Every rubber stopper product has different characteristics.

Some
of them could adsorb endotoxin. Inhibitory compounds may be leached
from some of them. To decide a sample preparation method, you need an
endotoxin challenge test to show suitable endotoxin recovery with your
condition. If you have an acceptable endotoxin recovery (usually 50% to
200%) when you spike a certain amount of endotoxin on the rubber
stoppers, you can use your extraction method. If not, you need to find
an appropriate extraction method for your rubber stopper. A detergent
or a protein solution could be an appropriate extracting solution.
Considering the information you provided, it is hard to judge
whether your sample preparation is suitable or not.

2. Endotoxin caluculation
The specification in endotoxin for rubber stoppers is expressed in
EU/each (rubber stopper). I guess you are using the gel-clot method,
and that you are testing dilutions of your extract. In this case, you
are getting an endpoint (EP) at a certain dilution of your extract on
the LAL test. You can obtain the endotoxin concentration in your
extract as below;

Endotoxin concentration in the extract (EU/mL) = Labeled sensitivity of

LAL (EU/mL) x Dilution rate of the extract at EP

If you use 100 mL of extract, the endotoxin level of the rubber stopper
is calculated as below;

Endotoxin on a rubber stopper (EU/each) = (Endotoxin concentration in

the extract (EU/mL) x 100 (mL))/Number of the rubber stopper extracted
(each)

I hope this information is helpful for you.

Regards,

KAZ Tsuchiya
Masakazu Tsuchiya, Ph. D.
Senior Research Scientist
Endotoxin and Microbial Detection
Charles River
TEL: 843-766-7575
FAX: 843-766-7576
E-mail: [log in to unmask]
Experience the new www.criver.com

Accelerating Drug Development. Exactly.

Notice - This email and any files transmitted with it are confidential
and may contain privileged and/or proprietary information. You must not
disclose this message to another party without Charles River's express
written consent. If you are not the intended recipient you must not
copy, distribute or use this email or the information contained in it
for any purpose other than to notify us. If you have received this
message in error, please notify Charles River immediately, and delete it
from your system.

-----Original Message-----
From: The Pharmaceutical Microbiology Forum Email List
[mailto:[log in to unmask]] On Behalf Of Khojema Ratlamwala
Sent: Tuesday, July 14, 2009 3:23 AM
To: [log in to unmask]
Subject: [PMFLIST] BET for the rubber closure

Dear all ,

During the method preparation of the bet testing of the rubber closure
:'
I had consider the following things

1. Sample preparation : transfer the no. of rubber closures to be

tested into the dehydrogenated test tube containing 100 ml of Water for
Injection.
2. calculation method used below

Endotoxin content EU/ml

=Lysate sensitivity x Dilution/No. of Rubber Closures x Average Surface

area
of one Rubber closures (cm2)

So I want to have the group opinion for the mehod I am using ,

Also I need to know what others follows at their end for the testing of
the
rubber stoppr or also is that any referance avaialble for the same

Regards

Khojema Ratlamwala.

Quality control

Unique Pharmaceutical Laboratories,

Plot No. 4,Phae IV,GIDC,Industrial Estate ,Panoli

Phone no:02646-272913/272914/272915

Fax:02646-272411

------------------
The PMFList (http://microbiol.org/PMFList_info.htm) is operated from
The Microbiology Network (http://microbiol.org) and supported by
our sponsors (http://microbiol.org/sponsor.htm) as a service to
the scientific community.

Please take a second to visit our sponsors' web sites and say thank you for
their support of this service.

Accugenix - http://www.accugenix.com

American Type Culture Collection - http://www.atcc.org

Applied Biosystems, MicroSeq Microbial ID System - http://www.microseq.com/

BD Diagnostic Systems - http://www.bd.com/ds/

Biolog - http://www.biolog.com

Biomerieux - http://industry.biomerieux-usa.com

EMD Chemicals - http://www.emdchemicals.com

MicroBioLogics, Inc. - http://www.microbiologics.com

Millipore - http://www.millipore.com

MODA Technology Partners - http://www.modatp.com

Pall - http://www.pall.com

NovaTek International - http://www.ntint.com

Rapid Micro Biosystems - http://www.rapidmicrobio.com (formerly GPS)

Raven Biological Labs - http://www.ravenlabs.com

Veltek Associates, Inc - http://www.sterile.com

=================================
The nature of this service is to provide a medium for communication. The
specific statements and endorsements of individuals participating in the
discussions are not necessarily those of The Microbiology Network, Inc., the
PMF, or the sponsors of the list.