LC – MSLC – MS

Liquid chromatography Liquid chromatography coupled with Mass coupled with Mass

spectroscopyspectroscopy

byby

Karnaker Reddy.TKarnaker Reddy.T

M.pharmacyM.pharmacy

LC-MSLC-MS

INTRODUCTIONINTRODUCTION

HISTORYHISTORY

PRINCIPLEPRINCIPLE

INSTRUMENTATIONINSTRUMENTATION

APPLICATIONAPPLICATION

IntroductionIntroduction

Why Liquid Chromatography?Why Liquid Chromatography?• Analysis of labile analytesAnalysis of labile analytes• Analysis of more polar compounds Analysis of more polar compounds

without derivatization.without derivatization.• Analysis of significantly higher massesAnalysis of significantly higher masses• Reduction of lengthy clean-upReduction of lengthy clean-up Using HPLCUsing HPLC

Mass spectroscopyMass spectroscopy Mass spectrometry probably is the most

versatile and comprehensive It measures the masses of individual

molecules, fragments of molecules and atoms.

It provides ultrahigh detection sensitivity

The mass spectrum of each compound is unique and can be used as chemical “fingerprint” together with its retention time to characterize the compound.

LIQUID CHROMATOGRAPHY COUPLED LIQUID CHROMATOGRAPHY COUPLED WITH MASS SPECTROSCOPYWITH MASS SPECTROSCOPY

LC with detectors like Refractive index, electrochemical, fluorescence, and ultraviolet-visible (UV-Vis) detectors generate two dimensional data; that is, data representing signal strength as a function of time

When coupled with MS In addition to signal strength, they generate mass spectral data that can provide valuable information about the molecular weight, structure, identity, quantity, and purity of a sample.

Historical Perspective

• Goldstein – 1886– Existence of positively charged particles• Wein – 1898– Positively charged ions can be deflected inelectrical and magnetic fields• J.J. Thomson – 1913– Demonstrated isotopes of Neon– “Father of mass spectrometry”• First GC-MS interface - 1960’s

• First LC-MS interface developed - 1969– 1uL/min flow into an EI source• Transport devices applied to LC/MS -

1970’s– Loss of volatile components– Thermally-reactive compound losses•

Thermospray (TSI) gains popularity - 1983– 1.0 – 1.5 mL/min– Mobile phases consist primarily of anaqueous buffer

Atmospheric Pressure Interfaces (API) Early 1990’s (commercialization)– Now most common interface– Electrospray (ESI) Initial interfaces required lower flows (1-5 uL/min)Able to produce multiply-charged molecules – Atmospheric Pressure Chemical IonizationSimilar to Thermospray -“Solvent-mediated” ionization

principleprinciple

LC/MS is a hyphenated technique LC/MS is a hyphenated technique combining the separation power of combining the separation power of HPLC, with the detection power of HPLC, with the detection power of mass spectrometrymass spectrometry

It uses an interface that will It uses an interface that will eliminate the solvent and generate eliminate the solvent and generate gas phase ions, transferred to the gas phase ions, transferred to the optics of the mass spectrometryoptics of the mass spectrometry

obtain spectra and molecular mass obtain spectra and molecular mass identification for each peak eluted identification for each peak eluted from the chromatography column. from the chromatography column.

Straightforward mass spectra of Straightforward mass spectra of directly infused samples won't directly infused samples won't distinguish between, buffer distinguish between, buffer components, contaminants and other components, contaminants and other components of a sample mixture. components of a sample mixture.

InstrumentationInstrumentation

LiquidChromatography

Ionization Mass AnalyzerDetector/

Data Collection

Schematic diagramSchematic diagram

MassSpectrometer

MassSpectrometer

LiquidChromatograph

LiquidChromatograph

Rough pumpRough pump

UVDetector

UVDetector

SamplerInjection port

SamplerInjection port

ColumnColumn

SolventsSolvents

Ion sourceIon source

PumpsPumps InterfaceInterface

ComputerComputer

Ion Sources Earlier LC/MS systems used interfaces that either

did not separate the mobile phase molecules from the analyte molecules (direct liquid inlet, thermospray)

The introduction of atmospheric pressure ionization (API) techniques greatly expanded the number of compounds that can be successfully analyzed by LC/MS.

In atmospheric pressure ionization, the analyte molecules are ionized first, at atmospheric pressure.The analyte ions are then mechanically and electrostatically separated from neutral molecules

Thermospray interfaceThermospray interface conventional flow rate (0.5 to 1.5 ml/min). conventional flow rate (0.5 to 1.5 ml/min). the effluent vaporized under reduced pressure by the effluent vaporized under reduced pressure by

heating a stainless steel tube of 0.10 to 0.15 mm heating a stainless steel tube of 0.10 to 0.15 mm inner diameter.inner diameter.

