Problem with Indole-3-carbinol chromotography

[kouroshh1]

Dear all,

I could kind of solve the problem. I tried the mobile phase solvent without any modifier. Since indole 3 carbinol can be converted into different products in an acidic condition so i though maybe it would be a good idea to run the standard in a non-acidic solvent.

I could still see 2 peaks however indole 3 carbinol peak intensity is much higher than a second peak (3,3'-diindolylmethane (DIM)). I do not know how can I completely stop the conversion of indole 3 carbinol into the condensation products inside a LC column even in an absence of acidic modifer.

In addition,I tired to run indole 3 carbinol in an acidic solvent at different temperature from 20 to 50 centigrade. By increasing the temperature I could see the DIM was a dominant product specially at 50 centigrade I could only see DIM peak (one single peak). So I am 100 % certain now that there is some column conversion problem.

If you have any comment I would appreciate it to hear.

Kourosh

[Tom Waeghe]

Maybe you might fare better with this compound at a higher pH.

http://onlinelibrary.wiley.com/doi/10.1 ... 201302/pdf

You could consider using a pH such as 8.2 to 9.2 using 10 mM ammonium acetate adjusted to desired pH with ammonium hydroxide.

The ACE UltraCore SuperC18 or SuperPhenylHexyl or ACE Excel SuperC18 columns can handle those conditions with no hysteresis and good stability at 40C or lower.

I would opt for a 50 or 75 mm length so you on-column time can be minimized using a 2.5 micron UltraCore column or a 2 or 3 micron ACE Excel column.

My thoughts,

Tom

[mattmullaney]

Wow, Kourosh,

Looking at Tom Waeghe's link again, it's hard to have much real hope in completely eliminating DIM to allow for non-interfered analysis of Indole-3-carbinol. Reminds me of what I did with DNPH-derivatized aldehydes...I had to treat standards and samples with like amounts of phosphoric acid to allow for a consistent conversion of each Z-form to E-form to allow for a better estimate of each derivative. Was a big pain in the behind...had to let all samples and standards sit for 24 hours prior to analysis.

I wish you well and will hold out hope that someone will have a better idea than me.

[Tom Waeghe]

Much more stable at higher pHs.

[mattmullaney]

Hi Kourosh, Tom,

Cool!

The data Tom kindly sent seems excellent news to me. Seems like the basic eluent approach is worth giving a whirl!

Best wishes to you both for a Happy and Prosperous New Year!

[kouroshh1]

Thank you Tom and Mattmullaney for your comments, links and information. I checked our column pH stability and fortunately it can be used at a pH from 1.5 to 10. So, I will run my analyte at the condition Tom suggested tomorrow and will get back to you very soon.

Happy new year too to everyone

Kourosh

[kouroshh1]

Dear All,

Today I tried running indole 3 carbinol through our LC-MS/MS system using solvent A: H20 + 10 mM Ammonium acetate (the pH was set to 8.5 by NH4OH) and solvent B: absolute methanol without any modifier.

Fortunately, I could see only one single peak. However, the intensity of the peak has reduced significantly (became 2000 cps) in comparison to the time I ran the analyte using solvent A: water without modifier and B: MeOH without modifier which the intensity was 20000 CPS in this case.

I am glad that now only one single peak can be seen but I will get problem with quantification so i really want to solve the problem regarding low signal intensity.

In addition, as I wrote above I didnt add modifier (ammonium acetate
) into MeOH because then adjusting of a pH in an absence of water didnt sound logical to me. Did I make a right decision that I didnt add ammonium acetate into my organic mobile phase solvent?

Can I also ask what do you also recommend to do to improve the signal intensity? My analyte is running in a positive mode of our MS system in a MRM mode.

Thank you in advance

Kourosh

[HPLC chemist]

You should have 10 mM Ammonium Acetate in both mobile phases otherwise you will develop an acetate or pH gradient. Adjust pH in the aqueous phase (since pH is an aqueous phenomenon) and before adding the organic solvent.

In addition most mobile phases are a combination of water and organic, like this;

Mobile A: 95% 10 mM Ammonium Acetate/ 5% Methanol
Mobile B: 5% 10 mM Ammonium Acetate/95% Methanol

Always use HPLC grade solvents (not absolute)!

[kouroshh1]

Sorry for using the wrong term. We are always using HPLC grade solvent.

The problem is that my organic solvent is 100 % Methaonl. So no water is present in my organic solvent that is the reason I couldnt adjust the pH while preparing my solvent. So, whenever i prepare my organic mobile phase solvent It is 100% MeOH.

That is the reason I didnt add ammonium acetate into my organic solvent. I hope I could explain clearly.

[mattmullaney]

Hi kouroshh1,

HPLC chemist is on to something. I have a different suggestion. The trouble is now not with the formation of multiple species, but with the fact that you're not getting enough ionization at this alkaline pH (at lower pH, where the conversion to DIM was happening because the system was in flux, ionization must be more readily possible).

Can you add to the eluent stream, post-column, some sodium or potassium acetate? This should help form an ion that will "light up" in positive mode via formation of an adduct.

Gee, Indole-3-carbinol is weird stuff to work with!

[kouroshh1]

your are right, this indole 3 carbinol made me so much trouble so far. I solve one problem then another one is popping up.

Unfortunately, we do not have the possibility of spiking sodium and potasium acetate into the post column. I will try to add ammonium acetate into 100 % methanol too,so it may help my analyte to be ionized better in a positive mode.

[mattmullaney]

Hi again,

Understood--HPLC Chemist's idea is sound and should work as well as a post-column addition of salt. Ionization process should be easier with more ammonium acetate present when the peak elutes.

Best Wishes for your trial!

[kouroshh1]

Thank you so much. I will get it a try and will get back to you all

[kouroshh1]

Dear all,

Today, I tried using ammonium acetate in both aquas and organic solvent (methanol) by adjusting the pH into 8.5 in both solvents.

Unfortunately, the peak intensity of an indole 3 carbinol became very low even lower than the time i added modifier into only aquas solvent. So my major problem at the moment is to improve the peak intensity. It seems to me that ammonia is stealing the protons and doesnt let my analyte to become positivily charged????

Another thing I may do is using of a shorter column. The length of the column i am using now is 25 cm so I am planning to use 15 cm. I was thinking maybe there is a less chance of condensation of indole 3 carbinol in a shorter column while i am using the mobile phase without any modifier.

If you have any recommendation I would really appreciate to hear it.

Kourosh

[mattmullaney]

Hi kouroshh1,

I am sorry to hear this news. The system just isn't basic enough to remove that very weakly-acidic proton on the indole-3-carbinol to help make it more ionizable.

Are you thinking of returning to the acidic mobile phase system with the shorter column? I suppose it's worth at least a try...I wish you well for it.