Wednesday April 5, 2017 10am Introduction to LC-MS Amy Patton, MS Laboratory Manager, Pinpoint Testing, LLC Little Rock, AR DESCRIPTION: Amy Patton, laboratory manager for Pinpoint Testing, will begin our discussion of compliant LC/MS procedures with the basics in this introduction to LC/MS. Overview of chromatography Overview mass spectrometry Types of mass spectrometry platforms Past and present technologies Coupling efficient sample prep techniques About the Speaker: Amy Patton is a native of Hot Springs, Arkansas. Following high school graduation, she completed the Emergency Medical Technician (EMT) course at National Park Community College and has been a Nationally-Registered EMT for ten years. She received her Bachelor s of Science degree in Biology from the University of the Ozarks in 2010 and a Master s of Science in Interdisciplinary Biomedical Sciences from the University of Arkansas for Medical Sciences (UAMS) in 2013. Amy began her work in synthetic cannabinoid research while as an undergraduate intern at the Arkansas State Crime Laboratory and the Arkansas Department of Health Public Health Laboratory, where she has held the positions Environmental Public Health Training Fellow and Chemist. Most of Amy s analytical experience centered on work conducted in the highly regulated ADH-Chemical Terrorism Laboratory. This laboratory was required to maintain the rigors of both CLIA and ISO17025. Amy currently works as the Laboratory Manager for PinPoint Testing, LLC where she maintains competency in a number of highly complex analytical techniques and mass spectral platforms, with a specialty in liquid chromatography-mass spectrometry (LC-MS). She has co-authored multiple peer-reviewed articles featuring synthetic cannabinoids and their metabolism and continues her research on synthetic cannabinoids and other emerging drugs of abuse. CRI and COLA do not endorse, directly or indirectly, the presentations given at this conference or the products or services provided by the exhibiting vendors. Presentations are intended to be free of bias. The use of any particular product is for demonstration purposes only, and does not imply an endorsement of the product by the presenter or the sponsors of the symposium. 2017 CRI
INTRODUCTION TO LC-MS AMY L. PATTON, M.S. LABORATORY MANAGER PINPOINT TESTING, LLC
OUTLINE Overview of chromatography Overview mass spectrometry Types of mass spectrometry platforms Past and present technologies Coupling efficient sample prep techniques
WHAT IS CHROMATOGRAPHY? Broad range of physical methods used to separate and or to analyze complex mixtures Components to be separated are distributed between two phases: a stationary phase bed and a mobile phase which percolates through the stationary bed Differential rates of migration occur as the mixture moves over adsorptive materials this provides separation Most modern applications of chromatography employ a column Stationary phase: material inside column Mobile phase: runs through the column and is adsorbed onto the stationary phase
TYPES OF ANALYTICAL CHROMATOGRAPHY Gas Chromatography Mobile phase: an inert gas. Stationary phase: an adsorbent or liquid distributed over the surface of a porous, inert support Liquid Chromatography Mobile phase: a liquid of low viscosity which flows through the stationary phase bed. Stationary phase: may be comprised of an immiscible liquid coated onto a porous support, a thin film of liquid phase bonded to the surface of a sorbent, or a sorbent of controlled pore size.
TYPES OF DETECTORS FID (Flame Ionization Detector) TCD (Thermal Conductivity Detector) ECD (Electron Capture Detector) IRD (Infrared Detector) UV/Vis (Ultraviolet-Visible Spectrophotometer) MS (Mass Spectrometer)
WHAT IS MASS SPECTROMETRY? MS is based on The generation of gas-phase analyte ions Separation of these ions according to their mass-to-charge ratio (m/z) The detection of these ions Three fundamental parts: Ionization source Ions created to enter MS Analyzer Separation of masses Detector Monitors the ion current, amplifies it, transmits to the data system
TYPES OF ANALYZERS ICP-MS Inductively coupled plasma mass spectrometry An inductively coupled plasma is a plasma that is energized (ionized) by inductively heating the gas with an electromagnetic coil Samples to be analyzed are introduced into this plasma, usually as a mist of liquid formed by passing the liquid sample into a nebulizer. The ions from the plasma are extracted through a series of cones into a mass spectrometer The ions are separated on the basis of their mass-to-charge ratio and a detector receives an ion signal proportional to the concentration
TYPES OF ANALYZERS ICP-MS
GAS CHROMATOGRAPHY
LIQUID CHROMATOGRAPHY
PARTS OF A MASS SPECTROMETER Source Analyzer Detector
IONIZATION SOURCES LC-MS APCI Atmospheric Pressure Chemical Ionization Ion production: Discharges on a solvent spray produce primary ions MALDI Matrix-Assisted Laser Desorption/Ionization Ion production: Laser fired at matrix causes matrix to absorb energy and ions are formed ESI Electrospray Ionization Ion production: High voltage is applied to a liquid to create an aerosol. GC-MS EI Electron Ionization Ion production: Electrons bombard entering molecules Hard Ionization CI Chemical ionization Ion production: Collision of the analyte with ions of a reagent gas Soft ionization All above techniques are soft ionization
IONIZATION SOURCES Electrospray Ionization
PARTS OF A MASS SPECTROMETER Source Analyzer Detector
MS ANALYZERS After ions are formed in the source region they are accelerated into the mass analyzer by an electric field Analyzer separates these ions according to their m/z value Selection of a mass analyzer depends upon the resolution, mass range, scan rate and detection limits Resolution: separation of two ions Mass range: the highest mass to charge ratio transmitted by the mass spectrometer Scan rate: how fast it scans a mass spectrum
TYPES OF ANALYZERS - QUADRUPOLE Most quadrupole instruments are limited to unit m/z resolution and have a mass range of m/z 1000 The analyzer consists of four rods or electrodes arranged across from each other As the ions travel through the quadrupole they are filtered according to their m/z value so that only a single m/z value ion can strike the detector m/z value transmitted by the quadrupole is determined by the Radio Frequency (RF) and Direct Current (DC) voltages applied to the electrodes RF Voltage rejects or transmits ions according to their m/z value by alternately focusing them in different planes DC potential combined with the RF potential acts like a low pass filter to reject high m/z ions.
