Mass spectrometry

• Analytical technique that assesses a compound/mixture via the conversion of its component molecules into ions which are subsequently analysed according to their mass

• QUALITATIVE and quantitative analytical method

• identity (ID) of a compound/drug can be determined or confirmed by means of its molecular weight

• highly sensitive technique utilised for trace analysis

• ANALYSIS/ID OF DRUGS, DRUG METABOLITES AND DRUG IMPURITIES

mass spectrometry

• TDM, research and forensic testing purposes. Normally utilised for qualitative/ID and/or ‘confirmatory’ purposes. But due to costs may not represent ‘routine’ analytical approach in some facilities for standard TDM and forensic wor

drug bionalaysis

• For analysis of drug substances and their formulations. Due to its high sensitivity is commonly applied for detection/ID of drug impurities (impurity profiling) and also drug formulation stability/stress testing.

drug quality control

• routinely employed analytical tool for several aspects/stages of drug candidate ‘preclinical’ studies. Most notably isynthesis, screening, stability and biological studies (e.g. drug metabolite ID) of drug candidates.

drug discovery and development

• Typically a very sophisticated and expensive instrument requiring a high level of operator skill

• Operates under high vacuum (<10-5 mBar i.e. very low pressure) in order to avoid any sample ion collisions/reactions with atmospheric molecules

• Basic system requires the sample to be presented in the vapour

• molecular ion generated is normally a cationic species (+ve ions) which if unstable may undergo fragmentation i.e. decomposes into smaller fragment

• Molecular ion (M +) and any fragments are analysed to provide a MASS SPECTRUM

• DESTRUCTIVE analytical technique

how mass spec works

• used depends on the compound and mass spectrometer type

ion generation

• Electron Ionisation (EI) Chemical Ionisation (CI) Fast Atom Bombardment (FAB) Matrix Assisted Laser Desorption Ionisation (MALDI

common ionisation methods

• represents the most basic and common ionisation method

• Sample must be vaporised prior to its ionisation thus sample must be vaporisable

• ionised with a high energy electron beam

• sample molecule (M) leads to an e- being 'knocked off' the molecule

• Induces the formation of a positively charged MOLECULAR ION/CATION

• represents a HARD ionisation technique - hitting the molecules fairly hard

• application - Analysis of relatively low MW organic compounds below 500

electron ionisation

• formed represents a high energy species hence usually very unstable in nature

• Energy transferred into M + can induce covalent bond breakage in the molecule leading to its extensive decomposition or FRAGMENTATION

M+

• unimolecular process resulting in the formation of smaller molecular ions known as fragment or daughter ions

• Fragmentation patterns are typically complex resulting in several fragment ions

• Each molecular ion has its own characteristic and unique fragmentation pattern

• M+ and its fragment ions are repelled from the ion chamber, accelerated then focussed by an array of charged slits onto a detector/analyser

fragmentation

• Involves the use of an ionised reagent gas

• Reagent gas is initially ionised by EI to yield reagent plasma

• Sample vapour is then added and becomes ionised by interaction with reagent plasma via a series of intermolecular reactions

• predominantly generates a PROTONATED molecular ion [M+H]+

• occasionally yields [M–H]+ ions via hydride ion abstraction

• represents a SOFT ionisation technique

• Application: Like EI, for analysis of relatively low MW organic compound

• Most standard MS analysis includes data from both EI and CI for comparative purposes

• CI, EI - most common

chemical ionisation

• dissolved in a viscous liquid matrix (e.g. glycerol) and placed on metal target

• target is then bombarded with high energy beam of inert neutral atoms

• Sample molecules are slowly 'spluttered off' the target and then collide with each other

• Collisions lead to sample 'self-ionisation' via a series of matrix induced protonation and deprotonation processes

• Stable 'low energy' [M+H] + are formed mostly and minimal fragmentation is observed

• More stable molecular ion usually permits the more rapid sample compound identification

• represents a SOFT ionisation technique

Fast Atom bombardment

• analysis of organic salts and high MW polar

application of FAB

• Sample is embedded in an organic crystalline matrix (e.g. carboxylic acids, urea) and dispersed onto a steel target

