Lcms) Milestones for Liquid Chromatography and Mass Spectrometry

C10G-E065A

Fundamental Guide to Liquid Chromatography Mass Spectrometry (LCMS) MILESTONES FOR LIQUID CHROMATOGRAPHY AND MASS SPECTROMETRY

Shimadzu Corporation focus on analytical and measuring instruments, Genzo Shimadzu Sr. founds medical systems, aircraft equipment and industrial machinery. Together with 1875 our partners around the world, we diligently strive to be a company that Shimadzu Corporation 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 © Shimadzu Corporation 2010 Delivers superior performance across a wider First Edition: October, 2018 Minimized cross talk application range with flexibility and reliability LCMS-8030 with UFsweeper™ Updated on: March, 2019 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 For Research Use Only. Not for use in diagnostic procedures. Achieved UFswitching™, Next-level method transfer, minimized environmental impact and easy maintenance UFscanning™ and UF-MRM™ quantitative Provides high resolution This publication may contain references to products that are not available in your country. Please contact us to check the detection with morphological imaging overlaid availability of these products in your country. UF-Qarray™ with molecular distribution images

The content of this publication shall not be reproduced, altered or sold for any commercial purpose without the written approval of Unified Chromatography (UC) 2015 UHPLC/SFC Switching System Shimadzu. LCMS-8060 World's 1st fully automated system that combines Company names, products/service names and logos used in this publication are trademarks and trade names of Shimadzu Corporation, its on-line SFE and SFC in a single flow path. subsidiaries or its affiliates, whether or not they are used with trademark symbol “TM” or “®”. Third-party trademarks and trade names may be used in this publication to refer to either the entities or their products/services, whether World’s 1st Automated LCMS or not they are used with trademark symbol “TM” or “®”. LCMS-8045 2016 Module for Clinical Applications CLAM-2000 Shimadzu disclaims any proprietary interest in trademarks and trade names other than its own. Cutting-edge UFsweeperIII™, DPiMS-2020 Quick and easy MS analysis The information contained herein is provided to you “as is” without warranty of any kind including without limitation warranties as to UF-grating™ and UF-FlightTube™ without sample preparation its accuracy or completeness. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating allows high-speed data acquisition to the use of this publication. This publication is based upon the information available to Shimadzu on or before the date of publication, MALDI-8020 and subject to change without notice. for high-throughput analysis LCMS-9030 Nexera Mikros Quadruopole Time-of-Flight 2018 With the added UF-Link, this low flow LCMS system Liquid Chromatography Mass Spectrometer improves operability and enables high-sensitivity MILESTONES FOR LIQUID CHROMATOGRAPHY AND MASS SPECTROMETRY

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

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 maintenance UFscanning™ and UF-MRM™ quantitative Provides high resolution detection with morphological imaging overlaid UF-Qarray™ with molecular distribution images

Uniﬁed Chromatography (UC) 2015 UHPLC/SFC Switching System LCMS-8060 World's 1st fully automated system that combines on-line SFE and SFC in a single ﬂow path.

World’s 1st Automated LCMS LCMS-8045 2016 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 Nexera Mikros Quadruopole Time-of-Flight 2018 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 he first chapter of Shimadzu’s Fundamental Guide to LCMS introduces the basic principles of LC and MS. Common LC techniques, namely Treversed-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 1900s 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

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5 Chapter 1: Introduction to LCMS

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 (RPLC) phase is used for RPLC. Based on the ‘like attracts (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, (NPLC) polar stationary phase and a non-polar mobile phase 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 Chromatography (HILIC) main difference is that water is added in the organic mobile phase to effectively separate and elute the strongly-retained polar molecules.

