High-Throughput Mass Spectrometry in Drug Discovery E-Book
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- Herausgeber: John Wiley & Sons
- Kategorie: Wissenschaft und neue Technologien
- Sprache: Englisch
High-Throughput Mass Spectrometry in Drug Discovery Apply mass spectrometry to every phase of new drug discovery with this cutting-edge guide Mass spectrometry is a technique that identifies and characterizes compounds based on their mass -- the fundamental molecular characteristic. It has become an invaluable analytical tool in various disciplines, industries, and research fields. It has become particularly central to new drug discovery and development, which broadly deploys mass spectrometry at every phase. The pharmaceutical industry has become one of the main drivers of technological development in mass spectrometry. High-Throughput Mass Spectrometry in Drug Discovery offers a comprehensive introduction to mass spectrometry and its applications in pharmaceutical discovery. It covers the foundational principles and science of mass spectrometry before moving to specific experimental methods and their applications at various stages of drug discovery. Its thorough treatment and detailed guidance make it an invaluable tool for pharmaceutical research and development. High-Throughput Mass Spectrometry in Drug Discovery readers will also find: * Detailed analysis of techniques, including label-free screening, synthetic reaction optimization, and more * An authorial team with extensive combined experience in research and industrial applications * Technical strategies with the potential to accelerate quantitative bioanalysis in drug discovery High-Throughput Mass Spectrometry in Drug Discovery is essential for analytical, bioanalytical, and medicinal chemists working in the pharmaceutical industry and for any researchers and graduate students interested in drug discovery and development.
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Table of Contents
Cover
Title Page
Copyright Page
List of Contributors
Preface
List of Abbreviations
Section 1: Introduction
1 Forty‐Year Evolution of High‐Throughput Mass Spectrometry
1.1 Introduction
1.2 Ionization Foundations of High‐Throughput Mass Spectrometry
1.3 High‐Speed Serial Chromatographic Sample Introduction
1.4 Parallel Chromatographic Sample Introduction
1.5 High Repetition Rate Lasers
1.6 Ion Mobility for High‐Speed Gas‐Phase Separations
1.7 Mass Spectrometer Sensitivity
1.8 High‐Speed Sub‐Microliter Volume Sampling
1.9 Conclusions and Future Prospects
References
Section 2: LC‐MS
2 The LeadSampler (LS‐1) Sample Delivery System
2.1 Introduction
2.2 Hardware and System Design
2.3 Software Integration
2.4 Enabling Emerging Techniques
2.5 Concluding Remarks
References
3 Evolution of Multiplexing Technology for High‐Throughput LC/MS Analyses
3.1 Introduction and Historical Developments
3.2 Developments Toward Fully Integrated Multiplexing Systems
3.3 Broadening Customer Options
3.4 Workflow and End‐User Considerations
3.5 Conclusion
References
Section 3: ESI‐MS Without Chromatographic Separation
4 Direct Online SPE‐MS for High‐Throughput Analysis in Drug Discovery
4.1 Introduction
4.2 History of the Development of Direct Online SPE‐MS
4.3 Hardware Details and Data Processing
4.4 Instrument Performance Highlights
4.5 Applications
4.6 Others
4.7 Future Perspectives
References
5 Acoustic Sampling for Mass Spectrometry
5.1 Introduction
5.2 Technology Overview
5.3 System Performance
5.4 Applications
5.5 Challenges and Limitations
5.6 Conclusion
References
6 Ion Mobility Spectrometry‐Mass Spectrometry for High‐Throughput Analysis
6.1 Introduction of Ion Mobility Spectrometry
6.2 IMS Fundamental and Experiment
6.3 IMS Analysis and Applications
6.4 High‐Resolution SLIM‐IMS Developments
6.5 Conclusions
References
7 Differential Mobility Spectrometry and Its Application to High‐Throughput Analysis
7.1 Introduction
7.2 Separation Speed
7.3 Separation Selectivity
7.4 Ultrahigh‐Throughput System with DMS
7.5 Conclusions
7.A Chemical Structures
References
Section 4: Special Sample Arrangement
8 Off‐Line Affinity Selection Mass Spectrometry and Its Application in Lead Discovery
8.1 Introduction to Off‐Line Affinity Selection Mass Spectrometry
8.2 Selected Off‐Line Affinity Selection Technologies and Its Application in Lead Discovery
8.3 Future Perspectives
References
9 Online Affinity Selection Mass Spectrometry
9.1 Introduction of Online Affinity Selection‐Mass Spectrometry
9.2 Online ASMS Fundamental
9.3 Instrument Hardware and Software Consideration
9.4 Type of Assays Using ASMS
9.5 Applications Examples and New Modalities of ASMS for Drug Discovery
9.6 Future Perspectives
References
10 Native Mass Spectrometry in Drug Discovery and Development
10.1 Introduction
10.2 Fundamentals of Native MS
10.3 Instrumentation
10.4 Application Highlights
10.5 Conclusions and Future Directions
References
Section 5: Other Ambient Ionization Other than ESI
11 Laser Diode Thermal Desorption‐Mass Spectrometry (LDTD‐MS)
11.1 A Historical Perspective of the LDTD
11.2 Instrumentation
11.3 Theoretical Background
11.4 Sample Preparation
11.5 Applications
11.6 Conclusion
References
12 Accelerating Drug Discovery with Ultrahigh‐Throughput MALDI‐TOF MS
12.1 Introduction
12.2 uHT‐MALDI MS of Assays and Chemical Reactions
12.3 Bead‐Based Workflows
12.4 Using Functionalized, Modified, and Microarrayed MALDI Plates for HT‐MALDI
12.5 Summary and Future Trends
Acknowledgment
References
13 Development and Applications of DESI‐MS in Drug Discovery
13.1 Introduction
13.2 Development of DESI and Related Ambient Ionization Methods
13.3 Applications in Drug Discovery
13.4 Conclusions and Future Outlook
References
Section 6: Conclusion
14 The Impact of HT‐MS to Date and Its Potential to Shape the Future of Metrics‐Based Experimentation and Analysis
14.1 Defining High‐Throughput Mass Spectrometry (HT‐MS)
14.2 HT‐MS: Impact to Date
14.3 Considering How HT‐MS Will Shape the Future of Drug Discovery
References
Index
End User License Agreement
List of Tables
Chapter 2
Table 2.1 Volumes associated with system components and plumbing.
