HPLC without extraction step ?

[KJO35]

Hi,

Is it possible to perform HPLC based on a cell lysate without extraction step ?
I would like to quantify intracellular Talinolol (β1-selective beta blocker) in Caco-2 cell line. And I use a guard column.

Thanks

[shemesh199]

Hi buddy,

I think you would find these lysates to be very impure and full of contaminants such as lipids. Ideally it would be nice if you had an internal standard to use. What you can do is a large dilution assuming the drug content is concentrated enough. You can dry down the sample, and reconstitute in your mobile phase, the problem is that you need to filter that before shooting in addition to a guard column, unless the dilution is large enough to remove most contaminants. Take care not to clog your HPLC lines with particulate.

Upon filtration, you might notice a large loss of your compound, but if you have a suitable IS then the loss will be about proportional.

Really depends what you are doing, are you doing qualitative work or quantitative work?

[Mr.Brown]

with ultra centrifugation (50.000 g) you can get rid of most of the solid stuff

we used standard centrifugation (15.000 g)
and some in-line 0.2 µm filter in addition to a pre column.
adds some dead volume and some back pressure however this additionally protects the column quite well

quantification will by tricky if some of the Talinolol is adsorbed to some cell compounds in the cell lysate

as shemesh199 suggested the right internal standard could help with this issue