PharmPK Discussion - RBC lysates Calibration Curve

• On 17 Oct 2002 at 08:59:13, "Louisa Alfazema" (LouisaA.at.wickhamlabs.co.uk) sent the message
  

  
  Does anyone in the group know or have an idea as to how one can do a
  calibration curve of analyte(s) using red blood cells lysates? and at
  what stage do you add the analyte to the RBC? Can it be done as an
  Internal standard or external standard calibration.
  
  If there is any references please let me know.
  
  Your views and ideas will be greatly appreciated.
  
  Louisa
  
  Dr Louisa Alfazema
  Bioanalyst Researcher
  Wickham Laboratories Limited
  Winchester Road
  Wickham
  Fareham
  Hampshire
  PO17 5EU
  
  

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• On 17 Oct 2002 at 21:02:23, eoconnor.at.Therimmune.com sent the message
  

  
  It is unclear to me what you are trying to do.  Is the lysate a
  preparation
  to examine distribution or metabolism?  Is it the matrix in which you
  intend
  to measure or is the red cell lysate the indicator of the presence or
  absence of your analyte? i.e. immunoassay using hemolysis as the end
  point?
  
  

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• On 18 Oct 2002 at 10:12:37, acardena.at.almirallprodesfarma.com sent the message
  

  
  I do not know exactly what is your purpose. Assuming that your idea is
  to
  determine the levels in RBC (not distribution plasma-RBC) I would try
  the
  following:
  
  Just after removing the plasma take a fix volume of RBC (for example
  250 µL)
  and ad then your analyte (at this time you can also ad your internal
  standard if you need to) and freeze the sample.  Of course your samples
  should be treated in the same way.
  
  The day of the analysis lyse the samples and your calibration curve in
  a way
  compatible with your analytical method  (for example we have used
  freeze-unfreeze three times, and ending with a osmotic shock and
  sonication,
  finally analysing the samples with a SPE-HPLC-UV procedure).
  
  You have to check the recovery and stability and taking into account the
  non-specific binding to RBC membranes (perhaps you should use an organic
  solvent to overcome this problem).
  
  I hope this can help
  
  Alvaro
  
  Dr.Alvaro Cardenas
  Drug Discovery Unit
  AlmirallProdesfarma
  Barcelona
  Spain
  
  

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• On 21 Oct 2002 at 10:07:56, BENECH Henri 137577 (BENECH.at.dsvidf.cea.fr) sent the message
  

  
  Dear all,
  An accurate description is also given for an LC/MS/MS in PBMCs in the
  following papers :
  
  Rapid Commun. Mass Spectrom. 16: 555-565 (2002).
  Rapid Commun. Mass Spectrom. 15: 1401-1408 (2001)
  
  Regards/Henri BENECH
  CEA
  FRANCE
  
  

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• On 22 Oct 2002 at 14:06:29, "Louisa Alfazema" (LouisaA.at.wickhamlabs.co.uk) sent the message
  

  
  The analyte(s) metabolism will be measured in the lysate and it is the
  matrix in which the calibration curve will have to be prepared in. i.e.
  the analytes will be prepared in blank RBC and then use LC-MSMS LC-UV
  for the analysis.
  
  I hope this is clear
  
  Thank you for all responses.
  
  regards
  Louisa
  
  

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• On 22 Oct 2002 at 17:42:28, sunil bajad (sbajad.-at-.yahoo.com) sent the message
  

  
  Hi everyone,
  I have few questions related to Loiisa's original
  question on RBC lysate.
  
  1. What matrix should be used for calibration curve if
  a) drug is highly metabolized in lysate both
  enzymatically and non-enzymatically (GSH, Ascorbic
  acid mediated reduction);b) if drug/metabolite
  strongly binds to haemoglobin which is ~ 90% of total
  RBC protein.
  
  Dr. Sunil bajad
  Research Associate
  Department of Medical Sciences
  School of Vet. Medicine
  University of Wisconsin-Madison,
  2015 Linden Drive west,
  Madison 53706, WI,USA
  
  

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