Table 2.2 Volumes of flow path components identified in Panel B.
Table 2.3 Resource savings and operational efficiency resulting from reconf...
Table 2.4 Summary of in vitro pharmacokinetic screens, associated methodolo...
Chapter 7
Table 7.1 Ratio of signal intensity for standards of mirtazapine to the sam...
Table 7.2 Comparison of peak areas for a panel of compounds with a DMS cell...
Chapter 12
Table 12.1 Examples of enzymatic assays, chemical reactions, and cell‐based...
List of Illustrations
Chapter 1
Figure 1.1 Histomap of LC/MS interfaces from 1967 to present and some emergi...
Figure 1.2 The evolution of electrospray LC/MS interfaces. (a) The ion evapo...
Figure 1.3 First demonstration of the feasibility of HT‐MS using the heated ...
Figure 1.4 Images and applications of the first ambient sampling (aka ambien...
Figure 1.5 Images and applications of the second ambient sampling (aka ambie...
Figure 1.6 High flow rate ESI ion source operating at 800 °C. Evaporative co...
Figure 1.7 Fast serial, staggered chromatography systems and autosamplers to...
Figure 1.8 MRM ion current trace using a monolithic stationary phase casted ...
Figure 1.9 Illustration of the four positions where signal can be toggled on...
Figure 1.10 Two examples of indexing systems prototyped and tested. (a) Flui...
Figure 1.11 Ion production indexing. (a) Photograph of the Medusa ion source...
Figure 1.12 Components of the multichannel autosampler (Parker‐Hannifin) and...
Figure 1.13 The prototype MALDI ion source adapted to a triple quadrupole in...
Figure 1.14 Example of the improvement in quantitative data when using a hig...
Figure 1.15 Illustrations of a differential ion mobility analyzer coupled to...
Figure 1.16 Ionograms created by scanning the CV voltage during a continuous...
Figure 1.17 Graph of the incremental gains in the sensitivity of commercial ...
Figure 1.18 Data from three compounds of varying ionization efficiency using...
Figure 1.19 (a) Diagram of a prototype Acoustic Ejection Mass Spectrometry s...
Figure 1.20 (a) Axial cross‐section of the OPI interface operating under con...
Figure 1.21 (a) Sonogram of 1536 samples dispensed as one 5 nL drop per samp...
Figure 1.22 (a) Data illustrating limits of quantitation and suppression eff...
Figure 1.23 Different modes of operating AEMS. (a) Data illustrating the abi...
Figure 1.24 (a) Chronograms illustrating the multiplexing method to increase...
Figure 1.25 Chronogram using an OPI interface modified to obtain maximum spe...
Chapter 2
Figure 2.1 Drug discovery LC‐MS‐based screening pathways. Although project t...
Figure 2.2 Prototype LeadSampler platform. (a) System overview. 1. Cavro
®
...
Figure 2.3 The current LeadSampler platform. The Sciex 6500 mass spectromete...
Figure 2.4 (a) LC trace with “standard” configuration: HALO C18 direct‐conne...
Figure 2.5 Bioanalytical data from high‐throughput in vitro pharmacokinetic ...
Figure 2.6 Flow of information through key touchpoints in the LeadScape bioa...
Figure 2.7 LeadScape Top‐level interface. Working from left to right the use...
Figure 2.8 The user can import a text file list of compounds and query the d...
Figure 2.9 Import text file with positions. Users can customize import and e...
Figure 2.10 LeadScape user interfaces for configuring input and data formatt...
Figure 2.11 LeadScape queue displays status of past, current, and future sub...
Figure 2.12 Comparison of sample‐consolidation strategies for enhancing oper...
Figure 2.13 LeadSampler system optimized specifically for micro‐flow LC‐MS/M...
Figure 2.14 Impact of post‐column system volume on microflow LC‐MS/MS chroma...
Chapter 3
Figure 3.1 Schematic representation of the general multiplexing process for ...
