Training Session Th1Thursday April 2, 2020 - Training Topic List

"Mass Spec Advanced Method Development - Peptides, Oligos, SM and Biomarkers: Solving Complex Challenges with Innovative Approaches on Sensitivity & Selectivity, Unstable/Sticky Compounds; Column Chemistry, Orthogonal Separations, Tissue Bioanalysis"

New level of Sensitivity & Selectivity (S&S); “Very” Unstable Metabolites/Pro-drugs how to catch them; Back to basic with adsorption of very “sticky” Compounds; Novel column chemistry and orthogonal separations. Novel strategies for tissue bioanalysis

Part 1: Method Development & Metabolites Quantification Challenges

Part 2: Biomarker Methods for new levels of Sensitivity & Selectivity (S&S)

Part 3: Peptides & Oligos - Solving Challenges with Innovative Approaches


Discussion Topic DETAILS of Th1 – Mass Spec Advanced Method Development

Part 1: Method Development & Metabolites Quantification Challenges


Part 2: Biomarker Methods for new levels of Sensitivity & Selectivity (S&S)
  • Lesson 7
    Novel Method Development Approaches for Tissue Bioanalysis of Biomarkers
    • Building on 2014-2016 White Paper in Bioanalysis recommendations on Tissue Bioanalysis of Biomarkers with new case studies and recent developments
    • “It is recommended to wash solid tissue samples with PBS during sample collection to remove possible blood contamination and flash freeze as soon as possible”
    • “When heterogeneity of the analyte distribution is a concern, use of pulverization and
    • homogenization techniques is recommended for the entire tissue sample to ensure a representative overall measurement”
    • Focus on Biomarkers Tissues analysis
      • Overcoming Sensitivity & Selectivity
      • development strategies
      • Tissue specific distribution
    • Adaptation of LCMS method for complex matrices
      • Increasing LCMS sensitivity for low level-analyte applications
      • Development of a novel extraction to isolate and enrich protein in tissues
      • Use of a selective, high throughput trapping LCMS method
    • Case studies on Novel developments and applications of a very sensitive LCMS assay for quantification of biomarkers in tissues

  • Lesson 8
    Novel Multiplex HRMS Approaches in the Quantification of Clinical Biomarkers of Transporter Mediated in DDI: Building on WRIB recommendations, new case studies, evolving trends
    • Building on 2018-2019 White Paper in Bioanalysis recommendations on Biomarkers of Transporter Mediated in DDI with new case studies and recent developments
    • Increased interest in utilizing endogenous probes for drug-drug interaction (DDI)
      • Limitation of current approaches
      • High false positive rates with
      • Organic cation transporters (OCT1 & OCT2)
      • Multidrug and toxin extrusion proteins (MATEs)
    • Focus on Clinical Small Molecule Biomarkers for FIH studies
      • HRMS Advanced Method development for
      • Isobutyryl-L-carnitine (IBC)
      • Carnitine
      • Thiamine
      • N1-methylnicotinamide (1-NMN)
      • Creatinine
      • Metformin
    • Chromatographic challenges
      • Developing a highly sensitive hydrophilic interaction chromatography (HILIC)
    • FFP Validation
      • Surrogate matrix approach
      • Avoiding interference from endogenous analytes
      • Parallelism to native plasma
    • Novel case studies on multiplexed assay utilized in clinical sample analysis

  • Lesson 9
    Method Development Challenges and Solutions in Biomarkers: Importance of thoroughly Optimized Chromatography and Column Chemistry
    • Building on 2017-2019 White Paper in Bioanalysis recommendations on Small Molecules Biomarkers Method Optimization & FFP Validation with new case studies and recent developments
    • Advantages of Surrogate Analyte approach
      • Stable isotope-labeled fructose and sorbitol
        • Surrogate standards
        • Internal standards
    • Fructose and Sorbitol in the clinical setting
      • Challenges to develop a reliable bioanalytical method
      • Multiple isomers of sugars present in human plasma
        • Interferences from glucose and mannitol
      • Low molecular weights of these monosaccharides
      • Challenging chromatographic characteristics.
        • No derivatization & Chromatographic separation on a hydrophilic interaction liquid chromatography
        • Importance to increase column temperature to increase the analytical throughput while maintaining acceptable separation efficiency
    • FFP Validation
    • Case studies on fructose as SM Biomarker for NASH (nonalcoholic steatohepatitis) and predictive biomarker for the total sugar intake

  • Lesson 10
    Challenges with Biomarkers Quantification in Rare Matrices: How to conduct method development for successful FFP BAV and sample analysis?
    • Issue in in developing biomarkers methods on Rare Matrices
      • Use of human cerebral spinal fluid (CSF)
        • High cost
        • Low availability of healthy subject CSF
    • Use as a surrogate matrix
      • Artificial CSF for method development
      • Experiments needed for comparison of authentic CSF with artificial CSF
      • Matrix to use to prepare QCs (authentic or artificial CSF).
    • Method development strategies for CSF
      • How to achieve pg/mL sensitivity
      • Assessment of parallelism
      • Matrix effect
      • Adsorption of analyte(s)
      • Acceptance criterion for slope of calibration curve
        • Surrogate matrix vs authentic biological matrix
    • Effect for the surrogate calibration curve was evaluated at three levels
    • Case studies to evaluate accuracy recovery after spiking analyte into endogenous analyte matrix and variation among authentic biological matrix lots


Part 3: Peptides & Oligos - Solving Challenges with Innovative Approaches


Agenda at a Glance Agenda at a Glance