Mastering LC-MS/MS: Essential Fundamentals and Theory with SCIEX (LC-MS/MS 101)

The session begins with an introduction to the fundamentals of chromatography and mass spectrometry (LC-MS) by Crystal Holt, a moderator from Psyx. Dr. Carl O'chin, a senior scientist at Psyx, is introduced as the speaker. His expertise lies in food, environmental forensics, clinical, and cannabis applications. The presentation aims to share knowledge on basic principles, key terms, and techniques associated with LC-MS technology. Carl discusses the widespread impact of mass spectrometry in everyday life, beyond its portrayal in media like 'Breaking Bad', and its applications in various fields such as environmental science, medicine, and food safety.

This paragraph delves into the history and development of chromatography, starting with its invention by a Russian botanist in 1906 for studying plant pigments. The discussion then branches into two main fields: Gas Chromatography (GC) and Liquid Chromatography (LC). The key difference between the two is the volatility of compounds for GC and solubility for LC. Carl highlights the importance of choosing the right chromatography technique based on the nature of the compounds being studied, with GC being suitable for smaller compounds and LC for larger ones.

The third paragraph provides an in-depth look at the components of an LC system, starting from the mobile phases, which are typically an aqueous phase (mobile phase A) and an organic phase (mobile phase B). The mobile phases are introduced into the system via a pump, with a degasser ensuring the removal of gas bubbles. The paragraph explains the role of the mixer, auto-sampler, and the analytical column where separation occurs. The importance of maintaining a consistent temperature for the column is emphasized to ensure accurate retention times.

Carl discusses the concept of chromatographic resolution, which is crucial for separating peaks in an LC analysis. The efficiency of separation is influenced by factors such as column length, particle size, and the interaction of analytes with the stationary phase. The paragraph introduces the equation for chromatographic resolution and explains how different factors contribute to the resolution. It also touches on the importance of selectivity in choosing a column and the role of hydrophobicity, steric influence, and hydrogen bonding in analyte interaction.

This paragraph explains how to interpret chromatograms, which display peaks representing individual analytes, and pressure traces, which indicate the organic pressure in the system. Carl describes a typical LC gradient, which includes an initial aqueous phase, a ramping phase for elution, a high organic phase to wash the column, and an equilibration phase. The importance of these phases in maintaining column cleanliness and consistent retention times is highlighted.

The focus shifts to the transition from LC to mass spectrometry, where the separated compounds are introduced to the mass spectrometer for analysis. Carl explains the data provided by mass spectrometry, which includes the mass-to-charge ratio (m/z) and intensity at specific times. The paragraph also introduces the concept of ionization, specifically electrospray ionization (ESI), which is crucial for transferring the liquid sample into the mass spectrometer.

Carl discusses different ionization techniques, emphasizing ESI for polar compounds and APCI as a bridge for nonpolar to polar compounds. The paragraph explains how ESI works, creating a fine spray that is charged and evaporated to separate the analytes of interest from the solvent. The importance of maintaining a good spray for consistent ionization is highlighted.

The paragraph introduces the quadrupole mass filter, a key component in mass spectrometers, which filters ions based on their mass-to-charge ratio. Carl explains how quadrupoles work by applying varying RF and DC voltages to filter out unwanted ions and retain the ion of interest. The explanation includes a description of the schematic of a triple quadrupole mass spectrometer and how it filters and fragments ions.

Carl discusses the use of Multiple Reaction Monitoring (MRM) for quantitative analysis, explaining how it involves selecting a parent ion, fragmenting it in the collision cell, and then filtering for a specific fragment ion. The paragraph illustrates the selectivity and sensitivity of MRM through an example where MRM was used to differentiate between pesticide isomers and matrix interferences in an algae protein powder sample.

This paragraph highlights the challenges of analyzing complex matrices, such as cannabis, where high concentrations of compounds and matrix interferences can complicate the detection of trace levels of pesticides. Carl uses the example of cyfluthrin, a pesticide with four isomers, to demonstrate the need for highly selective methods like MRM cubed to confidently identify and quantify target compounds in complex samples.

Carl introduces advanced MS/MS experiments, such as MRM cubed and MRM-EPI, which provide high selectivity and compound identification capabilities. The paragraph explains how MRM cubed can be used for ultra-selective analysis by fragmenting a fragment ion further, while MRM-EPI combines quantitative analysis with qualitative fingerprinting to identify unknown compounds with confidence.

The final paragraph of the script addresses common questions and challenges in LC-MS analysis, such as selecting additives for mobile phases, reducing sodium adduct formation, understanding pressure changes with organic composition, determining parent ions for MRM analysis, choosing precursor ions, selecting fragment ions, and dealing with matrix effects. Carl provides insights and strategies for method development and troubleshooting to improve LC-MS analysis.