particulates in end sample

Discussions about sample preparation: extraction, cleanup, derivatization, etc.

10 posts Page 1 of 1
Hi all,
We are having some issues with particulates. We currently use a modified quechers extraction to analyze for 160 or so pesticides in potatoes (and other matrices, but potatoes are our main focus and where we see this issue the most). We extract with acetonitrile, dry with NaCl, clean up with MgSO4 and LC-NH2, solvent exchange into MeOH and filter through a PTFE syringe filter into an autosampler vial for analysis on both GC/MS and LC/MS. In the past, when we did our solvent exchange we would reconstitute with MTBE for GC/MS analysis then we would do another solvent exchange in the autosampler vial and reconstitute in MeOH for LC/MS analysis. When we did this final exchange, there would be quite a bit of white floating particulate in the bottom of the vial. Once we vortexed the vial, the particulate would gather into one solid mass which we would then have to re-filter into another set of autosampler vials before analyzing on LC/MS. This was very costly since we would need an additional set of autosampler vials (not cheap), another set of syringes and syringe filters (not cheap), plus the time spent (another hour or so). We have since attempted to run GC/MS analysis with MeOH as our sample solvent which appears to work for the most part, but now we have some sort of black particulates floating in the autosampler vials after a couple of hours. This disappears completely if shaken or vortexed, but we are still perplexed as to where it is coming from in the first place. It doesn't seem like ANY kind of particulates would be good for the instruments, even if it does disappear/disolve so easily. We have spent many hours troubleshooting this problem by eliminating one part of the process at a time. We still cannot figure out where this is coming from. We use Optima or better quality solvents, we have used different syringe filter brands, different conicals, different solvent brands. We are at our wits end. Any suggestions?
Hello,

I'm not sure if I follow your procedure correctly. Could you write the steps down that happen after extraction to acetonitrile in a list perhaps?

Why do you introduce a solvent exchange from acetonitrile to MeOH (or whatever solvent) for GC injection? Most labs that runs Quechers inject acetonitrile. Nevertheless, do these particulates form only in samples or also in procedure blanks?

For (RP)LC acetonitrile is generally speaking too strong to inject (depending on several parameters). Changing to MeOH will not solve this. If you have the sensitivity, simple dilution with water is the easiest. If not, a solvent exchange to mobile phase starting conditions + filtering is your safest best.

To summarize, I think that a solvent exchange from acetonitrile to MeOH is useless for your analysis.
Rndirk wrote:
Hello,

I'm not sure if I follow your procedure correctly. Could you write the steps down that happen after extraction to acetonitrile in a list perhaps?

Why do you introduce a solvent exchange from acetonitrile to MeOH (or whatever solvent) for GC injection? Most labs that runs Quechers inject acetonitrile. Nevertheless, do these particulates form only in samples or also in procedure blanks?

For (RP)LC acetonitrile is generally speaking too strong to inject (depending on several parameters). Changing to MeOH will not solve this. If you have the sensitivity, simple dilution with water is the easiest. If not, a solvent exchange to mobile phase starting conditions + filtering is your safest best.

To summarize, I think that a solvent exchange from acetonitrile to MeOH is useless for your analysis.


I would stick with Acetonitrile also. Less vapor expansion in the GC inlet and better for LC injection as I have less problems with injecting ACN into a mostly aqueous mobile phase than I do Methanol. Plus any extra step can add places for errors to occur. It sounds as you have something dissolved in the ACN that is not as soluble in the Methanol and is falling out of solution.
The past is there to guide us into the future, not to dwell in.
Sorry, I guess I didn't make it clear that we concentrate our samples. This is where the solvent exchange comes in. Our analysis is pesticide residue in many different matrices and we routinely have MDL's in the low ppb range and occasionally the ppt range. We also do small volume injection on both our GC/MS and our LC/MS. So our process is as follows:

Add 15mL acetonitrile to 10g (typically)sample
Add NaCl
Take aliquot
Add MgSO4, LC-NH2
Take aliquot (9mL typically)
evaporate to dryness with assistance of MTBE
reconstitute to 1.5mL in MeOH and filter for analysis

The particulates show up in all of our controls as well as the samples. Sometimes we have the particulates in our standards and sometimes we do not. It is unclear what the difference is in the ones that have it and the ones that do not. Our standards are matrix matched.

Does this make a little more sense?
ifqal wrote:
Sorry, I guess I didn't make it clear that we concentrate our samples. This is where the solvent exchange comes in. Our analysis is pesticide residue in many different matrices and we routinely have MDL's in the low ppb range and occasionally the ppt range. We also do small volume injection on both our GC/MS and our LC/MS. So our process is as follows:

Add 15mL acetonitrile to 10g (typically)sample
Add NaCl
Take aliquot
Add MgSO4, LC-NH2
Take aliquot (9mL typically)
evaporate to dryness with assistance of MTBE
reconstitute to 1.5mL in MeOH and filter for analysis

The particulates show up in all of our controls as well as the samples. Sometimes we have the particulates in our standards and sometimes we do not. It is unclear what the difference is in the ones that have it and the ones that do not. Our standards are matrix matched.

Does this make a little more sense?


It makes more sense. I just wonder how does MTBE assist in evaporation to dryness?

