Hi,

I'm performing HPLC assays of testosterone in transdermal patches with a sample matrix of .1% phenol with .5% bovine serum albumin. I noticed after repeated injections, my chromatography started getting poor peak shape, split peaks and increased back pressure. I am using a YMC Basic c18 column. Could the bovine albumin be causing this? And is there any way to remove it from the column?

Thanks-Rose

I would say that the protein is the most likely culprit. There are a number of ways to get rid of the protein. You can precipitate with concentrated ammonium sulfate or alcohol. A method that I prefer for some of my own analyses involves solid phase extraction on a reversed phase cartridge. To do this, you would condition the cartridge per the manufacturer's directions, load a known volume of the sample, then elute the analytes with a suitable solvent (methanol or mobile phase) and assay as you normally would.

You will need to measure recovery carefully if you choose any of these techniques and (if practical) a well chosen internal standard can help measure recovery on a sample to sample basis.

Waters and varian have some good information on SPE. Contact your rep or check relevant web sites. I personally prefer Waters SPE products and have had good luck with them for a long time.

If you have further questions, feel free to drop a line at christopher.judd@collabo.com

Best of luck!

Chris

If you can afford it you may want to use a restricted access column.