11- MASS SPECTROMETRY

MASS SPECTROMETRY

• analytical technique based on the measurement of the mass-to-charge ratio of ionic species  related to the analyte under investigation 

MASS SPECTROMETRY

used to

determine the molecular mass and elemental composition of an analyte as well as provide an in-depth structural elucidation of the analyte

MASS SPECTRUM

displayed as a plot of m/z (mass-to-charge ratio) on the abscissa (x-axis) versus ion intensity as the ordinate (y-axis) and frequently is normalized to the most intense ion in the spectrum

PRINCIPLES OF MASS SPECTROMETRY

• Charged molecules or molecular fragments are generated in a high-vacuum region, using a variety of methods for ion production.

Two types of electron:

• core

• bonding

Core electron

- does not participate in a bond

• can be a part of a lone pair; recall that lone pairs do not participate in bonds

Bonding electron

- participates in a bond

The electron beam bombards propane (analyte) with electrons. 

• Electrons from the instrument and from the analyte will then collide with one another, then repel one another.

• Then, an electron will be expelled from the analyte.

Fragmentation .

refers to the breaking of the molecule into a cation or a radical

Propane: Core Electron

• The ejected electron is a core electron since no bond or element was removed from the whole molecule. From CH₃CH₂CH₃, it became CH₃CH₂CH₃⁺, the only difference is that the latter is charged. 

• This charged molecule is detected by the instrument, and will appear at 44 since its MW is 44 g/n.

Propane: Bonding Electron between C and H

• When an electron was ejected, C-2’s Hydrogen was also expelled. Hence, subtract one from the MW = 43 g/n.

Propane: Bonding Electron between 2 Carbons

• Ejection of an electron between two carbons led to two separate molecules: 1) charged ethyl and methyl; and 2) ethyl and charged methyl. 

• Charged ethyl is registered at 29 g/n.

• Charged methyl will appear at 15 g/n.

Single-stage MS

• one dimension

• provides a survey of all ions generated in the ion source

• no fragmentation

Tandem MS (MS/MS)

• carry out two sequential (consequent) m/z analysis events = can generate more fragments

• provides increased selectivity for detailed structure elucidation or quantitative analysis

The more fragmented the analyte or the more fragments generated, 

the more detailed the structure.

Multiple Stages (MSⁿ)

• perform multiple stages of MS to generate further fragment information

• capable of isolating an ion of interest and inducing fragmentation

INSTRUMENTATION

components

1. Sample introduction technique 

2. An ionization source to charge analyte (create ions)

3. A mass analyzer to separate the analytes (ions) on a mass/charge (m/z) scale 

4. A detector to measure the MW of ions

Sample Introduction

1. Direct Introduction

2. Infusion Introduction

3. Chromatographic Introduction

   1. Gas Chromatography

   2. High-Performance Liquid Chromatography

   3. Capillary Electrophoresis

Ionization Procedures

1. Electron Ionization (EI)

2. Chemical Ionization (CI)

3. Atmospheric Pressure Ionization

   1. Electrospray Ionization (ESI)

   2. Atmospheric Pressure Chemical Ionization (APCI)

4. Matrix-Assisted Laser Desorption Ionization (MALDI)

5. Ambient Ionization Procedures

Mass Analysis

1. Quadrupole

2. Magnetic Sector

3. Ion Traps and Ion Cyclotron Resonance

Direct Introduction

• direct insertion probe 

introduce the sample (typically 1 μL or less) into the mass spectrometer

Direct Introduction

sample

• usually a pure compound or relatively pure compound 

• dissolved in an appropriate solvent

• no matrix, impurities

Direct Introduction

probe 

consists of a metal filament, such as platinum, located at its tip

 Infusion Introduction

• done to provide

• a relatively long analysis time or perhaps to conduct a quick survey of a sample

Infusion Introduction

• done when

• analysts optimize the instrumental conditions for a specific analyte as well as to obtain greater numbers of spectra

Infusion Introduction

• may require

• more sample than conventional flow rates 

Infusion Introduction

• widely practiced for

• nanospray ionization applications

Infusion Introduction

• widely practiced for nanospray ionization applications

• for the analysis of proteins and peptides

• during specialized nanospray applications with small molecules in drug metabolism to determine structure or equimolar response ratios

Chromatographic Introduction

• generally for the separation of several analytes; requires pure cmpds 

GAS CHROMATOGRAPHY

• for nonpolar, volatile (or can be rendered volatile), and thermostable (heat-stable) analytes

• sample is volatilized, and a nonreactive, inert gas such as helium carries the sample through the GC column, which is contained in a temperature-controlled oven

HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

• for nonvolatile, thermolabile compounds

• analytes are separated based on the partitioning between the mobile phase and stationary phase and are introduced into the ion source of the mass spectrometer

CAPILLARY ELECTROPHORESIS
also called

capillary zone electrophoresis (CZE)

CAPILLARY ELECTROPHORESIS

• for ionic analytes

• exploits subtle differences in the ionic composition of analytes to separate them based on electrophoretic mobility in a conductive liquid

IONIZATION PROCEDURES

• Need ionized analytes

Electron Ionization (EI)

• Used mostly with

• GC applications when the analytes of interest are nonpolar and are easily volatilized

Electron Ionization (EI)

Procedures are characterized by

extensive fragmentation 

Electron Ionization (EI)

other details 

• Considered a hard-ionization mode

M + e^– → M⁺ + 2e^– 

• Highly reproducible(precise analytical method)

Electron Ionization (EI)

