Persistent issue with blank

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

11 posts Page 1 of 1
Hello everyone,

I have a serious, solvent-related issue with my LCMS. Since system installation, I ran 3-4 different methods, and I keep getting peaks in my blank (No volume injections). Initially I thought it was a contamination issue, and washed the system and column for weeks, but later it was pointed out to me that this is probably related to solvent purity.

I have LCMS-grade methanol, but used to have distilled water, instead of LCMS-grade water due to budget issues. I replaced the water with purified water from a Millipore machine and things got better. When I run blanks with no salt in the mobile phase (1 mM ammonium acetate, LCMS-grade), I now get no peaks. However, when I run blanks with 1 mM ammonium acetate, I keep getting really large peaks.

Any help would be greatly appreciated!
Which system are you using?

Can you post a no-injection chromatogram with the following information:

- Gradient
- M/z being recorded
I am using a triple quadrupole system, the gradient program is the following:
Time (min) %B
0,0 – 5,0 35
5,0 – 20,5 35->100
20,5 – 22,0 100
22,01 – 28,0 35

I monitor approximately 60 MRM, they are too many to mention them all, I have issues with approximately 10-15 compounds. Here are some examples: 345.1->109.1, 328.6->81.0, 443.2->109.1, 411.1->125.1, 401.1->271.3. It wasnt possible to embed a chromatogram in the post.
1) The compounds that you're measuring, have you recently injected them (for instance in a calibration curve or spiked sample)?

2) The blank peaks you're getting in no-injection runs, do they have the same retention time and ion ratios of the target compounds? Or in other words, would you identify those peaks as the target compounds?

4) Did you slice up the method in windows, monitoring each compound only in a window around its retention time?

5) Do the blank peaks go down in back-to-back no-injection runs? Do they go up after conditioning for a while and then running a no-injection?
1) I injected them a month ago

2) Yes, they have the same retention times and ion ratios of the target compounds, I would identify them as the target compounds

4) Yes

5) Yes, they go down in back-to-back no-injection runs and up after conditioning for a while and then running a no-injection

System contamination was the obvious and first hypothesis, however, I changed every piece of tubing, ESI probe and excluded the autosampler from the flowpath and the peaks were still present
Point 5, in my opinion/experience, strongly hints that you're continually introducing the compounds in the system. What happens during conditioning is that they build up on the front of the column - since the initial conditions won't let them elute - and then elute as the gradient goes up giving a nice peak (and larger the longer you condition).

I think you need to look for contamination in the mobile phase, especially the aqueous phase. A common source is the additive. I suggest to use a fresh, or at least different, bottle of ammonium acetate (and anything else you add to the water). Also make sure to take water fresh from your purification setup, in a recipient that is thoroughly rinsed with MeOH, followed by water.

While the additive is a likely source of the contamination, it can't be concluded by the lack of peaks if you don't use additive. The contamination might be still there, but perhaps it just doesn't ionize without the additive.

What you could do as a temporary 'fix' is to shorten both the initial and final conditioning of the method. Especially the final reconditioning seems a bit long, but I don't know the flow or column dimension.
Rndirk wrote:
Point 5, in my opinion/experience, strongly hints that you're continually introducing the compounds in the system. What happens during conditioning is that they build up on the front of the column - since the initial conditions won't let them elute - and then elute as the gradient goes up giving a nice peak (and larger the longer you condition).

I think you need to look for contamination in the mobile phase, especially the aqueous phase. A common source is the additive. I suggest to use a fresh, or at least different, bottle of ammonium acetate (and anything else you add to the water). Also make sure to take water fresh from your purification setup, in a recipient that is thoroughly rinsed with MeOH, followed by water.

While the additive is a likely source of the contamination, it can't be concluded by the lack of peaks if you don't use additive. The contamination might be still there, but perhaps it just doesn't ionize without the additive.

What you could do as a temporary 'fix' is to shorten both the initial and final conditioning of the method. Especially the final reconditioning seems a bit long, but I don't know the flow or column dimension.


Does the volumetric used to mix the mobile phase get used for any other part of the testing, such as making standards or sample prep?

I try to have a volumetric dedicated to each mobile phase I make and use it only for that mobile phase, to be sure no contaminates are introduced from other sources. Others in the lab usually get a stern warning if I catch them using my volumetrics for anything other that what I label them for.
The past is there to guide us into the future, not to dwell in.
It would be interesting to hear which kind of compounds you are measuring. Can they be found around the lab (for example: detergents, phtalates, perfluorinated compounds,...), or is the only possible source the analytical standards you're using?
I am measuring steroids and benzodiazepines, so the possible sources of contamination are the standards and samples
stdev wrote:
I am measuring steroids and benzodiazepines, so the possible sources of contamination are the standards and samples


I only had one course in biochem so don't know the answer, but could steroids come from algae growth in a water system?
The past is there to guide us into the future, not to dwell in.
As far as I know, this is not possible
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