I have been working on validating an LC-ESI-MS method for the analysis of neutral and acidic human milk oligosaccharides. While the recovery for the neutral oligosaccharides is good, I am having trouble recovering the acidic ones, with only 40-50% recovery.
In my sample prep, I centrifuge to remove the lipids and use a 10KDa molecular weight cutoff filter to remove the proteins. Then, I reduce the final extract with 0.25M NaBH4 and stop the reaction with 0.25M acetic acid. I inject the reduced oligosaccharides into the LC-MS without any cleanup.
I had previously validated this method on a different instrument and had an excellent recovery for all the human milk oligosaccharides, including both neutrals and acidics. This has me intrigued and I would appreciate hearing your thoughts on this.
I am wondering if the remaining boric acid in my sample is interfering with the acidic hMOS but not the neutrals. Do you have any thoughts on what might be causing this issue?
Thank you so much!
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