Ion Exchange IEC retains and separates charged species (ions) Proteins, amino acids, Chromatography based on electrostatic affinity of the analyte for the 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 Chromatography When sample of various sizes flow into the column, 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 Chapter 1: Introduction to LCMS

Detectors for LC Systems Intensity Chromatogram Mass

Chromatogram 0

Mass Retention Time m/z Spectrometry

Mass Spectrum

50 100 150 200 250 300 350 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,

Sensitivity refractive index, evaporative light scattering 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 optical detectors. LC coupled with optical Evaporative 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 Chapter 1: Introduction to LCMS

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mAU mAU 400 30 2 350 Standard Mixture Cream Sample 25 3 300 4 1 20 250

200 15 6 5 7 150 8 910 10 12 100 5 11 8 5 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 Chapter 1: Introduction to LCMS

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 Chapter 1: Introduction to LCMS

(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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10 Chapter 1: Introduction to LCMS Chapter 1: Introduction to LCMS

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 • Textile Testing • Protein and Glycan • Chiral Drugs Analysis • Industrial Discharge Analysis • 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 hapter 2 introduces the detailed configuration of the LCMS instrumentation. It generally consists of a LC separating system, Ca 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 Autosampler Mass Analyzers

Column (Vacuum) 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 Chapter 2: Integrating LC and MS

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 Chapter 2: Integrating LC and MS

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

80 256

(A) EI Mass Spectrum 60 171

Relative Intensity 156 61

20 285 116 185 74 77 91 103 198 130 143 227 270 345 360 301 329 333 341

0 50 100 150 200 250 300 350 m/z

120

OH 377 100 [M+H]+ OH Riboflavin (Vitamin B ) OH 80 2

Molecular Formula: C17H20N4O6 OH 60 + (B) ESI Mass Spectrum Formula Weight: 376.3639 NN O [M+Na]

40 Relative Intensity 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 Chapter 2: Integrating LC and MS

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 andliquid 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 Chapter 2: Integrating LC and MS

Sample & mobile phase I I I

Nebulizer gas+ Nebulizer gas I + + + +

I High Voltage + I +

+ + I I I +

+ +

I + + +ve -ve I + + I + + +

ESI Probe Tip + + + + + + + + + + + + + + + + + + + + + + + + +

Charged + + + + + +

Droplets + + + + + + + + + +

+ Drying gas + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +

+ Gas phase Ions + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +

Desolvation + + + + +

+ + +

+ + + + + + + + + + + + + +

+ +

+ + + + +

+ + + + + + + + + + +

+ + + + (Ion Evaporation + + + + + + ++ + + + + +

Model) + + + ++ + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +

+ 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 Chapter 2: Integrating LC and MS

120

1131.1 100 1060.5 998.2 1211.8 1305.0 80 942.8

1413.6 60 893.2

40 Relative Intensity 1542.0 848.7

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 are the 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 Chapter 2: Integrating LC and MS

Sample & mobile phase

Nebulizer gas Nebulizer gas

Heater

Drying gas

Desolvation Gas phase Ions + + + + + + + + + +

+ +

+ + + + +

+ +

+ + + + + + +

+ + + + + + + + + + + + + + + + + +

+ Ion source to + + + + + mass spectrometer + +

High-voltage Corona Needle + Analyte Solvent + Analyte + + Solvent Molecule Ion Ion

Proton Transfer 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 I I I + I I + + : Anion I + I + M

M m + + I I I + m I m I I

Vacuum + I + + + I I + I + I

+ M I

I m + + I

m I +

I M

m m I + I I + I +

Sample Slide Sample Slide Sample matrix mixture Sample matrix mixture before laser irradiation after laser irradiation 19 Chapter 2: Integrating LC and MS

Sample & mobile phase

Nebulizer gas Nebulizer gas

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 Chapter 2: Integrating LC and MS

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 Chapter 2: Integrating LC and MS

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 + Solvent Charged Ion M+

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

22 Chapter 2: Integrating LC and MS

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 Chapter 2: Integrating LC and MS

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 Chapter 2: Integrating LC and MS

