how do you analyse fecal samples?

[aniket]

I know this is pretty dirty, but i am working in Pharmacokinetics, lab, and for a molecule my boss wants me too find the conc. in rat fecal matter, it is very difficult for me t analyse these samples? would you all please suggest how to extract the frug from these dirty sample? i tried LLE bur got huge interferneces, will you suggest me some better cleanup procedure? any one has dirtier experience than this?

Thanks in Advance

Aniket

[bert]

You could try solid phase extraction after the liquid/liquid extraction.....

[aniket]

Thanks for the reply, have you ever followed this procedure,

can you please explain in deatil?

Thanks in Advance

[dr_Pyrex]

I agree with Bert sugestion SPE is the best solution for you. You can find many application in internet:
www.waters.com
www.phenomenex.com
www.jtbaker.com

What kind od molecule do you want to determine?

[Uwe Neue]

We can work out a protocol that gives you good results, but I need to know more about your analyte(s). Do you have one or more ionizable groups on your molecule? Which one(s)?

[aniket]

Sir
i can not revel much about the molecule(it is a Discovery Project), but it is a very non-polar molecule,
our current procedure is

1-Collect the Rat Fecal Matter
2-dry the collected matter,
3-grind the fecal matter, to a fine powder,
4-dilute this fine powder to 10 volumes of water
5-mix well
6-spike the drug in this liquified fecal,

ealier i tried spiking the drug into the weighed fecal, and adding 0.5 ml extraction solvent?
but my caliberation failed beacuse of rwicw because of igh interference,

so i tried the above mentioned procedure,

am i following a correct procedure?

how do you spike your caliberation stanards in to a solid samples?

how do you deal in your lab with such samples?

can we homogenise te fecal matter, (my lab attendent wont like this for sure)

Thanks for your Time Mr.Uwe

Regards
Aniket

[Uwe Neue]

You are starting with the right approach, but in order to truly copy an extraction, you need to homogenize your standard with the fecal matter. Next, I think centrifugation would be best to get rid of the solids.

Since your drug is soluble in water (you are able to spike it), it is possible that the recovery in the centrifuged water is high. I would load the supernatant water onto a conditioned solid-phase extraction cartridge and then follow standard protocols to clean up and elute.

This was where my question regarding the nature of the analyte came from. If the analyte has a basic function (an amino group), I would already in the extraction from the feces add acid, best probably phosphoric at about 0.1 N. Then I would use a mixed-mode ion-exchanger, Oasis MCX, for capturing the analyte, followed by washing and selective elution. If your analyte is not a base, but an acid, then an anion exchanger can be used. I cone give you more details on the protocol, if you tell me if the analyte contains an acid function, or a base function, or has none of these features.

[AA]

Some comments,

You are on the right track, I have done this messy business in the past and hated it, but still, it needs to be done.
1. Spike the fecal matter before anything else.

2. You dry the fecal matter (is your drug volitile in any way?) Unless I knew for sure, i would be freeze drying the fecal matter.

3. You say your moleculer is non-polar, why would you expect to see the parent non-polar molecule come out of the ground fecal matter with water? I might expect some some polar metabolites (in the real samples) perhaps but not the parent if it is truly as non-polar as you have said.

4. Mix well you say, I probably would be sonicating the material (unground) to give myself the highest chance of extraction without the potential loss on grinding and one less step.

5. After mixing (sonicating) filtering or centrifuging would be in order followed by a further clean up (if required) of the extract and probably a concentration step. A 10:1 extraction ratio ends up with a pretty dilute solution, assuming the drug or its metabolites are there at all in the real samples.

6. Solid phase extration is your best bet for further cleanup.

No one ever said this kind of thing was easy!

[Victor]

One way to deal with such samples is Soxlet extraction. If you take your dried fecal matter and weigh out a portion into the Soxlet cup, you can spike this dried powder with your drug (as a fairly concentrated solution in an organic solvent if you want) and mix it thoroughly in the cup. Then you can extract the stuff with hot solvent. If you place glass wool on top of your sample, and if you choose the solvent correctly, you may be able to extract a minimum of the matrix from feces. Of course this method falls down if as part of your procedure that you MUST dilute the sample with water, but I do not see from a scientific point of view why this is essential.
All these methods are subject to the criticism that the spiked sample may not truly replicate the situation which arises when the drug is fed to a rat (e.g. it becomes bound with matrix compounds etc)-exactly the same criticism applies to your own process.
I am not saying that Soxhlet extraction will solve your interference problems and you may well still need SPE, but it certainly makes handling the samples easier-you just come back in a few hours and you have the extracted sample in a flask in an organic solvent, already filtered. Of course you can also use solvents which are miscible with water e.g. methanol, which you cannot use in l/l extraction.

[aniket]

Thanks for the detailed replies to all of the experts, i just wanted to know how do deal with such samples in your lab? and how do we spike our drug into a solid matter? for doing this dont we need to liquify the sampleS? correct me if i am wrong? (i consider me still as an amature) but i think this forum has bridged the gap between two countries, i am siting and typing question from india getting response from all corenres of the globe, it is amazing for me, thanks all for answering my silly question, hope this will continue in future,

Have A Nice time
Aniket