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BioAnalysis Method Builder for an Oasis Ion Exchange Method         
To find the right Oasis Ion Exchange SPE Product for your method      
1) Select type of compound you want to isolate?
       
2) Select preferred SPE format?
     
3) Select use of µElution format to eliminate the evaporation step* (Y/N)
     
4) Select your usual plasma load volumes range (min-max)  ?
   
           
To calculate the optimized Method Development Protocol for your method          
5) Input the plasma load volume you expect to use for this new method (µL)      
6) Input the mid-point of the concentration range for this new method (ng/mL)?        
7) Select the number of Data points (replicates) used for your Recovery Calculations
       
           
  SPE Product Details        
  Compound Type        
  Oasis format chosen        
  Format details        
  Oasis Sorbent Selected        
  Waters Part Number to Order      
  © Copyright Waters Corporation 2014          
       
  SPE Method Calculated for selected SPE product      
  Plasma (sample) volume         
  Pre-SPE Treatment (diluent) volume      
  Condition      
  Equilibrate      
  Load      
  Wash      
  Elute 1      
  Elute 2      
  Total Elution volume per Elute        
  SPE Concentration or Dilution Factor  
  Flow Rate for SPE method      
           
* It is recommended to avoid the evaporation step for volatile and/or heat labile Compounds      
*The µElution format allows sample concentration up to 15x without evaporation (when all the compound elutes in the first 25 µL of the 2 elutions used in the method)  
OASIS IEX Recovery and Matrix Effects Experimental Set-up  
Mid-point of the concentration range for this method ng/mL    
Number of Data points (replicates) required  
STANDARDS PREPARATION    
Concentrated Standard Stock Solution preparation (S1)    
   
   
Standard Solution preparation (S2)      
     
   
   
   
   
 Pre-Treatment (diluent) Preparation       
   
   
   
  © Copyright Waters Corporation 2014  
PLASMA PREPARATION    
Estimated total volume of bulk Plasma needed    
Pre-Spike Plasma preparation (P1)    
   
   
   
       
   
       
   
   
Post-Spike Plasma preparation (P2)      
     
     
     
         
     
         
     
         
OASIS IEX Calculating Recovery and Matrix Effects        
  Pre-SPE Spiked Samples  
  Load Pre-Spike Plasma (P1)          
  Apply SPE Process      
  Elute 1   Elute 2    
  Add Water   Add Water    
  Inject onto LC/MS   Inject onto LC/MS    
  Final concentration (ng/mL)   Final concentration (ng/mL)    
  Enter LC/MS Data     Enter LC/MS Data      
       
  Post-SPE Spiked Samples  
  Load Post-Spike Plasma (P2)          
  Apply SPE Process          
  Elute 1   Elute 2    
  Add Standard Spike (S2)   Add Standard Spike (S2)    
  Inject onto LC/MS   Inject onto LC/MS    
  Final concentration (ng/mL)   Final concentration (ng/mL)    
  Enter LC/MS Data     Enter LC/MS Data      
       
  Standard Solution (S2)   Standard Solution (S2)  
  Used to Calculate matrix effects for Elute 1   Used to Calculate Matrix Effects for Elute 2  
  Standard (S2) concentration   Standard (S2) concentration  
  Take Aliquot S2   Take Aliquot S2  
     
  Inject onto LC/MS   Inject onto LC/MS  
  Final concentration (ng/mL)   Final concentration (ng/mL)  
             
  Enter LC/MS Data     Enter LC/MS Data    
     
         
   Results for Elute 1      
  (Reversed Phase bound components)      
  Recovery %      
  Matrix Effects % Calculations are only calculated    
  Matrix Factor   when all LC/MS data has been entered    
  Results for Elute 2      
  (Ionic components)      
  Recovery %      
  Matrix Effects % Blank Plasma    
  Matrix Factor          
      Diluted  
Understanding Charge States during  
OASIS Ion Exchange Solid Phase Extraction  
         
  Method Step Load   Wash   Elute 1   Elute 2    
  INPUT HERE                
Compound Type
      compound sorbent   compound   sorbent   compound   sorbent   compound   sorbent    
pH when loading
      ACIDS (pKa < 1)        
Sorbent       ACIDS (pKa 2-8)        
Protocol       BASES (pKa >10)        
    BASES (pKa 2-10)        
    NEUTRALS        
    ZWITTERIONS        
             
  Key       Wash   Elute 1   Elute 2    
Negative Charge -                    
Neutral Charge NC     Eluted Compounds        
Postive Charge +              
                 
           
     
                      © Copyright Waters Corporation 2014            
Optimizing the Wash and Elute steps in OASIS IEX Methods  
 
    Elution (% MeOH)  
  0 10 20 30 40 50 60 70 80 90  
  Compound 1  
  Compound 2  
  Compound 3  
   
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  Interpret Graph and Enter Data Below  
  % Methanol at Lift-off  
    % Methanol at Area Count Maxima  
 
 
 
  Wash with  
  Elute with  
  © Copyright Waters Corporation 2014  
 
    Numbers shown are for example. Overwrite them with your experimental results  
     
   
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