Chapter 4
Figure 4.1 Direct online SPE with a dual‐cartridge configuration, as first r...
Figure 4.2 RapidFire instrument (RapidFire 365) picture.
Figure 4.3 Valve positions and flow paths during RapidFire operation: (a) as...
Figure 4.4 Typical RapidFire‐MS data from a transporter inhibition assay, wi...
Chapter 5
Figure 5.1 (a) A – Source block of Waters SQDII, B – capillary transfer line...
Figure 5.2 (a) A – Echo 555 transducer, B – heated transfer capillary, C – c...
Figure 5.3 (a) Schematic of early AMI‐MS charging system. The resistive glas...
Figure 5.4 (a) Acoustic mist ionization (AMI) hardware used for high‐through...
Figure 5.5 CE‐compliant version of AMI‐MS system.
Figure 5.6 Diagram of an ADE‐OPI‐MS system. (A) Acoustic transducer. (B) Dro...
Figure 5.7 Well‐to‐well variability assessment for a 384‐well plate containi...
Figure 5.8 (a) Linearity of signal from example biomolecular analytes of int...
Figure 5.9 (a) TIC generated by AMI‐MS from 384‐well plates. Each packet of ...
Figure 5.10 Peptide and protein spectra from AMI‐MS. (a) Multiple‐charged sp...
Figure 5.11 HDAC inhibition screen. (a) Overview of the HDAC assay. (b) Exam...
Figure 5.12 HTS output for HDAC screening campaign. (a) Robust Z
′
fact...
Figure 5.13 A representation of the original data analysis workflow for AMI‐...
Figure 5.14 The application of the ADE‐OPI‐MS system for the DGAT2 screening...
Figure 5.15 Examples of data generated to support assay development using AM...
Figure 5.16 The application of the ADE‐OPI‐MS system for in situ kinetics st...
Figure 5.17 Use of metal ion chelator to identify zinc contamination in comp...
Figure 5.18 AMI‐MS spectra from cell lysates. (a) Spectrum from MCF7 cell ly...
Chapter 6
Figure 6.1 Schematics of a typical IMS‐MS instrument and conformation separa...
Figure 6.2 Example of an arrival time,
t
A
, versus
P
/
V
plot to determine the ...
Figure 6.3 Examples of IMS separation of isomeric species separation (a) and...
Figure 6.4
DT
CCS
N2
database for small molecules: (a) CCS values of small mol...
Figure 6.5 IM‐MS conformational space plots showing the regions occupied by ...
Figure 6.6 Coupling solid‐phase extraction, ion mobility spectrometry, and m...
Figure 6.7 (a) A schematic and photograph of the serpentine 13‐m SLIM IMS‐MS...
Figure 6.8 2D IMS‐MS plot of the reduced antibody–drug conjugate after a 4.5...
Chapter 7
Figure 7.1 Orthogonality of DMS separations versus
m/z
for three different c...
Figure 7.2 Q1 scans for chlordiazepoxide and temazepam.
Figure 7.3 (a) LC separation of 1. chlordiazepoxide and 2. temazepam. (b) MR...
Figure 7.4 Separation of the structural isomers: 1. morphine, 2. hydromorpho...
Figure 7.5 Separation of the structural isomers: 1. oxymorphone, 2. dihydroc...
Figure 7.6 Mirtazapine FIA signal taken with 5 μL injection for (a) standard...
Figure 7.7 Zoom of AEMS/DMS/MS data for Mirtazapine samples prepared in urin...
Figure 7.8 Analytical signal with increased injection volumes using an AEMS ...
Figure 7.9 Suppression effects for samples prepared in urine versus desaltin...
Figure 7.10 Schematic of an AEMS system with DMS system.
Figure 7.11 Coefficient of variation (CV) versus points across a peak for pe...
Figure 7.12 Raw data for four replicate AEMS injections using instrument con...
Figure 7.13 Switching between pseudo‐continuous signal mode and pulsed mode....
Figure 7.14 Comparison of separations for clozapine and midazolam using tee ...
Figure 7.15 Replicate measurements for reserpine injections with a DMS insta...
Figure 7.16 Zoomed view of the plot of reserpine signal intensity with a DMS...
Figure 7.17 Replicate measurements for minoxidil injections with a DMS insta...
Figure 7.18 Zoomed view of the plot of minoxidil signal intensity with a DMS...
Figure 7.19 AEMS data for injection of a mirtazapine standard while measurin...
Figure 7.20 AEMS data for injection of a desmethyldoxepin standard while mea...
Figure 7.21 Overlay of DMS ionograms for desmethyldoxepin (gray trace) and m...
Figure 7.22 AEMS data for mirtazapine and desmethyldoxepin with a DMS instal...
Figure 7.23 Calibration curves for mirtazapine in desalted urine. The inject...
Figure 7.24 Calibration curves for desmethyldoxepin in desalted urine. The i...
Figure 7.25 Overlay of Q1 spectra for midazolam (gray trace) and clozapine (...
Figure 7.26 Midazolam and clozapine example showing midazolam injection (a) ...
Figure 7.27 Separation of acetaminophen and phenacetin using (a) LC/MS and (...
Figure 7.28 LC/MS data for injection of a phenacetin sample: (a) MRM signal ...
