HPLC of active compounds in medicinal plants

[Clayton]

does any one have any experience with the extraction of and HPLC determination of active compounds in medicinal plants

for example Rosmarinic acid in Basil

[kiknos]

i am also very interested in this. hope someone here will kindly give some suggestions.

meanwhile, i am also interested in how to get(separate and collect enough) impurities from crude pharm. for identification. any one has done that?

[Uwe Neue]

While we have never tackled your specific problem, we have looked at generic methods for the extraction of compounds out of various matrices. Our typical issue to find an analyte in a blood sample, but ocaasionally we do french fries too

For the extraction and clean-up of your sample, I suggest a mixed-mode anion exchange packing for the sample preparation. With a bit of homework (instructions on how to do this in detail can be found on the Waters website under Oasis), you should find a regime with a reasonably clean sample that can then be analyzed by HPLC.

Assuming that your rosemarinic acid is an acid, and that it is available as a standard, you can do an exploration of the specific elution conditions required to elute it from a mixed-mode anion-exchange SPE device by varying the concentration of the organic and the pH. The protocol to do this takes no longer than 15 minutes of SPE work. Once you know the specific elution conditions, you can then apply them to an aqueous extract of the basil. The subsequent HPLC step is then used to remove remaining interferences with the usual HPLC method development techniques.

We apply such protocols routinely to many other complex samples.

[kiknos]

many thanks, though i am not sure whether the suggestion is for this topic or some topic else?

[Uwe Neue]

This was meant for Clayton.

Kiknos:

With respect to getting impurities from pharmaceuticals for identification, we have used preparative chromatography. Essentially, you develop a method (does not need to be perfect) that separates the impurities from the parent compound. Then you inject a LARGE amount the compound and collect fractions. I got an example in my book on "HPLC Columns" on pages 190 to 193.

You get the best results for late eluting impurities. Therefore it is often worth to do more than one method, say change the pH from 3 to 10 and similar tricks.

[remintola]

"Melissa leaf was ground to a fine powder (400) and 1.000 g of the powdered drug
was refluxed with 50 ml of methanol in a Soxhlet extractor for 3 h and evaporated to
dryness. The residue was dissolved in methanol, filtered and diluted to 25.0 ml with
methanol prior to analysis (solution A).
For sample pre-separation, 0.1 ml of solution A was diluted with 0.4 ml of methanol
in an Eppendorf tube and 0.2 ml of tetrachloromethane was added to give a clear solution.
Adding further 0.3 ml of distilled water to the solution causes the formation of a
two-phase system. The tube was shaken, and centrifugation (10 000 rpm, 10 min) was
used to obtain a sharp interface. The upper, water-methanolic layer was separated and
injected into the HPLC system."

from, "ROSMARINIC ACID – AN IMPORTANT PHENOLIC ACTIVE
COMPOUND OF LEMON BALM (MELISSA OFFICINALIS)"
1Tóth, J. – 1Mrlianová, M. – 1Teklovia, D., et.al.
_____________________________________________________________
see:http://www.fpharm.uniba.sk/Dokumenty/Ac ... A1%20M.pdf

[kiknos]

Kiknos:

With respect to getting impurities from pharmaceuticals for identification, we have used preparative chromatography. Essentially, you develop a method (does not need to be perfect) that separates the impurities from the parent compound. Then you inject a LARGE amount the compound and collect fractions. I got an example in my book on "HPLC Columns" on pages 190 to 193.

You get the best results for late eluting impurities. Therefore it is often worth to do more than one method, say change the pH from 3 to 10 and similar tricks.

many thanks for your comments, Uwe!
and wish you have a very auspicious Year of the Rooster! (as we are now celebrating the Chinese New Year...)

by the way, where to get your book? its name and contents? thanks!

[Uwe Neue]

Happy Year of the Rooster!

My book is available through Waters, or you can purchase it at Amazon.com.
The exact title is "HPLC Columns, Theory, Technology and Practice", author Uwe D. Neue, published by Wiley-VCH.

[kiknos]

Happy Year of the Rooster!

My book is available through Waters....

Thanks, Uwe! So the book is certain product available in Waters? now i fail to load on www.waters.com, so i can't do the searching.

[kiknos]

i found it in Amazon. Too expensive for me

hope later i can find it in the laboratory here in Shanghai.

anyway, thanks, and i believe the book is very practical tool.