I work in the area of bioanalytical determinations using HPLC coupled with UV or FLD detection. I would like to know about quick sample purification methods you can use to work with human plasma, specifically to precipitate proteins or eliminate other interferences. I do not want to use tedious steps like LLE or SPE because a need fast, cheap and simple determinations for routine clinical hospital laboratory. Currently I know about precipitation of proteins by organic agents such as ACN or MeOH, or employing salts of Zinc or Magnesium sulphate.
Are the alternatives techniques? What procedures do you use and demonstrate good clean-up results?
I would be grateful for any references on the subject.
I don't understand you Xinfeng. Can you, please, explain me that question please. I work on reversed phase LC employing a C18 column.
Thanks.
Actually, this assay requires two columns, one for on-line cleanup of the impurities and enrichment of the target compound while the other column for separation and determination of the compounds you interested. Details pls see: Journal of Chromatography B, 809 (2004) 237–242; Journal of Pharmaceutical and Biomedical Analysis 70 (2012) 549– 552; Chromatographia, 65(2007):149-153 and other literatures
Ok, Thanks but i refer to clean-up steps for eliminate interferences. Types of reactives used and description about how to use them. Is interesting the use of two columns for clean samples but i have never seen or heard it and it is a new topic for me.
I would not say column switching is cheap and simple...
An alternative to organic solvents to precipitate proteins is the use of strong acids such as TCA (2-10%). It may be useful for polar metabolites that might co-precipitate at high organic composition. Depending on the type of analytes, you could also consider QUECHERS methods.
I do not think exist something cheaper than precipitation with organic solvents. Regarding the speed, maybe online column switch could help you. Something like turboflow technologies are quite fast and not so complicated, but you need the equipment and of course the columns. Price depend on the number of sample, column lifetime etc (as usual).
Here an example from the system provided by Thermo
http://www.thermoscientific.com/en/product/transcend-ii-system-multiplexing-turboflow-technology.html
20% TCA works well for the precipitation of proteins in plasma or serum. The amount of TCA will depend on the volume of plasma you are trying to purify. The TCA method is quick and simple. After the proteins are precipitated just centrifuge and remove the upper supernatant for teh HPLC analysis. Good luck.
You can use simple treatments with methanol or acetonitrile, and centrifugation.
Read: Analytica Chimica Acta 6 0 8 ( 2 0 0 8 ) 204–210
Mixing 1:1 with 0.3 M ZnSO4 in 80 % MeOH and subsequent centrifugation works fine for me - although I do also use an online clean-up step as suggested in a previous answer to improve robustness of the method (Clin Chim Acta 419 (2013) 95-101).
TCA is OK, but 5-10% sulfosalicilic acid and centrifugation has been widely used as a convenient esay method to obtain deproteinized plasma. If you dont mind the acidic media for further study, this is a good, quick and easy method
Thanks to all. One question Francisco. What is the ratio sample/precipitation reagent do you usually use?
the best way to eliminate proteins is to do acid precipitation with TCA (as mentioned earlier) or also with sulfosalicylic acid. You can use between 20 and 50% of SSA
Thank you Olivier. And what is the usually used ratio sample/reagent for SSA?
The final concentration of 5%-10% TCA or SSA in the deproteinated plasma sample is enough. Try these 2 concentrations and choose the most suitable for you.
To avoid acidification, I performed the protein precipitation by adding ZnSO4 (25 g ZnSO4x7H2O plus 75 g water) water solution followed by equal volume of saturated Ba(OH)2 (5g/100 ml) water solution. E.g., 250 mkl of Zn SO4 solution was added to 150 mkl of protein-containing sample, followed by 250 mkl of saturated Ba(OH)2 solution, and after that 850 mkl buffer was added. Combined precipitate of BaSO4, Zn(OH)2, and proteins is easily removed by centrifugation, 10 min at 2500g .
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