Chromatography Forum

i am involved in the stability study of Imipenem and Cilastatin. For a couple of months i am wasting my time in method development for the same. my aim will be to develop the stability indicating method, which will well separate drugs as degradation products of the both drugs formed during different stress conditions, like acidic, hyydrolytic, oxidative, photolytic etc.

second thing is i will be transferring the method to LC-MS, so volatile buffer is essential.

till now, i tried, phosphate buffer with different pH (3.5, 4, 4.5, 6, 6.5, 6.7, 6.8, 7, 7.2) i.e. either K2HPO4 or KH2PO4. non gave better results. in fact, below, pH 4.5 at any pH i got two peaks of Imipenem fresh sample with the same uv-scan in PDA. as per literature report i perdicted as its rotamers (cis and trans), peak broadning and shape were horrific and also those rotamers never baseline separated.

then i moved to volatile buffers, first tried ammonium acetate, at pH of above adjusted with ammonia or acetic acid,

lastly i tried formate buffer, again results were disgusting, there is not a single LC-MS method for the drugs reported in literature.

i also tried BP assay method, it was just ok for assay but not suitable to proceed with to further develop stability indicating method

not yet tried USP method.

lastly i landes with the 25mM ammonium acetate and ACN method, again bad peak shape, surplus tailing and two peaks of fresh sample not being separated at base line of IMIPENEM.

i am using supelco discovery C-18 column., shimadzu HPLC system.

too much frustrated till now

i would be really indebted to the persons who help me to cast the prper way

I've heard that acetic acid/triethylamine is a good volatile buffer. What about TFA? I'm dealing with peptides, RP-HPLC, UV-detector so I'm not the expert.

I suspect TFA is pretty worthless at higher pH... w/ cis/trans enantiomers, wouldnt' you expect some separation? If they're a different shape, I would expect 2 peaks. It the relative compositions don't matter, just live w/ the unresolved pair and use a grouping event to quantitate them. When you have your forced deg. products separated from them, you're done... (unless your forced deg products wind up being cis/trans and poorly resolved too - if this is likely, I'd go right to MS detection so you don't waste more time worrying about resolving these pairs, unless they have differing biological activities which may require accurate, relative quantitation of ea.).

For my benefit, how is it that one quantitates numerous process intermediates, impurities and degradation products when the relative ionisation potentials are not going to be known? (if indeed that is what you are inferring by going "right to MS detection"). Do people actually do this - I was under the impression MS was only used for quantitation where isotopic standards were available.

Thanks and apologies for the thread-hijack.

thank you sir, i am very much agreed with your suggestion, and my aim is not to quantitate the drug as well as degradtion products. but just to separarte them, characterize in LC-MS, and to build the postulate the mechanism of degradation in different condition. But the two peaks are never base line separated, it seems as if a peak has got spllited and looks very ugly.

definitely at pH 6 or above TFA is of no use, whaT if we use ammonium carbonate buffer system to repalce K2HPO4?