introduction to lc-ms - eventrebelscompliant lc/ms procedures with the basics in this introduction...

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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.

Wednesday April 5, 2017 10am

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

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.

All above techniques are “soft ionization”

• 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”

LC-MS GC-MS

IONIZATION SOURCES

Electrospray Ionization

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

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


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 Factors to consider

Analyte molecular weight, volatility, and polarity

Sensitivity and linearity

Matrix/sample preparation requirements

Type of MS

AnalyteVolatility

Analyte Polarity Scope of Work Quant vs

ScreenGC-MS High Non-polar Volatile organics, hydrocarbons, untargeted

(unknown) compounds50/50

LC-MS Moderate-low Moderate polarity

Pesticides, drugs, thermally-labile compounds 90/10

LC-TOF Moderate-low Moderate polarity

Pesticides, drugs, proteins 10/90

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





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

introduction to lc-ms - eventrebelscompliant lc/ms procedures with the basics in this introduction...

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