Chromatography Forum

Hi everyone,

I am performing ethanol analysis on postmortem tissue samples using headspace GC. From what I've been told (I'm new to this job), only every once in a while would there be some interference resulting in broad peaks/poor sample duplicates in tissue samples (usually liver, brain, spleen, etc). But now it seems like a majority of the tissue samples are resulting in poor duplicates. Usually the first sample may have little to no EtOH present, but then the duplicate sample has a huge broad peak.

We started running the tissue samples separately from the rest of the postmortem blood samples to try to narrow down the problem. The first few attempts has been to significantly lengthen the run time, but that has only helped on a few samples.

What other conditions might be contributing to this poor output for tissue samples? All other samples always run just fine (blood, urine, vitreous, plasma, etc.).

The tissues are homogenized with a potassium phosphate buffer. Is there a better sample prep that should be done or is this an instrument parameter/set up issue?

Any help would be greatly appreciated!

Hi

Well have no experiance of the samples you run but a few thoughts.

Is the "fluids" (blood etc) typically lower in ethanol content? Might be a "borderline" instrument setting causing carry over at higher concentrations. So please state your instrument settings.

Tissue work up. In headspace GC it is essential that the matrix (sample and stadard vials) is as identical as possible, otherwise the partion coefficient will differ either between samples and/or between samples and standards. So is there a possibility that the first tissue sample for example contains less phosfate buffer or tissue than the duplicate?

Do you use standard addition or external standards?

Again have not worked with those tissues but instinctevely standard addition comes to mind.

We do not currently do standard addition, but rather external standards. It is something we are looking into for the future.

The samples are inverted several times prior to pipetting, so I would like to assume that the phosphate buffer is distributed evenly.

I was considering centrifuging the samples prior to pipetting...any thoughts? If this is a consideration, what would be the standard RPM and time?

Thanks!

Hi

Generally any thing that "simplefy" the matrix is good, however one have to work fast and as closed as possible to avoid loss by evapouration.

One thought that hit me, what is the pH of the phosfate buffer and what is the pH of the final sample solution?
This is a longshoot and my medical/anatomy knowledge is limited and old (like 15years), but at least liver tissue can contain esters and it will contain fatty acid ethyl esters (metabolites of ethanol) if the subject is a cronical drinker or has drinken "premortem".
Chemically speaking, if pH is either basic or acid you may get an hydrolysis of esters and form ethanol, this is known to have happnened in less complex headspace analysis when wrong pH has been used. As the second sample/duplicate likely is allowed to stand longer (30-60min while first injection is made?!) it might be possible that the reaction goes start/further there as it waits in roomtemperature(?).

Again that was a bit of a loongshoot/wild guess, glad to see that you consider standard addition for this complicated matrix.

A critical question is whether the huge broad peak that you see in the second replicate is ethanol, or is it something else ? The only way to be sure would be mass spectrometry - can you do this or get it done ?

Peter