Liquid chromatography coupled with Mass spectroscopy LC – MS Liquid chromatography coupled with Mass spectroscopy by Karnaker Reddy.T M.pharmacy

LC-MS INTRODUCTION HISTORY PRINCIPLE INSTRUMENTATION APPLICATION

Introduction Why Liquid Chromatography? Analysis of labile analytes Analysis of more polar compounds without derivatization. Analysis of significantly higher masses Reduction of lengthy clean-up Using HPLC

Mass 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 WITH 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 in electrical 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 an aqueous 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 Ionization Similar to Thermospray -“Solvent-mediated” ionization

principle LC/MS is a hyphenated technique combining the separation power of HPLC, with the detection power of mass spectrometry It uses an interface that will eliminate the solvent and generate gas phase ions, transferred to the optics of the mass spectrometry

obtain spectra and molecular mass identification for each peak eluted from the chromatography column. Straightforward mass spectra of directly infused samples won't distinguish between, buffer components, contaminants and other components of a sample mixture.

Instrumentation Detector/ Liquid Ionization Data Mass Analyzer Chromatography Ionization Mass Analyzer Detector/ Data Collection

Schematic diagram

Mass Spectrometer Liquid Chromatograph Rough pump UV Detector Sampler Injection port Column Solvents Ion source Pumps Interface Computer

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 interface conventional flow rate (0.5 to 1.5 ml/min). the effluent vaporized under reduced pressure by heating a stainless steel tube of 0.10 to 0.15 mm inner diameter. The resulting supersonic jet contains small droplets that vaporize further due to the hot gas in this low-pressure region of the ion source an auxiliary filament or low-current discharge device is used, complete evaporation of solvent from the liquid droplets produces gas phase ions from ionic compounds in the sample solution.

Thermospray

Electron impact ionization

The electron impact source consists of a heated filament that produces electrons which are accelerated to another electrode called the ion trap. Sample vapor diffuses into the electron beam and become ionized and fragmented.

Particle beam interface

Helium passed through organic liquid (4° C) into a bubbler. The resulting mixture is used as a dispersant gas by the particle-beam generator dispersant gas contacts effluent before a nebulizer causes the effluent to break up into droplets. A momentum separator removes helium, organic vapor, and solvent vapor from an analyte particle beam The analyte particle beam is directed to a MS for identification and quantification

Several common methods for ionozation Electrospray (ESI) Atmospheric Pressure Chemical Ionization (APCI) Atmospheric Pressure Photo-Ionization (APPI) New dual sources (ESI/APCI) or (APCI/APPI)

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 ionization

Atmospheric pressure chemical ionization 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 capillary sampling 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 PHOTON 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 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 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-ICR

Fourier transform-ion cyclotron 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

Detectors 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.

Applications Molecular Weight Determination Differentiation of Similar octapeptides

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

Structural determination of ginsenosides using MSn analysis

Pharmaceutical Applications Rapid chromatography of benzodiazepines

Identification of bile acid metabolites

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

Clinical Applications High-sensitivity detection of trimipramine and thioridazine In urine sample

Food Applications Identification of aflatoxins in food

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

Environmental Applications Detection of phenyl urea herbicides

Detection of low levels of carbaryl in food Pesticides in foods and beverages can be a significant route to human exposure. Analysis of the carbamate pesticide carbaryl in extracts of whole food by ion trap LC/MS/MS proved more specific than previous analyses by HPLC fluorescence and single-quadrupoles mass pectrometry

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