Liquid chromatography basics

[hts123]

Hi,

I have a question . I have a method A that I run on the lc. Using method A, I can get peaks for the standards but when I test the spike , I cannot get back the concentration . However , if I use method B, I can get peaks for the standards and also when I inject that same spike , I can get back the concentration.

I am confused . Why is it that one method can get back the spike and another cannot .

[Gerhard Kratz]

That maybe is a problem with the chromatographic conditions and it would be nice to get more information about your methods A and B, what buffer, what organic solvent, what column.

[Consumer Products Guy]

I agree: need significantly more information.

And (1) please confirm if this difference happens when the same sample preparation/same sample vial is used for the two methods.

(2) Are these two different methods run on the same HPLC system?

(3) Are different columns or different mobile phases used in these two methods?

[hts123]

Hi, yes.

The chemical is acetaminophen.

SPE extraction.

Mobile phase for one method is ACN and 0.1 % formic acid. I could get a linear calibration yet my extracted blank spikes got very low recovery .

Mobile phase for another method is 2mM ammonium acetate in MeOH and 2mM ammonium acetate in water. I could get a linear calibration too but the same blank spike that I had extracted could get good recovery when I use this method.

Both method uses the same LCMSMS and the same column.

Both method uses different flow rates which I had forgotten and will need to check again.

[Consumer Products Guy]

Acetaminophen in what matrix?

Acetaminophen at what levels in the sample? What I'm getting at is: is SPE or concentration REALLY needed?

[lmh]

If you're running exactly the same sample on two different methods, and in one you find the analyte, but you don't in the other, then the problem isn't how you prepared the sample, it's the method.
If you're quantifying by LC-MS then ionisation efficiency will depend on what else coelutes with your target compound, but in a typical MRM method you probably have absolutely no idea what's coeluting because your method will only detect the chemicals it's set up to detect - not everything else that happens to be hanging around.
Particularly, if a reverse phase method has poor retention, your target compound may coelute with a whole load of highly polar stuff that's basically unretained. Basically there aren't many alternative explanations if you're running the exact same vial on the exact same system, just with different solvents/gradient, because you know that both methods can detect paracetamol, because you can see it in your standard curve.
I'm guessing the buffer probably gives better retention. Certainly different retention! Good luck...

[James_Ball]

One method has ACN mobile phase, the other had Methanol and Water. What is the solvent in the sample vial? Is the same solvent in the standard used for the calibration? (are you using the same standards on both methods as well as the same extract?)

Is the first method running as 100% organic? If so I imagine there is a ton of interference passing through the column unretained along with the target analyte and you get ion suppression happening with the samples.