do for reaching this?
We perform quantitative analyses (bioanalytical measurements), and we have to do several calibrations between out samples because our signal is not stable. We know that the problem is not our HPLC system.
For feeding the MS instrument we use 99.999% nitrogen from gas tanks. We are not sure that the quality of this nitrogen is as specified, but we don't have an opportunity to test it.
Some colleagues say that nitrogen shouldn't be used for CID (as we use), and suggest to try argon, but in the instrument manual it is written that CID gas should be 99.999% nitrogen.
Some other colleagues advise to buy a nitrogen generator. But we are restricted in money and we can't risk and buy a nitrogen generator before we are sure that it would really help.

Please, help us with your kind advise.

Best regards,
Elena

[lmh]

Nitrogen generators provide a very low quality of nitrogen compared to what comes out of a cylinder. It's not suitable as a collision gas. Nitrogen generator gas should only be used as a drying gas/nebulising gas in the ion source, and nothing much else.

If you have varying peak areas in MS, there are many possible reasons, many of which may be nothing to do with your instrument, but have more to do with the sample. For instance the ion source may be becoming dirty with time, or there may be components of your mixture that coelute with the analyte of interest and change its ionisation efficiency.

You can keep the instrument cleaner by doing some sample clean-up before injecting, and by diverting sample to waste (if you have an appropriate valve) for the first minute or two of a run, and for the end of gradient runs where you are washing the "dirt" off the column. If you have a coeluting cosuppressor, unfortunately there may be little you can do, except use a proper internal standard to compensate.

But of course you are right to try to exclude instrument problems.

Good luck!

[chern_es]

Dear Imh, thank you for your reply.

Nitrogen generators provide a very low quality of nitrogen compared to what comes out of a cylinder. It's not suitable as a collision gas. Nitrogen generator gas should only be used as a drying gas/nebulising gas in the ion source, and nothing much else.

In the instrument we have there is one inlet called "Curtain gas", and from this inlet nitrogen is fed into ESI source as a curtain and into CID chamber as well. Our service engineer advised us to buy such N2 generator as this one: model NM40Z, Peak Scientific, and in specifications of this generator it is written: "The NM40Z is a specifically configured Nitrogen Generator designed to operate multiple Applied Biosystems/ MDS SCIEX LC/MS/MS instruments, producing the required flow rates, purities and pressures for curtain, source and exhaust gas with one generator." So as far as I understand it should be suitable... However, the purity of nitrogen is not indicated... I should think about it more.

If you have varying peak areas in MS, there are many possible reasons, many of which may be nothing to do with your instrument, but have more to do with the sample. For instance the ion source may be becoming dirty with time, or there may be components of your mixture that coelute with the analyte of interest and change its ionisation efficiency.

The problem is that we have varying peak areas not only for our samples, but also for our calibration points which are injected from the same vials.
The angle of calibration curve changes in time. It can move up and then down and then again up... And if we inject the same standard sample in ACN from the same vial for few hours, the signal drifts as well... Our column is rather new and it shouldn't be a problem such as washing of any impurities from column.

You can keep the instrument cleaner by doing some sample clean-up before injecting, and by diverting sample to waste (if you have an appropriate valve) for the first minute or two of a run, and for the end of gradient runs where you are washing the "dirt" off the column. If you have a coeluting cosuppressor, unfortunately there may be little you can do, except use a proper internal standard to compensate.

But of course you are right to try to exclude instrument problems.

Good luck!

Yes, we didn't try diverting sample to waste yet, and we should do it. As for the internal sample - we have performed some experiments, and normalization to IS peak area didn't help to eliminate the problem of signal instability completely (however, it helps to some extent).

Thank you again for your help.

Elena

[dharbour]

Nitrogen generators from Peak produce high quality nitrogen (used successfully on an API 3000) however, I would advise against buying one unless you can't stand using nitrogen cylinders. The problem that Peak generators have is that the filters within the system either break or need to be cleaned quite often (as often as twice a month) leading to continuous breakdowns. I haven't tried any of the other nitrogen generator manufactures but I might give them a look if you are looking for an alternative.

[lmh]

You may notice that the Peak specifications document doesn't tell you anything about the purity of the gas that comes out, merely that it is "high purity". I promise you, it won't be anything like as good as a cylinder, and if you've got a problem while using the cylinder, it's unlikely to be solved by moving to a generator. The main reason for using a generator is where you have an application (such as source drying gas) that eats cylinders too fast.

Nevertheless, if your instrument manufacturer says it is OK to use the Peak generator, they should know.

From my personal experience with Peak, I'd recommend you look seriously at alternative manufacturers.

[chern_es]

From my personal experience with Peak, I'd recommend you look seriously at alternative manufacturers.

Yes, now I also think that we should buy an N2 generator, but to use it only for the source, and not for CID cell. What other N2 generator manufacturer could you suggest?

[lmh]

We currently have one from Domnick Hunter, with which we're very satisfied. It's on a service contract, which is charged at a reasonable rate, and has given us minimal downtime. This was important to us, as we don't have a reliable house compressed air supply of adequate pressure, so we have to use a generator with compressor. The compressors are often the part that goes wrong.

I'd advise you to look around and ask for references from happy customers. Unfortunately very few people will have tried out multiple nitrogen generators, but if a company has no problem producing lots of happy customers who've been using their product for a few years, you should be safe.

If you choose not to have a service contract, check the prices of spare parts and allow for them in your costings.

Good luck!