Chromatography Forum

I am optimizing the LC-MS/MS parameters for PFAS compounds by infusion in method EPA533. Several of the method required MRM transitions have very narrow (and pointed) collision energy curves.

When the method is run, these same optimized MRM transitions lead to excessively jagged peaks (before smoothing). When the method is tested with varying degrees of Gaussian peak smoothing, these previously jagged peaks integrate to give unexpectedly high %RSD at all concentrations (not just near the DL).

Compounds of similar masses and structures within EPA533, having broader (more normal) collision energy curves, do not show the same jaggedness or instability before peak smoothing; therefore, I don't believe it is a source/spray issue.

Raising the cycle rate (from 12 to 20 data points per peak) and raising/lowering dwell time weighting hasn't corrected the issue.

What else can be tried to stabilize the signal from these particularly problematic MRMs?

Is NFDHA one of the problem children?
I found that using the alternate transition is much more stable.
I am using an Agilent 6470b.

PFBA and its isotopically labeled isomers are all problematic analytes, but surprisingly PFPeA and the longer perfluorinated acids are not as problematic. I want to point out that the problem is not intensity related. We see nearly a constant percentage of instability at all peak areas.

NFDHA is very sensitive to thermal breakdown at higher source temperatures, but otherwise no particular problems with it.

Is NFDHA one of the problem children?
I found that using the alternate transition is much more stable.
I am using an Agilent 6470b.

I know the NFDHA transition is sensitive to higher temperatures, but is there an alternate transition allowed under EPA 533, besides the listed 295 -> 201?

in method 533 section 9.3 allows modifications of MRM transitions. Section 10.1.2 allows for different product ions but does not mention precursor.
I collect for both the 295>201 and 201>85 transitions. The former does not meet QC requirements for reproducibility but the latter does. Both these transitions are listed in the manufacturer's guidance.
Only one transition is listed for each analyte in the method

Check with your accrediting authority as to whether they will allow it.

I see 295>85 listed in the draft EPA 1633 method as a confirmatory transition, but I haven't checked its optimized stability. I didn't think to look at 201>85 either, since that would be in-source fragmentation.

Thank you for the ideas.