• target is bombarded with a pulsed laser beam

• Matrix absorbs photons and then ionises the sample molecules

• Ions formed are slowly ‘desorbed off’ the target

• Very minimal fragmentation is observed

• Generally only M+ are detected

• represents a SOFT ionisation method

• Analysis of large high MW polar organic compounds

• Very sensitive technique and useful for drug bioanalysis

MALDI

• represents the spectral output of the mass spectrometer

• Presented as a HISTOGRAM of ‘Ion m/z value (Mass) vs % Relative ion abundance

• Ions represented as PEAKS along x-axis

• base peak - most intense peak and is arbitrarily assigned an intensity of 100% and usually denotes the most stable ion

• Other ion peaks on the spectrum are reported as a % of the base peak

• Presence and intensity of a M + peak depends on its stability and the ionisation method

Mass spectrum

• M+ peak = very low intensity or absent

• Molecular ion + H [M +1]+ peak = Base peak

• (M + H]+ or M+ peak = Base peak

• ID of an unknown M+ requires determination of molecular formula - necessitates ‘peak fitting' to a rational combination of elements

molecular ion peak

• atomic elements exist in various isotopic forms that give rise to isotope peaks in the mass spectrum

• typically low intensity and found as ‘paired’ or ‘shoulder’ peaks adjacent to larger intensity ‘major isotope’ peaks in the spectrum

• intensity is proportional to no. of isotope atoms present AND their natural isotopic abundance

• Certain elements can give rise to a characteristic ‘set of peaks’ with a readily identifiable isotope peak ‘intensity ratio’ between the major and minor isotopic forms

• arise due to the presence of specific elements may serve an important diagnostic function and prove beneficial in compound identification

• compounds with sulfur, bromine, chlorine - high molecular

isotope peaks

• Represents the decomposition of the molecular ion M + via a series of covalent bond breakages

• Dependent on the compound structure/type and ionisation method

• Degree and complexity of ion fragmentation is influenced primarily by: COVALENT BOND STRENGTH, M + AND FRAGMENT ION STABILITY

• Various molecules, ions and radicals undergo predictable/specific 'Fragmentation Patterns'

ion fragmentation

• promoted by 'electron rich' group(s) and/or atom(s)

• Occurs at 'single' covalent bonds

• Favoured at substituted alkyl C-atom

• Accompanied by loss of small, neutral molecules

• unsaturated bonds stabilise can ions and radicals

• certain cyclic, aromatic and heteroaromatic ring systems assist in ion/radical stabilisation

• 1 × Bond Cleavage → Cation + Radical

• 2 × Bond Cleavage (simultaneous) → Cation + Neutral

• Unsaturated bonds stabilise can ions and radicals

• Also certain cyclic, aromatic and heteroaromatic ring systems assist in ion/radical stabilisation molecul

ion fragmentation bond breakage

• involves a HPLC system fitted with a mass spectrometric (MS) detector

• Couples the high separative capability of a chromatographic system with the identification (detection) capacity of MS

• use of hybrid/hyphenated instruments

• Employed extensively for the identification of components within various mixtures

• Important analytical tool for the detection/ID of drugs and their metabolites in biological

• CANNOT be directly linked to the MS

• UT 'very low concn.' sample molecules must be separated from 'very high concn.' carrier solvent

• Requires the application of a LC-MS INTERFACE between the HPLC column and MS systems

• Interface connects both the systems, maintains vacuum and also undertakes sample ionisa

liquid chromatography mass spec

• most common interface, but both operate via a similar ‘jet separator’ system involving

• Ionisation of sample component molecules that occurs by spraying through a high voltage capillary tube

• Resulting ‘charged aerosol' is then stripped of carrier solvent using a stream of nitrogen gas

• Sample components are analysed separately

Electrospray ionisation

• column flow rates which tend to be lower for ESI

• Both are SOFT ionisation methods (cf. FAB/MALDI) - more expensive and newer

• ESI is used for more polar compounds (i.e. drugs)

• API is similar to CI and often referred to as APCI

differences and similarities in ESI and API

• Charged particles (ions) are pushed into a mass analyser, which sorts them based on their mass divided by their charge.

