Liquid Chromatography Mass Spectrometry (LCMS) : Fundamental Guide To

C10G-E065
Fundamental Guide to
Liquid Chromatography
Mass Spectrometry (LCMS)
© Shimadzu Corporation
First Edition: October, 2018
For Research Use Only. Not for use in diagnostic procedures.
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MILESTONES FOR LIQUID CHROMATOGRAPHY
AND MASS SPECTROMETRY
Genzo Shimadzu Sr. founds
Shimadzu Corporation focus on analytical and measuring instruments,
medical systems, aircraft equipment and industrial machinery. Together with 1875 Shimadzu Corporation
our partners around the world, we diligently strive to be a company that
meets the challenges faced by society and develop products which contribute
to the growth of industries and the enrichment of peoples’ lives.
WORLD’S 1ST MALDI-MS 1987

LAMS-50K
LCMS-2010
NOBEL PRIZE IN CHEMISTRY AWARDED TO KOICHI TANAKA 2002 AXIMA-CFR
LCMS-2010A
AXIMA-QIT
Koichi Tanaka Mass Spectrometry Research 2003

Laboratory established World-Class General Purpose
2004
World’s 1st Ion trap TOF LCMS
LCMS-IT-TOF 2006 Prominence UFLCXR
Opening up new worlds and expanding possibilities
LAUNCH OF UFMS TECHNOLOGY 2008 through ultra-fast liquid chromatography
MALD-TOF MS models
Shimadzu’s revolutionary technology Acquire mass spectra directly from
bringing next-generation analysis crude samples
performance
LCMS-2020
2010
Delivers superior performance across a wider
Minimized cross talk application range with flexibility and reliability
LCMS-8030 with UFsweeper™
MALDI-7090
Higher sensitivity with UF-Lens™ Ultimate performance in identification and structural
LCMS-8040 Improved CID efficiency with 2013 characterization of biomolecules
UFsweeperII™
2014
LCMS-8050 Integrated LC systems that provide high speed analysis, simplified
Achieved UFswitching™, Next-level method transfer, minimized environmental impact and easy
quantitative maintenance
UFscanning™ and UF-MRM™ Provides high resolution
detection with morphological imaging overlaid
UF-Qarray™ with molecular distribution images
2015 Unified Chromatography (UC)

UHPLC/SFC Switching System
LCMS-8060 World's 1st fully automated system that combines
on-line SFE and SFC in a single flow path.
LCMS-8045 2016 World’s 1st Automated LCMS

Module for Clinical Applications
CLAM-2000
Cutting-edge UFsweeperIII™, DPiMS-2020

Quick and easy MS analysis
UF-grating™ and UF-FlightTube™ without sample preparation
allows high-speed data acquisition
for high-throughput analysis MALDI-8020
LCMS-9030 2018 Nexera Mikros

Quadruopole Time-of-Flight
With the added UF-Link, this low flow LCMS system
Liquid Chromatography Mass Spectrometer
improves operability and enables high-sensitivity
Foreword
Hirano Ichiro
LCMS Product Manager
We are very happy to deliver you our first LCMS Primer which has very
well compiled the basic principles and theory of mass spectrometry. It also
describes the history of development of various innovative technologies
to enhance the performance of our LC-MS/MS systems. It serves both as
a textbook and a practical application guide, regardless of the scientific
background and working field of the readers.
All market figures indicate that Shimadzu is the fastest-growing mass

spectrometry company in the last decade. Since the product launch of
our first triple quadrupole LC-MS/MS, LCMS-8030 in September 2010, we
have been continuously rolling out the new LC-MS/MS systems to keep
up to date with increasing needs. This year (2018), we are celebrating our
landmarking launch of the LCMS-9030, our first ever Q-TOF instrument.
Given these achievements, it is now our obligation and responsibility
to deliver a primer to increase general understanding of the scientific
foundation that we stand upon, while also pointing to the future of the
LCMS that we are to realize.
I would like to express my gratitude for your eagerness to learn more

about LCMS and taking time to read this primer. I sincerely hope that it
satisfies your enthusiasm and gets you prepared for the new era of LCMS.
List of Contents
Chapter 1 Introduction to LCMS 4
Fundamentals of LC, MS and LCMS 5
Comparison of LCMS and other techniques 9
Applications of LCMS 11
Chapter 2 Integrating LC and MS 12
Basic instrumentation of LCMS 13
Interfaces for LCMS 15
Electrospray Ionization (ESI) 16
Atmospheric Pressure Chemical Ionization (APCI) 18
Atmospheric Pressure Photoionization (APPI) 18
Overview of Atmospheric Pressure Ionization (API) 21
Mobile phases compatible for LCMS 24
Chapter 3 Principles of MS 26
Introduction to mass analyzers 27
Magnetic sector MS 28
Quadrupole MS 30
Time-of-Flight (TOF) MS 33
Ion Trap (IT) MS 37
Comparison of mass analyzers 39
Chapter 4 Introduction to tandem MS systems 41

Core principles 42
Types of MS/MS systems and their key characteristics 46
Triple Quadrupole (TQ) MS 47
Quadrupole Time-of-Flight (Q-TOF) MS 47
Multistage MS (MSn) 50
Ion Trap Time-of-Flight (IT-TOF) MS 50
Comparison of MS/MS systems 51
Chapter 5 Challenges and development of LCMS 53

Analytical challenges and current limitations of LCMS 54
Key developments in Shimadzu’s LCMS 56
Ultra-Fast Mass Spectrometry (UFMS) 56
Clinical Laboratory Automation Module (CLAM) 58
Future trends of LCMS 59
Shimadzu’s Comprehensive LC and MS Solutions 60
Abbreviations
APCI Atmospheric Pressure Chemical Ionization
API Atmospheric Pressure Ionization
APPI Atmospheric Pressure Photoionization
CID Collision-induced Dissociation
CLAM Clinical Laboratory Automation Module
DUIS Dual Ion Source
EI Electron Ionization
ESI Electrospray Ionization
GC Gas Chromatography
GCMS Gas Chromatography Mass Spectrometry
HILIC Hydrophilic Interaction Liquid Chromatography
HPLC High-Performance Liquid Chromatography
ICP-MS Inductively Coupled Plasma Mass Spectrometry
IEC Ion Exchange Chromatography
IT Ion Trap
IT-TOF Ion Trap Time-of-Flight
LC Liquid Chromatography
LCMS Liquid Chromatography Mass Spectrometry
MALDI Matrix-Assisted Laser Desorption/Ionization
MALDI-TOF Matrix-Assisted Laser Desorption/Ionization Time-of-Flight
MRM Multiple Reaction Monitoring
MS Mass Spectrometry
MS/MS Tandem Mass Spectrometry
MW Molecular Weight
NPLC Normal Phase Liquid Chromatography
PDA Photodiode Array Detector
Q-IT Quadrupole Ion Trap
Q-TOF Quadrupole Time-of-Flight
RPLC Reversed Phase Liquid Chromatography
RT Retention Time
SEC Size Exclusion Chromatography
SIM Selected Ion Monitoring
SRM Selected Reaction Monitoring
TIC Total Ion Current
TOF Time-of-Flight
TQ Triple Quadrupole
UFMS Ultra-Fast Mass Spectrometry
UHPLC Ultra-High-Performance Liquid Chromatography
UV-VIS Ultraviolet-Visible Spectroscopy
Symbols
µ Micro
amu Atomic mass unit
B Magnetic flux density
E Electric field
e Elementary charge
g Gram
k Kilo
m Mass
m/z Mass-to-charge ratio
n Nano
Pa Pascal
V Voltage
v Velocity
z Charge
ω Oscillation frequency
Chapter 1
Introduction to LCMS
T
he first chapter of Shimadzu’s Fundamental Guide to LCMS introduces
the basic principles of LC and MS. Common LC techniques, namely
reversed-phase, normal phase and size exclusion chromatography, are
described. Next, we introduced LCMS and compared it with other ionization
and analytical techniques. Due to its superior sensitivity, high mass accuracy
and robust performance, LCMS plays a key role and is widely used in many
industries such as clinical, biopharmaceuticals, food safety and environmental.
Chapter 1: Introduction to LCMS
Fundamentals of LC, MS and LCMS

Liquid chromatography (LC) is a separation of each of the LC technique differs.
separation technique, first demonstrated Table 1 presents a list of the common LC
in the early 1990s by Russian botanist, techniques and their separation mechanism.
Mikhail Semyonovich Tswett. LC separates With current advancements in LC, it has
the components of a sample based on the evolved into technologies of smaller particle
differences in their affinity or retention sizes and higher pressure that are more
strength for the stationary phase and mobile efficient and of higher speed, sensitivity and
phase. This separation is illustrated in Figure resolution such as the high-performance
1 where the components in the sample are liquid chromatography (HPLC) and the ultra-
separated in a LC column. high-performance liquid chromatography
There are different techniques of LC (UHPLC).
available and the principles of retention and
From HPLC to UHPLC:

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5
Mobile
Phase Elution
Sample Separated components of the sample
Figure 1. An illustration of how LC works. The sample (in purple) is injected into the LC column and gets separated into
2 analyte bands (red and blue) and gets eluted from the column.
Table 1. Different types of LC techniques and their separation mechanism.
Type of LC Techniques Separation Mechanism Commonly used for
Reversed-Phase LC A non-polar stationary phase and a polar mobile Low molecular weight
phase is used for RPLC. Based on the ‘like attracts
(RPLC) (MW) compounds
like’ principle, the sample is separated based on the
molecule’s polarity preference to either the polar
mobile phase or the non-polar stationary phase.
For example, the non-polar molecules will prefer to
retain in the non-polar stationary phase rather than
the polar mobile phase. As a result, it gets eluted
later compared to the polar molecules.
Normal Phase LC NPLC works entirely opposite to RPLC. In NPLC, a Steroid hormones,
polar stationary phase and a non-polar mobile phase
(NPLC) phospholipids, saccharides
is used. Polar molecules are strongly retained by
the stationary phase as compared to the non-polar and tocopherols
molecules. As a result, the non-polar molecules
elute first.
Hydrophilic Interaction Liquid HILIC works on the same principle as NPLC. The Polar compounds
main difference is that water is added in the organic
Chromatography (HILIC)
mobile phase to effectively separate and elute the
strongly-retained polar molecules.
Ion Exchange IEC retains and separates charged species (ions) Proteins, amino acids,
based on electrostatic affinity of the analyte for the
Chromatography nucleotides, and inorganic
stationary phase containing a functional group of
(IEC) an opposite charge. Differential elution is induced ions
either by changing the pH of the mobile phase to
neutralize the analyte, or by increasing the ionic
strength (salt concentration) to competitively
displace the analyte.
Size Exclusion The SEC column used is filled with porous particles. Proteins and synthetic
When sample of various sizes flow into the column,
Chromatography high polymers
smaller molecules migrate more slowly because
(SEC) they penetrate deep into the pores, whereas large
molecules flow quickly as they do no enter the pores
as much. As a result, larger molecules elute from the
column sooner, which effectively sorts the samples
by molecular size.
6
Detectors for LC Systems Intensity
Chromatogram
Mass
Chromatogram 0
Mass Re 35
0
m/
z
te
Spectrometry
0
30
nt
ion 25
0
Tim 20
0
e 15
0
10
0 Mass Spectrum
50
Figure 3. A typical LC chromatogram (red) and a LCMS

mass chromatogram and mass spectrum (blue).
Upon separation by LC, the components

Fluorescence can be detected using optical properties such
as ultraviolet-visible (UV-VIS), fluorescence,
refractive index, evaporative light scattering
Sensitivity
or electrical conductivity based on the

analytes’ properties. Figure 2 shows the
various detectors for LC. When the analyte
passed through the detector, a change (e.g.
UV-VIS
increase or decrease) in the optical property
will be observed and recorded.
Chromatograms obtained using these
optical detectors primarily identify or qualify
substances based on the retention time and
Conductivity quantitate substances based on the peak
area and intensity. The LC chromatogram
(Figure 3, in red) shows an example of a
typical chromatogram obtained using these
Evaporative
optical detectors. LC coupled with optical
Light Scattering detection offers great quantitative accuracy
for analytes that can be chromatographically
resolved, where a detected peak comprises
only a single component. However, achieving
Refractive
Index
required resolution is challenging for complex
samples where multiple components elute
Figure 2. Various detectors for LC systems in
increasing sensitivity.
approximately at the same time.
7
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3
300
4
1 20
250
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5
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10
100 5 11 12
5 8
12
50 4 9
7 11
0 0
0.0 0.5 1.0 1.5 min 0.0 0.5 1.0 1.5 min
Chromatograms of parabens and preservatives in a standard mixture and cosmetic

cream sample.
In contrast, mass spectrometry (MS) With the coupling of LC with MS,

offers a highly sensitive detection technique the mass spectra obtained from these
that ionizes the sample components, measurements provide molecular mass
separates the resulting ions in vacuum based and structural information for eluted
on their mass-to-charge ratios (m/z) and components, which supplement the
measures the intensity of each ion. A mass qualitative information based on retention
spectrum plots the relative ion intensities times obtained using other LC detectors.
against the m/z values, and a series of mass Therefore, LCMS combine the outstanding
spectra are generated at each time point separation resolution of LC with the excellent
(Figure 3, in blue). This information indicates qualitative capabilities of MS.
the concentration level of ions that have
a given mass and is extremely valuable for
the unique identification of molecules, also
known as qualitative analysis. Moreover, MS
provides added specificity and sensitivity,
and the convenience of simultaneous multi-
component analysis.
8
Comparison of LCMS and other techniques