Figure 7.29 Raw data for injections of fentanyl samples prepared in desalted...
Figure 7.30 Calibration curves for fentanyl from a desalted urine sample acq...
Figure 7.31 Blank injections for norfentanyl from a desalted urine sample wi...
Figure 7.32 Overlay of a series of ionorams for determining the alpha curve ...
Figure 7.33 Alpha curves for amobarbital and pentobarbital.
Figure 7.34 Difference function for the alpha curves for amobarbital and pen...
Figure 7.35 DMS separation for a mixture containing pentobarbital and amobar...
Figure 7.36 Alpha curves for a group of five isobaric and near‐isobaric benz...
Figure 7.37 Alpha example of the separation with the separation field set to...
Chapter 8
Figure 8.1 Schematic diagram of on‐line versus off‐line affinity selection m...
Figure 8.2 Membrane ultrafiltration‐based affinity selection mass spectromet...
Figure 8.3 Pulsed ultrafiltration‐mass spectrometry (PUF‐MS) for screening o...
Figure 8.4 “SpeedScreen sandwich” assembly of three microtiter plates (in ei...
Figure 8.5 Relative binding affinity percentage %‐RBA (
y
‐axis) of 100 small ...
Figure 8.6 JQ1 and bromosporine profiling of the bromodomain panel. (a) Inte...
Figure 8.7 Schematic for the MagMASS approach for off‐line affinity selectio...
Figure 8.8 Schematic for ASMS workflow using self‐assembled monolayers and m...
Figure 8.9 Correlation of SAMDI ASMS data with an orthogonal biochemical bin...
Figure 8.10 Schematic of ultracentrifugation‐based strategy for multiplexed ...
Figure 8.11 Concentration distributions for proteins and tool compounds foll...
Chapter 9
Figure 9.1 Typical setup of the online ASMS with 2‐D LC/MS configurations....
Figure 9.2 The time‐dependent dissociation profiles simulated for a potent l...
Figure 9.3 Potential ASMS applications throughout the drug discovery and dev...
Figure 9.4 One successful example of identifying clinical drug candidates wi...
Chapter 10
Figure 10.1 Illustration of the electrospray ionization process of protein a...
Figure 10.2 Illustration of the inline‐desalting process. Non‐volatile salt ...
Figure 10.3 SEC‐IEX‐nMS chromatogram and spectra of reslizumab and bevacizum...
Figure 10.4 Representation of the nLC‐MS platform.
Figure 10.5 Depiction of the two modes of operation for the fluidic channel ...
Chapter 11
Figure 11.1 LDTD process. The laser path is shown heating the back of the sa...
Figure 11.2 The sample holder with standard dimensions (ANSI footprint) for ...
Figure 11.3 Schematic view of (a) the sample preparation deposited on the we...
Figure 11.4 The schematic view shows the two concentric tubes of the extract...
Figure 11.5 Logarithmic relationship between the desorption rate and the hea...
Figure 11.6 Normalized intensity of sulfadiazine compared to the temperature...
Figure 11.7 Maxwell–Boltzmann distribution.
Figure 11.8 Dried deposit of prednisone on a surface well magnified (a) 30 X...
Figure 11.9 Carrier gas flow and temperature profile. The carrier gas enters...
Figure 11.10 Relative abundance versus number of water cluster for different...
Figure 11.11 Relative signal intensity versus the rate of molecules desorbed...
Figure 11.12 Schematic of the signal intensity versus the volume deposited o...
Figure 11.13 Peak shape characterization when ionic saturation occurs, with ...
Figure 11.14 Typical laser pattern shape. The length of the power ramp and t...
Figure 11.15 LogD plot for three compounds with distinct behavior.
Figure 11.16 Correlation plot between the IC
50
values obtained form LDTD‐APC...
Figure 11.17 Area counts for different volumes deposited in the well for the...
Figure 11.18 (a) 384 samples of the same concentration of OH‐Midazolam acqui...
Figure 11.19 Calibration curve of dextrorphan acquired with a throughput of ...
Figure 11.20 Comparison of IC
50
curves obtained by LDTD and RapidFire, showi...
Figure 11.21 Caco‐2 model.
Figure 11.22 Cross validation of permeability analysis of Caco‐2/TCY between...
Figure 11.23 LDTD‐AMS SWATH method data for a full 384 well plate. (a) shows...
Figure 11.24 Dilution ratio optimization for a protein precipitate in plasma...
Figure 11.25 Results for the Quetiapine with a 250 nL volume deposited on th...
Figure 11.26 (a) Mean plasma concentration–time profiles of 27 subjects admi...
Chapter 12
Figure 12.1 Results from the cGAS HTS assay campaign and hit triaging. (a) Z
Figure 12.2 Workflow for uHT‐MALDI screening of chemical reactions. The firs...
Figure 12.3 uHT‐MALDI MS approach to screening potential reaction poisons. (...
Figure 12.4 Automated workflow of the cellular MALDI MS uptake assay: Adhere...
Figure 12.5 Scheme of MALDI‐TOF affinity selection mass spectrometry (AS‐MS)...
Figure 12.6 Bead‐assisted mass spectrometry (BAMS) workflow. Lysed and diges...
