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Hello,
I am extracting 3 hydroxy-N-methyl pyridinium (3-OH-NMP, metabolite of
pyridostigmine) with eserine (physostigmine as) an internal standard
through MCX column. But I am having lots of endogenous peaks and one coming
out at the retention time of 3-OH-NMP. Also the recovery is very low. The
steps which I follow are, conditioning the column with methanol and 2%
formic acid, then loading samples, rinsing the column with 2% formic acid
and 1:1 methanol and acetonitrile solution and lastly eluting with 5%
ammonium hydroxide in methanol solution. I have also tried using lower
concentration of ammonium hydroxude (0.5% v/v) but the recovery was even
lower.
Any help and suggestion will be greatly appreciated.
Thank you,
Manu
--
Manushree Bharadwaj
Graduate teaching assistant
CVHS, OSU, (OK)
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Check where your recovery loss is. Is it an incomplete loading of sample? A wash step that is too
strong? Or an incomplete elution of analyte from the column? An answer to these questions will tell
you where to focus your efforts.
As for interferences, you will either have to find a wash for your SPE which removes them or
separate them further with chromatography.
Lee
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Dear Manu,
Two possible options are available to increase the extraction recovery for 3-OHNMP.
1. Whether the matrix sample is treated with any buffer? If not, add 0.2% formic acid and continue
with the above procedure.
2. From the structure of 3-OHNMP it is evident that, it has OH group on pyridine ring, so you can
try with MAX as well.
Hope this will help.
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