Chromatography Forum

Hello:

I'm validating a methodology for tacrolimus ointment, but the assays of finished product, I'm loosing about a 3% of tacrolimus. The tacrolimus molecule is hidrophobic and it's disolved in several waxes. To extract it from the waxes I use methanol and heat, but when I inject the solution to the HCPL, it always 3% lower.

hi simael

i am stuck in pretty similar state as you are.

i am dispersing the ointment, first in dichloromethane and then adding Acetonitrile.If extraction is your issue you may try ethanol.

I use a zorbax XDB C8 column. But the response is vvvvvvvery low.
Increase in concetration or injection volume leads to split peaks.

Have you tried normal phase?
I am more worried about developing the method for 'related substances'.

PS: My process guy has just informed that tacrolimus changes form in Acetonitrile.......

Hello Simael and Bokoon javascript:emoticon(':)')
Smile
tacrolimus tends to form tautomeric isomers when dissolved in aqueous mixtures of acetonitril or methanol! So the content of tacrolimus in your sample solution is lower than 100%. But you should see the tautomeric isomers in your chromatogram otherwise your HPLC-method is not able to elute the isomers. Can you describe your detailed HPLC- method?

Dear Simael;

Remember: Tacrolimus is a good API but for us he is terrible!!! But we will be champions because we are Chromatographers.

Tacrolimus Lost
1 - I do not saw, yet, loss at 3% for Tacrolimus without degradation process. It is common on Stability (or Pre-Stability) Studies. It this last case we can see the peaks relative to Tacrolimus Degradation (with mass balance).

2 - You can evaluate the tautomers (and other Degradantion compounds) from Tacrolimus, so, know if your loss in 3% is one Degradation of your final product. But...

3 - Is your sampling procedure good? Can you change it?

Tautomerism Challenge of Tacrolimus Compund
1 - Tacrolimus in presence of Acn, MeOH e H2O can "tautomerizate" with good possibility. But I saw acceptable analysis with Acn/H2O: normally peoples around the world use MF = Acn:H2O (60-40) to (40-60).

2 - It is possible otimize the tautomerism with THF solvent in the Diluent and the Mobile Phase composition, but correct evaluation of the cut-off will be very important!!

3 - The common content of each Tacrolimus tautomer is NLT ~5% and on API, too.

Response of Tacrolimus Peak Improvement
1 - Tacrolimus is a very difficult analyte to UV-HPLC analysis, because all chromophors present in its molecule have weak UV-absorption.

2 - We can improve the injection volume or the concentration but if you are using reverse phase HPLC normally the new Tacrolimus peak profile will be worst. So, use particle size at 3u, and column with acceptable retention capacity.

3 - Bad Tacrolimus peak responses are not any relation with the analytical columns.


Good Elutions,


Carlos Teixeira

hi

earlier the method was buffer(phosphate+ionpairing pH2.5)and acn. C8 zorbax XDB 150mm
Sample Prep in Mecl2/ ACN
Now i have got rid of Acetonitrile.

I am trying with water:IPA:THF(trial runs are going on right now). C18 luna 150mm @ 50°C. The chromatographic parameters are working OK, sample preparation for ointment is still a HUGE worry. Trying ethanol or ethanol/water mixture. Right now working with API.