Chapter 13
Figure 13.1 Schematics of (a) desorption electrospray ionization (DESI), (b)...
Figure 13.2 (a) Schematic of a typical TLC‐DESI‐MS system for natural produc...
Figure 13.3 (a) Scanned optical image of a sagittal whole‐body tissue sectio...
Figure 13.4 (a) Schematic of liquid sample DESI‐MS for analysis protein–liga...
Figure 13.5 Workflow of a high‐throughput reaction screening system with DES...
Guide
Cover Page
Title Page
Copyright Page
List of Contributors
Preface
List of Abbreviations
Table of Contents
Begin Reading
Index
WILEY END USER LICENSE AGREEMENT
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High‐Throughput Mass Spectrometry in Drug Discovery
Edited by
Chang Liu
SCIEX
Concord, Canada
Hui Zhang
Entos Inc.
La Jolla
USA
This edition first published 2023© 2023 John Wiley & Sons, Inc.
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List of Contributors
Leigh BedfordSCIEX, Concord, ON, Canada
Aivett BilbaoEarth and Biological Sciences Directorate, Pacific NorthwestNational LaboratoryRichland, WA, USA
Thomas R. CoveySCIEX, Concord, ON, Canada
Sammy DatwaniBeckman Coulter Life SciencesSan Jose, CA, USA
Sergei DiklerBruker Scientific, LLCBillerica, MA, USA
Eva DuchoslavSCIEX, Concord, ON, Canada
Lucien GhislainBeckman Coulter Life SciencesSan Jose, CA, USA
John JaniszewskiNational Center for Advancing Translational Sciences (NCATS)Rockville, MD, USA
Mengxuan JiaPreclinical Development ADMET/BAMerck & Co., IncSouth San Francisco, CA, USA
Yang KangSCIEX, Concord, ON, Canada
Brendon KapinosPfizer Worldwide Research and Development, Groton, CT, USA
Adam LatawiecSCIEX, Concord, ON, Canada
Sylvain LetarteR&D Department, Phytronix Technologies Inc., QuébecQC, Canada
Chang LiuSCIEX, Concord, ON, Canada
Juncai MengDiscovery Technology and Molecular Pharmacology (DTMP)Janssen Research & DevelopmentLLC, Spring House, PA, USA
Olivier MozziconacciDiscovery Pharmaceutical SciencesMerck & Co., Inc, South San FranciscoCA, USA
Réal E. PaquinUniversité LavalQuébec, QC, Canada
Pierre PicardR&D Department, Phytronix Technologies Inc., QuébecQC, Canada
Elizabeth PiersonAnalytical R&D, Merck & Co., Inc.,Rahway, NJ, USA
Subhasish PurkayasthaSCIEX, Framingham, MA, USA
Jonathan RochonR&D Department, Phytronix Technologies Inc.,Québec, QC, Canada
Dylan H. RossEarth and Biological Sciences Directorate, Pacific Northwest National LaboratoryRichland, WA, USA
Bradley B. SchneiderSCIEX, Concord, ON, Canada
Wilson Z. ShouLead Discovery and OptimizationBristol‐Myers Squibb CompanyPrinceton, NJ, USA
Richard D. SmithEarth and Biological Sciences Directorate, Pacific Northwest National LaboratoryRichland, WA, USA
Aaron StellaSCIEX, Framingham, MA, USA
Christopher F. StrattonDiscovery Technology and Molecular Pharmacology (DTMP), Janssen Research & Development, LLCSpring House, PA, USA
Lawrence M. SzewczukDiscovery Technology and Molecular Pharmacology (DTMP), Janssen Research & Development, LLCSpring House, PA, USA
Matthew D. TroutmanHit Discovery and OptimizationPfizer, Inc., Groton, CT, USA
Andrew D. WagnerLead Discovery and OptimizationBristol‐Myers Squibb CompanyPrinceton, NJ, USA
Jianzhong WenPreclinical Development ADMET/BAMerck & Co., IncSouth San Francisco, CA, USA
Jonathan WingfieldMechanistic and Structural BiophysicsDiscovery Sciences, R&DAstraZeneca, Cambridge, UK
Hui ZhangEntos Inc.Department of Analytical Technologies, EntosSan Diego, CA, USA
Wenpeng ZhangState Key Laboratory of Precision Measurement Technology and Instruments, Department of Precision Instrument, Tsinghua UniversityBeijing, China
Xueyun ZhengEarth and Biological Sciences Directorate, Pacific Northwest National Laboratory, Richland, WA, USA
Preface
The automated and integrated high‐throughput sample analysis is critical to the drug discovery process. Traditional high‐throughput bioanalytical technologies such as colorimetric microplate‐based readers are often constrained by linear dynamic range. In addition, they need label attachment schemes with the propensity to modify equilibrium and kinetic analysis. On the other hand, mass spectrometry (MS) based methods can achieve label‐free, universal mass detection of a wide arrange of analytes with exceptional sensitivity, selectivity, and specificity. However, these techniques are limited by the speed of sample introduction. In recent decades, there have been a lot of efforts to improve the throughput of MS‐based analysis for drug discovery. Along with those developments, a dedicated book would be helpful to introduce the fundamentals, experimental details, and applications of a wide variety of technologies that enabled high‐throughput mass spectrometry‐based screens in supporting broad drug discovery applications. The key research areas include hit discovery by label‐free screen, synthetic reaction optimization, lead optimization and SAR support, ADME (absorption, distribution, metabolism, and excretion), toxicology screening, etc.
