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Dear all,
I would like to know a convenient way of breaking the emulsion that
forms during extraction of plasma with methylene chloride. We tried to add
NaCl, freezing and recenrifugation of the emulsion formed. We suspect
vigorous vortexing may be the reason for some emulsion formation in the tube
(plasma 0.1 ml, pH 2.0 buffer 0.25 ml and 5 ml of MeCl2). We need to break
the emulsion (about 2 ml of emulsion in each tube) for higher recovery of
the drug and internal standard. Thanks in advance, Delwar.
****************************
M. Delwar Hussain, Ph.D.
Assistant Professor of Pharmaceutics
School of Pharmacy
University of Wyoming
Laramie, WY 82071-3375
Tel:(307) 766 6129
Fax: (307) 766 2953
E-mail: delwar.aaa.uwyo.edu
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[A few suggestions - db]
From: "CHARLES"
Organization: School of Pharmacy
To: PharmPK.aaa.pharm.cpb.uokhsc.edu
Date: Thu, 20 Aug 1998 12:18:36 +1000
MIME-Version: 1.0
Subject: Re: PharmPK Breaking emulsion REPLY
X-Confirm-Reading-To: "CHARLES"
X-pmrqc: 1
Priority: normal
Vortex-mix 0.1 ml plasma + 1 ml chloroform or methylene chloride in a 13-15
mm diameter 10-15 ml tube then centrifuge for around 15 s at about 500 g then
simply decant the organic phase carefully away from the emulsified plasma.
Works like a charm, provided you have an internal standard.
(Other than this all I can suggest is adding an antifoaming agent)
Cheers,
BC
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X-Originating-IP: [194.95.249.25]
From: "Krishna Devarakonda"
To: PharmPK.at.pharm.cpb.uokhsc.edu
Subject: Re: PharmPK Breaking emulsion
Date: Wed, 19 Aug 1998 22:59:50 PDT
Dear Dr.Hussain,
This is rather a common problem. The emusion formation is seen more
often with chloroform and dichloromethane. Vigorous mixing definitely
contributes to it. I suggest a few tips for overcoming this problem,
please try (with or without NaCl):
1. Instead of vortexing use a rotary tube mixer, use longer time of
mixing with less number of rotations per min.
2. The problem is more severe when the volume of organic phase is less
or close to that of aq. phase. Therefore increase CH2Cl2 volume.
3. Instead of taking pure dichloromethane, try it with 20 - 30 % ethyl
acetate or methanol.
We tried the last method with success.
Good luck!
D.R.Krishna,Ph.D
AvH Scientist
Dr.Margarete-Fischer-Bosch-Institue
of Clinical Pharmacology
Auerbachstr.112
70376 Stuttgart, Germany
Tel. Off: +49-711-8101-3753, Res: +49-711-5590-273
Fax: +49-711-85 92 95
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From: "Anton Chadwick"
To: Delwar Hussain(by way of David_Bourne),
PharmPK.at.pharm.cpb.uokhsc.edu
Date: Thu, 20 Aug 1998 09:23:45 GMT
Subject: Re: PharmPK Breaking emulsion
Reply-to: achadwick.-a-.fs1.pa.man.ac.uk
Priority: normal
If the drug is heat stable, put samples in heated water bath.
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From: Charles.Gilbert.at.charnwood.gb.astra.com
To: PharmPK.aaa.pharm.cpb.uokhsc.edu
Subject: RE: PharmPK Breaking emulsion
Date: Thu, 20 Aug 1998 11:33:27 +0200
Mime-Version: 1.0
Have you tried freezing in dry ice following centrifugation (solid CO2
freezing mixture with ethanol for example) and tipping while still frozen?
Charles Gilbert
Bioanalysis and DMPK Development
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Date: Thu, 20 Aug 1998 07:42:50 -0400 (EDT)
From: Ahmad Mirfazaelian
To: PharmPK.at.pharm.cpb.uokhsc.edu
Subject: Re: PharmPK Breaking emulsion
Mime-Version: 1.0
Hello,
Have you tried ultracentrifugation for breaking the emulsion?
