Chromatography Forum

Dear Experts

We have tried to test Related Substances Test as per EP but unable to get RT or RRT of peaks mentioned in monograph .

Test conditions are kept exactly as per EP pharmacopoeia .

We used Column - DB-5 , 30 mtr length , 0.32 ID & 0.25 mm thickness.

Problem Faced :
1. Ref.soln a -- we get peak of Imp. B , Bupivacaine , Impurity E & Internal Std respectively ( meeting to EP pharmacopoeia )

2. Test Solution -- We get Bupivacaine Peak at retention time of Impurity E .( No separation between Bupivacaine & impurity E ) . Hence unable to calculate Imp. E in Test solution .

Could you please advise why this is happening or could you please send us the typical chromatogram of Related Substances test alongwith detail method of analysis .

Sunil Pandya

Dear Experts

We have tried to test Related Substances Test as per EP but unable to get RT or RRT of peaks mentioned in monograph .

Test conditions are kept exactly as per EP pharmacopoeia .

We used Column - DB-5 , 30 mtr length , 0.32 ID & 0.25 mm thickness.

Problem Faced :
1. Ref.soln a -- we get peak of Imp. B , Bupivacaine , Impurity E & Internal Std respectively ( meeting to EP pharmacopoeia )

2. Test Solution -- We get Bupivacaine Peak at retention time of Impurity E .( No separation between Bupivacaine & impurity E ) . Hence unable to calculate Imp. E in Test solution .

Could you please advise why this is happening or could you please send us the typical chromatogram of Related Substances test alongwith detail method of analysis .

Sunil Pandya

Hi
Need to put impurity E in controlled solution in high concentrations . To verify , that the substance is really not separated. Monday will be at work, look in more detail.

Check the typical hardware error (dimension column in the detector , ingector split ratio, detector make flow )
best regard , Dimitry, Moskow

Hi

If I remeber I will get back on monday, as I recall we reduced the flow sligtly from the default setting to improve resolution (consequently prolonged run time a bit) but it was still doeble at default setting.

Need to double check we also used DB-5, but yous filmthickness "feels" lower than default but need to double check.

Dear sunil
pls confirm what Retention time u got during analysis. confirm the gas type used is helium and then if u not getting the separation then please run on constant flow with initial flow as per pharmacopia.

Dear krickos

Tghanks for your reply .

To further more clarify the problem ple read below

1. We have applied method excatly as per mentioned in E.P. including column , carrier gas , Chromatographic Conditions etc...

2. In Ref.Soln a -- all peaks are getting separated as per mentioned in E.P. i.e. Imp. B (. Bupivacaine , Impurity E & Internal Std but in test solution , we get Bupivacaine Peak & Imp. E peak at the same Retention time .

3. In Reference Solution a --- Imp. B ( RT=7.5 min. ) , Bupivacaine ( RT=10.37 min.) , Imp. E ( RT= 11.6 min ) & Internal Std ( RT=14.9 min.)

4. In Test Solution ----- Imp. B ( RT=7.5 min ) , Bupivacaine ( RT= 11.6 min ) , Imp. E ( We donot know whether Imp. E is there in product but RT as per ref. solution a = 11.6 min. Hence Bupivacaine & Imp.E is merged at same RT ) , Internal Std ( RT= 14.9 min )

As per your suggestion we are trying the same with reduced flow rate & see .

You find an admixture of the rielated substancies E in solutions c, d?


Bupivikain overloads the column. Impurity E out on the falling shoulder of the big bupivicaine peak and not detected, the poor relation peaK/nouse.
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Try to make a mixture of 50 mg and 1 mg of substance impurity E is similar to testing a solution and achieve a complete separation. 1.1 is bad RT. To optimize the conditions for a particular instrument and column.

PS
Test solution can not contain impurities e! Chemical engineers are well cleaned substance specific party. Or do you use an alternate path of synthesis.
Then there is no reason to panic.

Hi Sunil

Yes I see that you followed EP to the letter.

What we did exactly was to reduce the flow rate (Helium) from 2,5 to 2,2ml/min, and we got an RT for Bupivacaine of 10,6min and 12,6 for impurity E.
Due to reduction of flow rate we had to expand final isothermal time slightly to allow for the internal standard to elute (RT 14,7min).

Hi Sunil

Yes I see that you followed EP to the letter.

What we did exactly was to reduce the flow rate (Helium) from 2,5 to 2,2ml/min, and we got an RT for Bupivacaine of 10,6min and 12,6 for impurity E.
Due to reduction of flow rate we had to expand final isothermal time slightly to allow for the internal standard to elute (RT 14,7min).

I agree with your suggestion . Work is going on . It will be more helpful if u can mention little more detail about chromatographic conditions u followed .

Hi Sunil

Yes I see that you followed EP to the letter.

What we did exactly was to reduce the flow rate (Helium) from 2,5 to 2,2ml/min, and we got an RT for Bupivacaine of 10,6min and 12,6 for impurity E.
Due to reduction of flow rate we had to expand final isothermal time slightly to allow for the internal standard to elute (RT 14,7min).

I agree with your suggestion . Work is going on . It will be more helpful if u can mention little more detail about chromatographic conditions u followed .

Hi
Apart from the flow reduction it is as per EP. The column was/is the same as yours ie a DB-5 with identical dimesion and filmthickness.

EP did not mention anything about constant flow so we use constant pressure.

Frankly I am not particular satisfied with EP choice of procedure, sure for a quick late stage in process control basic pH extraction to organic phase is somewhat convineant but for final drug substance testing I would rather seen something similar to the mepivacaine/ropivacaine EP monographs ie LC analysis instead of GC.

Dear krickos

Thank you so much for information shared with me till date .

I will get back to u if I get something positive in this matter .

Regards

Hi again

Found another thing, reference solution a (containing impurity B+E together with bupivacaine) may not be particular stable at room temperature over time. You may experiance over time a growing peak at RRT ~0,6 just prior to impurity B in the chromatogram.