LC-MS/MS Advanced Optimization & Method Development Strategies Course
Course Syllabus
Bring your optimization & method development problems to the course!!!
Advanced APCI and APPI Optimization
- Good reasons for using APCI in fast method development
- When to use APCI
- Case Study: Corticosterone and Hydroxyprogesterone optimization
- Advanced APCI optimization for polar compounds
- Corona current optimization for polar compounds
- Case Study: Verapamil and Reserpine optimization
- APCI limitations
- Leidenfrost Effect
- Optimal auxiliary gas optimization for reducing background noise
- Special and unique solvents for APCI to increase the chemical ionization (CI)
- APCI vs. ESI
- Choosing between electrospray and APCI
- What is the best source for each application?
- Normal Phase and APCI
- APCI and ethoxynonafluorobutane (ENBF)
- Case studies: Normal Phase APCI of steroids, benzodiazepines, central nervous system-active substances, nonsteroidal anti-inflammatory drugs, tricyclic antidepressants, and beta-adrenergic blocking agents.
- Good reasons for using APPI in fast method development
- Dopant APPI = Photoionization-induced APCI
- APPI and Negative Ions
- Ionization potentials (IP) strategies: solvents with high IP, low IP, medium IP
- Two commercially available APPI sources: Sciex vs. Syagen - pros/cons
- Dimers, Methanol, acetonitrile & APPI
- APCI vs. APPI vs. ESI
- Polarity (ESI, APCI, APPI, dopant-APPI)
- Charge competition (ESI, APCI, APPI, dopant-APPI)
- Ion suppression (ESI, APCI, APPI, dopant-APPI)
- Flow rates (APCI, APPI, dopant-APPI)
- Reverse phase vs. normal phase (ESI, APCI, APPI, dopant-APPI)
- Thermally unstable compound (ESI, APCI, APPI, dopant-APPI)
- Bioanalytical case study: Testosterone Quantitation and Ritonavir optimization
- Environmental case study: Carbamate and Phenyl urea
Advanced ESI Optimization
- pH and ESI
- Specific knowledge in using additives & buffers in ESI(+/-) method development
- Can you still use Ion Paring Chromatography in ESI?
- Use of TFA, TCA, HFBA and ion suppression
- Apffel et al. and the "TFA FIX"
- Environmental case study: Multiresidue Analysiss of Antibiotics in Food
- Atypical solvents used in ESI(+) and ESI(-)
- Use of DMSO in ESI (+): Imipramine & Amitriptyline optimization
- Use of DMF in ESI (+/-): Hydrophobic peptides and sugars
- Literature review and unique applications
- A fast review of the V.I.C. Rule and 5 Gold Roles in advanced ESI optimization
- Tricks and tips of using post-column additions to enhance sensitivity in ESI
- Application of post-column addition in ESI
- Case study: Sensitivity enhancement of ibuprofen by 2-(2-methoxyethoxy)ethanol
Advanced Optimization of New API Interfaces
- Optimization and advantages of new API interfaces in method development
- Head-to-head performance comparison among:
- Turbo V (Sciex)
- IonMax (Thermo)
- Orthogonal & Jet Stream (Agilent)
- ZSpray (Waters)
- Dual off-axis & Heated Auxiliary Vortex Gas (Varian)
- Flash vaporization in APCI
- Reduced background noise and memory effect
- Bioanalytical case study: Ion suppression for beclomethazone
- Are new ESI sources still concentration dependant?