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

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? TMany 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 20Ne 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 Chapter 3: Principles of MS

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 ions to be introduced only at a specific time Magnetic sector, a continuous MS, point. In a pulsed MS, ions from a continuous has been used historically the longest. As flow are usually accumulated and introduced the name implies, the mass analyzer uses together in pulses. magnetic field to separate ions of different Apart from a single MS, there are m/z values (Figure 13). High voltage is first tandem/hybrid arrangements, also known applied to the ions to accelerate them into as MS/MS systems. Single mass analyzers the magnetic sector. A continuous ion such as the magnetic sector, quadrupole, source is generated and supplied from the and time-of-flight (TOF) were commonly ionization unit to the magnetic sector. Once used for measuring organic compounds, but the ions enter, are exposed to the magnetic the quadrupole model has gradually been field. As a result, ions are deflected according 4 increasing their share due to its relatively to Fleming’s left-hand rule . The deflections 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 types of MS systems each take advantage magnetic field, resulting in a curved deflection path for the ions in the magnetic sector MS. of their respective features and are used Magnetic Sector Ion source Ion accelerators ) Centrifugal(f 2 force

) Acceleration Lorentz(f 1force Voltage

Detector Figure 13. Schematic of magnetic sector MS. 28 Chapter 3: Principles of MS

Equation 3: Ions experience a Lorentz force (f1) from the magnetic field that can be calculated according to Equation 3. As the direction f1 = Bzev 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 2 mv field region and reach the detector, it must f = –––– 2 r travel along a curved path of a given radius

(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), f = f = Bzev = –––– 1 2 r Equations 5 and 6 are simplified to give 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 KE = – mv2 = zeV ion 2 same charge). 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 2 2 m eB r the magnetic flux density (B) is scanned. –– = —— z 2V This means that ions with different masses (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 Chapter 3: Principles of MS

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 sector model or several tens of thousands The other MS which functions using a dual-focusing magnetic sector by scanning of ions and allowing ion model. Before the recent introduction of transmission is the single quadrupole mass the high-performance time-of-flight (TOF) analyzer. As its name suggests, it contains MS and ion-cyclotron-resonance (ICR) MS four parallel cylindrical metal rods (electrodes systems, the dual-focusing magnetic sector with a hyperboloidal interior surface) inside spectrometer was the only MS capable of a vacuum chamber, positioned equidistant such high-resolution measurements. from the center axis (Figure 14). Both a However, to improve the performance direct current (D.C.) and high frequency of the magnetic sector MS, the strength alternating current or radiofrequency (RF) of the magnetic field needs to be raised are applied to the quadrupole, so that only which means costly and larger systems are the ions with the target m/z successfully required. This limits the development of the pass through the quadrupole and get to the magnetic sector MS. In addition, magnetic detector. The quantity of ions that reach the sector MS systems require an extremely high detector is converted to a signal and output 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 Chapter 3: Principles of MS

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 is applied to adjacent poles as depicted by –– = K ——2 2 z r ω 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 thex- and y- ω: oscillation frequency directions. When a given set of parameters are applied to the poles, certain ions of

Direct current voltage a specific m/z range maintain a stable applied to electrode (U) Straight oscillation and pass through the quadrupole Scan Line (1) Ion Mass: m1 < m2 < m3 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, m Unstable 2 Stable Region for m Region causing them to collide with the poles, fly 3 m1 out of the system, and not be detected AB 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 Chapter 3: Principles of MS

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 Chapter 3: Principles of MS

