Poor repeatability (10-time repeat) (UPLC-MS/MS)

Discussions about GC-MS, LC-MS, LC-FTIR, and other "coupled" analytical techniques.

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Dear all,

I am developing a method for the determination of some psychiatric drugs through UPLC-MS/MS. There is a good sensitivity and separation for my analyses. When I proceed to do a 10-time repeatability by injection the same standard in succession, the trouble appears: the peak area of some of analyses may drop dramatically (10-time or 100-time lower than other injection) randomly in one or two injection out of the 10. And the peak-area dropping analyses are not the same in the 10 injections. And my internal standards do not go up or down with my native compound standards which results in a poor repeatability of Native/Internal standard value as well as poor R square of the calibration curve.

I have confirmed it is not a problem of the mass spec or the liquid nitrogen because other colleagues have just tried their method and the repeatability is quite good (less than 5%).

Has anyone who has encountered this problem? Or any suggestions?

Thanks so much for your attention.


Kenny
Not enough information supplied to make a diagnosis (no method, column or conditions provided), but perhaps something simple such as inefficient column washing after each run or column fouling.

Also, it is called "HPLC", not "UPLC" which is a Waters Trademark. ("UHPLC" too, but they are all forms of HPLC).

You may want to start here to troubleshoot it; "HPLC Retention Time Drift, Change, Variability or Poor Reproducibility. Common Reasons for it." https://hplctips.blogspot.com/2015/11/h ... hange.html
Where you say the peak area suddenly in one injection drops by a factor of 10 or 100, but only for one compound, not for the internal standard, I panic slightly. Can I just check what's maybe a silly question, but is this a peak area taken from the processed results in tabular form, or is this a peak area that you've confirmed by looking at the actual chromatogram?

The reason I ask is that with a given set of integrator parameters, quite often the integrator will find not only the correct peak, but also little bumps of noise on either side of it. Sometimes it will, sometimes it wont. If the expected retention time in your method isn't quite right (or if the peak moves very slightly) then there is a risk that if the integrator happens to find a tiny noise peak, and it happens to be a little closer to the stated retention time than the real peak, then the software will tabulate the (tiny) area of the noise peak in place of the correct peak.

The other common integrator issue is that if there is a little blip of noise superimposed on the correct peak, making a tiny dip, the integrator may decide that this is a double-peak and split it, quoting the area of only part of the peak.

Sorry if these are obvious things that you've already ruled out.
The trouble you describe with integration algorithms is especially true for Waters MassLynx (i.e. TargetLynx), which the original poster seems to use...
If you are looking at unsmoothed chromatograms with just about any data system you can have bad integration. Sciex Analyst defaults to unsmoothed and if you don't use at least a smoothing of 3 even some large peaks will be split. Always review the integration even when results look acceptable.

If all the peaks are integrating properly, then you have to look at the mass assignment. If you are set to high mass accuracy instead of unit or wide then you might be drifting just enough that it filters out the peak instead of passing it through if you are using MRM.
The past is there to guide us into the future, not to dwell in.
lmh wrote:
Where you say the peak area suddenly in one injection drops by a factor of 10 or 100, but only for one compound, not for the internal standard, I panic slightly. Can I just check what's maybe a silly question, but is this a peak area taken from the processed results in tabular form, or is this a peak area that you've confirmed by looking at the actual chromatogram?

The reason I ask is that with a given set of integrator parameters, quite often the integrator will find not only the correct peak, but also little bumps of noise on either side of it. Sometimes it will, sometimes it wont. If the expected retention time in your method isn't quite right (or if the peak moves very slightly) then there is a risk that if the integrator happens to find a tiny noise peak, and it happens to be a little closer to the stated retention time than the real peak, then the software will tabulate the (tiny) area of the noise peak in place of the correct peak.

The other common integrator issue is that if there is a little blip of noise superimposed on the correct peak, making a tiny dip, the integrator may decide that this is a double-peak and split it, quoting the area of only part of the peak.

Sorry if these are obvious things that you've already ruled out.


Dear lmh,

Actually the situation happens both for native and internal standards. I did take the peak area in tabular form. But when I found that some data was far from the normal ones, I checked the integration of the peak and there is no problems for those peaks.

Thanks anyway. I appreciate your help a lot.

Best regards,
Kenny
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