SINGLE-QUAD MASS SPECTROMETER
TYPES OF ANALYZERS TIME-OF-FLIGHT Time-of-flight (TOF) mass analyzer separates ions in time as they travel down a flight tube Velocity is inversely proportional to the square root of m/z High resolution exact mass Ions travelling through the source ions are accelerated and acquire approximately the same kinetic energy in this process Ions of different mass travel through the flight tube and will arrive at the detector sequentially Lighter ions = faster All masses, all the time
TIME-OF-FLIGHT MASS SPECTROMETER
RESOLUTION TOF VS QUADRUPOLE
TYPES OF ANALYZERS TRIPLE-QUADRUPOLE A tandem mass spectrometer consisting of two quadrupole mass analyzers in series, with a only quadrupole between them to act as a cell for collision-induced dissociation. Mass selection of a parent ion in the first stage of analysis within the instrument followed by analysis of the daughter ions often formed in a collision-induced dissociation (CID) process The ion selected by the MS1 is the parent ion. Dissociation occurs in the fragmentation region. The daughter ions are analyzed in the second spectrometer (MS2).
TRIPLE-QUADRUPOLE MASS SPECTROMETER
TRIPLE QUAD MS EXPERIMENTS Product ion scan The parent ion mass is fixed and the masses of all daughter ion formed from the parent ion are measured Precursor ion Scan The first spectrometer is scanned, while the second one is set to the mass of one of the daughters Neutral Loss Scan The multiplier can only be hit by ions that cleave the respective mass in the collision cell. Selected Reaction Monitoring An ion of a particular mass is selected in the first quad and an ion product of the precursor ion is selected in the third quad Provides best sensitivity and signal/noise ratio for quantitation
HYBRID MASS SPECTROMETERS Instruments constructed with at least two component mass analyzers (selectors) of different types arranged in sequence from ion source to ion detector QTOF Quadrupole time of flight QIT Quadrupole ion trap QIT/TOF Quadrupole ion trap time-of-flight
PARTS OF A MASS SPECTROMETER Source Analyzer Detector
MS DETECTORS Detection of ions is based upon their charge or momentum Detector records either the charge induced or the current produced when an ion passes by or hits a surface Typically, some type of electron multiplier is used Because the number of ions leaving the mass analyzer at a particular instant is typically quite small, considerable amplification is often necessary to get a signal
OVERVIEW Source Analyzer Detector
ANCILLARY COMPONENTS Vacuum system All mass spectrometers operate at very low pressure (high vacuum). This reduces the chance of ions colliding with other molecules in the mass analyzer. Any collision can cause the ions to react, neutralize, scatter, or fragment. Data System Converts detector signal to visual data for the user Mass Spectra: intensity vs. m/z plot Chromatograms
MASS SPECTRUM - TOF
MASS SPECTRUM - QQQ
CHROMATOGRAM
COMPONENTS OF A GOOD METHOD Chromatographic resolution and retention Adequate Dwell Time Adequate Cycle Time
SCHEDULED MRM The more times the instrument switches m/z assignments, the less time the instrument can devote to any given MRM Dwell time decreases Sensitivity decreases Scheduled MRM allows for maximizing the duty cycle of your instrument around your particular analyte
IDENTITY CONFIRMATION Triple-quadrupole mass spectrometers Retention time (relative to internal standard, if available) Signal/Noise ratio Two or more daughter ions from the parent mass ratio of one to another Quantitation ion vs confirmation ion Time-of-flight mass spectrometers Retention time (relative to internal standard, if available) Signal/Noise ratio Mass defect Isotope ratio and/or spacing
THE RIGHT SYSTEM FOR THE JOB Type of MS Factors to consider Analyte molecular weight, volatility, and polarity Sensitivity and linearity Matrix/sample preparation requirements Analyte Volatility Analyte Polarity Scope of Work Quant vs Screen GC-MS High Non-polar Volatile organics, hydrocarbons, untargeted 50/50 (unknown) compounds LC-MS Moderate-low Moderate Pesticides, drugs, thermally-labile compounds 90/10 polarity LC-TOF Moderate-low Moderate Pesticides, drugs, proteins 10/90 polarity ICP-MS Low n/a Metals 50/50
ALL SYSTEMS REQUIRE SAMPLE PREP: TYPES Urine Dilute and Shoot Liquid-Liquid Extraction Solid Phase Extraction Supported Liquid Extraction Blood Protein Precipitation Solid Phase Extraction Supported Liquid Extraction Supported Liquid Extraction Solid Phase Extraction
THE RIGHT METHOD FOR THE JOB METHOD DEVELOPMENT MS Selection - Having a robust LC-MS/MS method is just the beginning Select the right sample prep for the job It can only analyze what makes it to the instrumentation Sales pitches Having a well-designed sample preparation method allows for the maximum sample load to get to the instrument Good sample prep + appropriate MS system = hitting data quality objectives