• used to

• determine the structure (since it generates/elucidates many fragments) and to confirm the identity of unknown compounds

Chemical Ionization (CI)

• Rely on electron ionization of reagent ions such as methane, ammonia, or isobutene

• Reagent ions react with the analyte molecules (ion-molecule reaction) in the source of the mass spectrometer

• A softer ionization (less [or no] fragmentation of the molecular ions) mode than EI

Chemical Ionization (CI)

• Very useful for

• reactive and unstable compounds where a molecular mass determination is desired

ELECTROSPRAY IONIZATION (ESI)

• Produces fine charged droplets of a liquid phase that carries the analyte of interset

• Used with HPLC

• Involves the nebulization (turn to a fine mist) of the sample delivered at flow rates that range from nL/min to mL/min to produce a fine spray of droplets (radius = 0.5-1.0 μm)

ELECTROSPRAY IONIZATION (ESI)

• Referred to as a

• soft-ionization procedure because typically it does not result in fragmentation of the analyte during the ionization process

ELECTROSPRAY IONIZATION (ESI)

for 

analysis of large biopolymers (e.g., proteins)

ELECTROSPRAY IONIZATION (ESI)

ability to 

calculate the intact mass of the protein from a combination of all the charge states

ELECTROSPRAY IONIZATION (ESI)

allows

• more accurate average mass to be determined

Matrix-Assisted Laser Desorption Ionization (MALDI)

• Soft-ionization procedure used primarily for biomolecules that relies on the addition of a chemical matrix dried with the analyte of interest.

Matrix-Assisted Laser Desorption Ionization (MALDI)

• This matrix compound can

• absorb laser energy at a particular wavelength during the laser ablation process

Matrix-Assisted Laser Desorption Ionization (MALDI)

• Some of the ions generated in the matrix can

• transfer protons to the analyte, and the resulting gas-phase ions are focused into the mass spectrometer

Matrix-Assisted Laser Desorption Ionization (MALDI)’

• Produces

• lower charge-state ions, with singly charged ions being the most favorable.

Ambient Ionization Procedures

• Refers to a collection of MS procedures that permit direct sampling and interrogation of analytes from sample matrices or surfaces under ambient conditions with little or no pretreatment 

Quadrupole

• Consists of a set of four parallel rods

Quadrupole

When a combination of constant (DC) and alternating (AC) voltage are applied to the opposing rods respectively, 

the resulting electric fields allow ions of a specific m/z to stably transit the quadruple and to pass through to the detector

Quadruple mass spectrometers are relatively

low-cost instruments and provide good qualitative and quantitative analytical capabilities

• Limited to production of low-resolution mass spectra

Magnetic Sector

• Filter ions by the means of the application of magnetic field

• The magnetic field is varied, and ions are deflected to follow a curved path so that ions with different m/z ratios are separated

Ion Traps and Ion Cyclotron Resonance

• Ions can be trapped by

  • static electric fields (DC voltage),

  • dynamic quadruple electric fields [radio frequency (RF) AC voltage],

  • magnetic fields,

  • or some combination of the types

Ion traps are devices that 

“trap” ions in discrete, repeating orbits

Orbital paths are

complex and unique to each device

Ion Traps and Ion Cyclotron Resonance

• Detection of the ions may take place by

• sequential ion ejection (after trapping by m/z)

• detection of all ions simultaneously [while trapped, i.e., by image current detection and subsequent Fourier transformation (FT) of the data]

TIME OF FLIGHT (TOF)

• Time it takes for the particle to reach the detector

TIME OF FLIGHT (TOF)

uses differences in 

transit time through a field-free drift region to separate ions

TIME OF FLIGHT (TOF)

Ions generated in the ion source are

pulsed into the field-free drift region (flight tube) by an electric field

Lighter ions

have higher velocity and reach the detector sooner 

the TOF mass spectrometer has benefited significantly from the use of fast electronics and fast computers to

provide systems characterized by high speed, high sensitivity, and high resolution

QUADRUPLE ToF MS 

• TOF separation process in combination with electrospray and a quadrupole ion filter with reflectron making the lighter ions travel farther than the bigger ions 

APPLICATIONS

• used in trace analytical measurements (both qualitative and quantitative) 

• can provide molecular mass information via detection of the molecular ion or ions 

• can provide unique structural information via the generation of fragment ions

• used for the identification of a targeted compound, particularly when used in conjunction with an authentic standard or a chromatographic method 

• useful for the quantitative determination of actives or impurities in a drug substance or a drug product because of its selectivity and sensitivity

• provides a highly specified method for determining or confirming the identity or structure of drugs and raw materials used in their manufacture 

• GC-MS or LC-MS provides a method for characterizing impurities in drugs and formulation excipients

• an important tool in proteomics, which is currently a major tool in drug discovery

STRENGTHS

• best method for getting rapid identification of trace impurities 

• powerful method and important tool for structure identification because of its ability to provide information about the mass, elemental composition, and structural features of known and unknown molecular entities 

• method of choice for monitoring drugs and their metabolites in biological fluids because of its high sensitivity and selectivity 

• (ES-MS and TOF-MS) will be of major use in the quality control (QC) of therapeutic antibodies and peptides

LIMITATIONS

• not currently used in routine QC but is placed in R&D (Research & Development) used to solve specific problems arising from routine processes or in process development 

• expensive (one LCMS analysis run = 〜₱12,000; fun fact: 11M order nila ma’am for LCMS + free HPLC haha)

• requires support by highly trained personnel and regular maintenance