- ESI High flow applications
- New ESI sources ruggedness
- FAIMS
- Basic theory and ion mobility
- Noise reduction
- Bioanalytical case studies:
- Phase II metabolites
- Analysis of Testosterone by LC-FAIMS-MS/MS
Sample and Matrix Characterization: The First Step for a Successful Method Development
- Instrument Setup for Method Development
- Importance of having a flexible tool for method development
- Method development steps
- Gathering information on target analyte and matrix
- Problem definition and method design
- Molecular weight considerations and deductions from molecular structure
- Sample characterization
- Stability
- Volatility
- Polarity
- Choice of sample preparation according to matrix considerations
- Main targets of sample pretreatment
- Main extraction types:
- Matrix dilution
- Protein precipitation (PPT)
- Liquid-liquid extraction (LLE)
- Solid phase extraction (SPE)
- On-line extraction and Column Switching Applications
- Schematic of on-line SPE
- Turbulent flow: single and dual column mode
- High throughput applications in sample preparation
Developing the Right Chromatography for LC-MS
- LC parameters
- HPLC strategies for ESI and APCI
- Case Study: Lidocaine chromatographic method development
- Drawbacks of developing LC-MS/MS methods without good chromatographic separations
- Chromatographic parameters to consider when developing a method in LC-MS
- Analyte pKa and pH
- Stationary phases for LC-MS/MS
- Reverse phase (RP)
- Polar embedded phase
- Normal phase (NP)
- HILIC - Hydrophilic Interaction Liquid Chromatography
- HILIC benefit in LC-MS/MS
- Silica
- Coated silica
- Amino (NH2) and Amide
- Selectivity & Stability Comparison: Silica, NH2, Amide
- UHPLC - Ultra High Performance Liquid Chromatography
- Compatibility of UHPLC with MS, are we more productive?
- Efficiency vs. Selectivity: Case study: Positional Isomers of Nicotinamides
- Fuse-core columns vs. UHPLC vs. Conventional Columns
- Analytical Case Study: Desmosine and Isodesmosine quantification
- LC-MS/MS efficiency with Fused-Core columns
- Matrix interferences on Fuse-core columns
- Monolithic columns vs. UHPLC
- Metabolite ID Case Study: Loxapine
- Separation of Carnitine and Acylcarnitines
- Column Geometry and size
- Flow rate and LC Columns
- Speed & Sensitivity
- Mobile Phase composition and temperature effect
- LC column selection
- What column is best suited for a particular application?
- Optimal column chemistry for LC-MS/MS applications
- HPLC method development for screening and qualitative applications
- HPLC method development for quantitative applications
- Pre-column vs. filter frit: Dead volume issues in high throughput LC-MS/MS
- The importance of the Connecting tubes
- Effect Of Dead Volume On System Performance
- Particulate material in the sample flow stream
- Pre-column filter: troubleshooting
- Pressure vs. flow rate
- Fast chromatography but at least with K'>2
- Reduced chromatographic run time in LC-MS/MS
- Isocratic and gradient LC methods
- High pH separations and ESI(+)
- Is it really possible to get positive ions?
- Columns for high pH mobile phase
- The post-column addition approach
- Forensic toxicology case study: Morphine method development
- Case study: Doping agents in Urine
- Dwell time & fast chromatography in LC-MS
- What we have learnt from Acetonitrile (ACN) Worldwide Shortage: LC-MS/MS without ACN
- Problem identification: ACN is the preferred organic solvent for LC-MS due to its elution strength, low viscosity, low chemical reactivity, and good miscibility with water and mainly lower noise in MS
- Use of Shorter column with smaller particles
- Use Ultra High Temperature in LC-MS/MS
- Environmental Case Study: Herbicides
- Analytical Case Study: Phenoxy Acidis
- Reduction of the column internal diameter
- Column performance vs. column diameter
- Nano-LC and capillary columns
- Use of other organic modifiers in reversed-phase HPLC
- Pharmaceutical Case Study: Acetominophen, prednisolone, and corticosterone
- Chip Technologies
- LC-Chip-MS/MS –sensitivity & data quality
- DMPK applications
- Use of UHPLC
- Bioanalytical Case Study: Pindolol and Acebutolol in Human Plasma
- Can you use HILIC without ACN?
