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Isomers: Not So E-Z-Peasy

Posted by Stephanie A. Smith, Ph.D. and Sarah Dreheron Tue, July 8, 2014 @ 11:00 AM

Suppose you’re well on your way to purifying a microcystin toxin:  you have figured out your extraction protocols, you have tested what seems like dozens of columns and HPLC methods, and now, as you are running your final QC trace, the sample you could have sworn to be 99% pure no longer appears to be.   An annoying little peak right before the “right” peak keeps creeping up.  This actually happened to us with an early batch of Microcystin-RR, and fearing that the “extra” peak was a contaminant we were threatened with losing weeks of work and having to throw out the entire batch (several milligrams!).  This was our first lesson in microcystin isomers.

E isomer Z isomerFor those of you who may have spent a couple of years away from an o-chem textbook, an isomer is a molecule that shares the same molecular weight, but differs in the structural arrangement of the atoms.  In the case of mircocystin, the most common is a stereoisomer that occurs when the carbon 6 of the Adda moiety rearranges from E form to Z form (rotation of molecules around a double bond).  Without closely looking at the structure, you might not even notice a difference (look at the stuff to the left of C6).  Mass spectrometry cannot detect the difference (same molecular weight), and isomers tend to run similarly on HPLC gradients, depending on your column and mobile phase.  Isomerically-pure preparations are possible and identification via NMR can be performed.

If you’re using purchased microcystin as an analytical standard, the presence of an isomer will generally not have an impact.  In fact, if you’ve used microcystin-RR that was purified from laboratory-grown Microcystis cultures, you probably have already been using samples that have the minor isomer.  That turned out to be the case with our first batch of microcystin-RR, where we were using Microcystis cells grown and preserved by Wayne Carmichael’s lab in 2005, and extracted by Beagle Bioproducts in 2013.  We only noticed the isomer because of a minor change in one HPLC mobile phase.  It hasn’t cropped up in subsequent batches, which have come from different feedstocks.

Our experience led us to research this topic more deeply, and a white paper about isomers is available at our websiteWhile they are not problematic for analytical purposes, isomers may be very important for our understanding of toxicity of microcystins.  This is yet another area where study is needed as harmful algal blooms become more common, and more people and animals are exposed.

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