Apart from LCMS, there are various compounds undergo a hard ionization (i.e.
ionization techniques and analytical electron ionization (EI)) to form gaseous ions
instruments for chemical analysis such as and are further analyzed by MS. Today, GCMS
Inductively Coupled Plasma MS (ICP-MS), technologies provide a very effective means
Matrix-Assisted Laser Desorption/Ionization for separating, qualifying and quantitating
(MALDI-MS) and electron ionization (EI) with substances, but its applicability is limited to
gas chromatography MS (GCMS), MS was gases or volatile compounds with relatively
first introduced and used as a detector for GC low molecular mass, and samples with high
systems in late 1960s and their advantages thermal stability (Figure 4).
have been widely recognized. LCMS was only made possible in mid-
GC separates volatile compounds 1970s with the introduction of atmospheric
in a column where the mobile phase is an pressure ionization (API). With the use of
inert gas and the stationary phase is either API, the LC eluent vaporizes and ionizes to
a packed column or a coated liquid. The form gaseous ions, which are subsequently
compounds are retained and separated based introduced into the MS. API is a type of soft
on their polarity, volatility (boiling point) and ionization technique and examples include
their interaction with the stationary phase. atmospheric pressure photoionization (APPI),
After separation and elution from GC, the atmospheric pressure chemical ionization
Molecular Mass
100,000
LCMS
MALDI (ESI)
10,000
1,000
LCMS LCMS
(APPI) (APCI)
100
GCMS
(EI) ICP-MS
Non-Polar Polar Ionic

Polarity
Figure 4. Common ionization techniques (APCI, APPI, EI, ESI, MALDI and ICP) and their range of applicability.
9
(APCI) and the commonly used electrospray Other ionization techniques such as
ionization (ESI). These ionization techniques ICP and MALDI were later introduced in the
are discussed in greater detail in Chapter mid-1980s. ICP-MS is frequently applied
2. Unlike GCMS, one of the advantages of for elemental analysis while MALDI, a soft
LCMS is its broad applicability to a wide range ionization technique, is commonly used
of compounds (Figure 4). Once the sample for large molecules analysis (e.g. proteins,
is dissolved in a mobile phase, LCMS can peptides and polymers). In summary, with
analyze even the least volatile or thermally the introduction of API techniques, LCMS
unstable compounds that are difficult to provides broad applicability for an extensive
analyze using GCMS. range of substances (e.g. polar and non-
polar) and offers high selectivity.
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Refer to our compilation of application notes targeted for the
various industries.
10
Applications of LCMS
• Drug of Abuse • Water and Soil • Pesticide Residues
• Blood and Urine Analysis • Food Additives and
Analysis • Persistent organic Sweeteners
Pollutants • Veterinary Drugs
Forensics Environmental Food & Beverage
LCMS series
Biopharmceuticals Pharmaceuticals Industrial
• Antibody Drugs • Phospholipid Profiling

• Protein and Glycan • Textile Testing
• Chiral Drugs Analysis
Analysis • Industrial Discharge
• Bioanalysis
With its superior qualitative and LCMS is widely utilized for the qualitative
quantitative capabilities and robustness, and quantitative determination of known
LCMS is commonly used to meet the pollutants (e.g. pesticides, bacteria,
rigorous demands of the analytical market pharmaceuticals and personal care products)
and industries. LCMS is applied in many and trace-level emerging contaminants.
industries such as pharmaceuticals, Food safety and development have also
biopharmaceuticals, forensic, industrial, adopted the use of LCMS in their product
food and environmental sector. For clinical quality control such as the quantitation of
research, the analysis of drugs, vitamins and residual veterinary drugs, food additives and
minerals in whole blood, plasma, serum and the composition analysis of supplements and
urine is conducted routinely using LCMS. It organic foods. With high sensitivity, high
is also applied in metabolomics, proteomics detection selectivity and high qualitative
and genomics study. The use of LCMS in the capability, MS bring about the flexibility of
biopharmaceutical discipline have enabled simultaneous multi-component analysis,
the bioanalysis and characterization of improved productivity and efficiency to HPLC
antibody drugs. In the environmental field, analyses in these applications.
11
Chapter 2
Integrating LC and MS
C
hapter 2 introduces the detailed configuration of the LCMS
instrumentation. It generally consists of a LC separating system,
a mass analyzer and the LCMS interface API unit. Generation of
gaseous ions is crucial in LCMS and there are several factors limiting the
efficiency of this API process. In addition, there are many analytical parameters
to take note when switching from LC to LCMS, for instance, the flow rate and
the types of mobile phases compatible with MS. In this chapter, we discussed
in detail the API processes, their limiting factors and the key parameters to
take note when coupling LC and MS.
Chapter 2: Integrating LC and MS
Basic instrumentation of LCMS
Computer
HPLC MS
Pump Atmospheric Pressure

Ionization (API) Unit
Mobile
Phase
Ion Guide
(Vacuum)
Autosampler
Mass Analyzers
Column
Detector
Figure 5. Basic components of a LCMS system.
The basic components of a LC unit For a LCMS system (Figure 5), the
consist of: instrumentation comprises of:
(1) Pump − delivers the mobile phase at a (1) a LC unit,
required flow rate, (2) an interface between the LC and MS,
(2) Autosampler − injects the samples, (3) an ion source that ionizes samples (e.g.
(3) Column − for separation of sample, API unit),
(4) an ion guide (an electrostatic lens that
(4) Detector − for the analysis of the
efficiently introduces the generated ions
separated components in a sample.
into the MS,
(5) a mass analyzer unit that separates the
ions based on their mass-to-charge (m/z),
(6) a detector unit that detects the
separated ions.
Examples of common MS detectors are LC chromatogram, the quantity of ions that

electron multiplier and microchannel plate reach the detector is converted to a signal
(MCP) where they operate by the secondary intensity and output to a computer. The ion
electron emission process. Together with the guide, mass analyzer and detector are all
13
housed in a vacuum in the MS. By holding it achieve higher selectivity not possible with
in a vacuum, the generated ions are able to conventional LC alone. There are various
be introduced, analyzed and detected in the setups (e.g. online and offline) for multi-
MS with minimal collision and loss. dimensional LC and it can be coupled to
Besides the conventional LC which a MS to give a 2D-LCMS. The use of 2D-
utilizes only one chromatography technique LC in analyses can serve as a clean-up and
and column, there is the multi-dimensional pre-concentration step in a trap-and elute
chromatography which uses a combination system. Another setup, a trap-free 2D-LC-
of techniques (e.g. separation modes and/or MS/MS system, can allow the easy switch of
columns) for separation. One such system is involatile to compatible mobile phases for
the two-dimensional LC (2D-LC) which can detection using MS.
Multi-Dimensional LCMS
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14
Interfaces for LCMS

MS require ions to be in the gaseous energy provided, the larger the degree
phase and these ions are detected under of fragmentation. Figure 6 shows the
high vacuum in the MS. In the case of GCMS, difference in the mass spectrum of riboflavin
target compounds are already separated and (vitamin B2) when it undergoes EI verses
eluted in the gaseous state. In this case, they when it undergoes a ‘soft’ ionization like
can be directly introduced to the MS unit. ESI, where the molecular ion and its adducts
The gaseous compounds are ionized through are commonly observed. Given that the
various methods such as electron ionization energy provided by EI is uniform (e.g. 70 eV),
(EI) and chemical ionization (CI). the electron ionization and fragmentation
In EI, gaseous analytes are passed process is very reproducible, generating the
through a curtain of high-energy electrons same fragment ion species at pre-determined
where electron impact induces ionization intensity ratios. For this reason, the mass
of compounds. This high energy ionization spectra produced by EI is commonly used
fragments the compounds to produce to identify unknown samples and peaks by
ions of smaller m/z. This is considered matching acquired spectra to the spectrum
a ‘hard’ ionization due to the high library contained in the compound database.
degree of fragmentation. The higher the
120
242
100
Relative Intensity
80
256
(A) EI Mass Spectrum 60
171
40
156
61
20 285
74 77 91 116 185 198
103
130
143 227 270 345 360
301 329 333 341
0 50 100 150 200 250 300 350
m/z
120
OH
[M+H]+
377
100
HO
Riboflavin (Vitamin B2) OH
Relative Intensity
80
Molecular Formula: C17H20N4O6 OH

(B) ESI Mass Spectrum 60
Formula Weight: 376.3639 N N O [M+Na]+
40
NH
N 399
20 O
0 100 150 200 250 300 350 400
m/z
Figure 6. Mass spectra produced from (A) EI (hard ionization) and (B) ESI (soft Ionization).
15
However, EI is considered not applicable Electrospray Ionization (ESI)

to LCMS. In the EI chamber, high-energy ESI generates ions by first drawing and
electrons used for ionization are produced by spraying sample solutions at the tip of a
passing a current through a metal filament capillary tube, where a high voltage of about
in high vacuum. With LCMS, the LC flow is ± 3 to 5 kV is applied. This generates a fine mist
incompatible with the high vacuum system of charged droplets with the same polarity
required in EI and the heating of the metal as the applied voltage. To accommodate a
filament at atmospheric conditions causes larger LC flow rate, the nebulizer and heating
it to burn and evaporate (e.g. oxidation of gas flows from outside the capillary to speed
tungsten filament at high temperature). As up the solvent evaporation process. As this
a result, the use of EI in LCMS is not suitable process continues, the electric field on the
due to the vastly different conditions that droplet surface increases. When the mutual
they each operate in, which ultimately repulsive force of the charges exceeds the
created mechanical difficulties and liquid surface tension (i.e. repulsion), fission
interfacing issues. Therefore, it is crucial to occurs. It is thought that as this evaporation
have an interface to connect the LC outlet to and fission cycle is repeated, the droplets
the MS inlet that can efficiently transfer the eventually become small enough that
LC mobile phase to gas and at the same time the sample ions are liberated into the
ionize the analytes. gas phase (based on the ion evaporation
Various interfaces for LCMS were model). A schematic representation of the
developed, but issues with sensitivity, stability generation and desolvation processes in ESI
and user-friendliness were faced. After for positively charged ions are illustrated in
further improvements and developments, Figure 7. Similarly, negatively charged ion are
API, a type of soft ionization technique, generated by applying a negative voltage on
proved to be well-suited for use in LCMS. the ESI probe.
As its name suggests, it ionizes compounds ESI is one of the softest ionization
under atmospheric pressure conditions, method available, which means fragmenta-
which makes it especially useful for removing tion is minimal and it can be used for high-
solvents outside a vacuum. API serves as ly polar, least volatile, or thermally unstable
both the ionization source and the interface compounds. Since most of the compounds
in a LCMS system. In general, ions generated result in protonated (or deprotonated) mo-
by API are stripped of solvent, focused into lecular ions and adduct ions, without gen-
a beam using an ion guide, and finally erating complicated fragment ions, determi-
introduced into the mass analyzer. Three API nation of the molecular mass of compounds
techniques are described here, namely the is very simple.
electrospray ionization (ESI), atmospheric
pressure chemical ionization (APCI) and
atmospheric pressure photoionization (APPI).
16
Sample & mobile phase
I
Nebulizer gas Nebulizer gas
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Gas phase Ions
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+ +
Ion source to
++
+
++
+
+
+ +
+
+ +
+
+ +
++
++
+
+
+
+
++
+ +
++
+
mass spectrometer
+
+
+
+ +
+
+
++
+
Generation ++
of multivalent
+
+
ions
Figure 7. Schematic of the ionization and desolvation processes in ESI positive (+) mode.
In addition, ESI is known to occasionally mass spectrometer. It is also possible to use

generate molecular ions with multiple computer processing to predict the molecular
charges for compounds that have several mass from those multivalent ions. An example
potential charge-accepting functional of myoglobin is illustrated in Figure 8. For
groups. In the case of cations, this means myoglobin, ions with valences (n) from 10 to
multiple protons are added to form [M + 20 were detected and molecular mass was
nH]n+. The tendency to form multivalent calculated by deconvolution to be 16951.3.
ions is strongly influenced by the pKa of the This uniqueness of ESI-LCMS allows the
compound relative to the pH of the solution. analysis and measurement of extremely large
When the multivalent ion is observed, and highly-polar biological macromolecules,
molecular mass information can be obtained such as proteins and nucleic acids. This is
even for compounds with a molecular mass vastly different from GCMS, which usually
that exceeds the measurement range of the only provides peaks of z = 1.
17
120
1131.1
100
1060.5
998.2 1211.8
1305.0
Relative Intensity
80
942.8
1413.6
60 893.2
40
1542.0
848.7
20
1696.0
0 600 800 1000 1200 1400 1600 1800
m/z
Figure 8. Molecular mass of myoglobin calculated using ESI spetrum and deconvolution, multivalent ions (n = 10 to 20)
of myoglobin is observed in the ESI mass spectrum.
Atmospheric Pressure Chemical Atmospheric Pressure

