direct injection of biological samples?

[noam]

Hi,
I'm interested in injecting biological examples to LC systems (plasma, urine etc.).
I was wondering if someone had ideas for workable direct injections method.
I came across the "LiChrospher® ADS"(Merck), which sounds like a nice way to restrict access.
It seems like it wasn't very popular, and I wonder why?

[Bruce Hamilton]

ADS would seem to be SPE wearing different, and more expensive, clothes. Easier to process large number through an SPE system first.

My experience with fermentation broths is that ion interaction columns ( eg Aminex HPX-87H ) for sugars etc, and end-capped C18 columns for others, cope very well with biological samples that have been diluted and filtered or centrifuged, coping with 1000s of injections.

Other column chemistries, such as C8, are far less robust ( 10 - 20 injections, no rational regeneration process worked ).

SPE can be convenient for large numbers of samples, and liquid-liquid extraction, or even resin addition followed by centrifugation, are not too bad if your target separates quantitatively.

I suspect many biological fluids have direct methods for LC-MS applications for clinical and clinical trial samples.

Bruce Hamilton

[chhubert]

Lichrolut ADS is one of the restricted access media developed by Merck. Direct injection of plasma or urine into this media is able to remove large macromolecule whilst absorbing small molecules onto the media. After that, the small molecules trapped can be eluted with strong solvent e.g. mobile phase into analytical column for separation. In designing the system for direct injection, you may need a six-port switching valve and an additional pump for extraction (on-line spe). There are many publications using RAM for direct injection of plasma or urine and on-line clean-up. You may visit Rheodyne or Cohesive Technology for hints of system configuration.

[JMB]

Noam,

You could visit the Cohesive Technologies (developers of turbulent flow chromatography) website for ideas about this topic; they have done a lot of work on the separation of analytes from this, and other matrices.


www.cohesivetech.com

JMB

[SIELC_Tech]

Here is another Primesep approach for direct analysis of biofluids:

http://www.sielc.com/Technology_DirectP ... lysis.html

Regards,

Vlad

[Uwe Neue]

Noam,

What detection method are you planning to use?

[noam]

Thank you all for the information,
I would try to do more reading, though I was mainly looking for reasons why not to use a certain way- because that is the information that is less publicized.

I'm looking into the interaction of drugs with a mixture that represents the protein (suspected) targets in various biological conditions.
I had nice results (separating and identifying) my model mixture and I would like to compare it to the reactions in plasma and with cell extract (maybe urine later), to do so I would need to get rid of a lot of junk.
My detection is mainly UV and MS.

[Uwe Neue]

With UV and plasma, you will still have a ton of interferences with your drug. The restricted access packings exclude proteins, but not small molecules and peptides.

With MS, you got a better shot at it. Of course you need to divert the unretained peak to waste, and you will still have background interferences which are likely to affect your MS response. As long as you are aware that quantitation may be a bit shaky, it can be done.

[Bruce Hamilton]

I was confused, I thought the ADS came in different versions ( C2, C8, C18? ). similar to SPE. rather than just a way of knocking out large proteins.

I assumed that it was just the first column in series, and a multi-port valve effectively heart cut out the fraction of interest and dumped in onto the second column- in which case quite a bit of the other junk should also be lost.

If you have well characterised samples, it might be the best way to go, but the off-line simultaneous preparation of multiple samples using SPE, combined with a short analysis, might still be an alternative.

I need to hunt out a Merck Chrombook sometime and get up to speed, sorry for any confusion.

Bruce Hamilton

[james little]

I have done some work on plasma which minimizes steps in workup.

Put sample in 96 well plate, precipitate with acetonitrile, centrifuge, inject above pellet. Fairly simple,

see top topic at..

http://users.chartertn.net/slittle/

[HW Mueller]

If Sailor´s method works the restricted access should also do it, namely, when the analyte "towers" over all the gunk.
In my experience it appears that one can get overloading phenomena with the gunk, apparently even with the proteins (not sure). Certainly, one can have a complete plug (frit) with one injection if one is "lucky" enough to get blood from the "right" (usually catabolic) patient (mentioned that before). Also, we had cases in analyzing cortisol in blood where even a 3-step (also strangely called 3-dimensional) HPLC was swamped with dirt even on the third column (one injection). It all depends.