Dear all,Back to the Top
can you pls tell me how to precipitate the proteins in serum for hplc of
a polar molecule
what difference does it make with Acetonitrile, perchloric acid 70%,
hexane, dichloromethane
thanking you
amrutesh
The best method to precipitate plasma proteins in order to analyze aBack to the Top
polar (!!!) compound by HPLC is precipitation by HClO4 7% (0.2 ml acid
for 0.5 ml plasma) or with TCA 20%. This is only for polar compounds
that in acidic medium are either positive ionized or unionized. With
acetonitrile or methanol probabely you will have problems in
chromatography. With hexane and dichlormethane you cannot precipitate
proteins.
Regards,
Vlase Laurian
Dept. of Pharmaceutical Technology and Biopharmaceutics
Faculty of Pharmacy
University of Medicine and Pharmacy "Iuliu Hatieganu"
13, Emil Isac
Cluj-Napoca, Cluj 3400, Romania
vlaselaur.-at-.yahoo.com
Dear Amrutesh,Back to the Top
The selection of the extracting solvent depends on the polarity of
the compounds. To extract a polar compound from serum, solvents
such as methanol (3-4 times), acetonitrile (2-3 times), perchloric
acid 70% normally work perfectly. However, non-polar solvents like
hexane, dichloromethane, ether etc. are immiscible in aqueous part
of serum. Therefore, you will not get a polar drug in these
solvents or may be some amount in aqueous part and some amount in
non-aqueous part depends on polarilty of drug and v/v ratio
between solvents and serum. So it is advisable to use polar
solvents for a polar drug for protein precipitation method.
I hope it would be helpful to you.
Jai
Protein precipitators are ususally required to be miscible with water,Back to the Top
compete water with protein and then precipitate protein. Therefore,acetonitrile
and perchloric acid are good options, however, hexane and dichloromethane
are immiscible with water and mostly used for extraction of lipophilic or
unionized polar drugs. Also the stability of drugs need to be
considered when you plan to use perchloric acid.
--
314 Room, 19 Russell Street
Faculty of Pharmacy
University of Toronto
Toronto, Ontario M5S2S2
(Tel) 416-978-6996
(Fax)416-978-8511
I would like to add one more thing for protein precipitationBack to the Top
method. If you have problems in eluting the peaks on HPLC after
extracting from acetonitrile or methanol, you can adjust pH of
your eluents. For pH (2-3) you can use 0.1% trifluroacetic acid
and for pH (7.4-8.5) some buffer e.g. 0.1M borate buffer,
phosphate buffer etc. with other eluents.
Jai
Jai Prakash
Department of Pharmacokinetics and Drug Delivery,
University of Groningen,
Ant. Deusinglaan 1,
9713AV, Groningen
The Netherlands.
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