Ionization (APCI) Photoionization (APPI)
The other API technique is APCI, which APPI ionizes analytes by irradiation of
is a type of chemical ionization. Though short-wavelength vacuum ultraviolet (VUV)
the interface design is similar to ESI, the light. The interface design in APPI (Figure
ionization principle differs, making it more 10) is very much the same as APCI, only
suitable for low- and medium-polarity with the swap of the high-voltage corona
compounds (non-polar molecules). As discharge needle to the VUV lamp. Similarly,
illustrated in Figure 9, APCI vaporizes the the nebulizer and heater are used to create
solvent and sample molecules by spraying droplets and to vaporize the solvent. Upon
the sample solution into a heater (about 400 VUV light irradiation, the analytes absorb
°C) using a gas, such as N2. Solvent molecules
a photon and get electronically excited. If
are ionized by the corona discharge needle the analyte’s ionization energy is lower than
to generate stable reaction ions. Protons arethe photon energy, the analyte ion that was
transferred between these stable reaction ionized due to the photons may receive a
ions and sample molecules (ion-molecule proton from the hydrogen in the solvent, to
reaction) and this leads to ionization. become a protonated cation. This ionization
These ion-molecule reactions are known technique achieves good sensitivity ionization
to involve several patterns, such as proton- with low to moderate polarity compounds
transfer reactions and electrophilic addition(e.g. polycyclic aromatics).
reactions. Unlike ESI, APCI involves a higher In summary, ions can be generated
energy process and does not have the through either the continuous or pulsed
tendency to form multiply-charged ion [M (discontinuous) modes. The three API
+ nH]n+. Consequently, it is commonly used techniques (ESI, APCI and APPI) that were
for analyzing highly fat-soluble compounds introduced operates in the continuous
or compounds that do not ionize in solution. mode, giving a constant flow/supply of
18
Heater
Drying gas
Desolvation
Gas phase Ions
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
Ion source to
+
+
+
+
+
+
+
+
+
mass spectrometer
+
+
+
+
+
ge
+
lta dl
e
-vo Ne
e
gh
Hi na
ro
Co
Analyte Solvent Analyte

+ + Solvent
+
Molecule Ion Ion
nsfer
n Tra
Proto
Figure 9. Schematic of the ion-molecular reaction (e.g. proton transfer) in APCI.
CLICK HERE
Understand more about the principles of MALDI,
a discontinuous ionization source suitable for coupling to MS.
This technique is commonly used for the identification and analysis of large
biomolecules (e.g. structural characterization of proteins).
M : Compound of
Laser Interest
m : Matrix
: Cation
+
m
+
: Anion
I
I
I
I
+
I
+
M
+
I
+
M m
+
Vacuum
I
I
+
m
I
I
I
+
I
+
+
I
I
+
I
+
M m
+
I
I
+
I
m
+
I
+
m M
+
m
I
I
I
I
+
I
+
+
Sample Slide Sample Slide

Sample matrix mixture Sample matrix mixture
before laser irradiation after laser irradiation
19
Heater
Drying gas
Desolvation
Gas phase Ions
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
Ion source to
+
+
+
+
+
+
+
+
mass spectrometer
+
+
+
+
+
Analyte VUV Radical

Molecule
+
Light
+ Cation
+ e−
Analyte Solvent VUV Analyte
+ + + Solvent
+
Molecule Ion Light Ion
Figure 10. Schematic of the ionization process by UV light in atmospheric pressure photoionization (APPI).
ions to the MS. On the other hand, the the various API options for LCMS interface.
pulsed mode generates a discontinuous Next, we examined the efficiency of these
source of ions such as the Matrix-Assisted ionization processes and the importance of
Laser Desorption/Ionization (MALDI). In selecting appropriate analytical conditions
this section, we introduced the LCMS for LCMS.
components and discussed the principles of
20
Overview of Atmospheric Pressure is crucial to achieve high sensitivity. it is

Ionization (API) important to obtain extremely fine droplets
Several parameters affect the efficiency and liberate ions from these droplets
and sensitivity of these API processes (i.e. ESI, by reducing droplet surface tension and
APCI and APPI). Apart from the instrument optimizing droplet pH. For example in
specifications and parameters, the limiting Equation 1, a basic compound (R-NH2) is
factors are: commonly detected as positive ions [R-
• Flow rate of LC or liquid inlet NH3]+. By adding an acidic reagent (AH), it
• Solvents / mobile phases (e.g. types, shifts the equilibrium to the right, which
pH and additives used) increase the production of the positive ions
• Properties of analytes (e.g. volatility, and thereby increase sensitivity. In general,
thermal stability and ability to form a desired mobile phase has a pH value 1 or
charged species) 2 lower than the pKa value of the sample.
• Matrix effects (e.g. suppression and Conversely, for acidic compounds (Equation
enhancement) 2), sensitivity can be increased by adding a
• Output from LC (e.g. peak width) basic reagent (B) or by using a mobile phase
with a higher pH than the sample pKa. In
Flow rate is one of the key parameters the case of neutral compounds without an
to achieve high sensitivity, especially for ionic functional group, adding a volatile salt
ESI. In LCMS, a semi-micro (2 mm internal such as ammonium acetate can sometimes
diameter (ID)) LC column is preferred over increase ionization efficiency as adduct ions.
the conventional (4.6 mm ID) LC column
commonly used in HPLC. Using a smaller Equation 1:
ID column, the optimum flow rate for
chromatographic separation is lower, and R-NH2 + AH → [R-NH3]+ + A-
lower flow rate generally increases the
ionization efficiency of ESI. For this reason, Equation 2:
a standard ESI ionization unit is designed to
achieve an optimal sensitivity for the flow R-COOH + B → [R-COO]- + BH+
rate of 0.2-0.8 mL/min and sensitivity is
typically compromised at higher flow rates. In addition, it can be effective to
Next, changes made to the mobile increase the proportion of organic solvent
phases such as the types and pH of mobile to reduce surface tension and accelerate
phases and using additives can particularly solvent evaporation. If using these additives
affect the sensitivity of these API. Particularly affects the LC separation, the solution can be
for ESI, the formation of droplets and ions added post-separation (prior to MS analysis)
as well.
21
In the case of APCI, it requires a compound (dopant), which has lower

analytes to be thermally stable and volatile. ionization energy than the analytes.
It is ideal for the ionization of low to The dopant facilitates and increases the
medium molecular mass compounds (e.g. ionization of the analytes, thereby increases
polyaromatic hydrocarbons, carbohydrates the sensitivity.
and triglycerides) but not suited for In general, the selection of API
macromolecules due to high boiling point. techniques in LCMS is based on the analytes’
Ionization efficiency of APCI is governed polarities and properties. Table 2 summarizes
more by the chemical property of solvent the analyte properties required for the three
than analyte. APCI requires that the solvent API techniques. These API techniques can
is protic, i.e. has sufficiently high proton be further optimized by varying the mobile
affinity to display hydrogen bonding, for phases and flow rate to reduce matrix effects
it to mediate ionization of the analyte. If and improve efficiency and sensitivity. As
the use of a protic solvent is not suited for described (Figure 4), ESI is generally selected
chromatographic separation, other non- for high-polarity compounds which can
protic solvents may be used with a few be typically found in drugs and pesticides,
percent of protic solvent added to assist while APCI and APPI are usually selected for
ionization. In general, APCI can be used compounds having lower polarity, such as
in reversed-phase, normal phase and size polycyclic aromatics and mycotic toxins.
exclusion modes and is less affected by salts In the event where analysis of multi-
compared to ESI. ple components with differing polarities and
For APPI (Figure 11), sensitivity properties is required, some samples may
can sometimes be increased by adding need to be analyzed by multiple settings to
+
e−
+ Radical Cation
Dopant
Analyte +H
Charged Ion
+
Solvent
M+
Figure 11. Illustration of the use of dopant to increase sensitivity in APPI.
22
Table 2. Summary of ionization process and analyte properties of API techniques.
API techniques ESI APCI APPI
Ionization process Ions in solvent Ionization occurs in Ionization occurs in gas

transition to gas phase gas phase by corona phase by UV irradiation
by electrospray discharge
Types of ions formed Singly charged ions Singly charged ions Singly charged ions
Multiply charged ions
Volatility of analyte Do not need to be Require some degree of Require some degree of
volatile volatility volatility
Stability of analyte Do not need to be Must be thermally Must be thermally

thermally stable. Can stable stable
be thermolabile.
cover the entire range of compounds. To ESI and APCI. With this technology, it gives
shorten the total time required for analysis higher sample throughput where the both
and method development, and to improve ionizations (i.e. ESI and APCI) can be com-
both laboratory efficiency and confidence in pleted in a single run.
results, Shimadzu have developed the Dual
Ion Source (DUIS) that enables simultaneous
CLICK HERE
Dual Ion Source (DUIS)
for simultaneous ESI and APCI measurement
MS Chromatograms
ESI Method APCI Method DUIS-2020
23
Mobile phases compatible for LCMS

As MS operates in high vacuum, it is LCMS. As mentioned in Chapter 1, there
necessary to ensure that the LC parameters are a variety of separation modes for LC
and mobile phases (liquid inlet) are (Table 1) and the types of stationary phase
compatible to MS. Usually when switching and mobile phase can be modified based
from LC to LCMS analysis, the current LC on sample characteristics, the desired level
conditions can be used if the mobile phase is of separation, ionization level and MS
volatile. If involatile (e.g. phosphate buffer), compatibility.
modifications (e.g. chromatography mode, A great starting point would be to use
column, type and volatility of mobile phase) volatile mobile phases. A list of mobile phases
is required. This should be investigated step- suitable for API and LCMS are summarized
by-step to allow better interfacing with in Table 3. In addition to the fundamental
Table 3. Mobile phases suitable for API interface and LCMS.
Fundamental Mobile Phase Solvents pH Adjusting Reagents (volatile, ≤10mM)
a) Alcohols (e.g. methanol, ethanol, Acids

propanol) a) Acetic acid
b) Acetonitrile (ACN)1 b) Formic acid
c) Water (pH adjusted, if necessary) c) Trifluoroacetate (TFA)
Base
d) Aqueous ammonia
Buffers
e) Ammonium acetate
f) Ammonium formate
Relatively Volatile Ion Pair Reagents2 Usable Organic Solvent3
To retain basic compounds a) Dimethylsulfoxide (DMSO)

