Sunday, September 13, 2009

Protein precipitation

As for my MP, i have to analyze a drug in plasma sample. However, there are many impurites in plasma sample. In order to obtain as little interfering peaks when analyzing by HPLC, we have to remove the protein in the plasma.

General theory of using an organic solvent to precipitate out proteins in plasma:
- organic solvent reduces water activity
-thus water cannot dissolve the hydrophilic proteins

This is done by:
1. Adding acetonitrile to plasma sample in the ratio of 2:1
2. Shake well
3. Centrifuging at 6000rpm for 10 minutes
4. Supernatant is then spiked in with the drug for analysis
5. Vortex for 2 minutes

_____________________EDIT________________________

Advantages of using acetonitrile for protein precipitation as compared to other method:
- it is a common solvent used in HPLC as mobile phase
- it has very low to no absorbance at wavelength more than 190nm


eriko
0700477C
Posted by at

8 comments:

  1. MedScientists of Grp 6September 14, 2009 at 4:53 PM

    Hey ERIKOOOOOO! :D how are you?

    anywaes, what is HPLC and can the organic solvent precipate away all the proteins present?

    Thank you berry muchie!!

    Joanna Yeo!
    0702054H

    ReplyDelete
  2. TG01-Group 2September 15, 2009 at 10:19 PM

    Hello eriko! :D

    Can I know what is the purpose of adding acetonitrile?

    Thanks!

    Siew Ming
    0702862D
    TG01 Grp 2

    ReplyDelete
  3. emadtechsSeptember 16, 2009 at 5:45 PM

    Hi eriko

    May i know what do you mean by "interfering peaks"?

    Liyana
    0703827F

    ReplyDelete
  4. BordetellasSeptember 17, 2009 at 2:48 PM

    Hi! joanna!
    with acetonitrile, it can remove about 95% of the protein present, the last 5% will be removed by the guard column to prevent the clogging of the main column. HPLC is high performance liquid chromatograohy. I did an introductory post a while back:)

    @siew ming - acetonitrile is an organic solvent, the purpose of adding it is to precipitate the proteins in plasma:)

    @liyana - interfering peaks refer the peaks that are not derived from your analyte and they are very close to your analyte peak:)

    eriko 0700477C

    ReplyDelete
  5. TG01-Group 2September 17, 2009 at 8:33 PM

    hi eriko!

    i want to ask,
    what are some eg of impurities in the plasma samples that can affect results of HPLC? is there any specific reason why you must add acetonitrile in ratio of 2:1? what will happen if you add in a higher concentration of acetonitrile??

    cheers
    JIA HUI (JOEY)
    tg01 group2 0703605f

    ReplyDelete
  6. BordetellasSeptember 22, 2009 at 11:46 PM

    hi eriko!
    is there any difference between using acetonitrile and ethanol?

    nyzah 0702888i

    ReplyDelete
  7. BordetellasSeptember 22, 2009 at 11:47 PM

    what i mean is, could u use ethanol instead of acetonitrile?will the results differs?

    nyzah

    ReplyDelete
  8. BordetellasSeptember 30, 2009 at 5:29 PM

    hi joey, example of some impurities are the dissolved solutes in the plasma like amino acid, glucose, metabolic wastes.. There is no specific reason to why i use the ratio 2:1, I have used the ratio of 1:1 and it worked, as long as it reduces the water solubility of plasma proteins.

    Hi nyzah, i don't think there is any difference in the protein precipitation but since you are going to inject the plasma into HPLC, it's recommended to use a organic solvent that has as low absorbance as possible at the wavelength you have set:)

    SORRY for the late reply!!!

    thanks~~

    eriko 0700477C

    ReplyDelete

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