Course Information
Course Title: HPLC Method Development for LC/MS
Categories: 1 - Atmospheric Pressure Ionization
2 - Liquid Chromatography
3 - Liquid Chromatography-Mass Spectrometry
4 - Mass Spectrometry
Instructor(s): Shane Needham Course Number: 48
Affiliation: Alturas Analytics, Inc.
Course Date: 03/14/2011 - Monday Course Length: 1 1/2 Day Course
Start Time: 08:30 AM End Time: 05:00 PM
Course Date 2: 03/15/2011 - Tuesday    
Start Time: 08:30 AM End Time: 12:30 PM
Fee: $660 ($960 after 2/14/11) Textbook Fee:

Course Description
This practical course on HPLC method development for LC/MS covers the specifics of how HPLC methods are developed for interfacing to ESI and APCI. The course discusses the importance of HPLC when interfaced with mass spectrometry including the optimization of HPLC methods for mass spectrometry. Specific examples are included for each topic discussed including “real-world” problem solving and applications. Compound classes that are presented include, small molecules, peptides, zwitterions, amines, acids, etc. A step-wise method development tutorial of developing methods for specific compound classes based on the structure will be included in the final summary.

Target Audience
Beginner and Intermediate LC/MS users familiar with chromatographic principles will benefit from this course.

Course Outline
1. Introduction
1.1. Brief History of Coupling HPLC and Mass Spectrometry
1.1.1. Early Pitfalls of LC/MS/MS without Good HPLC Separations
1.1.2. Why is a separation important with the selectivity of MS? Ionization effects in the source of the MS Separation of labile molecules from parent Separation of isobaric drugs or metabolites
2. Interfacing HPLC with Atmospheric Pressure Ionization
2.1. Electrospray Ionization (ESI)
2.1.1. Optimum Operating Conditions Apparent Concentration Sensitivity
2.2. Atmospheric Pressure Chemical Ionization (APCI)
2.2.1. Optimum Operating Conditions
3. Developing HPLC Methods for MS Detection
3.1. Define final intended use for method
3.2. Obtain basic chemical information on analytes
3.3. Obtain basic information on biological matrices to be analyzed
3.4. HPLC Column Chemistry Choice
3.4.1. Reversed-Phase Columns
3.4.2. Normal-Phase Columns
3.4.3. Other Columns Ion-Exchange Columns HILIC Columns Monolithic Columns Polar Embedded Phases Columns for use at high pH and high temperature
3.4.4. On-Line Extractions
3.5. Optimum HPLC Column Size Choice
3.6. Buffer and Solvent Choice for LC/MS
3.6.1. How to optimize MS signal by choosing the correct additive and solvent
3.7. Choice of Mode of HPLC Separation
3.8. Column Switching Applications
4. HPLC/MS Methods: Experiments and Results
4.1. Where to start for a typical HPLC/MS method
4.2. What is adequate retention and peak shape?
4.2.1. Calculating retention factors (k’) with LC/MS
4.2.2. Calculating chromatographic resolution (Rs)
4.3. Desired Results and What to Expect
4.3.1. Analysis Time
4.3.2. Accuracy and Precision
4.3.3. Peak shape
4.3.4. Resolution
5. Typical Problems, Solutions and Troubleshooting HPLC for MS Detection
5.1. Matrix Interferences and Ion-Suppression in LC/MS
5.1.1. How to determine the occurrence of ion-suppression 
5.1.2. How to minimize ion-suppression
5.2. The Transfer and Improvement of HPLC Methods
5.3. How to improve analysis time without sacrificing quality data
5.4. How to improve chromatographic peak shape
5.5. How to improve chromatographic retention
5.6. Simultaneous HPLC/MS analysis of molecules with different properties
5.7. LC/MS Survival Kit-Tips and Tricks for What you really need to know
5.7.1. Maximizing MS “uptime” with good HPLC practices
5.7.2. Identifying when something goes wrong
6. Group Problem Solving-Case Studies in LC/MS Method Development
6.1. Dissecting some case studies for specific problems encountered by attendees
6.2. Example method development schemes based on select molecules
7. Questions and Answers

Course Instructor's Biography
Shane Needham manages all scientific aspects of the HPLC/MS/MS bioanalytical contract laboratory at Alturas Analytics, Inc. The course instructor has taught similar LC/MS courses on the national and international level at various conferences and venues. Shane has over 80 publications and external presentations in the area of LC/MS. He has over 19 years of LC/MS and analytical lab experience in the drug discovery through drug development stage in the pharmaceutical industry. He previously worked for Pfizer in Groton, CT where he developed and validated GLP and non-GLP LC/MS/MS methods for the trace analysis of drugs and metabolites in biological fluids. He also performed in-vivo and in-vitro structure elucidation studies of drugs and metabolites using HPLC/MS/MS and HPLC/MSn instrumentation. Shane continued his work in assay development by developing high-throughput HPLC/MS/MS methods for the quantitative analysis of drugs and metabolites in support of high-throughput screening. He also developed novel methods for the on-line extraction of drugs from biological fluids when interfaced to HPLC/MS. Shane has LC/MS experience working with triple quadrupole, single quadrupole, ion trap, time-of-flight and quadrupole time-of-flight instrumentation. His area of academic research included the development of optimized stationary phases to improve the assay of pharmaceuticals by HPLC/MS. Shane is a member of the American Chemical Society, American Society for Mass Spectrometry, American Association of Pharmaceutical Scientists and the International Society of the Study of Xenobiotics. Shane is also a reviewer on several analytical and bioanalytical science journals.