a) Perfluorocarbonate, C2 to C8 b) Dimethylformamide (DMF)
c) Tetrahydrofuran (THF)
To retain acidic compounds d) Acetone
b) Dibutylamine, e) Esters
c) Triethylamine (TEA) f) Chloroform
g) Benzene
h) Hexane
1
Acetonitrile is not compatible with APCI due to the reduction of nitrile to carbon for negative ionization. In
this case, methanol should be used instead.
2
Use minimally as these substances can remain in the LC and MS system even after changing mobile phase. It is
necessary to flush the LC system to remove any traces of these ion-pairing agents.
3
If a “fundamental mobile phase solvent” is present, it usually does not pose a problem if the mobile phase
contains some of these organic solvents. (However, the ionization effect decreases as the concentration of
usable organic solvents increases.)
24
mobile phases such as water, methanol and may pose a challenge to the LC separation,
acetonitrile, acetic acid is also commonly used post-column addition or modifications
to adjust the pH level. For buffer solutions, (prior to MS) may be a good alternative. In
volatile salts such as ammonium acetate and addition, there is the trap-free 2D-LC-MS/
ammonium formate are used. Also, volatile MS technique that can be used to change
ion-pair reagents can be added to the mobile the mobile phase to ensure compatibility
phase to facilitate the separation of polar with MS. Also, better LC separations and
compounds using reversed-phase LC. These easier interfacing to LCMS can be achieved
reagents, which have a long hydrophobic by switching the type of LC columns. For
tail and a polar ionic group, tend to attach to example, if the C18 (octadecyl) column
the stationary phase of the column with the for reversed-phase mode is used, we can
ionic group sticking out. In the presence of try switching to C30 column to increase
ion-pair reagents, polar compounds interact retention or C8 or C4 to reduce retention, or
with the charged ionic groups of the ion-pair -phenyl or -CN column to increase separation
reagent and gets retained and separated in selectivity. If the difference in elution times is
the reversed-phase column. With focus on too great or peaks are too broad, gradient
the ionization efficiency, protic solvents are elution program can be utilized. If the mobile
essential for generating reaction ions for phase and salt concentration are suitable for
APCI, and polar solvents are essential for ESI API, then different chromatography mode
since they are required to dissolve polar or (e.g. size exclusion or ion exchange) can be
ionic compounds. used as well.
A key point to note is involatile salts,
such as phosphate buffer solution, is not
CLICK HERE
suitable for use in LCMS. Involatile salts
Practical Tips for LCMS Analysis
form precipitates at the LCMS interface, We have collated additional tips
which immediately causes sensitivity drop and more in-depth discussion on LCMS
by affecting the electrical fields applied for method development and analyses.
ionization and ion transfer. It can further cause
physical damage, such as contamination on
the needle electrode for APCI or by inducing In summary, this chapter covered the
electrical discharge (spark). Also, nonpolar basic components of a LCMS system and
solvents, such as hexane, contribute very the various ionization methods used in
little to ionizing sample molecules using LCMS. The factors that limit the sensitivity
APCI. Therefore, analytical conditions that of the API techniques and the need to select
use such mobile phases cannot be used appropriate analytical conditions for LCMS
without modification. are discussed to aid in your analyses and
In the event that they may be limitations give a better understanding of the LCMS
to the mobile phase choices and changing interface.
25
Chapter 3
Principles of MS
T
hus far, we have only described MS as a method used to measure
the mass of atoms and molecules, but how does it measure mass?
Many types of mass analyzers are available depending on the method
preferred to separate ions. In this chapter, we shift our focus to MS and describe
the principles and key features of these mass analyzers.
Chapter 3: Principles of MS
Introduction to mass analyzers
From ion source To mass analyzer
Desolvation
Line UF-Qarray™ UF-Lens™
Figure 12. Schematic of Shimadzu’s ion optical system (e.g. ion guide).
Mass spectrometry involves the control started in 1912, where the English physicist,
of ion movement by applying electrostatic J. J. Thomson, utilized the fact that the flow
fields. Ion optics is the term given to this of charged particles bends in an electric or
electrostatic manipulation of ion flow in magnetic field to develop an instrument that
analogy with light manipulation by optical could separate charged particles by its mass
lenses. Figure 12 presents Shimadzu’s ion number. In his instrument, which used a
optics system. It is used to focus the ions cathode ray tube, cations with identical ratios
generated at the ion source into a beam. of charge (z) and mass (m) converged along
Simultaneously, it removes non-ionic gas the same parabola. When he measured the
particles from the system by progressive neon (Ne) gas molecule, the parabolas for
pumping and partitioning. This is important 20
Ne and 22Ne (both are monovalent cations)
for achieving high-sensitivity analysis as were slightly different, which proved the
residual particles interfere with the ion beam. existence of isotopes. Therefore, with the
Ion transmission and focusing is achieved use of this electromagnetic interaction, ions
by applying the required electric fields or
radiofrequency voltage at the quadrupole CLICK HERE
ion guide. Development of UF-Qarray:
How does the mass analyzer work? RF Ion Guide with Improved
Normally when we measure mass (m), we Ion Focusing Capability
Learn about the specifics
use a mass scale or balance, which relies on
of Shimadzu’s high frequency
the Earth’s gravity. So, how do we measure quadrupole ion guide (Qarray). It is
the mass of a molecule, which is so extremely designed to enhance ion focusing and
small that its gravitational force is almost minimize contamination.
too small to measure? The first occurrence
27
can be separated and measured according according to various analytical objectives.

to m/z. This chapter elaborates on the separating
There are a variety of mass analyzers and operating principles of the single mass
and they can be classified by how the ions analyzers and compares the characteristics,
are being introduced such as continuous or pros and cons for each of these mass
pulsed modes. Continuous MS allows an analyzers.
uninterrupted supply of ions to enter the
mass analyzer while pulsed MS requires the Magnetic sector MS
Magnetic sector, a continuous MS,
ions to be introduced only at a specific time
has been used historically the longest. As
point. In a pulsed MS, ions from a continuous
the name implies, the mass analyzer uses
flow are usually accumulated and introduced
magnetic field to separate ions of different
together in pulses.
m/z values (Figure 13). High voltage is first
Apart from a single MS, there are
applied to the ions to accelerate them into
tandem/hybrid arrangements, also known
the magnetic sector. A continuous ion
as MS/MS systems. Single mass analyzers
source is generated and supplied from the
such as the magnetic sector, quadrupole,
ionization unit to the magnetic sector. Once
and time-of-flight (TOF) were commonly
the ions enter, are exposed to the magnetic
used for measuring organic compounds, but
field. As a result, ions are deflected according
the quadrupole model has gradually been
to Fleming’s left-hand rule4. The deflections
increasing their share due to its relatively
lower cost. Besides these mass analyzers, differ based on their m/z where lighter ions
an ion trap MS system that temporarily (of the same charge) will experience more
accumulates ions of a selected range before deflection.
separating them by mass, and a tandem/ 4
Fleming’s left-hand rule can predict the direction/
hybrid MS system that combines multiple force of the movement when there is an electric current
moving in an applied magnetic field. As a result, ions are
MS units have been developed as well. These accelerated in a direction perpendicular to the current and
magnetic field, resulting in a curved deflection path for the
types of MS systems each take advantage ions in the magnetic sector MS.
of their respective features and are used
Magnetic Sector e
forc
Ion source Ion accelerators
i f u gal
r
Cent (f 2)
rce
n t z fo
e
Acceleration Lor (f 1)
Voltage
Detector
Figure 13. Schematic of magnetic sector MS.
28
Equation 3: Ions experience a Lorentz force (f1) from

the magnetic field that can be calculated
f1 = Bzev according to Equation 3. As the direction
of the ion changes, a centrifugal force (f2),
Equation 4: expressed by Equation 4, acts on the ion.
For the ions to pass through the magnetic
mv2 field region and reach the detector, it must
f2 = –––– travel along a curved path of a given radius
r
(r) where f1 and f2 are balanced (Equation
Equation 5: 5). Furthermore, the kinetic energy of ions
accelerated by voltage V is shown in Equation
mv2 6. By eliminating the velocity of the ion (v),
f1 = f2 = Bzev = –––– Equations 5 and 6 are simplified to give
r
Equation 7. By keeping the ion acceleration
voltage V constant and varying the magnetic
Equation 6: flux B (or keeping B constant and varying V),
a detector placed on the corresponding path
1 radius r could detect any mass m (given the
KEion = – mv2 = zeV same charge).
2
In reality, only one ion detector is used
Equation 7: and both the acceleration voltage (V) and
curve path radius (r) are kept constant while
m eB2r2 the magnetic flux density (B) is scanned.
–– = —— This means that ions with different masses
z 2V
(m) all pass along the same path through
the magnetic field, one after another, and
reach the detector. One mass spectrum is
B: magnetic flux density obtained from each scan of the magnetic
z: charge of the ion field. That is to say that magnetic sector
e: elementary charge mass analyzer function by ion transmission
v: velocity of the ion and scanning mode. Besides the described
m: mass of the ion single magnetic sector MS, there are also
r: path radius models of MS with both the electric sector
V: acceleration voltage applied to ions and magnetic sector in a single MS, which is
known as a dual-focusing MS. This setup is
able to focus and converge ions of different
energy and identical mass thereby obtaining
29
higher mass resolution. mass analyzers are now rarely used in LCMS
Some of the key features of magnetic systems. On the other hand, they interface
sector MS are its high resolution and high relatively easily to a GC unit. Consequently,
dynamic range. The measurement range such GC-MS systems are used for dioxin
of magnetic sector MS systems typically is analysis due to the outstanding high-
about 10 to 10,000, though it depends on resolution selected ion monitoring (HR-SIM)
the acceleration voltage V and instrument capability of magnetic sector MS.
design. Resolution of about 2000 can be
obtained using a single-focusing magnetic Quadrupole MS
The other MS which functions
sector model or several tens of thousands
by scanning of ions and allowing ion
using a dual-focusing magnetic sector
transmission is the single quadrupole mass
model. Before the recent introduction of
analyzer. As its name suggests, it contains
the high-performance time-of-flight (TOF)
four parallel cylindrical metal rods (electrodes
MS and ion-cyclotron-resonance (ICR) MS
with a hyperboloidal interior surface) inside
systems, the dual-focusing magnetic sector
a vacuum chamber, positioned equidistant
spectrometer was the only MS capable of
from the center axis (Figure 14). Both a
such high-resolution measurements.
direct current (D.C.) and high frequency
However, to improve the performance
alternating current or radiofrequency (RF)
of the magnetic sector MS, the strength
are applied to the quadrupole, so that only
of the magnetic field needs to be raised
the ions with the target m/z successfully
which means costly and larger systems are
pass through the quadrupole and get to the
required. This limits the development of the
detector. The quantity of ions that reach the
magnetic sector MS. In addition, magnetic
detector is converted to a signal and output
sector MS systems require an extremely high
to a computer.
vacuum level of 10-7 Pa, causing difficulty in
Continuous ion source generated
the LCMS interface. Furthermore, they have
in the ionization unit are first accelerated
the disadvantage of a slower scan speed
in the z-direction (Figure 14, green arrow)
than other MS systems. Therefore, these
by a relatively weak voltage of only a few
y
+
-
z
x
-
+ Ion Detector
Quadrupole
Ion Source
Resonance ion Non-resonance

ion
Figure 14. Schematic of how Quadrupole MS works.
30
dozen volts. These ions pass through a tiny Equation 8:

orifice and enter the quadrupole. Voltage of Mathieu Equation
the same polarity is applied to diagonally-
opposite poles and opposite voltage polarity m v
–– = K ——
is applied to adjacent poles as depicted by z r 2ω 2
the blue and red rods in Figure 14. When a
combination of the direct current voltage and m: mass of the ion
high-frequency alternating current voltage is z: charge of the ion
applied to each pole, an electric field with a K: constant
rapidly varying phase is generated within the V: voltage applied
quadrupole. r: effective distance between the
Consequently, ions passing through electrodes
this electric field oscillate in the x- and y- ω: oscillation frequency
directions. When a given set of parameters
are applied to the poles, certain ions of
a specific m/z range maintain a stable Direct current voltage
applied to electrode (U)
Straight
oscillation and pass through the quadrupole Ion Mass: m1 < m2 < m3
Scan Line (1)
to reach the detector (Figure 14, resonance

Unstable
ion). On the contrary, the oscillations of ions Region
m3
with other m/z values become unstable, m2 Unstable
Stable Region for m3 Region
causing them to collide with the poles, fly m1
out of the system, and not be detected

A B C Straight
(Figure 14, non-resonance ion). High Frequency Voltage Scan Line (2)
Applied to Electrode (V cos wt)
The oscillation of ions within the
Figure 15. Mathieu stability diagram for the stable regions
quadrupole MS is known to occur according for ions in Quadrupole MS system.
to the Mathieu Equation (Equation 8). The
motion of the ion in a quadrupole follows The region of stability is different for ions with
this equation regardless of its initial velocity masses m1, m2 and m3. If the voltage is varied
or position. Figure 15 illustrates how the while keeping the ratio between the direct
equation is solved which is also the Mathieu current voltage (y-axis) and high-frequency
stability diagram for the stable regions for alternating current voltage (x-axis) constant,
ions in a quadrupole MS system. a straight scan line 1 is obtained. This scan
As illustrated in the shaded areas line passes through respective regions of
in Figure 15, the conditions required for stability for ions with masses m1, m2 and m3.
stable ion oscillation are determined by Consequently, these ions are passed through
the mass and oscillation frequency of the the quadrupole consecutively in the same
ion as observed in the Mathieu Equation. order (m1, m2 and m3). In this way, a mass
31
spectrum is obtained for ions with masses in the specified range of the mass spectrum.
ranging from small to large. For the SIM mode (Figure 16B), only the
For a single quadrupole MS system, it selected m/z (red ion) is monitored, passed
can operate in two modes: (A) Scan and (B) through the quadrupole and detected.
Selected Ion Monitoring (SIM). In scan mode, This SIM mode offers higher sensitivity and
the voltages to the quadrupoles is configured avoids the effects of unwanted analytes and
such that the entire mass range specified is impurities.
scanned sequentially with appropriate dwell
time5 for each m/z value. As illustrated in 5
Dwell time is the time allocated for measuring or
Figure 16A, the blue ions, followed by red acquiring the data of an ion of a particular mass-to-charge
ratio in a mass spectrometer. It is the acquisition portion of
ions and lastly yellow ions passed through the the LCMS duty cycle. The longer the dwell time, the greater
quadrupole sequentially and gets detected. the number of target ions detected. In simultaneous multi-
component analysis, dwell time may be shortened for each
The result is a record of the ion abundance target component.
Ion Guide
+ +
+ +
+ + + + Resonance ion
+
+ + + + + +
Non-resonance
ion
(A) Scan mode (B) SIM mode
Increasing Quadrupole Constant Quadrupole

