LC-MS/MS

INTRODUCTION:
The need for faster and more efficient analysis of larger and more
complex samples has driven the development of advanced
instruments in the lab, leading to the creation of improved
instruments capable of handling increasing demands for
productivity and accuracy.
For the identification and quantification of compounds, liquid
chromatography (LC) coupled with mass spectrometry (MS) has
become the technique of choice due to their separation capabilities
and resolving power.
Triple quadrupole mass spectrometers (LC/MS/MS) are established
in labs dealing with challenging matrices, as they offer very low
detection limits with specificity and selectivity.
Additionally, LC/MS/MS solutions have been improved to add
automation and reduce the complexity of the analytical workflow
while increasing lab uptime and reducing maintenance needs.
PRINCIPLE:
Mass spectrometric analysis is based on the separation of ions by
their mass to charge ratio (m/z).
There are several different types of mass analysers used for mass
spectrometric analyses including quadrupole, magnetic sector,
radio-frequency ion trap, time of flight (TOF), orbitrap and ion
cyclotron resonance.
There are also numerous approaches to create ions in the gas
phase prior to analysis by mass spectrometry including electron
ionization, chemical ionization, electrospray ionization (ESI),
photoionization, inductively coupled plasma and matrix-assisted
laser desorption ionization.
 LC-MS-MS is the combination of two selective techniques that
allows the analyte(s) of interest in highly complex mixtures to be
isolated and measured.
 LC differentiates compounds by their physico-chemical
properties and MS differentiates compounds by mass
(specifically their mass- to-charge ratio).
 Though an LC/MS/MS system is known as a triple quadrupole
instrument, it is technically a tandem quadrupole mass
analyzer.
 The first and third quadrupoles are mass analyzers, while the
second quadrupole functions as a collision cell. This method
yields greater structural information, enhanced selectivity, and
sensitivity and ensures a robust and clean signal.
■ Why MS/MS?
❑ Additional structural elucidation
❑ Further reduction of clean-up
❑ Specificity
❑ Useful MS modes
 Mass
LC Inlet Ionize Detect
Analyze

