Accelerating the Implementation of Mass Spectrometry in the Clinical Lab

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Practical Training

Practical Training: Lectures

From MSACL 2016 EU:

Compound Optimization
Daniel Holmes
St. Paul's Hospital, Vancouver

After ensuring your instrument’s resolution and mass accuracy are appropriately set, the next step in developing a quantitative LC-MS/MS multiple reaction monitoring (MRM) assay is to perform compound-specific tuning. Well-defined signal-optimization experiments are used to determine appropriate ion source electronics and gas flow parameters to specifically quantify the compound of interest. This overview will introduce basic concepts of so-called compound optimization. In other words, we will explain what is meant when people say that they have developed a mass spectrometric method.


Developing MRM Transitions
Stephen Master
Weill Cornell Medical College

Now that you understand how to perform compound-specific tuning, you are ready to build your assay. In this session, we will explore how to take a compound that you're interested in measuring by mass spectrometry, break it apart, and pick appropriate product ions for quantitation. This is the final step that allows you to sensitively and specifically measure the abundance of your desired analyte.


System Suitability Testing
Mike Wright
LGC

Slide Deck 01 - Intro (PDF, 1.3 MB)
Slide Deck 02 - Case Studies (PD, 2.2 MBF)

System Suitability Tests (SST) are a valuable tool for ensuring data quality in clinical diagnostic testing. The information gleaned from running an SST prior to analysis can dictate whether a batch of samples is run and, if not, acts as an immediate troubleshooting guide. Additionally it provides data over time to determine preventative maintenance required to develop a robust platform. Focusing on LC-MS/MS this presentation will cover creating an SST material, a number of different SST strategies and deciding on acceptance criteria to use.

This talk will also cover a series of "real world" case studies of LC-MS/MS assays where the system suitability test (SST) injection has failed the acceptance criteria. Starting from the presentation of symptoms displayed by the SST chromatogram, through the process of determining the next troubleshooting experiments to be run, and finally to the diagnosis and treatment of the problem it will soon become apparent why many call the SST “the Doctor”.


Challenges in Chromotagraphy
Grace van der Gugten
St. Paul's Hospital, Vancouver

Slide Deck - Challenges in Chromatography (PDF, 1.5 MB)

One of the critical decisions during LC-MS/MS assay development is selection of the analytical column. This is particularly important when the analyte(s) of interest must be separated from similarcompounds (particularly isobars) that cannot be uniquely identified on the basis of the mass-to-charge ratio. Often the suggested starting point for method development is a C18 column. However, for some compounds the C18 stationary phase may inadequate for baseline separation except by use of a gradient that is impractically long. Column manufacturers are routinely developing new stationary phases and improving existing ones. Selecting the best column can be overwhelming to newcomers, especially considering the vast array of vendors and stationary phases available. Although manufacturers have slick advertising materials demonstrating the strengths of their products, it is often advisable to rely more on recommendations of colleagues and our own experience to make unbiased decisions. This presentation will review the basics of reversed phase LC and discuss column chemistries and how they affect retention and selectivity. Examples of standard solutions and extracted biological samples will be shown run on a range of vendor columns with different column chemistries.


Surviving Matrix Effects Experiments
Grace van der Gugten
St. Paul's Hospital, Vancouver

Slide Deck - Surviving Matrix Effects Experiments (PDF, 1.8 MB)

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is highly specific and sensitive, allowing for the measurement of endogenous compounds such as steroids and proteins, and xenobiotics and their metabolites in biological matrices. However, biological matrices are “dirty”, containing a vast spectrum of potentially interfering compounds, many of which can be co-extracted during the sample preparation process. These compounds can cause problems for LC-MS/MS analysis suppressing or enhancing signal of the analyte(s) of interest: these phenomena are generically referred to as ‘matrix effects’. These matrix effects must be assessed during LC-MS/MS method development and validation.

Post-column infusion and phospholipid transition monitoring are techniques to qualitatively assess matrix effects. The post column infusion visually demonstrates areas of ion suppression in the LC gradient. Phospholipid transition monitoring shows where phospholipids elute during the chromatographic run. These visual qualitative assessments can indicate if the extraction procedure or LC method need further development.

Quantitative matrix effects spiking experiments are the determination of the percentage of losses or gains of signal as a result of ion suppression or enhancement from co- extracted matrix compounds. This experiment was introduced in 2003 by Matuszewski [1] and consists of spiking pre and post extraction and using the results along with non- extracted samples to calculate % matrix effect. Even for those of us who have done many quantitative matrix effects experiments, the calculations for the concentration of analyte in the pre and post spiked samples can be challenging.

This presentation will describe matrix effects in detail, discuss the qualitative and quantitative matrix effects experiments, and give examples of each type. Detailed examples of the quantitative matrix effects pre and post spike calculations as well as results from matrix effects experiments performed in our laboratory will be shown.


Troubleshooting 101: The Visual Basics of being an Effective Troubleshooter
Will Thompson
Duke University

Troubleshooting Peak Shapes (starting at 49:00)
Judy Stone
Center for Advanced Lab Medicine at the University of California, San Diego

Often the most intimidating portion of troubleshooting an instrument problem is getting started. It doesn’t have to be scary! A planned, systematic troubleshooting approach can make all the difference. In this session, we will open with a description of a few strategies to begin isolation of a wide variety of instrument problems, then we will use case studies to demonstrate the use of these tactics to quickly diagnose the most likely cause of a problem.