Voltage Voltage
Time Time
Mass Spectrum Mass Spectrum
Figure 16. Schematic of the (A) scan mode and (B) SIM mode in Quadrupole MS.
32
Since the separating principle of the SIM mode in a quadrupole MS, it can deliver
quadrupole MS systems is straightforward, a high-sensitivity quantitative analysis of a
they are comparatively easier to operate and large number of target compounds, making
maintain. Also, the quadrupole is compact it a widely recognized system among MS.
in design, robust and relatively inexpensive.
Consequently, they are widely adopted as Time-of-Flight (TOF) MS
Unlike magnetic sector and quadrupole
a general-purpose analytical instrument.
MS, Time-of-Flight (TOF) MS is a pulsed
Furthermore, unlike other MS which require
and non-scanning MS. It has a simple
high vacuum levels, quadrupole MS can
construction, consisting of an accelerator, a
adequately function at lower vacuum levels
field-free region, a reflectron and detector
(≈ 10-2 to 10-3 Pa). Even if they are interfaced
inside a high vacuum chamber called a flight
with a GC or LC unit, the drop in vacuum
tube (Figure 17).
level caused by the interface has minimal
TOF MS separates and detects ions of
effect on the mass separation performance,
different m/z by measuring the time taken for
making it the best suited for interfacing with
the ions to travel through a field-free region.
chromatographic techniques.
First, ions generated in an ionization unit are
Quadrupole MS demonstrates good
accumulated and introduced in pulses to a
scan speed and sensitivity. With a maximum
flight tube. These ions are accelerated by
scan speed of 15,000 amu/second, it is
applying a high acceleration voltage between
capable of measuring at higher scan speeds
the electrodes. The corresponding kinetic
than the magnetic sector MS. Its mass range
energy is obtained as described in Equation
can reach up to 2,000 m/z which enables
9. Given a constant acceleration voltage
the qualitative analysis in a practical range of
as well as kinetic energy, each ion flies at
molecular masses. In addition, it allows high-
its unique velocity inside the flight tube to
speed polarity switching, which facilitates
reach the ion detector, which is higher for
simultaneous monitoring of multiple selected
ions with smaller masses and lower for ions
ions of different polarity. With the use of the
with larger masses.
33
C146-E119B
LCMS-2020
Liquid Chromatograph Mass Spectrometer
Seei ng i s Bel ie v i n g .
UFscanning
15,000 u/sec high-speed scanning
UFswitching
15 milliseconds high-speed polarity ionization switching time
UFsensitivity
High-speed analysis with high sensitivity
UF s c a n n i n g & U Fswitch in g
UFLC/MS Measurement
UFscanning and UFswitching are critical for ultra-fast analysis.
In ultra-fast analysis where multicomponent may elute within 1 minute,
ultra-fast (MS measurement) detection is also required. The UFswitching and
UFscanning functions are what make possible such ultra-fast MS
measurement.
15 milliseconds 15 milliseconds
polarity switching polarity switching
Positive ion measurement Negative ion measurement Positive ion measurement

15,000 u/sec 15,000 u/sec 15,000 u/sec
UF s e n s i ti v i t y
Ultra-fast analysis with excellent sensitivity Calibration curve
Newly developed ion optical system and new Qarray®
optics provide excellent sensitivity, repeatability and
linearity, even in ultra-fast analysis.
Hard wa re featu res th a t p o we rfully s upport 3 types of UF func tionali t y

Toughness against dirty samples Easy Maintenance
In order to check toughness of LCMS-2020 against dirty samples, The DL capillary (desolvation line), which transfers the sample
plasma samples simply precipitated with only acetonitrile were into the vacuum chamber from the ion source, can be installed
injected 2,500 times over 10 days (1L volume per injection). and removed without breaking the vacuum, greatly speeding
Excellence reproducibility of peak area was demonstrated and its maintenance operations.
RSD was 2.26%.
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www.shimadzu.com/an/ © Shimadzu Corporation, 2012
Printed in Japan 3295-11103-30ANS

Ion Source
Ion Acceleration Ion Detector

Electrode
Flight Tube
Ion Mass Ion Speed
+
+
++
+
Reflectron
+++
+
Figure 17. Schematic of a TOF MS.
Equation 9: As shown in Equation 10, Time of flight

(T) is proportional to the square root of m/z,
1 i.e. for a fixed flight distance (L), ions with
KEion = – mv2 = zeV smaller m/z reach the detector sooner than
2
those with larger m/z. Therefore, by keeping
Equation 10: all other parameters constant, the time of
flight (T) can be converted directly to m/z,
distance m L which is how a mass spectrum is generated
T = ––––––– = –– X ––– in a TOF MS. Since there is no limit to the
velocity √ z √2eV
time of flight in TOF MS, it can theoretically
T: Time of flight measure an unlimited mass range.
m: mass of the ion Due to its operating principle, TOF
v: velocity of the ion MS systems do not introduce ions into the
z: charge of the ion analyzer until after the previous group has
e: elementary charge reached the detector. Therefore, it has good
V: acceleration voltage applied to ions compatibility with ionization methods that
L: flight distance in TOF ionize molecules in pulses, such as laser
36
ionization. This analytical technique has can be thought of as the entrance and
been extremely useful for proteomics using exit of a quadrupole connected in a ring
MALDI-TOF MS systems, where proteins are shape. Normally, the IT MS is used without
identified by comparing measurements of applying the direct current voltage (U) to the
fragmented peptides with a database. With electrodes, which corresponds to movement
advancements that minimizes the differences and operation along the horizontal axis.
in the kinetic energy of ions, such as the use To measure a spectrum, end-cap
of reflectron and pulsed extraction methods, electrodes are first grounded, then a low
TOF MS systems are currently being utilized high-frequency voltage is applied to the ring
for high-resolution MS. electrode. The ions are introduced into the
IT MS in a pulse mode, where they are all
temporarily trapped inside the electrode. This
CLICK HERE
state, where the ions with varying mass are
Learn more about experiencing stable oscillations, is indicated
MALDI-TOF in Figure 18A. Subsequently, to detect a
applications on compositional
and structural analysis specific ion, the high-frequency voltage is
gradually increased (Figure 15, straight scan
line 2), while keeping the direct current (U)
Ion Trap (IT) MS to zero. As the voltage increases (Figure
IT MS is based on an ion trapping 15), the oscillation of the blue ion becomes
mechanism and pulsed MS. There are several unstable at point B in the Mathieu diagram
variations of IT MS, for example the 2D linear and the red ion becomes unstable at point
quadrupole IT MS and the 3D ring IT MS and C, at which time these ions are discharged
the components and system configurations via the hole in the end-cap electrode (Figure
may vary slightly. These IT MS employ the 18B). Quadrupole MS systems separate and
same principle as the quadrupole MS and detect masses by letting oscillating ions pass
the motion of ions within the mass analyzer through the quadrupole to reach a detector,
follows the Mathieu Equation (Figure 15). In whereas ion trap MS systems separate and
this section, the 2D linear quadrupole IT MS detect masses by discharging ions with
are described in greater detail. It generally unstable oscillations from the system.
consists of a donut-shaped ring electrode As the name implies, ion trap MS
sandwiched between two end-cap electrodes systems trap the generated ions before
(Figure 18). An ionization unit is located separating them by mass. Consequently,
at the entrance and a detector at the exit. they cannot perform selected ion monitoring
Just like a quadrupole system, the internal (SIM) transitions and measurements for
surface of electrodes is hyperboloidal, which transmission type MS. Furthermore, they
operate by pulsed mode and only a limited
37
(A) Stable ion oscillations Ring Electrode
Ion detector
Ion source
End-cap End-cap
Electrode Electrode
Ring Electrode Cooling/ CID Gas
(B) Discharging of ions

with unstable
oscillations
Ion detector
Ion source
Figure 18. Diagram of an IT MS.
quantity of ions can be trapped, resulting in similar mechanism and principles. In the
a narrower dynamic range than quadrupole FT-ICR setup, the use of both the electric
MS systems. However, all trapped ions and magnetic field generates the stable
are detected in the IT MS, this provides oscillation and motion of the ions. To detect
higher sensitivity in scanning analysis than the ions, the selected ions are accelerated
quadrupole models. In addition, it enables such that its radius of oscillating motion
the trapping of a specific ion, fragmenting increases, the oscillation becomes unstable
it and then trapping a specific product ion and eventually the ion gets removed. By
for further fragmentation, and so forth. determining the cyclotron frequency, it can
Therefore, IT MS is considered a mass be Fourier transformed and the ion mass is
spectrometer specialized for elucidating deduced. For Orbitrap MS systems, it only
the fragmentation pathway for structural requires the use of electric field to trap
determination of a target molecule. and separate the ions. These MS systems
In addition to the two kinds of IT MS, demonstrate excellent mass resolution and
there are other similar trapping type of MS mass accuracy.
such as the Fourier Transform Ion Cyclotron
Resonance (FT-ICR) and Orbitrap. They adopt
38
Comparison of mass analyzers of these single mass analyzers are listed in

It is important to note that no single Table 4.
mass analyzer is excellent for all analyses. Mass spectrometers are now used for
Therefore, it is important to understand an extremely diverse range of applications,
the different principles, features and each with its own characteristics. Therefore,
characteristics of these mass analyzers and it is not easy to decide in a simple manner
choose the one suitable for your needs. which type of MS is optimal. In terms of cost
Some of the key advantages and limitations and ease-of-operation, quadrupole mass
Table 4. Advantages and limitations of the various mass analyzers.
Mass Analyzer Description Advantages Limitations

Magnetic Sector Scanning • High resolution • Expensive and bulky
Continuous • High dynamic range • Slow scan speed
• High reproducibility • High vacuum
• High sensitivity required
• Difficult to
couple with
pulsed ionization
techniques and LC
Quadrupole Scanning • Compact and simple • Limited mass range

Mass Filter • Relatively cheap • Low resolution
• Good selectivity (SIM) • Little qualitative
Continuous
• Moderate vacuum information
required → well suited
for coupling to LC
Time-of-Flight Non-scanning • High sensitivity and ion • Requires pulsed

Pulsed transmission introduction to MS
• High resolution • Requires fast data
• Excellent mass range acquisition
• Fast scan speed
Ion Trap Trap • Small and relatively • Limited dynamic

Pulsed cheap range
• High sensitivity • Limited ion trap
• Good resolution volume
• Compact • Limited resolution
• Requires pulse
introduction to MS
39
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Investigation of Synthetic Compounds in Drug Discovery by
Nexera-i and LCMS-2020.
Read more about the analysis of pharmaceutical substances in a workflow
that is typically used for pharmaceutical synthesis confirmation analysis. The
use of Nexera-i integrated UHPLC system with high-scan-speed LCMS-2020
offers rapid and comprehensive qualitative and quantitative analysis.
Sample Name: Trimethoprim
log P : 0.91
pKa : 7.12
MS Chromatogram MS Spectrum
Inten. (×1, 000 ,000 ) Inten. ( ×1,000 ,000 )