Separation
MS Identification

Mass Mass
Inlet Ionize Fragment Detect
Analyze Analyze

MS1 Collision MS2

Cell

MS/MS
Mass analysers can be combined to perform tandem mass
spectrometry (MS/MS), which is a relatively simple concept —
two mass analyses occur in series, often with a fragmentation step
in between.
One approach to MS/MS is to combine two or more identical
types of mass analysers in series, such as a triple quadrupole
analyser or a TOF/TOF analyser.
Mass spectrometry analysis is accomplished by performing the
first mass analysis or filtering, passing selected ions into a
collision cell where fragmentation occurs, followed by a second
mass analysis.
For quantitative analysis, a specimen is pretreated via dilution
or by purification to remediate some complexities of the matrix,
and then injected onto the liquid chromatography system.
The liquid chromatography eluent carrying the analyte is
introduced into the source of the mass spectrometer, where gas
phase ions are produced.
The first mass analyser filters incoming ions, only
allowing parent ions to move forward towards the collision cell.
In the collision cell, precursor ions are fragmented to daughter
ions, which are analysed in the last stage of the tandem mass
spectrometer.
The resulting mass spectrum reflects the daughter ions only;
when focusing only on particular precursor/product ion pairs,
quantitative analyses can be performed using a technique called
selected reaction monitoring.
INSTRUMENTATION:
Sample preparation
Sample preparation is required before LC-MS/MS
analysis. This can be simple (for example, dilution or
protein precipitation) or more complex (for example, solid
phase extraction (SPE), liquid–liquid extraction (LLE) or
supported liquid extraction (SLE))
Samples can be diluted with water or a mobile phase
before injection onto the LC-MS/MS system. The diluted
sample should be centrifuged and/or filtered to remove
particulates before injection.
Liquid chromatography
Liquid chromatography is a technique that separates analytes
based on the interaction between a stationary phase (the
chromatography column) and a liquid phase (mobile phases).
These interactions are based on multiple mechanisms including
polarity, ionic interactions and size exclusion. Reversed-phase
chromatography is most commonly used whereby the stationary
phase is less polar than the mobile phase so non-polar molecules
adsorb to the stationary phase until a less polar mobile phase
passes through. In normal-phase chromatography, the stationary
phase is more polar than the mobile phase at sample introduction4.
When coupled with mass spectrometry, chromatographic mobile
phases also may contain a proton donor species that contributes to
ionization of the analytes.
Ion sources
In order for mass spectrometry analysis to take place, the
components of the flow from the liquid chromatography system
have to be ionized. There are three main types of atmospheric
pressure ionization: ESI, atmospheric pressure chemical
ionization (APCI) and atmospheric pressure photoionization
(APPI).
ESI is the most common type of ionization used in clinical
diagnostics, as it is most useful for polar compounds that form
ions in solution.
■ The mechanism of electro spray ionization involves the
emission of ions from a droplet into the gas phase at
atmospheric pressure, a process known as ion evaporation.
■ In ESI, the eluent passes through a stainless steel capillary that
carries a high potential, typically 2 to 4 kV. The strong
electric field generated by this potential and a concentric
nebulizing nitrogen gas flow cause the formation of a fine spray
of highly charged droplets at the tip of the capillary (hence
ESI).
APCI tends to be used for analysis of more non-polar compounds
that do not form ions in solution but are able to be ionized in the
gas phase, such as steroids or cannabinoids.
■ APCI uses analyte desolvation and charge transfer reactions
in vapour phase analyte ions.
■ Eluent introduced into interface using capillary similar to ESI, no
potential applied to capillary.
■ Liquid emerges from the capillary surrounded by a flow of inert
gas into a heated region.
■ Combination of gas and heat forms an aerosol that begins
to rapidly evaporate.
■ A pin is placed within the heated region that has a high potential
applied to it and produces an electrical discharge that ionises
eluent molecules via charge transfer reactions.
 Electrospray Ionization (ESI)
Summary: The sample solution is sprayed from a
hollow capillary needle across a high-potential
difference (a few kilovolts) into an orifice in the
interface. Heat and gas flows are used to desolvate
the ions existing in the sample solution.
Electrospray ionization can produce multiply
charged ions with the number of charges tending to
increase as the molecular weight increases.
Sample Introduction
• Flow injection
• LC/MS
• IC/MS
• Typical flow rates are from 50 µL/min up to >1 mL/min
Benefits
• Good for charged, polar, or basic compounds
• Excellent for IC or reversed-phase LC
• Permits detection of high-mass compounds at low
mass-to-charge ratios(m/z less than 2000)
• Very low chemical background yields excellent
detection limits
• Can control presence or absence of fragmentation by
varying interface potentials
Atmospheric Pressure Chemical
Ionization (APCI)
Summary: A corona discharge is used to ionize
the analyte in the atmospheric pressure
region. The gas-phase ionization in APCI is
more effective than ESI for analyzing less-
polar species. ESI and APCI are
complementary methods.
Sample Introduction
• Flow injection
• LC/MS
• Typical flow rates are from 200 µL/min up to over
1 mL/min
Benefits
• Good for less polar compounds
• Excellent liquid chromatographic interface for
normal phase separations
• Complementary to ESI
• Can control presence or absence of
fragmentation by varying the interface
potentials
APPI can be important for weakly polar or non-polar compounds
that are challenging with ESI or APCI. APPI greatly reduces matrix
effects and background signal; however, it is rarely used.
Types of mass analysis
Triple quadrupole mass spectrometers (or tandem mass
spectrometers) are most commonly used for clinical diagnostics.
Two mass-filtering quadrupoles can transmit ions with a
specific m/z value based on the applied radio frequency and direct
current voltage. They can also allow all ions to pass through (radio
frequency only).
Commonly, another quadrupole is employed to act as a collision
cell where fragmentation of ions can take place (radio frequency
and collision gas applied).
Quantitative clinical LC-MS/MS methods commonly employ
selected reaction monitoring.
In this type of analysis, quadrupole 1 provides a stable
trajectory for ions of a specific m/z value, and only ions of the
specific m/z value enter quadrupole 2 where they are
fragmented to form a charged product and a neutral loss. These
fragment ions enter quadrupole 3, which is set to filter ions of a
specific m/z value, and these ions strike the detector.
 Collision
cell MS2
MS1
Application Areas for LC/MS/MS
Environmental Analysis
For environmental analysis, LC/MS/MS has become the analytical
technique required by Official Methods, for detecting and
quantifying contaminants in soil, water, and air samples. It can be
used to measure a range of environmental contaminants, pesticides,
and industrial chemicals. For example, LC/MS/MS can be used to
detect and quantify per- and polyfluoroalkyl substances (PFAS).
Food and Beverage Analysis
For the analysis of the composition of food and beverage samples,
LC/MS/MS is used to identify and quantify various compounds,
such as flavor components or contaminants. In the case of
mycotoxins, LC/MS/MS can be used to detect and quantify them in
food samples. Regulatory guidelines for maximum mycotoxin
limits have now been implemented, with current global regulatory
limits requiring a reliable, robust LC/MS/MS method for
monitoring a range of mycotoxins.
Forensic Analysis
LC/MS/MS instruments are also used in forensic analysis to identify
and quantify drugs of abuse in biological samples, such as urine or
blood. They can be used to detect a range of drugs, including
opioids, cocaine, and amphetamines.

Pharmaceutical Analysis
The pharmaceutical industry leverages the analytical capabilities
of LC/MS/MS systems for drug discovery, development, and quality
control.
The identification and quantification of small molecules, peptides,
and proteins in complex biological matrices are performed with
LC/MS/MS systems. For example, LC/MS/MS can be used to
measure drug levels in plasma or urine samples or to analyze the
composition of protein therapeutics.
In toxicology, LC-MS or tandem mass spectrometry (MS/MS) is so
frequently used because firstly it can be used for non-volatile and
heat-labile compounds, unlike gas-chromatography mass
spectrometry (GC-MS).
LC-MS/MS method is used to quantify antibiotics for eaxample
piperacillin, meropenem, linezolid, and teicoplanin.
LC-MS/MS, in addition to its common role for analyzing lawful
drugs and illicit drugs, can also detect metabolites that come from
phase I or II metabolisms. This characteristic differs from other
instruments and represents the main advantage related to the use of
LC-MS/MS in Pharmacokinetics and Pharmacodynamics studies or
for pharmacotoxicological purposes.
Clinical Diagnostics
LC/MS/MS instruments are increasingly being used in clinical
diagnostics to measure biomarkers in biological fluids, such as blood
or urine. They can be used to diagnose diseases, monitor disease
progression, and evaluate the effectiveness of treatments. For
example, LC/MS/MS can be used to measure the levels of various
metabolites, hormones, or drugs in patient samples.