The resulting supersonic jet contains small The resulting supersonic jet contains small droplets that vaporize further due to the hot gas droplets that vaporize further due to the hot gas in this low-pressure region of the ion source in this low-pressure region of the ion source

an auxiliary filament or low-current discharge an auxiliary filament or low-current discharge device is used, complete evaporation of solvent device is used, complete evaporation of solvent from the liquid droplets produces gas phase ions from the liquid droplets produces gas phase ions from ionic compounds in the sample solutionfrom ionic compounds in the sample solution..

ThermosprayThermospray

Electron impact ionizationElectron impact ionization

The electron impact source consists The electron impact source consists of a heated filament that produces of a heated filament that produces electrons which are accelerated to electrons which are accelerated to another electrode called the ion trap. another electrode called the ion trap.

Sample vapor diffuses into the Sample vapor diffuses into the electron beam and become ionized electron beam and become ionized and fragmented.and fragmented.

Particle beam interfaceParticle beam interface

Helium passed through organic liquid (4° C) into a Helium passed through organic liquid (4° C) into a bubbler. bubbler.

The resulting mixture is used as a dispersant gas The resulting mixture is used as a dispersant gas by the particle-beam generatorby the particle-beam generator

dispersant gas contacts effluent before a dispersant gas contacts effluent before a nebulizer causes the effluent to break up into nebulizer causes the effluent to break up into droplets.droplets.

A momentum separator removes helium, organic A momentum separator removes helium, organic vapor, and solvent vapor from an analyte particle vapor, and solvent vapor from an analyte particle beam beam

The analyte particle beam is directed to a MS for The analyte particle beam is directed to a MS for identification and quantification identification and quantification

Several common methods for Several common methods for ionozationionozation

Electrospray (ESI)Electrospray (ESI) Atmospheric Pressure Chemical Atmospheric Pressure Chemical

Ionization (APCI)Ionization (APCI) Atmospheric Pressure Photo-Atmospheric Pressure Photo-

Ionization (APPI)Ionization (APPI) New dual sources (ESI/APCI) or New dual sources (ESI/APCI) or

(APCI/APPI)(APCI/APPI)

Electrospray Electrospray

Electrospray ionization

The LC eluent is sprayed (nebulized) into a chamber atmospheric pressure in the presence of a strong electrostatic field and heated drying gas is passed.

The electrostatic field causes further dissociation of the analyte molecules.

The heated drying gas causes the solvent in the droplets to evaporate. As the droplets shrink, the charge concentration in the droplets increases.

Eventually, the repulsive force between ions with like charges exceeds the cohesive forces and ions are ejected (desorbed) into the gas phase.

These ions are attracted to and pass through a capillary sampling orifice into the mass analyzer.

useful for analyzing large biomolecules such as proteins, peptides,and oligonucleotides,

analyze smaller molecules like benzodiazepines,sulfated conjugates.

electrospray can be used to analyze molecules as large as 150,000 u even though the mass range (or more accurately mass-to-charge range) for a typical LC/MS instruments is around 3000 m/z.

Atmospheric pressure chemical Atmospheric pressure chemical ionizationionization

Atmospheric pressure chemical Atmospheric pressure chemical ionizationionization

In APCI, the LC eluent is sprayed through a heated (typically 250°C – 400°C)

vaporizer at atmospheric pressure. The resulting gas-phase solvent

molecules are ionized by electrons discharged from a corona needle.

The solvent ions then transfer charge to the analyte molecules through chemical reactions (chemical ionization).

The analyte ions pass through a capillarysampling orifice into the mass analyzer.

APCI is applicable to a wide range of polar and nonpolar molecules. typically used for molecules less than 1,500 u for analysis of large biomolecules that

may be thermally unstable. APCI is used with normal-phase

chromatography more often than electrospray is because the analytes are usually nonpolar.

ATMOSPERIC PRESSURE ATMOSPERIC PRESSURE PHOTON IONIZATIONPHOTON IONIZATION

A discharge lamp generates photons in a narrow range of ionization energies.

The range of energies is carefully chosen to ionize as many analyte molecules as possible while minimizing the ionization of solvent molecules.

The resulting ions pass through a capillary sampling orifice into mass analyzer.

APPI is applicable to many of the same compounds that are typically analyzed by APCI.

highly nonpolar compounds and low flow rates (<100 μl/min), where APCI sensitivity is sometimes reduced.

Mass Analyzers

• Quadrupole • Time-of-flight • Ion trap • Fourier transform-ion cyclotron

resonance

Quadrupole

The analyte ions are directed down the center of four parallel rods arranged in a square.

Voltages applied to the rods generate electromagnetic fields. These fields determine which mass-to-charge ratio of ions can pass through the filter at a given time.