TIC(+) 4 Positive
TIC(-) 291.2
7.5 291 .2(+)
3
5.0
2
2.5
1
0
0
0.0 0.5 1.0 1.5 min 250 .0 275 .0 300 .0 325 .0 350 .0 m/z
analyzers have been increasing in market it sensitivity, peak resolution, or a compact

share for LCMS applications. Likewise, ion general-purpose system. In summary, it
trap and TOF MS systems offer performance is important to consider and choose a MS
not obtainable from quadrupole models, so system that allows benefiting from the
their usage and popularity is also continuing advantages offered by each ionization
to increase. This means the MS system method and mass separation technique.
must be selected based on objectives, be
40
Chapter 4
Introduction to tandem
MS systems
C
hapter 4 introduces the core concept and key features of a MS/MS
system. It includes the basic instrumentation, the collision-induced
dissociation and the various scan modes in MS/MS. Tandem/Hybrid
MS systems (e.g. Triple Quadrupole MS, Q-TOF, and IT-TOF) is a versatile and
powerful instrument used in many applications. To gain a better understanding
and to find the most suitable system for your application, the resolution,
sensitivity, capabilities and strengths of these MS/MS are compared.
Chapter 4: Introduction to tandem MS systems
Core principles
MS is a very useful and powerful tool proteins and (3) metabolomics identification
in quantitative and qualitative analysis. and quantitation research. Notably, the
Furthermore, it can identify analytes based on use of MS/MS provides detailed mass
m/z and provide accurate mass information information and allows the reduction of
on elemental composition, isotopic analysis matrix interferences and background giving
and structural information. However, there superior selectivity and sensitivity.
are limitations of a single mass spectrometer. The basic components in a MS/MS
A single MS may not provide reliable system are illustrated in Figure 19. Like
quantitative and qualitative information in LCMS, MS/MS system can be coupled to a LC
cases where resolution is insufficient (hard to or GC for chromatographic separation prior
separate) for both chromatography and m/z to MS analysis. The main difference between
(e.g. isomers). This is particularly the case a LCMS and a LC-MS/MS is the addition of
where the sample matrix is complex and the a collision cell and a MS2. The single mass
target analytes are in trace concentrations. analyzers described earlier (e.g. quadrupole,
Therefore, a technique that provides a TOF, ion trap or magnetic sector MS) can be
higher selectivity, specificity and sensitivity integrated into a MS/MS system.
and gives additional unique mass and 6
Tandem MS refers to MS/MS systems that use
structural information of the target analytes the same mass analyzers (e.g. triple Quadrupole MS/MS)
while hybrid MS refers to MS/MS systems with different MS
is required. systems (e.g. Quadrupole-TOF MS).
A MS/MS system, also known as a
tandem/hybrid6 MS (denoted MS/MS),
consists of two mass analyzers connected
in series with a collision or fragmentation
cell in between. Ions are separated in the
first mass analyzer (MS1), enter the collision
cell and undergo fragmentation, resulting
in generation of ions called product ions
which are separated in the second mass
analyzer (MS2) and detected. MS/MS serves
as a solution for the challenges faced by a
single MS analysis. Since its introduction,
MS/MS has been a versatile and powerful
instrument for many applications such as
(1) drug discovery and development, (2)
structural analysis of polysaccharides and Awarded with
The Analytical Scientist
Innovation Award
42
Mass Collision Mass

HPLC Ion source and guide Detection
Analyzer 1 Cell Analyzer 2
Figure 19. Basic instrumentation of a LC-MS/MS.
Collision-induced dissociation (CID) is energy supplied because some bonds

the preferred method of fragmentation in the require higher activation energy than others
collision cell. Fragmentation of the precursor for breakage. As shown in Figure 21, with a
ions occurs and this provides unique MS data CID collision energy of 0 volts, the molecular
of the fragment ions. Figure 20 illustrates the ion of Osteltamivir (m/z = 313) is of highest
processes and fragmentations that occurs in abundance with no product ions. As collision
the collision cell. energy increases, the abundance of the
The precursor ions of m/z selected molecular ion decreases and fragmentation
by MS1 enter the collision cell filled with occurs to generate a variety of product ions.
chemically inert gas (e.g. He, Ar, Xe and N2) At even higher collision energies (e.g. >50 V),
and collisions between the precursor ions the degree of fragmentation is more extensive
and inert gas are induced by applying an resulting in the mass spectrum showing
oscillatory field (giving a ‘shake’). Collisions no molecular ion and higher abundances
cause conversion of kinetic energy into of product ions of lower m/z. Besides CID,
molecular excitation (internal energy) that there are other less common alternatives
cause chemical bond breakage to generate to induce fragmentation such as electron
product ions. The degree of fragmentation capture/transfer dissociation, surface-
and product ion species depend on the induced dissociation and photodissociation.
Collision gas
Collision Cell
Percursor ion Collision-induced Dissociation Product ions MS2
Figure 20. Illustration of CID in the collision cell of MS/MS systems.
43
208
Chapter 4: Introduction to tandem MS systems 166
120
208
243
296 94 136 179 225
166 50 100 150 200 250 300 m/z

225
313 50 100 150 200 250 300 m/z
50 100 150 200 250 300 m/z
166 > 120
H 50
ESI - MS
100 150 200 250 300 m/z
ESI - MS 2120
225 313
O
225 225 > 179 313
(CID
ESI - 0MS
Volts) 166 2
ESI - MS 2 2 (CID 10 Volts) ESI - MS
243 O
(CID 10 Volts) (CID 30 Volts)
(CID 20 Volts) 94
O 166 208
208
166
166 > 136
HN 166
H
137
NH 2 120 243
225 > 208
O 296 243 208 109
296
296
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179 > 136 225 > 208
94 136 179 67 161
225
Osteltamivir (Tamiflu™) 50 100 150 200
225
250 300 m/z
313
50
50
100
100
150
150
200
200
250
250
300 m/z
300 m/z
50 100 150 200 250 300 m/z 50 100 150 200 250 300 m/z
ESI - MS 2 166
Structure
2
ESI - MS
ESI - MS 2
166 (CID 10 Volts)
120 65
(CID 20 Volts)
ESI - MS 2 ESI - MS 2
(CID 20 Volts) 94
166
208 (CID 30 Volts) (CID >50 Volts)
elucidation of
pharmaceuticals
166
120 137
243
296 77 92
120
208 using ultrafast
triple quadrupole
208 94 136 179
109 225
94 136 179 120
225 161
50 67 100 150 200 250 300 m/z
50 100 150 200 250 300 m/z 50 100

ESI - MS 2
150 166 200 250 300 m/z
50
50
100
100
120
150
150
200
200
250
250
300 m/z
300 m/z
MS.
ESI - MS 2
120 (CID 20 Volts) (CID 30 Volts)
ESI - MS 2 65
2 94
ESI - MS
Figure 21. CID of Osteltamivir (Tamiflu) at various collision
(CID >50 Volts)energies (0, 10, 20, 30, >50 V).
166
94 (CID 30 Volts)
166
120 137
208
137 109
77 92 136 179
94
Figure 16 describes the scan and historically preferred by IUPAC, while

109 225
67 161
67 161 120
50 100 150 200 250 300 m/z
50 100 150 200 250 300 m/z
50
selected ion monitoring (SIM) modes
100 150 200
ESI - MS
250
of a 300 m/z
50 100
120
150 200 250 2 300 m/z 65 instrument ESI vendors
- MS
including Shimadzu 2
65 (CID 30 Volts) 94
2 (CID >50 Volts)
quadrupole (CIDMS.
>50 Volts)Likewise in MS/MS, the MS1
ESI - MS 166
preferred the alternative term, Multiple
and MS2 can either acquire the scan or SIM 137
Reaction Monitoring (MRM). Now IUPAC
77 92
77 92 (fixed) mode. The different arrangements 67

109
161
recommends that the term SRM be used to
120
allow the system to operate at various

120
50 100 150 200 250 300 m/z
50 describe the technique applied to a single
100 150 200 250 300 m/z
50 100 150 200 250 300 m/z 65
scan and monitoring modes (Figure (CID >5022). It target and MRM for plurality of SRM. In
2
ESI - MS
Volts)
is important to note that the actual scan this document hereafter, the technique is
and monitoring modes available in MS/MS 77 92 referred to as MRM regardless of the number
systems depends on the mass analyzers (MS1 120
of precursor/product pairs monitored
50 100 150 200 250 300 m/z
and MS2) used. Some of these scan modes simultaneously.

(e.g. precursor ion and neutral ion scan) may Prior to performing MRM, product
not be available in time-based7 MS (e.g. ion ion scan is usually conducted to determine
trap and FT-ICR). the product ion of highest abundance. In
For a long time, Selected Reaction conclusion, MS/MS can not only remove
Monitoring (SRM) used to be the term noise and interference but also selectively
target particular ions for quantitation to
deliver a higher specificity and sensitivity
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analysis.
Find out about how
precursor ion scan, Time-based MS refers to ion trap and FT-ICR MS,
7
neutral loss scan and MRM is used in where ions are separated temporally and gets detected
Simultaneous Analysis of Amino Acids sequentially at a fixed point. On the other hand, space-
and Acylcarnitines in Dried Blood Spot based MS separates ions spatially based on their deviation
with LCMS-8040 in terms of path radius or velocity. Examples of space-based
MS are magnetic sector, quadrupole and TOF MS.
44
(A) Product Ion Scan:
MS1 is fixed at selected m/z while MS2 operates at scan mode. This scan acquires all the product ions from the fragmentation
of a selected precursor ion. The intensity and m/z of all product ions are captured and displayed in the mass spectrum.
MS1 Collision Cell MS2
Fixed Scan
(B) Precursor Ion Scan:
MS1 operates at scan mode while MS2 selects the product ion of a particular m/z formed by CID in the collision cell. This
mode is especially useful for determining the precursor ions that produce fragment ions of a particular m/z.
Collision Cell
Scan Fixed
(C) Neutral Loss Scan:
MS1 and MS2 operates at scan mode while keeping a specific m/z difference. This scan can determine the precursor ions that
lose a specific neutral molecule (e.g. hydroxyl group and phosphate group) during fragmentation.
Collision Cell
Scan Scan
(D) Selected Reaction Monitoring (SRM):
The transition of a selected precursor ion to a selected product ion is monitored where MS1 and MS2 are fixed at the specific
m/z.
Collision Cell
Fixed Fixed
Figure 22. Types of scan and monitoring modes in MS/MS systems.
45
Types of MS/MS systems and their key

characteristics
With the combination of two mass TQ, Q-TOF, IT-TOF and multistage MS) and
analyzers in MS/MS systems, several tandem discuss in detail its applications, strengths
and hybrid configurations consisting of and limitations.
quadrupole, magnetic sector, TOF and/or Other than these MS/MS systems, there
ion trap MS are obtained (Table 5). There are also other tandem/hybrid MS that utilizes
are no changes to the separating principles more than 2 mass analyzers. However, this
of these mass analyzers in a MS/MS system. configuration is not commonly used due
The following sections elaborate on the key to the higher cost and complexity of the
characteristics of these MS/MS systems (i.e. equipment.
Table 5. Various tandem/hybrid MS/MS systems.
Triple Quadrupole (TQ)
Tandem MS 2 Dual-focusing MS (combination of magnetic & electric

fields)
Quadrupole Time-of-Flight (Q-TOF)
Ion trap Time-of-Flight (IT-TOF)
Quadrupole Ion Trap (Q-IT)
Quadrupole Ion-Cyclotron-Resonance (Q-ICR)
Hybrid MS Ion trap Ion-Cyclotron-Resonance (IT-ICR)
Ion trap Orbitrap (IT-Orbitrap)
2 Time-of-Flight (TOF-TOF)
Multistage MS (MSn)
46
Table 23. Instrumentation of Triple Quadrupole (TQ) MS.
Triple Quadrupole (TQ) MS Quadrupole Time-of-Flight (Q-TOF)

The simplest and most common MS/ MS
MS system is the TQMS. It consists of three By switching the last quadrupole mass
quadrupoles arranged in series with the analyzer in a TQ MS to a TOF mass analyzer,
first and third quadrupole acting as MS1 we will get the Q-TOF hybrid MS (Figure
and MS2 respectively and the CID taking 24). With the inclusion of a TOF, this hybrid
place in the second quadrupole (Figure 23). system provides excellent dynamic range,
With the use of precursor ion scan, neutral high mass resolution and mass accuracy.
loss scan and MRM, it can achieve superior In addition, it can perform good quality
selectivity, specificity and sensitivity with quantitative analysis. With the full scan and
minimal background. As a result, TQ MS full ion transmission capability in Q-TOF MS,
is an excellent instrument for quantitative it captures all the ions in a single run and
analysis and is commonly employed for allows the reinvestigation of data for new
routine targeted analyses. and unknown compounds without the need
On the contrary, TQ MS falter in terms for reacquiring. With these properties, it is
of mass accuracy and resolution as compared commonly used for high resolution accurate
to other types of MS/MS. It is not commonly mass analysis such as in the identification
employed for untargeted analyses. Another of unknown molecules for proteomics and
reason is that prior knowledge of the metabolomics research.
compound (e.g. molecular ion mass) is
required to utilize the high sensitivity MRM
modes in TQ MS. In this case, other MS/MS
systems such as Q-TOF and IT-TOF are used.
47
C146-E287B
Liquid Chromatograph Mass Spectrometer
LCMS-8060
The LCMS-8060 is the latest in Shimadzu’s Ultra-Fast Mass Spectrometry product line, designed to deliver you the highest
sensitivity and fastest analysis speed of any LCMS on the market today.
Sensitivity Highest Sensitivity

A newly developed UF-Qarray boosts ion intensity but suppresses
Heated ESI Probe
noise. By improving the ion sampling device, the ion guide, and
vacuum efficiency, Shimadzu has achieved an unprecedented
sensitivity in LCMS. Drying Gas
Speed
Fastest Speed Heating Gas
DL (Desolvation Line) UF-Qarray UF-Lens