Ahmad M.
PK student
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From: "Tata, Prasad"
To: "'PharmPK.at.pharm.cpb.uokhsc.edu'"
Subject: RE: PharmPK Breaking emulsion
Date: Thu, 20 Aug 1998 08:21:43 -0400
MIME-Version: 1.0
I would recommend either Sodium Sulfate or Ammonium sulfate or add 0.1% Iso
amyl alcohol. Adding first two salts offers the advantage some how they
reduce solvent evaporation time and assists ionization(in case you are using
Mass Spec). Addition of Amyl alcohol by some unknown mechanism assists in
improved recovery of polar metabolites into org. solvents.
Hope this helps.
Prasad Tata
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Mime-Version: 1.0
Date: Thu, 20 Aug 1998 08:53:21 -0500
To: PharmPK.aaa.pharm.cpb.uokhsc.edu
From: shoaf.at.clinpharm.niaaa.nih.gov
Subject: Re: PharmPK Breaking emulsion
Don't vortex! cap tube and put on a reciprocating shaker, the one that
tilts back and forth. Set it on slow and extend your shake time to 15 min
or more. We have also used a horizontal shaker, like used for gels, but the
tilt works faster.
Susan Shoaf, Ph.D.
shoaf.at.clinpharm.niaaa.nih.gov Nat'l Institute on Alcohol Abuse and Alcoholism
I don't speak for NIAAA, Unit of Pharmacokinetic Studies
That's for the PR office
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Date: Fri, 21 Aug 1998 21:52:57 +0200
From: "W.J.J. Krauwinkel"
Reply-To: wjj.krauwinkel.-at-.wxs.nl
MIME-Version: 1.0
To: PharmPK.-a-.pharm.cpb.uokhsc.edu
CC: Multiple recipients of PharmPK - Sent by
Subject: Re: PharmPK Breaking emulsion
X-Priority: 3 (Normal)
There are several things you can try. Sometimes it helps to add
sodium chloride or sodium sulphate. With toluene I have the experience
that it can form a kind of 3-dimensional web attached to the glass wall.
Silanizing the glass tube before extraction helped in this case. Maybe
it also helps with methylene chloride.
With kind regards
Walter Krauwinkel
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From:
Date: Sun, 23 Aug 1998 00:14:20 EDT
To: PharmPK.-at-.pharm.cpb.uokhsc.edu
Mime-Version: 1.0
Subject: Re: PharmPK Re: Breaking emulsion
Dear Dr. Hussain:
Actually, a better alternative is extracting with hexane with about 10%ethyl
acetate, This way the organic phase is on top and can easily be pipetted out
from the lower emulsified plasma.
For about 0.1 ml plasma, add 2 x 0.5 ml hexane with 0.05 ml ethyl acetate.
Quickly centrifuge in a microfuge (polyethylene) for about 5 min at 500 g. Of
course, an internal standard will monitor recovery. This allows for effective
separation of highly lipophilic compounds.
I hope this helps your PK experiments.
Yours,
Melinda Guzman-Harty, Ph.D.
Senior Research Scientist
Battelle Memorial Institute
guzman-hartym.-at-.battelle.org
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Can you precipitate plasma proteins and remove the pellet prior to
extracting without losing drug?? This will probably reduce emulsification.
K. Anderson
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Once your plasma extract has formed an emulsion, it is very hard
to eliminate. My suggestion would be to eliminate the cause of
the emulsions. Substitute a horizontal shaker for the vortex and
do your extractions with less vigorous mixing. You'll need to
re-establish your equilibrium time for this extraction step. I
hope this helps.
Hank Pieniaszek
DuPont Pharmaceutical Company
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Yes, the plasma proteins may be precipitated in many different ways eg.
acetonitrile, methanol, trichloroacetic acid. The important thing to remember
is to centrifuge at least 12,000 rpm or 500 g for about 10 min so that the
supernate will contain most of the drug.
It will definitely help to use an internal standard to monitor recovery.
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