Chromatography Forum

Hi,

I am interested in LC-MS detection of SAH and SAM in brain tissue at nmolar levels. I am following a paper where this is achieved using phenylboronic acid SPE cartridges for extraction followed with LC using a 2 x 100 Waters Symmetry shield column with water, methanol, 0.02% acetic acid as solvents. SAM and SAH elute at about ~ 0.5% methanol. In this paper SAH and SAM are detected down to nM levels (20 uL injection of 5 nM solution) and the calibration curve is linear up to ~800 nM. Flow into the source is split four-fold.

I am using a TSS uplc column, 2.0 x 100 mm, and similar solvents and gradients. SAM peak looks broad but symmetrically tailing. SAH peak is terribly broad. Sensitivity is very bad.

I switched to an old 1 x 150 mm column and noticed that the SAH peak was better but still terrible.

Any suggestions with this analysis is greatly appreciated.

Thanks,
Mona

Hi,

I am interested in LC-MS detection of SAH and SAM in brain tissue at nmolar levels. I am following a paper where this is achieved using phenylboronic acid SPE cartridges for extraction followed with LC using a 2 x 100 Waters Symmetry shield column with water, methanol, 0.02% acetic acid as solvents. SAM and SAH elute at about ~ 0.5% methanol. In this paper SAH and SAM are detected down to nM levels (20 uL injection of 5 nM solution) and the calibration curve is linear up to ~800 nM. Flow into the source is split four-fold.

I am using a TSS uplc column, 2.0 x 100 mm, and similar solvents and gradients. SAM peak looks broad but symmetrically tailing. SAH peak is terribly broad. Sensitivity is very bad.

I switched to an old 1 x 150 mm column and noticed that the SAH peak was better but still terrible.

Any suggestions with this analysis is greatly appreciated.

Thanks,
Mona

Just wanted to mention that my analyses are working quite well now. Three things helped: switching from TSS column to a Symmetry BEH, adding some TFA to the sample, modifying source conditions (a lot hotter) on the ESI source. Thanks to those who read the post.