Shimadzu’s proprietary technologies allow acquisition of up to 555
MRM channels per second, ultra-fast polarity switching, and ultra-fast
scanning, all with high data quality. Solutions
Fusion of sensitivity and speed
UFscanning: Max. 30,000 u/sec
Don’t miss an exciting performance results in next page!!
UFswitching: 5 msec
High-Sensitivity Quantitation of Catecholamines in Plasma
Catecholamines in plasma, namely norepinephrine (NE),
epinephrine (EP) and dopamine (DA), are routinely measured
in the research of such diseases as hypertension or
neuroblastoma. Since plasma samples contain endogenous ×106
Quantitative range of neat and matrix-matched calibration curves
2 1. Norepinephrine
catecholamines, it is difficult to evaluate the LLOQ in plasma 3.0
2. Epinephrine
3. Dopamine Neat standard curve Matrix-matched
matrix. Here we used deuterated catecholamine compounds Standard Compound name Range Linearity Range Linearity
Deuterated
as standards to estimate the LLOQ in plasma matrix, rather 2.0 (pg/mL) (r2) (pg/mL) (r2)
3
Norepinephrine-d6
than as internal standards for quantitation. (158.1 > 111.1)
2.5 – 2000 0.9999 2.5 – 2000 0.9997
1
A neat standard curve was prepared by serial dilution in 1.0
Epinephrine-d6
10 – 2000 0.9999 10 – 2000 0.9994
HPLC solvent, whereas a matrix-matched standard curve was (190.1 > 172.1)
Dopamine-d4
prepared by dilution with pooled plasma sample treated with 0.0
(158.1 > 95.1)
5 – 2000 0.9999 10 – 2000 0.9995
1.0 2.0 3.0 4.0 min
SPE. The table on the right summarizes the quantitation
Representative MRM chromatograms
results, which convincing demonstrate the capability of of 3 catecholamines
LCMS-8060 to detect catecholamines at ultra-high sensitivity
without matrix interference.
Norepinephrine Norepinephrine-d6 Epinephrine Epinephrine-d6 Dopamine Dopamine-d4
×104 ×104 ×105 ×105 ×105 ×105
2.0 2.0
In the actual quantitation assay,
7.5 5.0 5.0
deuterated catecholamines are 7.5 300 pg/mL
spiked
500 pg/mL
spiked
500 pg/mL 1.5 1.5 500 pg/mL
spiked spiked
spiked as internal standard at 500 5.0 5.0 300 pg/mL
blank spiked 1.0 300 pg/mL 1.0
pg/mL in plasma and analyzed by blank
2.5 2.5 spiked
2.5
LCMS-8060. The figures on the right 2.5
blank blank
0.5
blank
0.5
blank
show the MRM chromatograms of 0.0 0.0 0.0 0.0 0.0 0.0
spiked and endogenous 0.5 2.5 min 0.5 2.5 min 1.0 2.5 min 1.0 2.5 min 2.5 5.0 min 2.5 5.0 min
catecholamines in plasma. Detection of Norepinephrine, Epinephrine and Dopamine and their deuterated internal standards in plasma.
×104
7.0
100 pg/mL Alprazolam in human plasma with internal standard
1.4
Outstanding Durability
6.0 m/z 309.05 > 281.10 1.2
The robustness of the LCMS-8060 and modified ion
A week
1st day 2nd day 3rd day 4th day 5th day 6th day
5.0 1.0
optics were assessed by injecting 2400 samples of
Area Ratio
Peak Area
4.0 0.8
Area Ratio 3.46 %RSD femto-gram levels of alprazolam spiked into

3.0 0.6
2.0 0.4
protein-precipitated human plasma extracts over a 6
1.0
Peak Area 4.97 %RSD
Vacuum 0.2
day period (over 400 samples were injected each day).
Pause in batch Pause in batch system Pause in batch Pause in batch
0.0
analysis analysis vented analysis analysis
0.0
The RSD of peak area response was 5% over this test
0 400 800 1200 1600 2000 2400
Injection number period; using a deuterated internal standard
Long term stability study on LCMS-8060 (alprazolam-d5) the RSD was 3.5%. As part of the
1.00
(×10,000)
1.00
(×10,000)
1.00
(×10,000) robustness test the vacuum system was vented to
1st injection 1200th 2400th injection
0.75 0.75 injection 0.75
model a transient power failure with no effect on
0.50 0.50 0.50 signal response or baseline noise level.
0.25 0.25 0.25
0.00 0.00 0.00

0.8 0.9 1.0 1.1 1.2 1.3 min 0.8 0.9 1.0 1.1 1.2 1.3 min 0.8 0.9 1.0 1.1 1.2 1.3 min
MRM chromatograms for the 1st, 1200th and 2400th measurements of Alprazolam
Intraday and interday variations on LCMS-8060

Intraday Variation (%RSD) Interday Variation (%RSD)
Compound 6 Day
1st day 2nd day 3rd day 4th day 5th day 6th day Days 1–3 Days 4–6
Total
Alprazolam 5.04 4.94 5.06 5.38 4.55 4.83 3.19 1.63 2.74
Alprazolam-d5 (ISTD) 5.04 4.68 5.48 5.31 4.26 4.91 2.62 1.89 2.18
Area ratio 3.48 3.11 3.48 3.44 3.71 3.54 1.79 0.26 1.40

This publication may contain references to products that are not available in your country. Please contact us to check the availability of these
products in your country.
Third-party trademarks and trade names may be used in this publication to refer to either the entities or their products/services, whether or not
they are used with trademark symbol “TM” or “®”.
www.shimadzu.com/an/ The contents of this publication are provided to you “as is” without warranty of any kind, and are subject to change without notice. Shimadzu
does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the use of this publication.
© Shimadzu Corporation, 2016

First Edition: May 2015, Printed in Japan 3655-09621-30ANS
Figure 24. Instrumentation of the Quadrupole Time-of-Flight (Q-TOF) MS.
Multistage MS (MSn) temporarily trapped and then subjected to

Multistage MS (MSn) is a technique of either the detection system or to precursor
performing multiple mass analysis in a single ion isolation for further fragmentation. The
instrument and usually requires a time-based entire process can be repeated unlimitedly (n
MS. Using ion trap MS as an example, the times) and hence denoted MSn. Multistage
introduction of ions, selection of precursor MS provide unique structural elucidation
ions, fragmentation and analysis of product and qualitative analysis. However, when
ions are all performed in a single mass interpreting mass spectrum from ion trap
analyzer. Ions are first introduced in the ion MS, it is important to note that undesirable
trap, the selected m/z precursor ion is isolated artifact ions may be generated due to
by applying a suitable voltage. The precursor long trapping conditions and ion-molecule
ions oscillate in stable motions in the trap reactions in the ion trap.
while the rest of the ions are subjected to
unstable motions and are ejected from the Ion Trap Time-of-Flight (IT-TOF) MS
system. The parameters in the ion trap are The IT-TOF MS system provides MSn
then adjusted such that the precursor ions capability with enhanced sensitivity and
are given vigorous oscillation and collide is coupled with high resolution and high
with pulses of inert gas introduced into mass accuracy capability of TOF MS. The
the ion trap. The range of product ions are components in an IT-TOF MS is illustrated in
50
Figure 25. Instrumentation of an Ion Trap Time-of-Flight (IT-TOF) MS.
Figure 25. With the inclusion of the ion trap Comparison of MS/MS systems
MS in a IT-TOF MS, there is no need for a In the previous sections, we introduced
collision cell as collisions and fragmentations several MS/MS systems and covered their
are possible in the time-based MS itself. The key features. With all things considered, it
IT MS also double up as a focusing guide is evident that there is no universal MS or
to concentrate the ions to the center of MS/MS for all applications and analyses.
the trap before ejecting into the TOF MS. Each instrument has its own strength and
LCMS-IT-TOF enables the identification of limitations and it is necessary to understand
compounds using high accuracy MSn data and weigh the pros and cons to determine
and is also frequently applied in impurity the most suitable system for your needs.
analysis, metabolic profiling and biomarker
research.
Inten. (x1,000,000)
5.0 364.9496 MS1 spectrum

297.0472
2.5 223.0267
239.0586 313.0780 491.0042

165.0410 199.1703 372.0667
273.0254 328.9901 431.0352
0.0
50 100 150 200 250 300 350 400 450 m/z
Inten. (x1,000,000)
2.0
313.9469 MS2 spectrum
CLICK HERE
1.5
1.0
Development of a
0.5
spectrum library of
285.9549 363.9534
200.9846
103.2363 160.9496 249.9785 328.9700
0.0
endocrine disruptors
50 100 150 200 250 300 350 400 450 m/z
Inten. (x100,000)
3.0 285.9537 MS3 spectrum

by LCMS-IT-TOF
2.0
249.9762
1.0
67.0836 136.0645 189.9831 225.9813 277.9719

0.0
50 100 150 200 250 300 350 400 450 m/z
MSn Spectra for Tetrachlorobisphenol A
51
Table 6. Comparison of MS/MS systems (TQ MS, Q-TOF MS and IT-TOF MS).
MS/MS Systems Strengths Limitations Applications
• Highest sensitivity • Low mass • Quantitative

(MRM) resolution analysis (MRM)
• Wide dynamic • Targeted
range of detection analysis
TQ MS • Lower cost
• High mass • Low • Qualitative

resolution sensitivity analysis
• Wide mass range than TQ • Structural
Q-TOF MS MS MRM elucidation
• Medium dynamic mode
range of detection • Sequencing
• High sensitivity
• Multistage MS • Limited in • Qualitative

(MSn) scan modes analysis
• High mass • Structural
IT-TOF MS resolution elucidation
• Sequencing
Depending on your objectives, the use then single quadrupole MS (SIM) mode in
of LC, LCMS, LC-MS/MS and even several terms of quantitative analysis performance
MS systems may be required. We have earlier (e.g. sensitivity, limits of detection,
provided a comparison table of the various selectivity). While for qualitative analyses,
single mass analyzers (Table 4). Now, we TOF MS is superior based on the accurate
compared the performance and limitations mass and high resolution. The ranking is as
of these MS/MS systems (Table 6). follows: TOF > Ion Trap > Quadrupole.
In summary for LCMS single and
tandem/hybrid MS systems, TQ MS (MRM
mode) ranks first, followed by TOF MS and
52
Chapter 5
Challenges and
development of LCMS
I
n this final chapter, we discussed the needs of industries today and the
current challenges of analytical LCMS, with special emphasis on the
factors that limit the efficiency and performance of LCMS. We elaborated
on how Shimadzu overcome these challenges and develop new technologies
to increase the performance and enhance the sensitivity of these analytical
instruments. Notably, Shimadzu developed the Ultra-Fast Mass Spectrometry
(UFMS) and other key analytical techniques for present and future global needs.
Chapter 5: Challenges and development of LCMS
Analytical challenges and current limitations

of LCMS
Excellent
Performance
NEXT
GENERATION
LCMS
Optimized Maximized
Throughput Expandability
Since its introduction, LCMS has proved safety and development, environmental,
to deliver high quality data and robust energy and chemical.
performance. As discussed in earlier chapters, These industries are also constantly
LC and MS technologies have evolved to evolving and their demands are growing.
meet the needs of the industries. With Some of the main issues faced by the
these dynamic developments, the demand industries include having complex sample
for these techniques for both quantitative matrices, trace-level analytes, multiple
and qualitative analyses have increased component analysis of targets and
and LCMS has progressively become an unknowns, and complicated and time-
essential and routine analytical instrument consuming sample preparation procedures.
in many sectors. It has established itself Thus, there are needs for more advanced
to be a key tool in clinical, pharmaceutical instruments or software that give higher
and biopharmaceutical industries for drug throughput, greater efficiency, lower limits
discovery and protein analysis. It has also of detection, higher mass accuracy and more
set foot in other applications such as food user-friendly interfaces.
54
Besides working to meet these needs, These parameters affect the LCMS
LCMS providers are also striving to improve results. By optimizing these processes,
their analytical products in terms of its the ion abundance and signal-to-noise
throughput, performance and expandability ratio will increase and there will be lesser
in areas such as: contamination issues. In summary, LCMS
a) To increase the efficiency and manufacturers are looking to achieve these
reproducibility of the ionization key end-outcomes: high sensitivity, efficiency,
process reproducibility and durability, and producing
b) To create an efficient LCMS interface a simple and easy-to-use LCMS instrument.
and ion optics
8
Crosstalk may occur during MRM analysis where
c) To reduce unnecessary matrix effects the product ions of the previous MRM transition (residual
(unwanted suppressions and product ions in the collision cell) is erroneously detected as
product ions of the next MRM measurement.
enhancement)
d) To minimize sample carryover or
crosstalk8 effects
e) To increase the efficiency and
reproducibility of the fragmentation
in collision cell
f) To enable sufficient chromatographic
separation
g) To provide more options and
flexibility in terms of LCMS sample
preparation and analysis
Editors’ Gold Award
U n i fi e d C h r o m a t o g r a p h y
Winner of 2015 R&D100 Award
55
Key developments in Shimadzu’s LCMS

