Hi, everyone:

I am involved in troubleshooting an LC-MS/MS method in which the column is Waters XTerra MS C18 and the mobile phase is ACN : 0.05% of formic acid (40:60). The flow is 0.25 ml/min. Moexipril is used as ISTD, which contains an amide and an ester group,along with a secondary amine and carboxylic group (in our condition, it might partially exist as zwitter ions). When using a new column, we need more than 10 hours time to get stable RT for moexipril. Its peak shows very good symmetry but is very broad. For the two other analytes (both are quite hydrophobic carboxylic acids), the peaks are sharp and symmetric and much less equilibration time is needed. I am puzzeld. If seconadry interaction caused very broad moexipril peak, why didn't we see peak tailing? Why is Moexipril peak so broad but other peaks very sharp? I also doubt that in our conditions silanol is partially ionized. We see from many other projects that XTerra C18 columns generally need much long equilibration time than Symmetry and Zorbax C18. IS this the nature of the hybrid silica columns?

Thank you very much for sharring your thought with me.

Hi Anonymous,

I think your broad peak is due to analyte complexing with metal ion impurities. Anytime that an analyte has lone pair electrons that can complex with metal ions by forming 5 or 6 member rings you need to think that in solution there could be an equilibrium set up between the complexed and free forms. I think your amino nitrogen and amide oxygen will form a 5 member complex and the amino nitrogen to the ester oxygens will form 5 and 6 member complexes.

These forms will have different retention times and the peak is broadening because the time to interconvert between forms is slow enough to have an effect but not so slow that you see different peaks.

All of this is assuming that Moexipril is an ACE inhibitor and is structurally similar to other "...pril" drugs. Hopefully someone with some experience will post regarding what can be done to sharpen peaks if your analyte is forming metal complexes.

This article from the Agilent reference library says: "A phosphoric acid wash has been shown to be effective at reducing tailing caused by the sample complexing with metals in the HPLC system. Typically a 1% phosphoric acid wash of the system and column is suggested to eliminate this tailing and it works. It is perfectly reasonable to use these recommended wash conditions with Agilent ZORBAX StableBond reversed-phase HPLC products."

I was thinking maybe you could use EDTA or some other chealating agent in your mobile phase.

Also see this article for some data on Lisinopril forming complexes with metal ions in solution. Good Luck.

Sorry about the broken second link I'll try again . If that doesn't work the url is http://www.rsc.org/CFmuscat/intermediate_abstract.cfm?FURL=/ej/DT/1997/C9701500.PDF&TYP=

I have not yet found the structure of the stuff, but the name indicates that it's a ...pril.

Captopril and enalapril have a proline structure with an amide-bond to the nitrogen of the proline. They exist as two sterically different conformers, that interconvert slowly - on the time frame of chromatography. The consequence of this is that you get a broad peak, or even double peaks with a bridge in between.
For the other ...prils the fix is to go to a still lower pH. This is not possible with LC/MS. You could go to a higher temperature, or you could declare that you just need to live with it. It is not a problem of the column.
If you want to brush up on this, the ...pril phenomenon is described in my book (HPLC Columns) on page 362.

Hi Uwe,

Thanks for the alternative perspective, I sure you are right if you have studied this. My take was just a WAG based on recalling that moexipril is a dimethoxy analogue of quinapril and that the mechanism of action is to complex with zinc in the active site of ACE.

There is no proline structure but there is a isoquinolinecarboxylic acid with a similar amide bond that is sterically hindered. Thanks again, I've ordered your book :-)

Question to Uwe Neue:

You are speaking about your book, what kind of book is this, were can we order that book?

It is called "HPLC Columns, Theory, Technology, and Practice", Wiley-VCH 1997. Available directly from Wiley or through Amazon.com or similar book dealers.
It covers the different chromatographic techniques with focus on the column and the packing materials. Also covers the different chromatographic techniques in detail (SEC, normal phase, RP, HIC, IEX, HILIC, prep). Sections on method development and column troubleshooting, column design and column chemistry as well as theory of chromatography.

A customer called about this in the last 6-12 months, and I found this short application note, which shows the impact of column temperature on peaks shape for these compound types. See the following hyperlink:

http://www.chem.agilent.com/temp/rad1956F/00015179.pdf

Tom Waeghe
LC Marketing Applications Group
Agilent Technologies