Course 1:
LC-MS/MS Bioanalytical Advanced Method Development Course
8am - 5pm, March 26, 2012
Instructor: Dr. Fabio Garofolo, Algorithme Pharma
Course Description:
LC-MS/MS Bioanalytical Advanced Method Development course has been specifically designed for scientists and experts who work in drug discovery and bioanalytical laboratories desiring advanced training in bioanalytical method development.
Original method development strategies and difficult method development cases are discussed.
Course Syllabus:
- Improving selectivity and sensitivity in bioanalytical method development: Chromatographic & Ionization strategies
- Impact of high pH mobile phase on the ESI(+)
- How is it possible to analyze basic compounds in ESI(+) if they are not in their ionized state under high pH mobile phase?
- Wrong-Way-Round Ionization
- Post Column Addition and high pH mobile phase
- Ammonium Hydroxide vs. Formic and Acetic Acids
- Ammonium Acetate pH 9.3 vs. Ammonium Acetate pH 6
- Ammonium Bicarbonate pH 7.8 and pH 10.5 vs. Ammonium Acetate pH 9.3
- Advanced use of Atypical Modifiers to Perform High pH Chromatography by LC-ESI(+)-MS/MS
- Signal Enhancement of Pyrrolidine vs. Formic Acid
- System Stability Detection / Chromatography
- Method development of High pH Mobile Phase in LC-ESI(+)-MS/MS Under HILIC Mode
- HILIC Columns Durability/Robustness
- Case studies: Acyclovir; Morphine; Candesartan; Ramipril and Ramirilat; Hydromorphone
- Improving selectivity and sensitivity in bioanalytical method development: Clean up/Extraction strategies
- Impact of phospholipids and lipemic plasma on bioanalytical method development
- Importance of removing phospholipids during sample clean-up
- Source of matrix effect and its dependency on the nature of the analyzed matrix
- Matrix effect and the presence of phospholipids in plasma samples
- Focus on different extraction techniques preventing the extraction of phospholipids
- Matrix Effect: Co-eluting matrix components that affect the efficiency of ionization of the analyte of interest
- Phospholipids cause matrix effect under reversed-phase (RP) conditions
- Phospholipids cause matrix effect under HILIC Conditions
- Phospholipids MS/MS Fragmentation
- Phospholipids Removal in SPE
- Evaluation of Different SPE Elution Conditions and different SPE: silica-based vs. polymer-based
- Case Studies: Diclofenac and Etodolac
- Phospholipids Removal in LLE
- Testing Different Extraction Conditions: Extraction pH; Buffer Concentration; Extraction Solvent
- Case Studies: Risperidone and Ziprasidone
- High Throughput Phospholipids Depletion Plate
- PPT vs.Captiva ND Lipid Removal Plate
- Case study: Tolterodine
- Impact of unstable metabolites on bioanalytical method development
- Drug Metabolism: Fundamentals
- Metabolite Conversion During Sample Extraction
- Metabolite Conversion During Instrumental Analysis
- Challenge: Metabolite may be extracted from Human matrices with its parent drug
- General Strategy for checking method selctivity
- Acylglucuronides: Method Development Strategies
- Measurement of acylglucuronides and strategies to test them when the reference standards are not available
- Case Studies:
- Isotope distribution of Chlorines
- MS/MS Strategy Apply to Ramipril and Ramiprilat and Beta-Glucuronidase Procedure
- Impact of Acyl Glucuronide and N-Oxide Metabolites During Sample Processing
- The "whole" Clopidogrel saga (Episode I:The Acyl Glucuronide degradation; Episode II: Clopidogrel Acid conversion; Episode III: Clopidogrel & SPE; Episode IV: The Transesterification )
- Etodolac and its conjugated acyl glucuronide metabolite
- Repaglinide and its conjugated metabolite acyl glucuronide
- N-oxide conversion & the evaporation step
- Impact of Hemolysis on bioanalytical method development
- Effect of Hemolysis on Matrix Effect
- Effect of Hemolysis on Recovery
- Effect of Hemolysis on Ionization
- Importance of method development strategies to minimize or eliminate the impact of hemolysis on LC-MS/MS methods in Bioanalysis
- Internal standard evaluation
- Chromatographic adjustements in order to better control the suppression caused by the hemolyzed matrix
- Post-Column Infusion Profile
- Case studies: Entacapone; Amlodipine
- New case study: Hemolysis & Stability
- Method development challenges & strategies correlated to the Implementation of Dried Blood Spots (DBS) In Bioanalysis
- DBS & Bioanalysis
- Challenges in Punching a 3 mm disc from the DBS card: Manually vs. Using robotic system
- Challenges in Extracting the disc in organic solvent
- Challenges with Chromatography: DBS vs. Plasma
- Impact of the hematocrit; Effect on dilutions;Internal standard techniques to add it earlier; Considerations on stability
- Multiple DBS cards evaluation during method developmet : Treated vs. Untreated and their effect on recovery, ionization suppression/enhancement
- Post-Column Infusion Profile
- Important considerations on DBS validation
- Recovery evaluation with DBS
- Dilution integrity evaluation with DBS
- Long-term on-card at room temperature stability
- Metabolite stability: acylglucuronide metabolite
- Case Study: Naproxen
- Method development challenges with Macrolide Immunosuppressant Drugs
- Structures & Considerations: Sirolimus, Tacrolimus, Everolimus and Ascomycin
- Whole blood extraction & possible solutions
- Ionization & Fragmentation issues
- Choice of Internal Standard
- System Stabilization: matrix and type of extraction vs. different system parameters
- Chromatography: Very sharp peaks and close elution
- MRM Limitation vs. MRM3
- S/N: MRM vs. MRM3: pros and cons
- Systematic Method Development: Cytidine Analog & Matrix Effect: "the perfect storm"
- Impact of nucleoside metabolism on method development strategies
- Suppression due to endogenous interferences
- APCI vs. ESI approach
- RP vs. HILIC approach and column screening
- Decision tree : Acetonitrile vs. methanol
- Extraction efficiency and matrix effect