This book starts with an overview of the 40 years of efforts to improve the analytical throughput of MS‐based approaches (Chapter 1). Then, technologies with high‐speed sequential and parallel chromatographic sample introduction, high repetition rate lasers, ion mobility, and low‐volume MS samplings were summarized.
Due to its high specificity and high sensitivity, the LC‐MS technology has been widely used in various steps of the drug discovery workflow. In Part 2 (Chapter 2–3), the efforts to improve the LC‐MS analytical throughput are introduced. The development of the high‐speed sample introduction for LC‐MS and its application on ADME and HTS applications is described in Chapter 2. Another approach for throughput improvement utilizing paralleled multiplexing LC is described in Chapter 3.
Following the conventional LC‐MS‐based technologies, other electrospray ionization (ESI)MS‐based high‐throughput platforms without chromatographic separation are summarized in Part 3 (Chapter 4–7). Direct online solid‐phase extraction (SPE) MS and its application in ADME and HTS workflows are described in Chapter 4. The utilization of the acoustic energy for non‐contact transfer samples from microplates to MS for high‐throughput analysis, including the acoustic mist ionization (AMI) and through the open‐port interface (OPI), is summarized in Chapter 5. By skipping the chromatographic separation process, these approaches demonstrated higher analytical throughput than the conventional LC‐MS approach. However, there would be the risk of potential isomeric/isobaric interference. Ion mobility spectrometry (IMS) and differential mobility spectrometry (DMS), described in Chapters 6 and 7, respectively, provide the additional dimension of the selectively, potentially solving the specificity issues of these high‐throughput technologies for some drug discovery assays.
Part 4 (Chapters 8–10) summarized the MS‐based high‐throughput hit identification technologies based on the drug‐target interaction. Affinity‐selection mass spectrometry (ASMS) is a rapidly developing technology for high‐throughput hit identification. The off‐line and in‐line ASMS approaches are introduced in Chapters 8 and 9. In addition, as a direct confirmation tool for the protein‐drug binding, native MS has been rapidly developed in the past decade, which is described in Chapter 10.
Part 5 (Chapter 11–13) introduces developments of ambient ionization technologies other than the conventional ESI and their applications in the high‐throughput drug discovery workflows, such as Laser Diode Thermal Desorption (LDTD, Chapter 11), Matrix‐Assisted Laser Desorption/Ionization (MALDI, Chapter 12), and Desorption Electrospray Ionization (DESI, Chapter 13).
The last chapter (Chapter 14) provides perspectives for future development opportunities after a brief reflection of the realized impacts of high‐throughput MS on drug discovery and the pharmaceutical industry.
We believe our goal in this book is accomplished through the extensive coverage of fundamentals, experimental details, and applications of state‐of‐art technologies that enable high‐throughput MS‐based screens in supporting drug discovery. We hope it could benefit scientists in pharmaceutical/biopharmaceutical companies and CROs who design and perform the studies and provide analytical support throughout drug discovery processes. We would like to acknowledge the commitment and contributions of all authors of the book chapters and the support and valuable discussions with colleagues and collaborators in the SCIEX research team and Pfizer Discovery Science department. In addition, we sincerely thank the editorial team at John Wiley & Sons, especially Adalfin Jayasingh, Stacey Woods, Jonathan Rose, Andreas Sendtko, and Sabeen Aziz, for their generous support of this book. Finally, we are grateful to our family members for their understanding and support for our editing work in the evening and on weekends.