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 a general-purpose analytical instrument. Unlike magnetic sector and quadrupole Furthermore, unlike other MS which require MS, Time-of-Flight (TOF) MS is a pulsed high vacuum levels, quadrupole MS can and non-scanning MS. It has a simple adequately function at lower vacuum levels construction, consisting of an accelerator, a (≈ 10-2 to 10-3 Pa). Even if they are interfaced field-free region, a reflectron and detector with a GC or LC unit, the drop in vacuum inside a high vacuum chamber called a flight level caused by the interface has minimal tube (Figure 17). effect on the mass separation performance, TOF MS separates and detects ions of making it the best suited for interfacing with different m/z by measuring the time taken for chromatographic techniques. the ions to travel through a field-free region. Quadrupole MS demonstrates good First, ions generated in an ionization unit are scan speed and sensitivity. With a maximum accumulated and introduced in pulses to a scan speed of 15,000 amu/second, it is flight tube. These ions are accelerated by capable of measuring at higher scan speeds applying a high acceleration voltage between than the magnetic sector MS. Its mass range the electrodes. The corresponding kinetic can reach up to 2,000 m/z which enables energy is obtained as described in Equation the qualitative analysis in a practical range of 9. Given a constant acceleration voltage molecular masses. In addition, it allows high- as well as kinetic energy, each ion flies at speed polarity switching, which facilitates its unique velocity inside the flight tube to simultaneous monitoring of multiple selected reach the ion detector, which is higher for ions of different polarity. With the use of the ions with smaller masses and lower for ions with larger masses.

33 C146-E119B

LCMS-2020 Liquid Chromatograph Mass Spectrometer

Seeing is Believing.

UFscanning 15,000 u/sec high-speed scanning

UFswitching 15 milliseconds high-speed polarity ionization switching time

UFsensitivity High-speed analysis with high sensitivity UFscanning & UFswitching

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

UFsensitivity

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.

Hardware features that powerfully support 3 types of UF functionality

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%.

Company names, product/service names and logos used in this publication are trademarks and trade names of Shimadzu Corporation and its affiliates, whether or not they are used with trademark symbol “TM” or “®”. Third-party trademarks and trade names may be used in this publication to refer to either the entities or their products/services. Shimadzu disclaims any proprietary interest in trademarks and trade names other than its own.

For Research Use Only. Not for use in diagnostic procedures. 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. www.shimadzu.com/an/ © Shimadzu Corporation, 2012 Printed in Japan 3295-11103-30ANS Chapter 3: Principles of MS

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 KE = – mv2 = zeV ion 2 smaller m/z reach the detector sooner than 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 Chapter 3: Principles of MS

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 Chapter 3: Principles of MS

(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 Chapter 3: Principles of MS

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 Chapter 3: Principles of MS

CLICK HERE 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

hapter 4 introduces the core concept and key features of a MS/MS system. It includes the basic instrumentation, the collision-induced Cdissociation 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 6Tandem MS refers to MS/MS systems that use structural information of the target analytes the same mass analyzers (e.g. triple Quadrupole MS/MS) is required. while hybrid MS refers to MS/MS systems with different MS 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 Chapter 4: Introduction to tandem MS systems

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.

43 313 ESI - MS (CID 0 Volts)

50 100 150 200 250 300 m/z

225 313 313 ESI - MS2 ESI - MS (CID 10 Volts) (CID 0 Volts) 208

166

243 296

50 100 150 200 250 300 m/z 50 100 150 200 250 300 m/z 313 ESI - MS 166 313 225 313 ESI - MS2 ESI - MS ESI - MS2 (CID 0 Volts) (CID 20 Volts) (CID 0 Volts) (CID 10 Volts)

208

Chapter 4: Introduction to tandem MS systems166

120 208 243 296 136 94 179 225

166 50 100 150 200 250 300 m/z 225 50 100 150 200 250 300 m/z 50 100 150 200 250 166 > 123000 m/z 50 100 150 200 250 300 m/z313 225 313 H ESI - MS ESI - MS2 225 225 > 179 313 120 O 166 2 ESI - MS2 (CIESID - 0MS Volts)2 (CID 10 Volts) ESI - MS 243 O (CID 10 Volts) (CID 20 Volts) 94 (CID 30 Volts) O 166 208 208 166 166 > 136 HN 166 H

