LC-MS/MS Advanced Optimization & Method Development Strategies Course

"Everything you need to know about your LC-MS/MS optimization and method development that may not be written in instrument manuals and books"

May 4-6, 2011, Somerset, New Jersey, USA
September 26-28, 2011, San Francisco, California, USA

About This Course | Course Syllabus | Course Agenda | Instructor Biography

Registration Fees | Online Registration | Hotel Accomodation

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
    - Xanthine's case study
  - 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.