Chang Liu and Hui Zhang
List of Abbreviations
%‐RBA
relative binding affinity percentage
μFLC
microflow liquid chromatography
2d
two‐dimensional
2‐HG
2‐hydroxyglutarate
3CLpro
3‐chymotrypsin‐like cysteine protease
4EBP1
Eukaryotic translation initiation factor 4E‐binding protein 1
A
pre‐exponential factor constant
ACE50
affinity competition experiment 50% inhibitory concentration
AChE
acetylcholinesterase
ADC
antibody−drug conjugate
ADE‐OPI‐MS
acoustic droplet ejection‐open port interface‐mass spectrometry
ADME
adsorption, distribution, metabolism, and excretion
AEMS
acoustic ejection mass spectrometry
AMI‐MS
acoustic mist ionization‐mass spectrometry
AMS
affinity mass spectrometry
ANSI
American National Standards Institute
APCI
atmospheric pressure chemical ionization
API
atmospheric pressure ionization
APIs
active pharmaceutical ingredients
APPI
atmospheric pressure photo ionization
ASAP
atmospheric solids analysis probe
ASMS
affinity selection mass spectrometry
ASMS
American society mass spectrometry
Asp
aspartic acid
ATD
arrival time distribution
ATP
adenosine triphosphate
AUC
analytical ultracentrifugation
AUC
area under the curve
BACC
bacterial acetyl coenzyme‐A carboxylase
BACE
beta‐site APP cleaving enzyme
BAMS
bead assisted mass spectrometry
Bcl‐xL
B‐cell lymphoma‐extra large protein
bdf
batch data file
BE
buffer exchange
Bead‐GPS
bead‐based global proteomic screening
BFA
bound fraction analysis
BKM120
Buparlisib
BSA
bovine serum albumin
BTE
Boltzmann transport equation
C18
octadecyl stationary phase
C8
octyl stationary phase
CCS
collision cross section
CD
circular dichroism
CDMS
charge detection mass spectrometry
CEM
chain ejection model
cGAMP
cyclic GMP‐ATP
cGAS
cyclic GMP‐AMP synthase
CHCA
α‐cyano‐4‐hyroxycinnamic acid
CHK1
checkpoint kinase
CID
collision induced dissociation
CIU
collision induced unfolding
CN
cyano stationary phase
CoV
compensation voltage
CPATI
cytosolic proteome and affinity‐based target identification
CRIMP
Compression Ratio Ion Mobility Programming
CRM
charged residue model
CV
coefficient of variation
CYP
cytochrome P450
Da
Dalton, measurement unit used in mass spectrometry
DAR
drug‐to‐antibody ratio
DART
direct analysis in real time
DDI
drug–drug interaction
DEC
desorption enhancing coating
DEL
DNA‐encoded library
DESI
desorption electrospray ionization
DHAP
2,5‐dihydroxyacetophenone
DHFR
dihydrofolate reductase
diCQA
dicaffeoylquinic acid
DI‐GCE/MS/MS
direct injection/on‐line guard cartridge extraction/tandem mass spectrometry
DIMS
differential IMS
DIOS
desorption ionization on silicon
DLS
dynamic light scattering
DMA
differential mobility analyzer
DMS
differential mobility spectrometry
DP
declustering potential
DQ
DiscoveryQuant
DSF
differential scanning fluorimetry
DT
drift time
DTIMS
drift tube IMS
DUB
deubiquitilase
E
a
energy of activation
ebox
electronics box
E
d
bound dissociation energy
EDTA
ethylenediaminetetraacetic acid
EHDI
electrohydrodynamic ionization
EI
electron impact
EM
electron microscopy
ERK1/ERK2
extracellular signal‐regulated kinase 1 and 2
ESI
electrospray ionization
ESI‐MS
electrospray ionization mass spectrometry
E
λ
energy associated with the vibrational wavelength
FAB
fast atom bombardment
FAIMS
high field asymmetric waveform ion mobility spectrometry
FAK
focal adhesion kinase
FASN
fatty acid synthase
FIA
flow injection analysis
FLD
fluorescence detector
FP
fluorescence polarization
FTE
full‐time equivalent
FTICR
Fourier‐transform ion cyclotron resonance
FWHM
full width at half maximum
GABA
γ‐aminobutyric
GC
gas chromatography
GLP
good laboratory practice
GPC
gel permeation chromatography
GST
glutathione S‐transferase
GWAS
genome‐wide association studies
HBSS
Hank's buffered salt solution
HCV
hepatitis C virus
HDMA
high‐density micropatterned array
HEPES
4‐(2‐hydroxyethyl)‐
1
‐piperazineethanesulfonic acid
HIC
hydrophobic interaction chromatography
HLM
human liver microsomes
HMW
high molecular weight species
HPLC
high‐performance liquid chromatography
HRMS
high‐resolution mass spectrometry
HT‐ADME
high‐throughput absorption, distribution, metabolism, excretion
HTE
high‐throughput experimentation
HT‐LC/MS/MS
high‐throughput mass spectrometry
HT‐MALDI
high‐throughput matrix‐assisted laser desorption/ionization
HT‐MS
high‐throughput mass spectrometry
HTRF
homogenous time‐resolved fluorescence
HTS
high‐throughput screening
IC
50
half maximal inhibitory concentration
ID
internal diameter
IDH1
isocitrate dehydrogenase 1
IEX
ion exchange chromatography
IM
ion mobility
IMAC
immobilized metal ion affinity chromatography
iMALDI
immuno‐matrix‐assisted laser desorption/ionization
IMS
ion mobility spectrometry
IR‐MALDESI
infrared matrix‐assisted desorption electrospray ionization
IS
internal standards
isoAsp
isoaspartic acid
ITC
isothermal titration calorimetry
ITO
indium tin oxide
IVIVC
in vitro to in vivo correlations