137 120 243 225 > 208 NH 296 2 109 O 296 243 208 179 > 136 296 225 > 208 136 161 94 179 225 67 Osteltamivir (Tamiflu™) 50 100 150 200 250 300 m/z 50 100 150 200 250 300 m/z CLICK HERE 50 100 150 200 250 300 m/z 225 313 50 100 150 200 250 300 m/z 50 100 2 150 200 250 300 m/z 166 ESI - MS ESI - MS2 166 2 (CID 10 Volts)120 65 Structure ESI - MS ESI - MS2 (CID 20 Volts) ESI - MS2 (CID 20 Volts) 94 208 (CID 30 Volts) (CID >50 Volts) 166 elucidation of 166 pharmaceuticals 120 243 208 using ultrafast 120 137 296 77 92 208 136 109 94 179 225 94 136 179 120 triple quadrupole 225 161 50 67 100 150 200 250 300 m/z 50 100 150 200 250 300 m/z

50 100 150 200 250 300 m/z 166 50 100 150 200 250 300 m/z MS. 50 100 2 150 200 250 300 m/z 120 ESI - MS ESI - MS2 120 2 65(CI D 20 Volts) ESI - MS 2 94 (CID 30 Volts) ESI - MS 166 94 (CID 30 Volts) Figure 21. CID166 of Osteltamivir (Tamiflu) at various (CIcollisionD >50 Volts) energies (0, 10, 20, 30, >50 V).

120 137 208 137 109 77 9294 136 179 109 225 67 161 16Figure1 16 describes the scan and historically preferred by IUPAC, while 67 120 50 100 150 200 250 300 m/z 50 100 150 200 250 300 m/z

50 100 150 200 250 300 m/z 120 selected ion monitoring50 100 (SIM)150 modes200 ESI 250- MS 2 300of m/z a 65 instrument vendors including Shimadzu ESI - MS2 65 94 (CID 30 Volts) 2 ESI - MS 166 (CID >50 Volts) quadrupole(CID MS. >50 Volts) Likewise in MS/MS, the MS1 preferred the alternative term, Multiple and MS2 can either acquire the scan or SIM Reaction Monitoring (MRM). Now IUPAC 137 77 92 109 77 92 (fixed) mode. The different161 arrangements recommends that the term SRM be used to 67 120

120 50 100 150 200 250 300 m/z allow the system to operate at various50 describe100 150 200the 250technique 300 m/z applied to a single 50 100 150 200 250 300 m/z 65 ESI - MS2 scan and monitoring modes (Figure(CID >50 22). Volts) It target and MRM for plurality of SRM. In is important to note that the actual scan this document hereafter, the technique is

and monitoring modes77 92available in MS/MS referred to as MRM regardless of the number systems depends on the mass120 analyzers (MS1 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 CLICK HERE analysis. Find out about how precursor ion scan, 7Time-based MS refers to ion trap and FT-ICR MS, 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 Chapter 4: Introduction to tandem MS systems

(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

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 Chapter 4: Introduction to tandem MS systems

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 Chapter 4: Introduction to tandem MS systems Chapter 4: Introduction to tandem MS systems

