Bioanalytical method development


An analytical method used for quantification of analyte(s) or metabolite(s) in a biological matrix is reliable and reproducible to achieve its purpose, to determine the analyte or metabolite with a degree of accuracy and precision.
                                 How to perform bioanalytical method development for estimation of  drug(s) and              
                                 metabolite(s) in human plasma by LC-MS/MS method.         
                                    1. Literature survey for drug and metabolites.
      • Collect information on physicochemical properties, pharmacokinetic parameters, regulatory aspects, chromatographic and extraction procedures of drug from possible literature sources like drug bank, pub chem, internet, physician desk reference, OGD guidance and other available sources.
      • Collect the information regarding the stability of the analyte(s} in solution (pH dependent stability and photo sensitivity) and in biological matrices.
      • Review the literature for major active and inactive metabolites/isomers/epimers which may contribute to analyte estimation.
      • Report the physicochemical parameter (structure, molecular weight and solubility), dosing strength, tentative cc range, pharmacokinetics (Cmax, food effects) parameters in the method development.
2.                                 2.  Selection of initial Conditions 
  • To determine suitable internal standard based on the structure / category from stable labeled compound or literature.
  • To determine equipment considering the sensitivity required.
  •  To determine conditions based on the properties of the drug.
  • To determine initial chromatographic conditions to start with like column, buffer, mobile phase, flow rate, detector, and others, if any.
  • Select the extraction procedure like liquid phase extraction, solid phase extraction, precipitation methods
                             3.     Optimization of drug and metabolites for mass spectrometry analysis
      • Calculate the exact molecular weight of analyte(s) or metabolite(s) I internal standard(s) with the help of mass calculator from instrument software or in some COA’S it is mentioned or from online like merck index, drug bank etc.
      • Prepare main stock in which drugs solubility is mentioned in COA or from literature or drug bank etc.
      • From main stock prepare tuning solution of about 200 ng/mL to 1000 ng/mL it depends upon the sensitivity of instrument which we are using.
      • Tune the drug or metabolite with suitable polarity on mass spectrometer to get the molecular ion peak in Q1 mode.
      • Identify the product ion(s) (Q3) in product ion scan mode.
      • Acquire scans averaged for 1 minute for Q 1 and Q 3 ions
      • Create acquisition method for quantification of drug and metabolites.
                             4.      Chromatography
        Select the mobile phase and column and identify the retention time for drug(s), metabolite(s)            and internal standard(s).Follow the above step for drug/metabolites, internal standards                       individually and     as mixture. Modify the chromatographic conditions based on the peak                    shapes,retention time and     resolution as required.
                                    5.   Extraction procedure
      • Spike drug and metabolite solution/s in the biological matrix and extract the drug as per proposed extraction procedure.Optimize the extraction procedure and chromatographic conditions.
      •  Process to extract the blank biological matrix as per the selected extraction method. Check the interferences at the retention times of analyte(s)/metabolite(s) and internal standards.
      •  Prepare a set of unextracted and extracted samples in the expected range of the calibration curve based on the pharmacokinetic parameters and inject on to the chromatographic device. Check the preliminary chromatographic data, linearity and recovery.  
      • Check for interconversion between analyte and metabolite(s) wherever applicable.
      • Check for consistent recovery, matrix effect and selectivity. Fine-tune the chromatographic conditions and sample clean up procedure.
      • Check for late eluting peaks in the extracted samples by extending the run to at least 5 times the run time.

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