Quadrupoles tend to be the simplest and least expensive mass analyzers.

two modes: Scanning (scan) mode&Selected ion monitoring

(SIM) mode In scan mode, the mass analyzer monitors a

range of mass-to-charge ratios. In SIM mode,the mass analyzer monitors only a

few mass to-charge ratios. SIM mode is more sensitive than scan mode but

provides information about fewer ions. Scan mode is typically used for all analyte

masses are not known in advance. SIM mode is used for quantitation and monitoring

of target compounds

Time of flightTime of flight

Time-of-flight

a uniform electromagnetic force is applied to all ions at the same time, causing them to accelerate down a flight tube.

Lighter ions travel faster and arrive at the detector first, so the mass-to-charge ratios of the ions are determined by their arrival times.

Time-of flight mass analyzers have a wide mass range and can be very accurate in their mass measurements.

Ion trap analyzerIon trap analyzer

Ion trap

consists of a circular ring electrode plus two end caps that together form a chamber.

Ions entering the chamber are “trapped” there by electromagnetic fields. Another field can be applied to selectively eject ions from the trap.

Ion traps have the advantage of being able to perform multiple stages of mass spectrometry without additional mass analyzers.

FT-ICRFT-ICR

Fourier transform-ioncyclotron resonance (FT-ICR)

Ions entering a chamber are trapped in circular orbits by powerful electrical and magnetic fields.

When excited by a radio-frequency (RF) electrical field, the ions generate a time dependent current.

This current is converted by Fourier transform into orbital frequencies of the ions which correspond to their mass-to charge ratios.

They also have a wide mass range and excellent mass resolution.

They are, however, the most expensive of the mass analyzers

DetectorsDetectors

3 different types of detector are used with the analysers

Electron multipliers, dynolyte photomultiplier, microchannel plates.

Electron multiplier A conversion dynode is used to convert

either negative or positive ions into electrons. These electrons are amplified by a cascade effect in a horn shape device, to produce a current. This device, also called channeltron, is widely used in quadrupole and ion trap instruments.

Dynolyte photomultiplier Ions are converted to electrons by a conversion

dynode.These electrons strike a phosphor ,excited, emit photons. The photons strike a photocathode at the front of the photomultiplier to produce electrons and the signal is amplified by the photomultiplier. The photomultiplier is sealed in glass and held under vacuum. This prevents contamination and maintain its performance for a considerably longer period than conventional electron multipliers.

Microchannel plate multichannel plate (mcp) detectors which have a time

response < 1 ns and a high sensitivity (single ion signal > 50 mV). The large and plane detection area of mcp's results in a large acceptance volume of the spectrometer system. Only few mcp channels out of thousands are affected by the detection of a single ion i.e. it is possible to detect many ions at the same time which is important for laser ionisation where hundreds of ions can be created within a few nanoseconds.

ApplicationsApplications

Molecular Weight DeterminationMolecular Weight Determination Differentiation of Similar octapeptidesDifferentiation of Similar octapeptides

Green fluorescent protein (GFP) is a Green fluorescent protein (GFP) is a 27,000-Dalton protein with 238 amino 27,000-Dalton protein with 238 amino acidsacids. .

Structural determination of ginsenosides Structural determination of ginsenosides using MSn analysisusing MSn analysis

Pharmaceutical ApplicationsPharmaceutical Applications Rapid chromatography of benzodiazepines Rapid chromatography of benzodiazepines

Identification of bile acid metabolitesIdentification of bile acid metabolites

Biochemical Applications Biochemical Applications Rapid protein identification using capillary LC/MS/MSRapid protein identification using capillary LC/MS/MS

Clinical ApplicationsClinical Applications High-sensitivity detection of trimipramine and thioridazine High-sensitivity detection of trimipramine and thioridazine

In urine sampleIn urine sample

Food ApplicationsFood Applications Identification of aflatoxins in foodIdentification of aflatoxins in food

Determination of vitamin D3 in poultry feed Determination of vitamin D3 in poultry feed supplements using supplements using MSn:a poultry feed extract yields a MSn:a poultry feed extract yields a peak at m/z 385 suggesting the presence of vitamin D3 peak at m/z 385 suggesting the presence of vitamin D3

Environmental ApplicationsEnvironmental Applications Detection of phenyl urea herbicides Detection of phenyl urea herbicides

Detection of low levels of carbaryl in food Detection of low levels of carbaryl in food Pesticides in foods and beverages can be a significant route to Pesticides in foods and beverages can be a significant route to

human exposure. human exposure.

Analysis of the carbamate pesticide carbaryl in extracts of whole Analysis of the carbamate pesticide carbaryl in extracts of whole food by ion trap LC/MS/MS proved more specific than previous food by ion trap LC/MS/MS proved more specific than previous analyses by HPLC fluorescence and single-quadrupoles mass analyses by HPLC fluorescence and single-quadrupoles mass

pectrometrypectrometry

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