Triclosan Repeatability and reproducibility

[nageshwar]

Hi to all,

I am trying to analyse triclosan for the related substances as per the USP monograph method. I have little bit doubts to carryout the test. The procedure says to prepare the test solution as follows:

Test Solution- Transfer about 2.0 g of Triclosan, accurately weighed, to a screw-capped centrifuge tube, add 5 mL of 2 N potassium hydroxide, and shake for 10 minutes to dissolve. Add 3 mL of n-hexane, shake for 10 minutes, and allow the phases to separate. Transfer the organic layer to a suitable container, add another 3 mL of n-hexane to the aqueous layer, shake for 10 minutes and allow the phases to separate. Transfer the organic layer to the previous extract, discard the aqueous layer, add 3 mL of 2 N potassium hydroxide to the combined organic layers, shake for 10 minutes, and allow the phases to separate. Transfer the organic layer to a suitable container, and evaporate with the aid of a stream of nitrogen to dryness. Dissolve the residue in 1.0 mL of methanol, and mix.
In the above said procedure how i can acheive repeatability and reproduciblity results and what type of apparatus can i use to dry the organic layer.
Please help if any body concern with the subject.

nageshwar

[danko]

I don’t think you need any apparatus for evaporation of the organic part of the described mixture.
Just direct a stream of nitrogen into the test tube, containing the liquid and give the process the time needed for complete evaporation to take place.
Regarding repeatability and reproducibility, there are slightly diverse opinions on the reproducibility matter. Some describe reproducibility as the ability to obtain similar (within the predefined acceptance criteria) results when test is carried out in different laboratories.
My idea of reproducibility is slightly different and it sounds like: The ability to obtain similar (within the predefined acceptance criteria) results when 2 or more samples (prepared independently from one another i.e. separate weightings, dissolving procedures, dilutions etc.) are analyzed following the same instructions/sample preparation procedure.
Whereas the repeatability is a simple repetition of the analytical part of the procedure of analysis (f. ex. 2 ore more injections from the same sample solution/vial).
So, the repeatability would show/document the analytical equipment related variation/uncertainty whilst the reproducibility would document the overall variation/uncertainty.
Please note that there could be (I’m almost sure that there would be) different opinions on the reproducibility matter.

Best Regards

[nageshwar]

Dear Dancho Dikov,

Thanks for your response.

I agree with you regard repeatability and reproducibility. However in the method the sample extraction, separation and evoparation is a long process and where I unable to get reproducible results. What I feel the results are varying based on the efficiency of extraction of impurities from the sample.

Thank you once again.
Nageshwar

[danko]

Hi again,

Maybe I didn’t quite understand your query from the beginning. I thought you asked how you could determine/estimate the reproducibility and the repeatability of the analytical procedure.
I realize now that you needed a second opinion on how the reproducibility could be improved.
I assume that the repeatability – as we agreed on – is OK (i.e. if you inject twice ore more times from the same sample solution you see very similar results).
If the assumption above is true, then the reproducibility, minus the repeatability, is the actual problem. That is to say the sample preparation needs more attention. As I see it, there are roughly speaking 2 potential sources of error in the sample preparation procedure, that are serious enough to influence the reproducibility of the results:

1. “Transfer about 2.0 g of Triclosan, accurately weighedâ€

[mohan_2008]

Nag -

You are basically performing a sequential extraction of the active (and/or impurities) from the aq.phase into the organic phase.

Using KOH, you are breaking up the sample (saponification) and dissolving the components into the aqueous phase. Sequential extraction with hexane is performed to concentrate your active/impurities into the hexane layer.

The concentrated hexane layer is then evaporated (by nitrogen flow) to obtain your concentrated residue, which is solubilized in methanol.

Procedure:

Use a PTFE centrifuge tube (say about 20 ml) -
Use a SHAKER for shaking purpose as manual shaking tends to be inefficient for this purpose.

1) Weigh 2.0 g (accurately) into the tube, add 5 mL of KOH and shake well to dissolve.

2) To this add 3 mL of hexane - Shake for 10 minutes. The phases will separate. You can see a distinguishing line between these phases.

Now, very critical step. Keep the centrifuge tube UNDISTURBED in a holder, and using a plastic pipette dropper carefully pipet out the top hexane layers into another Centrifuge tube or container. Leave a small layer of hexane intact with the aqueous phase and do not suck it out.

3) Add 3 mL of hexane again and continue step 2). Transfer the hexane layer into the hexane container (above). Again leave a small thin layer of hexane intact with the aqueous phase.

4) Repeat step 3) atleast four to five times to get a representative hexane extract.

5) There is no need to do a final shake up with the KOH since there is no reason to do so.

6) Remember all this time with hexane extraction, leave a very thin layer intact with the aqueous phase and do not suck out the aqueous phase.

7) Discard the aqueous phase.

Now there is a choice. You can dry-out hexane layer, solubilize in Methanol for your Reversed phase seperation.

9) Or/ make up your extracted hexane layer to a final volume say 100 mL. And perform a direct Normal phase method, with Hexane/IPA mobile phase.

Reproducibility shoudn't be a problem.

[Teodor]

For the evaporation step you can use an instrument like a dry evaporator, or a rottary evaporator. One company that makes these instrument is Heidolph.

[nageshwar]

Thanks to all for the best suggestions.

Nageshwar