Chromatography Forum

Respected Forum,

I am a newcomer to bioanalysis and use of LC-MS-MS, i am having some problems higher system pressure and rheodyne port blockage, i am predominantaly using Protien precipitation as my sample celanup procedure??
I am using 400 µl of the precipitating solvent for 100 µl of plasma, is this volume sufficient for sample clanup??
i just wnated to know from all of the experts, which is the best cleanup procedure for bioanalysis? Should i go SPE or LLE? Also i am working for Discovery lab, so i am on lookout for a high throughput method, can you give some suggestions?

Hoping for a positive reply from your side,
Aniket

4:1 dilution with acetonitrile followed by centrifugation is the standard approach for protein precipitation. However, the result is not a clean sample. Commonly, the problem is interference in the C-gram. Anyway, I am a strong advocate of sample prep via mixed-mode SPE. The samples are signficantly cleaner, resulting in less quantitation problems afterwards, whether you analyze by LC/UV or LC/MS, and definitely no injector plugging or column clogging.

From my experience, the order of better sample clean-up is SPE>LLE>precipitation, but in general, the amount of time each clean-up process takes is the same relation. Is the port blockage you speak of your autosampler valve, or a column switching valve? If it is occuring in your autosampler valve, then do you see any particulates in your sample when you transfer the solution to an autosampler vial? I would go with LLE or SPE then.

If the blockage is occuring after the autosampler injection valve, then I would invest in some inline frits (0.5um) and install it before your column. When your pressure goes up too high, and/or retention times start changing, then change the frit. This way the block will occur on this frit, and $8 and 1 minute later you're back in business.

http://www.upchurch.com/PDF/Lit/Frits.pdf Product #A-102X

I forgot your question about a high throughput method.

I suggest SPE with 96-well plates. You process a lot of samples in parallel, and there are very well developed fast generic protocols around. You can either look at the Waters website, or I can send you an (already older) book chapter on SPE methods.

Thank you all for replying,

Uew can you please send me the copy of the book, my mail id is anaik@nicholaspiramal.co.in or aniketmpharm@gmail.com.

I am not seeing any particulate particles in my autosampler vials, still i am getting high backpressure?

You told to use 1:4 ratio of precipitation solvent can i use a higher ratio like 1:9 or 1:15, would it be useful?

Can you please elaborate on the 96 well plate SPE??

Aniket

Regarding 96-well plates: they contain adsorbents just like a single SPE device, and they are available with different adsorbent volumes, for processing different sample volumes. The protocols are the same as with a single SPE device, but you process 96 wells in parallel. Since you said that you are interested in high-throughput methods, the assumption was that this means that you have a lot of samples of the same type that can be processed this way.