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

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 3.0 2 1. Norepinephrine catecholamines, it is difcult to evaluate the LLOQ in plasma 2. Epinephrine Liquid Chromatograph Mass Spectrometer 3. Dopamine Neat standard curve Matrix-matched matrix. Here we used deuterated catecholamine compounds Standard Compound name Range Linearity Range Linearity as standards to estimate the LLOQ in plasma matrix, rather 2.0 Deuterated (pg/mL) (r2) (pg/mL) (r2) 3 Norepinephrine-d6 than as internal standards for quantitation. 2.5 – 2000 0.9999 2.5 – 2000 0.9997 LCMS-8060 1 (158.1 > 111.1) 1.0 A neat standard curve was prepared by serial dilution in 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 5 – 2000 0.9999 10 – 2000 0.9995 (158.1 > 95.1) 1.0 2.0 3.0 4.0min 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-d6Dopamine Dopamine-d4 ×104 ×104 ×105 ×105 ×105 ×105 In the actual quantitation assay, 2.0 2.0 5.0 5.0 300 pg/mL 7.5 500 pg/mL 7.5 500 pg/mL 500 pg/mL deuterated catecholamines are spiked spiked 1.5 1.5 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 2.5 2.5 spiked blank 2.5 LCMS-8060. The gures on the right 2.5 0.5 0.5 blank blank blank blank show the MRM chromatograms of 0.0 0.0 0.0 0.0 0.0 0.0 spiked and endogenous 0.52.5 min 0.52.5 min 1.02.5 min1.0 2.5min 2.55.0 min2.5 5.0min catecholamines in plasma. Detection of Norepinephrine, Epinephrine and Dopamine and their deuterated internal standards in plasma.

×104 7.0 1.4 100 pg/mL Alprazolam in human plasma with internal standard Outstanding Durability 6.0 m/z 309.05 > 281.10 1.2 A week 1st day 2nd day 3rd day 4th day 5th day 6th day The robustness of the LCMS-8060 and modied ion 5.0 1.0

Area Ratio optics were assessed by injecting 2400 samples of 4.0 0.8 femto-gram levels of alprazolam spiked into 3.0 Area Ratio 3.46 %RSD 0.6 Peak Area protein-precipitated human plasma extracts over a 6 2.0 0.4 Peak Area 4.97 %RSD day period (over 400 samples were injected each day). 1.0 Vacuum 0.2 Pause in batch Pause in batch system Pause in batch Pause in batch analysis analysis vented analysis analysis The RSD of peak area response was 5% over this test 0.0 0.0 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 (×10,000) (×10,000) (×10,000) robustness test the vacuum system was vented to 1.00 1st injection 1.00 1200th 1.00 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

The LCMS-8060 is the latest in Shimadzu’s Ultra-Fast Mass Spectrometry product line, designed to deliver you the highest 0.00 0.00 0.00 sensitivity and fastest analysis speed of any LCMS on the market today. 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 Sensitivity Highest Sensitivity 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 A newly developed UF-Qarray boosts ion intensity but suppresses Alprazolam 5.04 4.94 5.06 5.38 4.55 4.83 3.19 1.63 2.74 Heated ESI Probe noise. By improving the ion sampling device, the ion guide, and Alprazolam-d5 (ISTD) 5.04 4.68 5.48 5.31 4.26 4.91 2.62 1.89 2.18 vacuum ef ciency, Shimadzu has achieved an unprecedented Area ratio 3.48 3.11 3.48 3.44 3.71 3.54 1.79 0.26 1.40 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 For Research Use Only. Not for use in diagnostic procedures. This publication may contain references to products that are not available in your country. Please contact us to check the availability of these MRM channels per second, ultra-fast polarity switching, and ultra-fast products in your country. Solutions Company names, products/service names and logos used in this publication are trademarks and trade names of Shimadzu Corporation, its scanning, all with high data quality. Fusion of sensitivity and speed subsidiaries or its afliates, whether or not they are used with trademark symbol “TM” or “®”. 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 UFscanning: Max. 30,000 u/sec they are used with trademark symbol “TM” or “®”. Don’t miss an exciting performance results in next page!! Shimadzu disclaims any proprietary interest in trademarks and trade names other than its own. UFswitching: 5 msec 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 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 3.0 2 1. Norepinephrine catecholamines, it is difcult to evaluate the LLOQ in plasma 2. Epinephrine 3. Dopamine Neat standard curve Matrix-matched matrix. Here we used deuterated catecholamine compounds Standard Compound name Range Linearity Range Linearity as standards to estimate the LLOQ in plasma matrix, rather 2.0 Deuterated (pg/mL) (r2) (pg/mL) (r2) 3 Norepinephrine-d6 than as internal standards for quantitation. 2.5 – 2000 0.9999 2.5 – 2000 0.9997 1 (158.1 > 111.1) 1.0 A neat standard curve was prepared by serial dilution in 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 5 – 2000 0.9999 10 – 2000 0.9995 (158.1 > 95.1) 1.0 2.0 3.0 4.0min 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-d6Dopamine Dopamine-d4 ×104 ×104 ×105 ×105 ×105 ×105 In the actual quantitation assay, 2.0 2.0 5.0 5.0 300 pg/mL 7.5 500 pg/mL 7.5 500 pg/mL 500 pg/mL deuterated catecholamines are spiked spiked 1.5 1.5 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 2.5 2.5 spiked blank 2.5 LCMS-8060. The gures on the right 2.5 0.5 0.5 blank blank blank blank show the MRM chromatograms of 0.0 0.0 0.0 0.0 0.0 0.0 spiked and endogenous 0.52.5 min 0.52.5 min 1.02.5 min1.0 2.5min 2.55.0 min2.5 5.0min catecholamines in plasma. Detection of Norepinephrine, Epinephrine and Dopamine and their deuterated internal standards in plasma.

