Which is the best of extraction method for bioanalysis??

[aniket]

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

[Uwe Neue]

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.

[JJG]

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

[Uwe Neue]

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.

[aniket]

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

[Uwe Neue]

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.