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Dear all,
As I have read from many articles about method of protein precipation
before drug extraction, mostly acetonitrile rather than methanol is used
for this process. Please anyone tell me the differences using these 2
organic solvents. Someone mentioned in PK discussion that methanol can
cause problem during HPLC analysis. I don't really understand.
Thank you so much
Benjamas
Asst. Prof. Benjamas Janchawee, Ph.D.
Department of Pharmacology
Faculty of Science, Prince of Songkla University
Hat Yai, Songkhla 90112
Tel/Fax: 66 74 446678
Email: benjamas.j.-a-.psu.ac.th
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Way back there was a classic paper in Journal of Chromatography, you
can accomplish protein precipitation by organic solvents or using
acids, bottom line being protein precipitation by denaturalization. TCA
was a time tested chemical for protein precipitation but sudden shifts
in pH is not good for labile compounds like cephalosporins, therefore
organic solvents are a general choice acetonitrile is a better protein
precipitant compared to methanol. Relative efficacy of each solvent was
rank ordered by determining the percentage of proteins left in plasma
after the precipitation. Usually 1:2 plasma:solvent (methanol or
acetonitrile) are used for getting best drug recovery. Choice of
methanol or acetonitrile depends on your compounds preferential
solubility - which usually have marginal difference when it comes
between methanol and acetonitrile. Other factor that decides which one
you go for is the composition of your mobile phase if your mobile phase
consists of acetonitrile better to use acetonitrile as your precipitant
likewise for methanol. Other factor you have to remember is UV cut off
for these two solvents.
Hope this answers your question.
Prasad NV Tata
Mallinckrodt, Inc.
Saint Louis, MO 63134
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I think Dr. Tata has the following paper in mind: it is excellent work
and should be studied carefully.
1: J Chromatogr. 1981 Dec 11;226(2):455-60.
Evaluation of the relative efficacy of various techniques for
deproteinizing plasma samples prior to high-performance liquid
chromatographic analysis.
Blanchard J.
Angus McLean Ph.D,
8125 Langport Terrace,
Suite 100,
Gaithersburg,
MD 20877
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The following message was posted to: PharmPK
Hi, Dr Benjamas,
Actually, both of them can be used for protein precipitation in HPLC
coupled with UV or fluorescence detector. However, these two will
produce a quite different effect. Generally, acetonitrile is a better
choice due to the big difference of their lipophilicities. You can
easily "sense" this difference when you develop a analytical method
using LC-MS. Hope this is helpful. Thanks!
Zhi
PhD candidate
Department of Pharmcology
National University of Singapore
Tel: (65)68745493
Email: g0305850.-at-.nus.edu.sg
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The following message was posted to: PharmPK
I had several references to protein precipitation that I found very
useful..
"Optimization of protein precipitation based upon effectiveness of
protein removal and ionization effect in
liquid chromatography-tandem mass"
Polson, C.; Sarkar, P.; Incledon, B.; Raguvaran, V.; Grant, R.
spectrometry, J. Chrom. B, 2003. 785, 263-275.
"Isolating Drugs from biological Fluids Using "Solvent First" Protein
Precipitation"
see http://www.argotech.com/products/analytical/isolute_ppt/
"Precipitation of Large, High-Abundance. Proteins from Serum with
Organic Solvents"
http://www.abrf.org/Other/ABRFMeetings/ABRF2003/Alpert.pdf
James Little
Tel. No. 423-229-8685 office
Tel. No. 423-229-8022 lab
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The method chosen depends on analyte stability, extration, separation, and
detection methods. Orgainic solvent, acid, salt and metal sion methods
were examed for protein removal and effect on ionization for LC-MS assay
in the paper bolow :
Polson C et al. Optimization of Protein precipitation based upon
effectiveness of protein removal and ionization effect in LC-MS. J
chromatogr B 785 (2003) 263-275
Xiaowei Teng (Shirley)
UBC
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Dear all,
I am working on protein precipitation of highly polar drug and I have
tried ACN, Methanol, IPA and DCM (in combination) percloric acid but
in all the cases the recovery is not more than 50%. Can any one
suggest any suitable solvent for polar drugs?
Second can anyone suggest me What should be optimum speed of vortex
and centrifugation?
Can anyone suggest me suitable references for protein precipitation
technique?Any suggestions as to how I can follow up on this idea
(suitable references for protein precipitation technique) would be
reatly appreciated.
Also are there any pharmacokinetics sites where I could post this
question as well?
thanks
raahul
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The following message was posted to: PharmPK
A leading reference..
Optimization of protein precipitation
based upon effectiveness of protein removal and ionization effect in
liquid chromatography-tandem mass Polson, C.; Sarkar, P.; Incledon, B.;
Raguvaran, V.; Grant, R.
spectrometry, J. Chrom. B, 2003. 785, 263-275
James Little
Tel. No. 423-229-8685 office
Tel. No. 423-229-8022 lab
Tel. No. 423-367-0914 Cell
"A Little Mass Spectrometry and Sailing":
http://users.chartertn.net/slittle
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The following message was posted to: PharmPK
Dear Raahul,
If nothing is working, you should switch to solid phase extraction.
As long as the recovery is always comparable (validate!!) and the
extracted fraction can still be quantitated, there is no problem.
Rob
--
Rob ter Heine, MSc, PharmD
Department of Pharmacology, Slotervaart Hospital
Amsterdam, The Netherlands
E: aprth.aaa.slz.nl
T: 020-5124737
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