Is the situation that after reconstitution in MeOH and filtering, there are no particles in the vial but they start appearing later on? I've seen this happen before. Have you tried changing to a different (smaller) filter size? Could waiting an hour before filtering fix the issue?

I know there are a lot of modifications to quechers. I personally like to add water in the first step when i'm modifying a quechers method. The idea is that after salting out, stuff that you don't want/need in the acetonitrile phase (salts, sugars, starch,..) has a place to go to (next to precipitating). I am not sure about this, take it with a grain of salt. It depends of course if your samples are wet or dry.

Edit: Note that I don't have experience with the concentration factors you're applying. I dare to say it's high compared to common quechers procedures. I'm used to work with intakes of a couple of gram to ~10mL acetonitrile, without further increase of the concentration. Your procedure uses a factor 50-100x higher.
I have done the concentration step with Quechers before also, but stop short of dryness at about 500-700ul, then bring back to 1ml volume with acetonitrile. Less chance to lose more volatile analytes that way. Acetonitrile is compatible with both GC and LC so this makes a good final solvent without needing to go to dryness. Dryness is only mandatory if you are switching to a solvent with lower boiling point, which is needed for going from acetonitrile to ethyl acetate or methylene chloride or methanol.

If there are any impurities in the salts you are using that are soluble in acetonitrile but not soluble in methanol or ethyl acetate then they would appear in the final solution, even for the matrix matched standards.

As far as autosampler vials are concerned, if they are standard 2ml crimp or screw cap vials those are less than 1$ each so we don't consider using two for a process expensive. Syringe filters though can cost a few dollars each but still not expensive compared to what the test is normally billed for.
The past is there to guide us into the future, not to dwell in.
James_Ball wrote:
If there are any impurities in the salts you are using that are soluble in acetonitrile but not soluble in methanol or ethyl acetate then they would appear in the final solution, even for the matrix matched standards
I'm leaning this way too. The fact that the particulate only appears when going to a polar solvent, and that the particulate coagulates into a single blob, sounds an awful lot like lipids to me. Fortunately, this is easy to test. Once the blob is observed, evaporate to dryness under N2, reconstitute in hexane or chloroform, and analyze on GC. To avoid the particulate, control your solvent polarity or do a lipid extraction at some point in the process.

James_Ball wrote:
As far as autosampler vials are concerned, if they are standard 2ml crimp or screw cap vials those are less than 1$ each so we don't consider using two for a process expensive. Syringe filters though can cost a few dollars each but still not expensive compared to what the test is normally billed for.
This is absolutely true. Moreover, the vial septa/caps are more expensive than the vials by about 2-3x. If cost savings are crucial, use a new vial but reuse the cap between filtering steps. Alternatively consider using integrated filtering vials (Filter Vials by Thompson Scientific) if your solvent is compatible; the Filter Vial + cap may be cheaper than a vial+cap+syringe filter and will likely have lower co-extractables.
Remember that labor costs are always the highest cost of any procedure.

Remember that labor costs are always the highest cost of any procedure.

Remember that labor costs are always the highest cost of any procedure.
James_Ball wrote:
I have done the concentration step with Quechers before also, but stop short of dryness at about 500-700ul, then bring back to 1ml volume with acetonitrile. Less chance to lose more volatile analytes that way. Acetonitrile is compatible with both GC and LC so this makes a good final solvent without needing to go to dryness. Dryness is only mandatory if you are switching to a solvent with lower boiling point, which is needed for going from acetonitrile to ethyl acetate or methylene chloride or methanol.

When you concentrate but stop short of dryness during the blow down procedure, how do you measure back to 1mL accurately? We are using 15mL polyconicals for this evaporation step and our final volume of 1mL is critical to our calculations.


As far as autosampler vials are concerned, if they are standard 2ml crimp or screw cap vials those are less than 1$ each so we don't consider using two for a process expensive. Syringe filters though can cost a few dollars each but still not expensive compared to what the test is normally billed for.



We normally analyze 48 samples at a time and our final volume is split into 2 sets of autosampler vials to be run on 2 separate GC/MS (we look for ~120 compounds on GC/MS so these are split into 2 different methods on 2 different instruments to shorten run time and avoid as much co-elution as possible). After GC/MS analysis, one set of vials is then evaporated and reconstituted in MeOH for LC/MS analysis (where we analyze for ~80 compounds). This is when the particulates show up and we have to use another set of vials/syringes/filters. If the vials are $1 each and the syringes/filters are $3 each, we're looking at an additional $192 for 48 samples. This doesn't take into account the time spent on the additional steps (which can take a little over an hour depending on how many samples there are). So it truly does add up and is definitely an area that if we could figure this out it would save us quite a bit of money. And we all know how hard funding is to come by anyway!
Yes, it is different when running samples that are your own versus running samples in a contract lab.

If you can do the Quechers with Acetonitrile and inject that into both the GC/MS and LC/MS then you eliminate the need for a second vial and filter, and the need for Ethylacetate and Methanol as sample solvents. This should further reduce costs since you would be able to inject the same final solution in both GCMS and LCMS without any need to exchange.
The past is there to guide us into the future, not to dwell in.
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