Ar-CONH2 and Its Degradant

[syx]

Dear chromatographers,

I need suggestion for my developed method to separate an active pharmaceutical ingredient with its main degradation product.
The formula of the api:Ar-CONH2, and the degradant: Ar-COOH. Both are very polar compounds.

The latest chromatographic condition:
Column : Atlantis dC18 (5 μm) 150 mm x 4 mm
Mobile phase: 25 mM (NH4)2PO4 , 0.1% TEA, adjusted to pH 7.0 with 10% H3PO4 .
Oven temperature: 25°C
Flow rate : 0.5 ml/minute

On that condition, I get results:
Rt Ar-COOH = 12.807 minutes

Ar-CONH2:
Rt = 13.893 minutes
Resolution = 1.67
k’ = 6.82
Asym (10%)= 1.38
Tp = 7096

Decreasing pH of mobile phase up to 2.5 will decrease the resolution.
How to get more adequate condition for this method? My targets are better resolution and shorter run time.



Best regards,
SYX

[Yury Zelechonok]

To shorten the run time you can add organic modifier in your mobile phase. To increase resolution you can go to higher pH since when you decrease pH you did observed decreased resolution. Phosphate buffer is not healthy for the column to go on pH above 7 so choose something else and check Waters for appropriate buffer at higher pH on this column. By changing buffer you may see small increase in resolution (or decrease).
The ultimate solution though for your problem is to use a mixed-mode column with an anion-exchange/RP surface chemistry like Primesep B from SIELC Technologies. Your Ar-COOH starts to interact with column by ion-exchange mechanism while the Ar-CONH2 will not. By changing amount of buffer you can have any resolution you want for this pair. Interesting that acid will retain more than amide in this system while on RP column the order usually is reverse. See some examples:
http://allsep.com/makeChr.php?chr=Chr_053
http://allsep.com/makeChr.php?chr=Chr_063
In last example neutral (or slightly basic) riboflavin retain more when pH is low enough to suppress ionization of ascorbic acid.

[Mark]

syx,

Another easier solution without changing your current conditions might be to go to a 3 micron packing and a shorter column. The improvement in efficiency with the smaller particle size will more than offset the few plates you will lose going to a shorter column.

Regards,
Mark
P.S. I have found an EXCELLENT new column made by Imtakt called Cadenza from Silvertone Sciences. Very high plate count and much lower backpressure than comparable 3 micron columns. Their website is www.silvertonesciences.com

[Uwe Neue]

A smaller particle size in a shorter column might help, but I would try something els before this. If I understand you correctly, you do not have any organic solvent in the mobile phase. You could do two experiments by adding around 1% acetonitrileand adding 1% methanol to see if the selectivity shifts.

The Atlantis dC18 column can be used at pH 8, but I would guess that this additional change in the pH will have no influence on retention, since I expect your analyte to be already fully deprotonated (Ar-COOH). Therefore I do not recommend to go there.

[syx]

Which one better to choose between these conditions?
Condition 1:
-Rt ArCOOH (degradant) 8.5 min
-Rt ArCOONH2 (main component) 10.0 min
-Tp > 8000
-Tf 1.56
-Res 3.65
Condition 2:
-Rt ArCOOH (degradant) 11.2 min
-Rt ArCOONH2 (main component) 13.4 min
-Tp > 11000
-Tf 1.43
-Res 4.4
The first is faster, but the other has better condition.

[Einar Ponten]

You are at the dead end for RP columns... Why don't you try another approach.

While using the ZIC®-HILIC column you will have a mobile phase containing approx 80% acetonitrile and 20% buffer. If the retention is too long you increase (!) the water content of the mobile phase.

You can retreive more information at http://www.sequant.com

[syx]

Good alternative, but I have use 5% - 10% methanol in my latest chrom cond with different column dimension from previous condition. Which one better, the first or the second cond?

[DR]

I'd go with the shorter one, provided it isn't too particular about the conditions (does resolution drop off a lot if your pH, temp etc. are off just a bit?).

[syx]

I have found a method from BP2004 for Piracetam related substances. But the resolution just based on 2-pyrrolidone, not (2-oxopyrrolidin-1-yl) acetic acid (ArCOOH). It uses C-18 column and a mixture of acetonitrile and phosphate buffer pH 6.0 (10:90). Retention time of Piracetam is about 4 minutes.
We should separate it with ArCOOH which is a significant degradant of the piracetam in the dosage form. In the case of stress by base or acid, it was showed that the acid or base caused significant degradation. The degradation rate is higher in base, producing (2-oxopyrrolidin-1-yl) acetic acid and ammonia.

ArCOONH2 + H+/H2O --> ArCOOH + NH4+

ArCOONH2 + OH-/H2O --> ArCOO- + NH3

(2-oxopyrrolidin-1-yl) acetic acid has higher absorbance than piracetam.
In my method, the resolution between them will be drop off a lot when we use higher flow rate, temperature, or solvent strength.