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

For Research Use Only. Not for use in diagnostic procedures. 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. Company names, products/service names and logos used in this publication are trademarks and trade names of Shimadzu Corporation, its subsidiaries or its afliates, whether or not they are used with trademark symbol “TM” or “®”. 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 “®”. Shimadzu disclaims any proprietary interest in trademarks and trade names other than its own. 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.

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 n system. The parameters in the ion trap are The IT-TOF MS system provides MS 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 Chapter 4: Introduction to tandem MS systems

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 372.0667 491.0042 165.0410 199.1703 431.0352 0.0 273.0254 328.9901 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 0.5 363.9534 200.9846 285.9549 249.9785 328.9700 0.0 103.2363 160.9496 spectrum library of 50 100 150 200 250 300 350 400 450 m/z endocrine disruptors Inten. (x100,000)

3 by LCMS-IT-TOF 3.0 285.9537 MS spectrum

2.0 249.9762 1.0 277.9719 0.0 67.0836 136.0645 189.9831 225.9813 50 100 150 200 250 300 350 400 450 m/z

MSn Spectra for Tetrachlorobisphenol A

51 Chapter 4: Introduction to tandem MS systems

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

n this final chapter, we discussed the needs of industries today and the current challenges of analytical LCMS, with special emphasis on the Ifactors 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 Chapter 5: Challenges and development of LCMS

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 c) To reduce unnecessary matrix effects Crosstalk may occur during MRM analysis where the product ions of the previous MRM transition (residual (unwanted suppressions and product ions in the collision cell) is erroneously detected as enhancement) product ions of the next MRM measurement. 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

Uni ed Chromatography

Winner of 2015 R&D100 Award

55 Chapter 5: Challenges and development of LCMS

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 9Measurement 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 Chapter 5: Challenges and development of LCMS Chapter 5: Challenges and development of LCMS

End of previous MRM

Pause time Dwell time Conventional design Ions lose momentum due to collision with gas. Ion count (CPS)

Time

shorter pause time & dwell time

Pause Dwell time UFsweeper™ time UFsweeper efﬁciently More time for accelerates ions out of the measuring other compounds collision cell without losing momentum. Ion count (CPS)

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 10Separate 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”.

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Quantitative Analysis of 646 Pesticides (1,919 MRMs) by LC-MS/ MS with a Fast 10.5 minute Cycle Time

57 Chapter 5: Challenges and development of LCMS

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 Chapter 5: Challenges and development of LCMS Chapter 5: Challenges and development of LCMS

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

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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 Chapter 5: Challenges and development of LCMS

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 Chapter 5: Challenges and development of LCMS

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

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