[gingi85]

Hey All,

I'm am running some LCMS samples and I'm having trouble getting a consistent mass chromatogram (TIC). Injecting the same sample three times under identical conditions, I get an identical UV signal each time, but very inconsistent TIC signals -- both in terms of the absolute intensity of the peaks and the intensity of the peaks in relation to each other. See attached TIC chromatograms. (Ignore the difference in retention times. I am using separate instrument to control the LC and the MS and they start collecting data at slightly different times. The retention time for the UV peak was identical for all three injections.)

Conditions:

Channel A: Water
Channel B: Acetonitrile
Column: 4.6x50mm C18 3.5um
Flow rate: 0.5mL/min
Post-column modifier: none
Ionization: ESI

Any ideas?

[MOTOBALL]

If the LC-UV trace is identical (number of peaks/ relative peak heights/relative peak intensities), then the injection volumes are identical and the issue is in either the HPLC-MS interface or purely in the MS.

Inspection of the three TIC traces shows an immediate problem---you should NEVER see a trace that has flat-lined (i.e. flat, level trace with absolutely no signal, not even noise in large stretches).

1) your data acquisition parameters need to be reset so that the detector is picking up noise all the way across the TIC.

2) whereas UV is a non-performance destroying (of the analyte) detector the ESI-MS is very much a performance-destroying detector.  Each ESI analysis slightly reduces the sensitivity of the system, by leaving deposits on tuning lenses etc.

3) the MS system (ESI needle tip, tuning lenses, quadrupole rods) may need cleaning to restore optimal sensitivity.

4) the MS detector may need replacing.

Address (1) using just the HPLC flow (no injection) to detect the MeCN/H2O clusters; perform replicate (n = 3) injections to compare area counts.

Perform (3) if necessary to get at least your initial 30,000 counts.

Check (4) by performing the sensitivity test outlined in user manual.

Finally, send a check to...no, just kidding !!!!!!

PS. What is MeCN/H2O ratio ??

[gingi85]

Thanks for your response, MOTOBALL!

The TIC I showed are acquiring only acquiring data from 500-2000m/z, because that is where my analytes will be. Is that reason I am not seeing the noise that you were talking about? If so, to what value should I set those parameters?

Did you mean to inject the sample three times without chromatography?

My MeCN/H20 ration is about 30:70. That is the mobile phase composition at which the compound elutes from the LC column. I am considering adding 70:30 MeCN:H20 w/ 0.1% formic acid post-column to try to increase the organic content of the mobile phase and enhance ionization. Do you think that will give me a more consistent signal?

Thanks for your *delete me* The check is in the mail.

[MOTOBALL]

The TIC I showed are acquiring only acquiring data from 500-2000m/z, because that is where my analytes will be. Is that reason I am not seeing the noise that you were talking about? If so, to what value should I set those parameters?

Don't change your SCAN acquisition parameters (m/z 500-2000 in X sec), but do adjust the DATA processing parameters (minimum peak width, minimum peak area, and one other that I cannot remember (Peak Threshold ??)) to acquire low-level noise.  Try setting these while you scan m/z 150-500 in X secs-----N.B. do note the data processing params. before changing---it may only be the peak threshold that needs to be lowered to pick up the required noise.

Did you mean to inject the sample three times without chromatography?

No, I did mean with chromatography; however, to save time and to observe the base-line, you could initially do three shots without a column.

My MeCN/H20 ratio is about 30:70. That is the mobile phase composition at which the compound elutes from the LC column. I am considering adding 70:30 MeCN:H20 w/ 0.1% formic acid post-column to try to increase the organic content of the mobile phase and enhance ionization. Do you think that will give me a more consistent signal?

Since ESI signal is concn.-dependent I would not add a post-column diluent, UNLESS sensitivity is a real issue and the increased organic content may overcome the dilution effect.  In your case here, I would change from MeCN/H2O to MeOH/H2O as a first step; methanol is a weaker eluent, and you will probably need 40:60 rather than 30:70 to achieve the same R.T.  This should improve sensitivity considerably, and 0.1% formic acid would not hurt.

Keep us posted.

[thelastone]

I know this may seem stupid, but... Have you given enough time to your analysis? Maybe you are having incosistent TIC because there are some compounds that have enormous retention times? Have you tried for example doing one analysis of 45 min?

I know that sounds idiot, but when I started at HPLC I had that problem. : My analysis should have lasted 12 min but there were componds with Rt = 15 min. so I had overlapping.