• Most ions carry a charge of $+1$ (unipositive cations).

• Sorted by Mass: Because the charge is almost always (1) the sorting ratio (m/1) ends up being equal to the ion’s exact weight (mass).

• cations are separated by their weight.

ion separation and detection

• Field Analyser

• Time-Of-Flight (TOF)

types of mass analysers

• ions fly through a curved tube surrounded by a magnetic or electromagnetic field. The strength of the magnet bends the paths of the ions.

• by sweeping the magnetic strength from low to high, ions hit the detector one after another in order of increasing mass (lightest ions first, heaviest ions last).

  • Offers extremely high resolution (down to $0.00001 amu) / 5–6 decimal places).

  • Ideal for Accurate Mass Measurements (AMM) to determine the exact identity of a molecular ion

magnetic sector analyser

• Uses 4 voltage rods with two electric fields (Direct Current, DC + Radio Frequency, RF) set at right angles.

• As ions travel down between the rods, they wiggle/oscillate. Only ions with a specific (m/z) ratio achieve a stable oscillation to pass all the way through to the detector; others crash into the rods.

• Adjusting the Direct current /radiofrequency fields scans across different masses to generate a full spectrum.

  • Provides higher sensitivity (great for detecting lower concentrations or a wide range of ions).

  • Offers lower resolution compared to magnetic sector instruments (down to $0.10\amu).

quadpoler analyser

• Divided into Linear TOF and Reflectron TOF.

• Ions are accelerated down a vacuum tube (called the drift region).

• Separation depends on ion velocity:

  • Lighter ions fly faster and hit the detector first.

  • Heavier ions fly slower and arrive later.

• High Sensitivity & Applications: Extremely sensitive (down to picogram levels). Often combined with MALDI (MALDI-TOF) to analyze large biomolecules like proteins.

• Detectors Used: Employs an electron multiplier or photomultiplier.

• Signal Output: When ions strike the detector, it generates an amplified electrical current or photon signal that software processes.

• Quantification: Signal strength (intensity) is directly proportional to ion abundance (how many ions are present).

• Mass Spectrum: Software plots Ion Abundance vs. Ion m/z generating the final Mass Spectrum

Time-of-Flight (TOF) Analyser

• Involves bond breakage where a single, individual electron is returned to each of the two separating atoms.

• Splits an odd-electron molecular ion (M+) into a free radical and a cation fragment.

• The process is typically started by a heteroatom such as oxygen, nitrogen, sulfur, or a halogen.

• It is the primary pathway that generates the base peak and major fragments in a spectrum.

homolytic cleavage

• Involves the transfer of an entire electron pair toward the most electron-deficient (charged) atom.

• Also breaks an (M+ ion) into cation and free radical fragments, but via a paired-electron movement.

• This process depends heavily on the specific analyte molecule and typically produces minor-intensity ions rather than major peaks.

heterolytic cleavage

• technique using a trap analyser to isolate and monitor only specific mass-to-charge (m/z) values.

• can track a single target m/z value (such as a drug's molecular ion, M+), making it ideal for analyzing complex mixtures and biological samples.

• Capable of detecting target molecules down to the low picogram per milliliter (pg/mL) range.

• Assays at low concentrations can suffer from unpredictable sample loss, such as drug molecules sticking to glassware (adsorption).

• To maintain high sensitivity and specificity, an Internal Standard (IS) must be added.

• Using a labelled analogue of the target compound accounts for any sample loss and ensures accurate quantification.

selective ion monitoring

• Pairs a Selective Ion Monitoring (SIM) selection step with a secondary diagnostic testing step for the chosen ion.

• A specific target ion—known as the "parent" molecular ion —is filtered out and directed into a collision cell.

• Induced Fragmentation: Inside the collision cell, the parent ion collides with a neutral inert gas, which breaks it apart into smaller pieces (induced dissociation).

• Identification: The resulting smaller fragments form a unique fingerprint used to verify the exact identity of the original parent ion.

• Key Application: Excellent method for identifying unknown compounds, such as identifying drug metabolites in biological samples.

Tandem Mass Spectrometry (MS-MS)