Chromatography Forum

I tried post column infusion using LCMS for the first time and got the below chromatograms:





Would those tiny dips in baseline (e.g. a dip starting around 7.25 min in the second chromatogram) be considered as ion suppression due to matrix effect? The large dips are obvious, but to what extent should the suppression be, to be considered significant?

Blank milk extracts were injected. 100ppm cyanuric acid and 100ppm melamine were infused at 5 uL/min for each separate run. Is that infusion rate considered to be too low? It is obvious I am limited by the volume of the infusion syringe and analysis runtime, but what other parameters should I consider?

Pardon these elementary questions. I am not too sure what to expect, given my limited experience with LCMS. Thanks!

Wan,

I wouldn't consider the dip at 7.25 min in the second TIC to be significant ion suppression, unless it happens every time and/or gets worse over successive injections. But if it causes variability in your method, then obviously it is unacceptable and method adjustment for your eluting compound would need to be made.

100ppm at 5uL/min is a fine amount to infuse as long as it is a sufficient amount that can be detected on your HPLC-MS method. Consider the total uL/min flow rate.

I do have one concern: if you have an isocratic method, you have to be especially wary of late eluters from your matrix off of the column. Your best bet would be to continue the injections back-to-back to see if the suppression/enhancement changes over time. Ideally you would want to investigate this for the entire time that you expect to have an analysis run (i.e. if you have an average of 35 samples, 8 calibrators, and 3 QCs, you would do the injections at least 46 times in a row).

Kerri

Thank you, Kerri

I am running a gradient elution based on FDA LIB4422 method, so I don't worry too much abt late eluters. I might try an isocratic method if I am trying to determine only 1 of those analytes, so I will try out what you mentioned if I do.

Even if the method is a gradient, it is still good practice to do the post-column infusion with multiple injections as I described. If you spend one morning/day performing this experiment, it may save you LOTS of time and troubleshooting in the future. Many times the method is perfectly sound and very late eluters do not interfere... but once in a while you will run in to problems. Milk is a very complex matrix.

Good luck!!
Kerri

I understand what you are trying to do, but your chromatograms leave me with a question.

What is one looking at when they look at the chromatograms?
I see no label for what mass or masses you are scanning although I do see what looks like TIC in the upper right hand corner.
I assume this is total ion count?
If so, what ions are you scanning in each c-gram?

If you are looking at cyanuric acid and melamine are you scanning a range of several AMU or are you scanning specific ions eg A- and B+ (or A+ and B+) or just A+ in one c-gram and B+ in the other....

Alp

Hi Alp,

Yes, those are TIC. Given that I am using a LCMS, I scan only for 128 and 127 for cyanuric acid and melamine respectively.

I am using a span of 0.2 as recommended. Should I be increasing that?

Thanks.

I think I would go about it a little differently.

I'd first optimize my system so I was getting the most intense signal for my analytes (while keeping the background low). As I am using ms/ms I would then see what fragments these ions gave and I would optimize parameters for a good parent/fragment pair for each compound.

Then i would run ms/ms mode, infusing ONE analyte while injecting the matrix onto the column. I would do this for both analytes separately.

If you cannot do ms/ms I would urge you to optimize for the "exact mass" of your analyes (the mass the spectrometer sees, as the system may be calibrated a bit off from optimal) and only scan that ion and not a span or range.

eg, your system sees melamine at m/z 127.06 then use that value and scan for 127.06. (unless operation of your mass spec requires a range, but all the mass specs I have used you can zero in on a specific mass without spanning a range.

Then repeat for other analyte.

I assume the analytes elute at different retention times? If so they shouldn't be co-eluting in your method so you would be right to infuse them separately in this case.

You want to get your analytes eluting away from interferences caused by all the junk in the milk you are injection. You'll need to work on chromatography for this.

Alp

Hi Alp,

Thanks! If I can use the LCMSMS, I will. Right now, I am only allowed to use the LCMS as per lab policy.

Thanks for your advice re LCMS. What I got with the MS is 126.90 and 128.0 and will used those for further analysis.

Yes, cyanuric acid and melamine are eluting at different times and I am infusing them separately.

Thanks for your advice!