Dynamic equilibrium of Girard T-derivatized oxysterols

This is a question relating to organic chemistry, moreso than chromatography.


I am running LC-MS on various hydroxycholesterols (HC) and dihydroxycholesterols (diHC). To increase ionization efficiency with the electrospray, I derivatize my oxysterols with Girard T reagent. The picture below shows the two forms, syn and anti, that the derivatized oxysterols take (here showing derivatized 7α,24S-dihydroxycholesterol).


[![enter image description here](https://i.stack.imgur.com/p0xCR.png)](https://i.stack.imgur.com/p0xCR.png)


It is common knowledge that these isomers often have different retention times through an LC column, leading to split peaks. Some of my oxysterols do show splitting, but my group believes this not to be caused by the syn/anti-situation, as they claim the stationary phase I am using (phenylhexyl from ACE) does not show this. Rather, they say it is probably contamination of our standards (perhaps of some precursor).


I have observed that the split peaks converge when column temperature is increased. Below is a chromatogram of 7α,24S-diHC splitting at 40 C, but becomes a single broad peak at 70 C. This is consistently true for all my diHCs, but only one (22R-HC) of my HCs (the rest show no splitting at 40 C).
[![enter image description here](https://i.stack.imgur.com/c95jB.png)](https://i.stack.imgur.com/c95jB.png)


The mobile phase moving the oxysterols along is around 67% water, 26.7% acetonitrile and 6.3 % methanol. The pH is 2.8 via 0.1 % formic acid.


**My question is this: Could there be a dynamic/active equilibrium ongoing between the syn and anti conformations**, or is this unlikely given the presumably rigid hydrazone bond? This could, perhaps, explain the "bridge" between the two split peaks.


