By Cecília on Thursday, April 29, 2004 - 07:27 am:
Does anybody have an efficient method to determine carbocystein by HPLC? All methods I have seen, the peak of the carbo appears at the same time as the dead volume (usually in 2 minutes)with 0,8 mL/min. (250 x 4,6). And the worst part is that all methods that i have seen were validated.. how come?
well, hope you can help me...
By MG on Thursday, April 29, 2004 - 10:33 am:
Maybe HILIC would work. Search this forum, and you'll find quite a few HILIC posts.
By Cecília on Thursday, April 29, 2004 - 10:38 am:
Sorry, but what's HILIC?
By MG on Thursday, April 29, 2004 - 11:56 am:
Hydrophilic interaction chromatography.
It's like normal phase, but uses more polar solvents like acetonitrile and water. In HILIC, acetonitrile is the weak mobile phase. Aqueous buffer is the strong mobile phase. The stationary phase is polar, either bare silica or silica derivatized with polar functionality. I have used the Waters Atlantis HILIC Silica column most successfully this purpose, although other companies manufacture columns for this purpose. I have only used HILIC for a couple of different projects, but I can say that it will certainly retain polar compounds that cannot be retained by reverse phase. An added bonus (for me at least) is that the mobile phases are LC/MS friendly.
By Jan on Friday, April 30, 2004 - 06:25 am:
Cecilia,
Carbocystein is too polar for classical RP.
We determine carbocystein in various formulated drug products with an amino column (Lichrosorb NH2 250 x 4.6) and a phosphate buffer at pH 3.5 (no organic phase).
At a flow of 2 ml/min, retention time is about 7 min (solvent front at 2 min).
We haven't particularly validated this method, but it should be possible.
Hope it helps
Jan
By AllsepTech on Friday, April 30, 2004 - 10:20 am:
Here is the method for retention of cysteine. You carbocysteine will behave similarly at this conditions (ACN-water-TFA).
http://www.allsep.com/makeCmp.php?cmp=Cmp_025
You can control retention by both hydrophobic and ion-exchange mechanisms and elute your compound in 2-20 minutes depending on conditions
By Einar Pontén - SeQuant AB on Sunday, May 2, 2004 - 02:45 pm:
Yes, HILIC will work!
We have experience of running cysteine and cystine on ZIC®-HILIC with good retention, i.e., opposite to RP where these normally elute close to the void. The S-(carboxymethyl) group on carbocysteine will not make the column and technique less suitable.
By Einar Pontén - SeQuant AB on Sunday, May 2, 2004 - 02:48 pm:
Cecilia, send us an e-mail and you will receive a short application note.
You can learn more about ZIC®-HILIC at http://www.sequant.com/pdf/SeQuant.ZIC-HILIC.column.pdf [pdf-file]