In this section, we shift our focus to improved with the use of a heated ESI probe.
Shimadzu’s ongoing efforts to overcome these Together with the enhanced desolvation line
analytical challenges. New technologies, and ion optics system, there is an increase
notably Ultra-Fast Mass Spectrometry (UFMS) in ion production and transmission, thereby
and Clinical Laboratory Automation Module generating a high-intensity and focused ion
(CLAM), are developed to increase efficiency beam leading to higher sensitivity in LCMS.
and enhance performance of LCMS. The next component of the UFMS
consists of the UFsweeper. It is a high-
Ultra-Fast Mass Spectrometry sensitivity and high-speed collision cell that
(UFMS)
enables ultra-fast ion sweeping. With the
Shimadzu has improved the ion optics,
new pseudo potential surface (Figure 27),
collision cell design and data acquisition
ions entering the collision cell are accelerated
systems and developed the UFMS. With
and maintain their momentum upon collision.
the use of UFMS, it can obtain unrivalled
Under these circumstances, the efficiency of
sensitivity and performance, that arise due
the fragmentation or CID is improved. This
to ultra-fast (UF) scanning, UF switching, UF
technology allows quicker and better ion
MRM and UF sensitivity.
transmission in the collision cell, maintaining
Firstly, the ion optics system consists of
signal intensity and dramatically suppressing
the UF-Qarray and UF-Lens (Figure 26). With
crosstalk, even when shorter dwell and
the new design, ions are precisely converged
pause9 times are used. Furthermore, this
and signal losses are minimized. In addition,
results in the possibility of high-speed MRM.
the efficiency of the ionization process is
Together with the developments
9
Measurement conditions must be switched
to perform simultaneous measurements of multiple
compounds, (multi-component analysis). The time needed
for this switching is termed as “pause time”. As data cannot
Heated ESI Probe be acquired during the pause time, it should be as short as
possible.
Desolvation
Line UF-Qarray™ UF-Lens™
Figure 26. Ilustration of the ion source and ion optics (UF-Qarray™ and UF-Lens™) in UFMS.
56
End of previous MRM
Pause time Dwell

time
Ion count (CPS)

Conventional design
Ions lose momentum due
to collision with gas.
Time
shorter pause time & dwell time
Pause Dwell
time time
UFsweeper™
Ion count (CPS)

UFsweeper efficiently More time for
accelerates ions out of the measuring other
compounds
collision cell without losing
momentum.
Time
Figure 27. Comparison of a conventional collision cell and UFsweeper™ collision cell.
in data acquisition system, UF polarity to achieve the world’s highest levels of

switching and UF scanning is made possible. sensitivity.
It results in shorter pause time and polarity
switching time10 and bring about more time
10
Separate positive ionization and negative
ionization modes may be used in LCMS. However,
for data collection. By combining all the UF switching between the positive and negative ionizations
technologies, the UFMS system delivers UF during analysis is required to and the time taken to switch
scanning, switching, sensitivity and MRM between these modes is known as the “polarity switching
time”.
CLICK HERE
Quantitative
Analysis of 646
Pesticides (1,919
MRMs) by LC-MS/
MS with a Fast
10.5 minute Cycle
Time
57
UFsweeper
Collision Cell UF Scanning
+
UF-Qarray
UF-Lens UF MRM UF Sensitivity
Ion Optics
+
UF Switching
LabSolutions
Data acquisition
System
Furthermore, with the use of UFsweeper Clinical Laboratory Automation

and high-speed scan analysis in UFMS, Module (CLAM)
simultaneous scan and MRM analysis can be Besides enhancing the performance
achieved where it alternates rapidly between and increasing the efficiency of the MS,
the scan and MRM modes at high speeds. Shimadzu has looked into innovative
Both scan and MRM data are obtained from solutions that help to simplify the analytical
a single analysis. Target compounds can be workflow. Pertaining to clinical research,
quantitated from the MRM analysis results Shimadzu was the first to develop the CLAM,
while unknown compounds can be identified a fully automated sample preparation module
by comparing the mass spectra to library for LCMS. From the initial pre-treatment to
search results. It maintains resolution and analysis, it is fully automated and individual
achieves high ion transmission even at high samples are analyzed successively in parallel.
scanning speeds. Therefore, with the use of Clinical scientists working with numerous
Shimadzu’s UFMS, triple quadrupole MS can samples can now utilize this instrument for
be used not only as an excellent instrument increased efficiency of existing workflow and
for quantitative analysis but also qualitative improves data reproducibility and accuracy.
untargeted analysis.
58
CLICK HERE
Learn more about features and
application of CLAM
Awarded with 2016 The Analytical Scientist

Innovation Award
Future trends of LCMS

Although there are lots of ongoing it is expected to break into widespread
efforts and developments on LCMS, there industrial use in the near future. There are
is still more to be explored to fully unleash also efforts from LCMS manufacturers in
the capacity and potential of LCMS. Future designing a more compact, lighter and
trends and developments of LCMS are greener LC system for LCMS, that requires
expected to still focus on achieving better less amount of materials, consume less
performance in terms of higher throughput, volume of solvents and having less dead
quality, efficiencies and robustness. volumes.
One such development is the Another trend aims to get more while
integration of various LC separation modes doing less, that is to move towards minimal
such as two-dimensional LC (2D-LC) and sample preparation without compromising
nano/capillary flow LC into LCMS platform. on data information and quality. As discussed
Progress is ongoing and it can improve the earlier, current sample preparation processes
separating power and sensitivity of LCMS. tend to be a laborious, time-consuming
This technology is promising for impurity and repetitive. Therefore, the simplification
analysis in pharmaceuticals and protein of these processes and even the use of
identification. If made easily comprehensible, automation for LCMS is favored and
CLICK HERE
Nexera Mikros
Micro flowrate LCMS
Previously, nano-flow rate compatible LCMS systems often suffer from complications in terms of
instrument operability and processing speed. With Shimadzu’s Nexera Mikros, a micro-flow rate LCMS, a
wide range of flow rates, from semi-micro-flow rates (100 μL/min to 500 μL/min), to micro-flow rates (1
μL/min to 10 μL/min) can be accommodated. This system achieves both durability and operability while
enabling analysis with at least ten times the existing sensitivity.
59
future developments are expected to head multitude of benefits that LCMS can bring
towards this direction. Another approach to their analysis, LCMS providers will need
to achieve greater throughput would be a to reach out to them and knock down the
development of sophisticated software to markets’ perceived barriers to LCMS.
enable multiplexing. For example, by shifting In conclusion, LCMS is a powerful and
from the original serial data acquisition to versatile tool equipped with both separating
parallel data acquisition, allowing more and detection capabilities. This fundamental
samples to be analyzed in a shorter period. guide covers the key concepts and principles
With the combined efforts in sample of LCMS and describe the various types of
preparation, software developments and LCMS and LC-MS/MS instruments in details.
data integration, the streamlining of LCMS We hope that the contents provide you
workflow from the initial sample preparation with a comprehensive overview and assist
to the final data analysis enables LCMS to in your LCMS analyses. Driven by our policy
be a one-stop, highly reliable, efficient and of contributing to society through science
automated process. and technology, we strive to further improve
Given its strengths and versatility, LCMS our technologies, create unique solutions
is expected to continue to be the analytical and innovative breakthroughs with a diverse
technique of choice for both quantitative and product range to protect and restore the
qualitative analyses in many applications. environment, and to deliver better health
However, to allow the market to enjoy the and lifestyles to people.
Shimadzu’s Comprehensive LC and MS Solutions

HIGH RELIABILITY AND EXCEPTIONAL PERFORMANCE LC SYSTEMS
Shimadzu provides a wide variety of LC systems
(HPLC, UHPLC, integrated, UC, 2D-LC) to meet
user requirements in nearly every market and
applications.
MASS SPECTROMETRY
With our outstanding capabilities in MS, we
produce various MS and tandem MS techniques
specifically catered for your quantitative and
qualitative analyses.
60
AUTOMATION
With our promise in innovation, Shimadzu
constantly develops new methods and products
in automation and integration to simplify your
workflow and achieve high productivity and
accurate results.
DATA MANAGEMENT AND SOFTWARE
Our LabSolutions analysis data system features

an innovative operating environment and
provides complete data management to ensure
secure information in networked laboratories.
APPLICATION SUPPORT AND DATABASE
Shimadzu is equipped with extensive libraries

and databases, application notes, method
development and solution system packages to
support all your analyses.
VERSATILE COUPLING WITH SPECTROSCOPY TECHNIQUES
Shimadzu provides a variety of spectroscopic

techniques and detectors available for coupling
with LC (e.g. fluorescence, photodiode array,
UV-Vis and conductivity).
FULL RANGE OF LABORATORY CONSUMABLES & SERVICES
We offer a wide range of laboratory

consumables such as LC columns and vials,
sample preparation products. Labtotal Smart
Service Net provides extensive assistance which
includes laboratory asset management and
application support.
61
Contact us
https://www.shimadzu.com/an/contact/index.html
First Edition: October, 2018

This publication may contain references to products that are not available in your country. Please contact us to check the availability of these
products in your country.
The content of this publication shall not be reproduced, altered or sold for any commercial purpose without the written approval of Shimadzu.
Shimadzu Corporation Third-party trademarks and trade names may be used in this publication to refer to either the entities or their products/services, whether or not
they are used with trademark symbol “TM” or “®”.
www.shimadzu.com/an
The information contained herein is provided to you “as is” without warranty of any kind including without limitation warranties as to its
accuracy or completeness. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the
use of this publication. This publication is based upon the information available to Shimadzu on or before the date of publication, and subject
to change without notice.
© Shimadzu Corporation, 2018

Liquid Chromatography Mass Spectrometry (LCMS) : Fundamental Guide To

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For Research Use Only. Not for use in diagnostic procedures.

WORLD’S 1ST MALDI-MS 1987

NOBEL PRIZE IN CHEMISTRY AWARDED TO KOICHI TANAKA 2002 AXIMA-CFR

Koichi Tanaka Mass Spectrometry Research 2003

2015 Unified Chromatography (UC)

LCMS-8045 2016 World’s 1st Automated LCMS

Cutting-edge UFsweeperIII™, DPiMS-2020

LCMS-9030 2018 Nexera Mikros

All market figures indicate that Shimadzu is the fastest-growing mass

I would like to express my gratitude for your eagerness to learn more

Chapter 4 Introduction to tandem MS systems 41

Chapter 5 Challenges and development of LCMS 53

Fundamentals of LC, MS and LCMS

From HPLC to UHPLC:

Shimadzu’s extensive LC lineup fulfills a wide range of analytical needs, from

Sample Separated components of the sample

Table 1. Different types of LC techniques and their separation mechanism.

Type of LC Techniques Separation Mechanism Commonly used for

Detectors for LC Systems Intensity

Figure 3. A typical LC chromatogram (red) and a LCMS

Upon separation by LC, the components

or electrical conductivity based on the

Chromatograms of parabens and preservatives in a standard mixture and cosmetic

In contrast, mass spectrometry (MS) With the coupling of LC with MS,

Comparison of LCMS and other techniques

Non-Polar Polar Ionic

Forensics Environmental Food & Beverage

Biopharmceuticals Pharmaceuticals Industrial

• Antibody Drugs • Phospholipid Profiling

Basic instrumentation of LCMS

Pump Atmospheric Pressure

Figure 5. Basic components of a LCMS system.

Examples of common MS detectors are LC chromatogram, the quantity of ions that

Peak capture loop

Interfaces for LCMS

(A) EI Mass Spectrum 60

0 50 100 150 200 250 300 350

Molecular Formula: C17H20N4O6 OH

0 100 150 200 250 300 350 400

However, EI is considered not applicable Electrospray Ionization (ESI)

Sample & mobile phase

In addition, ESI is known to occasionally mass spectrometer. It is also possible to use

0 600 800 1000 1200 1400 1600 1800

Atmospheric Pressure Chemical Atmospheric Pressure

Sample & mobile phase

Nebulizer gas Nebulizer gas

Analyte Solvent Analyte

Molecule Ion Ion

Figure 9. Schematic of the ion-molecular reaction (e.g. proton transfer) in APCI.

Sample Slide Sample Slide

Sample & mobile phase

Nebulizer gas Nebulizer gas

Analyte VUV Radical

Molecule Ion Light Ion

Overview of Atmospheric Pressure is crucial to achieve high sensitivity. it is

In the case of APCI, it requires a compound (dopant), which has lower

Figure 11. Illustration of the use of dopant to increase sensitivity in APPI.

Table 2. Summary of ionization process and analyte properties of API techniques.

API techniques ESI APCI APPI

Ionization process Ions in solvent Ionization occurs in Ionization occurs in gas

Stability of analyte Do not need to be Must be thermally Must be thermally

Chapter 4 Introduction to tandem MS systems 41

Chapter 5 Challenges and development of LCMS 53