Waters Oasis HLB Extraction plate

[tlili]

Hi,

I am currently using the Waters Oasis HLB Extraction plate to extract my plasma samples.

I will condition the 96 well plate with 100% methanol, follow by equilibrate with MilliQ water. Once finish, I will load in my diluted plasma samples follow by 2x washing and elution of samples and complete the whole extraction. Each of my plasma samples is done in duplicate. All these samples will be analyzed using HPLC, PDA.

The problem that I encounter is:
- not all samples will go through. Some will take a longer time while other will be faster. As a result, the final analyte result of the same sample will vary quite a lot.

I will appreciate greatly if anyone can give me some suggestions on how to overcome the above mentioned problem.

Can anyone enlighten me the principle in this type of extraction method? What is the purpose of doing conditioning follow by equilibration?

Thanks for all your time.

[sassman]

The sorbent in the cartridge is hydrophobic. So if you do not perform the conditioning step, it will not be fully hydrated when you load your sample. The equilibration step is done to remove excess solvent from the cartridge. If the equilibration step is not done, some of your sample will be eluted prematurely because of the strong solvent that is present in the cartridge.

The cartridge works by sorbing compounds in your sample that are hydrophobic (kind of like liquid-liquid extraction). Then you wash the cartridge to remove everything that is not sorbed to the cartridge. Then you elute your sample with some strong solvent which sorbs to the cartridge and kicks everything else off.

It should not be a huge problem if some of the samples go faster than others. Just make sure that the fast ones are not going TOO fast. 3mL/min is a good rate I think (although I use cartridges and not plates). If you have an internal standard that will help with precision.

[Uwe Neue]

The main purpose of the conditioning is to get liquid into the pores of the packing. If you put a plasma sample onto a dry packing, the liquid will go into the pores, and the protein components of the plasma will stay outside. This creates a very high viscosity, and it would be very difficult to get the sample through the cartridge.

When you dilute your plasma sample with the internal standard solution, you accomplish also a reduction of the sample viscosity, which once again makes it simpler to get the sample through the bed. You did not specify, by how much you dilute the plasma, or what the solution is. You may get somewhat better results by diluting the sample more. Don't forget, the analyte gets enriched on the cartridge, therefore a dilution of the initial sample is rarely a problem (only for very very polar compounds), and you benefit from a more uniform flow of all samples.