I am trying to decrease the amount of time it takes me to prepare and analyse invitro metabolism samples. Currently It takes about six hours to prepare 24 samples before they can be injected onto an LC column. The samples I analyse consist of a TRIS buffer, liver microsomes, NADPH and an organic compound of some sort. Usually the compound has very low solubility in water and is added to the buffer and microsomes as a stock solution in acetonitrile. Usually not more than 5µl of the stock solution is added to the sample mixture. Before injecting these samples onto the LC, the compound is extracted using a liquid-liquid technique. This extraction process is a huge bottleneck and takes up alot of time. Therefore I would like to delete it from the sample prep procedure entirely. This would mean shooting a mixture of microsomes, buffer and compound onto the column. Currently I am using a Waters Symmetry C18, 250mm X 4.6 column which would not be suitable for this application. My run time is 28min and I use an acetonitrile:0.02%TFA mobile phase. It is important that I get good resolution of metabolites and parent compound.What I want to do is reduce the run time to about 10min without sacrificing resolution. I also want to shoot sample onto the column without extracting. WHAT COLUMN WOULD BE BEST TO USE FOR THIS APPLICATION? I am using an HP1100, binary pump LC. The detector is a DAD. Any suggestions would be greatly appeciated.

Try using a solid phase extraction cartridge. I have had good results doing a similar type of analysis using the Oasis cartridges from Waters. Ask them for the information on how to use them and an applications booklet. That info may be available on thier web site. www.waters.com

Working out the sample prep part of an assay for biological samples is why we bio-analytical method developers get paid so much.... if it were easy, anybody could do it! :-)

The solid phase extraction (SPE) idea is okay though I hate doing that work off line on the bench. If you've got the hardware and software to do it, I'd go for a column switching method with an SPE or RAM precolumn followed by a good analytical column as the second column. Try doing a lit search for Karl-Siegfried Boos, he is my hero on this technique. He has published several "dilute and shoot" methods for biological sample analysis via column switching.

As to the analytical column, I'd simply try cutting down the size of the column. The Symmetry comes in 4.6x150 as well as some narrow bore sizes. Another option might be to switch to a nonporous support. (Shameless plug to follow....) A truly awesome ;-) method paper for a small molecule method in rat plasma on a nonporous C18 column is by Paasch et al, JoC/B, 704 (1997) 231-242, which includes a comparison of porous and nonporous C18 columns. The nonporous column made for faster runs and, in this case, better separation of analyte from residual matrix peaks.

Good luck!

-brian paasch
Genentech, Inc.

SPE and RAM columns are good choices, but another easy way to do it is to precipitate the microsomes with acetonitrile (use three or at least two volumes of ACN), spin the precipitate down and inject the supernatant. Microsomes aren´t near as complex as plasma or urine and this approach has worked very well for several compounds in my lab, especially for very hydrophobic ones.

Why not try a 10 cm column with particles (available if You want to stay with the Symmetry family)? And if You do, why not try a Symmetry Shield column? This column has very good chromatographic properties for a wide variety of compounds.

Good luck!

Magnus Olin / Active Biotech Reaearch