PharmPK Discussion - Nicotine LC-MSMS method development

• On 9 Mar 2014 at 18:36:57, Inas Ali (nosa_ebraheem.-at-.hotmail.com) sent the message
  

  
  I am developing an LC MSMS method for nicotine quantification and it's metabolite (cotinine) in
  plasma using ritonavir as internal Standard.
   The System is as follows:
  mobile phase: (ACN:water:formic acid)(750:250:1)
  Agilent C18 column.
  Tuning of the three compounds were made easily by Empower's Intellistart.
  the method of extraction is Liquid - liquid extraction using ethyl acetate.
  
  the lowest conc that can be detected for nicotine is 0.25 ng/mL
  Rt for nicotine is 1.05 and cotinine 1.1 and IS 1.7 min
  
  The Problem is that when I started method validation and I started with
  Linearity; there were a huge fluctuations on the same set of concentration and and when I calculate
  peak area ratio there were no linear response.
  
  the second problem, I started to increase the concentration of cotinine , I found that this lead to
  an increase on the detected nicotine area.
  
  I don't know where is the problem with this method, because I am short out of time and I had to
  proceed on method validation because, later I will receive plasma samples that I have to analyze.
  
  Thanks,
  Inas
  

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• On 9 Mar 2014 at 21:00:46, David Bourne (david.aaa.BOOMER.ORG) sent the message
  

  
  Please try to retain the drug more on column. Separate drug and IS retention time. Try gradient
  method.
  Thanks,
  Sudipta Basu
  
  sudipta basu 
  --
  
  You do not have a method to validate and should not proceed to sample analysis.  It seems that your
  choice of ion for nicotine may need to be changed since it is influenced by cotinine.   How well did
  you scout selectivity?  There are dozens of methods for nicotine and cotinine.  How does yours
  differ?
  
  Edward O'Connor 
  --
  
  I think nicotine is endogenous do the base line correction
  Regards
  Tushar
  
  tusshar_ank.-at-.yahoo.co.in
  

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• On 10 Mar 2014 at 09:10:56, David Bourne (david.at.BOOMER.ORG) sent the message
  

  
  Hello Inas,
  
  It is preferable to use internal standards that have physicochemical/LC-MS properties similar to the
  analyte(s).  Nicotine and its derivatives are small (MW ~160), polar (LogP ~1) compounds while
  ritonavir is >4x big (MW 721) and much greasier.  Assay variability in nicotine will be unlikely to
  be compensated by changes in ritonavir.  Stable label nicotine is available from multiple suppliers,
  as are structurally similar analogues.  For something potentially this tricky I'd suggest moving to
  a more relevant IS.
  
  All the very best,
  
  Bernard
  
  Bernard Murray, Ph.D.
  Senior Research Scientist, Drug Metabolism
  Gilead Sciences, Foster City CA
  
  Bernard Murray 
  --
  Dear Inas.
  Did you calculate the AUC for everyone separate? If so add one to the nicotine and check linearity.
  If ok, it means you can not combine them in one run. change the IS.  I have these experience when
  more drugs included in one run.
  
  Mahasen Radwan 
  --
  Dear Anas
  
  I think you have to do the following:
  1. Change the mobile phase to:  ACN : H2O: Formic acid  (850:150: 1 ), in order to shift the analyte
  away from the solvent interference i.e. 2.5 min RT and more.
  2.   Try to use extracting solvents of less polarity than ethyl acetate such as ethyl ether,
  di-chloromethane and / or isopropanol ....etc
  
  Sincerely yours
  
  DR.ELTAYEB E.M.EID, Ph.D.
  (Drug Development and Clinical Proteomics )
  Assistant Professor
  Department of Pharmaceutics
  College of Pharmacy, Qassim University
  Qassim, Saudi Arabia
  
  ELTAYEB EID 
  
  

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• On 18 Mar 2014 at 07:13:34, Jiangeng Huang (jiangenghuang.at.gmail.com) sent the message
  

  
  Dear Anas,
  
  1. Is there any info. available regarding matrix effect?
  2. How about the cross-talk between nicotine and it's metabolite?
  
  Best,
  
  Jiangeng Huang
  
  

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• On 18 Mar 2014 at 21:56:16, Jagdish Jaiswal (jagdish207.at.gmail.com) sent the message
  

  
  Hi Inas,
  
  Please refer to a recent paper published in Drug Metabolism and Disposition: 42:282-293 (Feb 2014).
  Although the authors used a stable isotope (deuterated) internal standards but would give you good
  idea about the method, mobile phase, extraction etc of nicotine and its four metabolites. They
  reported MRM 163>117 for nicotine and 177>80 for cotinine. Do you see these transitions in your mass
  spec for nicotine and cotinine?
  
  cheers,
  
  Jagdish
  
  --
  Jagdish Jaiswal, PhD
  Auckland Cancer Society Research Centre
  The University of Auckland
  New Zealand
  
  

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