- Other solvents for HILIC: MeOH, EtOH, IPA, THF, Acetone, DMF, MTBE
- Bioanalytical Case Studies: Muraglitazar in Human Plasma; Cisplatin; Epirubicin
- Temperature and HILIC: Hydrazine’s case study
Main Challenges in LC-MS/MS Method Development and how to overcome them
- Matrix effects and Ionization effects in the source
- Matrix Effect, Ion suppression and S/N on Different API sources
- API3000 vs. Quattro Ultima vs. TSQ 7000
- API4000 Qtrap vs. Premier vs. TSQ Quantum/Classic
- General criteria to fight matrix effect
- Ion suppression
- Instrument set-up
- How to minimize ion-suppression
- Differential suppression
- Carryover
- Definition
- Guidelines
- Fighting carryover
- Special washing solutions
- Accumulation
- Carryover & contamination
- Sensitivity Enhancement
- Improving instrument response and reduce noise and interferences (S/N)
- How to deal with Isobaric species
- When the MS/MS selectivity is not enough
- Chromatographic separation
- FAIMS
- High resolution
- Mass accuracy and resolution
- Solving problems with high resolution
- GLP Bioanalytical case study: High resolution in SRM
- GMP Product development case study: Use of high resolution separations during Sulfonamide method development
- Interference evaluation
- Elimination of Interferences in LC-MS/MS
- GLP Bioanalytical case study: 5-HETE
- Enantiomeric Separations
- Separation or analysis of optically active compounds
- Best available options when confronted with the need for chiral separations in LC-MS/MS
- Normal phase vs. reverse phase chiral chromatography
- In vivo conversion R/S: (R,S)-Thalidomide, (R,S)-Oxybutynin, (R,S)-Verapamil & Norverapamil and new NCEs
- High throughput Chiral Separation Case Study: Metoprolol
Qualitative Analysis
- Overview of drug discovery and development in pharmaceutical industry
- Professor Mc Lafferty's 4 S
- Advantages in pharmacokinetic screening
- Comparison among: HPLV-UV; Radiochemical detection, GC-MS, LC-MS/MS
- Advantages in pre-clinical
- Advantages in in vitro drug metabolism
- Strategies in Identification of Metabolites
- Case Study: Buspirone metabolites
- Sciex API4000 Qtrap
- Thermo LTQ-OrbiTrap
- UHPLC
- Data-dependent scan
- Metabolite ID
- GMP/CMC – Impurity Detection
- Identification of Flavonoids
- Ammonium Bicarbonate for analysis of basic substance in LC-MS/MS
Quantitative Analysis
- Approaches to Quantification
- Standard curve
- Limit of Quantification (LLOQ & ULOQ) and other Key Terms
- Quantitative Relationships in MS Instruments
- Nature of Regression Errors
- Standard addition
- Internal standard
- Important feature of an internal standard
- Homologues and Isomers
- Stable-labeled internal standards: D, 13C and 15N
- Isotopic purity and problem with deuterated internal standards
- Ursodiol and Usodiol-d5
- Oxymorphone and Oxycodone-d3
- What is the best internal standard?
- The Validation Process and the Validation Goals
- Uncertainty in Quantitative Analysis
- Validation Parameters and Method Validation
- Accuracy and Precision
- Analysis of Variance (ANOVA)
- Assessing Accuracy and Precision
- Stability Tests
- Recovery
- Defining Acceptance Criteria
- Essential Documentation
- System Suitability and its Importance
- The Steps in a Typical MS Quantitative Analysis
- Routine Sample Analysis
Actual Case Studies and Troubleshooting: Pharmaceutical, Environmental & Forensic (recognition, resolution of problems and approaches to systematic troubleshooting)
- Real world approaches to assessing problems and improving method ruggedness
- Strategies to reduce or eliminate interferences from metabolite and matrices
- GLP Bioanalytical case study: Gemcitabine method re-development (HILIC vs. RP Chromatography)
- GMP Product Development Case Study: Identifying Impurities by LC-MS
- Sulfachloropyridazine impurities
- Salbutamol impurities
- Forensic Toxicology Case Study: Screening of Amphetamines in Urine
- Sample preparation
- Confirmation of amphetamines
- Environmental Analysis Case Study: Pharmaceuticals in Surface Water
- Sample pre-treatment and enrichment
- UPLC-MS/MS analysis
- Optimization by infusion:
- Erythromycin
- Vitamin D2
- Minoxidil
- Naproxen
- Progesterone
- Method development for the identification of polyphenols
- Best Instrumental conditions for:
- Acetylmorphine
- Procainamide
- Caffeine
- Propanolol
- Nortriptyline
- Oxazepam
- Ampicillin
- Clavulanic Acid
- Ketoralac
- Triphenyl methane
Your Method Development Problem/Challenge
- Please send to the instructor your "real-world" method development problems in advance so that they could be discussed during the course interactive sessions or if you prefer in 1:1 meetings during the course coffee breaks.