k
rate constant
LC
liquid chromatography
LC/MS/MS
liquid chromatography tandem mass spectrometry
LC‐MALDI
liquid chromatography‐matrix‐assisted laser desorption/ionization
LC‐MS
liquid chromatography mass spectrometry
LDLR
low‐density lipoprotein receptor
LDTD
laser diode thermal desorption
LESA
liquid extraction surface analysis
LLE
liquid–liquid extraction
LOD
limits of detection
LogD
distribution coefficient
LOQ
limit of quantitation
LPS
lipopolysaccharides
M3
microfabricated monolithic multinozzle
mAbs
monoclonal antibodies
MagMASS
magnetic microbead affinity selection screen
MALDI
matrix‐assisted laser desorption ionization
MALDI‐2
laser‐induced postionization
MALDI‐FTICR MS
matrix‐assisted laser desorption/ionization Fourier‐transform ion cyclotron resonance mass spectrometry
MALDI‐TOF MS
matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry
MetAP2
methionyl aminopeptidase 2
MnESI
microflow‐nanospray electrospray ionization
MPS
mesoporous silica
MRM
multiple reaction monitoring
MRO
medical review officer
MS
mass spectrometer
MS/MS
tandem mass spectrometry
MSI
mass spectrometry imaging
MTBE
methyl tert‐butyl ether
MTP
microtiter plate
MuRF
muscle RING‐finger protein
NADPH
nicotinamide adenine dinucleotide phosphate
NALDI
nanostructure‐assisted laser desorption/ionization
Nano‐DESI
nanospray desorption electrospray ionization
NAPA‐LDI
nanopost array‐laser desorption/ionization
NDM‐1
New Delhi metallo‐lactamase1
NDX
native‐denatured exchange
nESI
nano electrospray ionization
NHS
N
‐hydroxysuccinimide
NIMS
nanostructure‐initiator mass spectrometry
nL
nanoliter
NMR
nuclear magnetic resonance
nMS
native mass spectrometry
NSAID
nonsteroidal anti‐inflammatory drugs
NSP14
nonstructural protein 14
OATP2B1
organic anion transporting polypeptide 2B1
OIMS
overtone IMS
OPSI
open port sampling interface
PAH
polycyclic aromatic hydrocarbon
PBED
polybrominated diphenyl ether
PBS
phosphate‐buffered saline, buffer solution about pH 7.4
PCB
polychlorinated biphenyl
PCB
printed circuit board
PC‐mass‐tags
photocleavable mass‐tags
PFAS
per‐ and polyfluoroalkyl substances
PK
pharmacokinetic
p
K
a
acid dissociation constant
PKCα
protein kinase C‐α
PMF
peptide mass fingerprinting
PoC
percentage of control
POE
percent of enrichment
PPT
protein precipitation technique
PROTAC
proteolysis targeting chimera
PTP1B
tyrosine phosphatase 1B
PUF‐MS
pulsed ultrafiltration‐mass spectrometry
PVDF
polyvinylidene difluoride
QA/QC
quality assurance and quality control
qPCR
quantitative polymerase chain reaction
qTOF
quadrupole time‐of‐flight
QuEChERS
quick easy cheap effective rugged and safe
R
universal gas constant
R
2
coefficient of determination
RAM
restricted access media, usually a type of filtering or extraction media
RAM
restricted access medium
RF‐MS
RapidFire – mass spectrometry
ROI
return on investment
RXRa
retinoid X receptor‐a
S/N
signal‐to‐noise ratio
SALLE
salt assisted liquid–liquid extraction
SAM
S
‐adenosyl‐
L
‐methionine
SAMDI
self‐assembled monolayers and matrix‐assisted laser desorption ionization
SAR
structure‐activity relationship
SEC
size‐exclusion chromatography
SEC‐TID
size‐exclusion chromatography for target identification
SEM
scanning electron microscope
SESI
secondary electrospray ionization
SEZ
staggered elution zone chromatography
SIK2
salt‐inducible kinase 2
SIMS
secondary ion mass spectrometry
Sirt3
Sirtuin 3
SISCAPA
stable isotope standards and capture by anti‐peptide antibodies
SLIM
structures for lossless ion manipulations
SLS
static light scattering
SME
small molecular entity
SmyD3
SmyD3 histone methyltransferase
SNP
single‐nucleotide polymorphism
SPE
solid phase extraction
SPE‐MS
solid‐phase extraction mass spectrometry
SPME
solid‐phase microextraction
SPR
surface plasmon resonance
SRM
selected reaction monitoring
SSP
surface sampling probe
SUPER
Serpentine Ultralong Path with Extended Routing
SV
separation voltage
SWATH
sequential window acquisition of all theoretical mass spectra
T
absolute temperature in Kelvin
TCP
tumor cell percentage
THC
tetrahydrocannabinol
TIMS
trapped ion mobility
TLC
layer chromatography
TMA‐lyase
trimethylamine‐lyase
TM‐DESI
transmission mode DESI
TM‐IMS
transversal modulation IMS
TOF
time‐of‐flight
TR‐FRET
time‐resolved fluorescence energy transfer
TRIS
Tris (hydroxymethyl) aminomethane
TWIMS
traveling wave ion mobility
UFA
unbound fraction analysis
UHPLC
ultrahigh‐performance (or pressure) liquid chromatography
UHPLC/MS
ultrahigh‐performance liquid chromatography‐mass spectrometry
uHT‐MALDI
ultrahigh‐throughput matrix‐assisted laser desorption/ionization
uHTS
ultrahigh‐throughput screening
UPLC
ultra performance liquid chromatography
UV
ultraviolet, usually meant to describe absorbances between 190 and 400 nm
UVPD
ultraviolet photodissociation
WBA
whole‐body autoradiography
XRD
X‐ray diffraction
Δ
9
‐THCC
Carboxylic Δ
9
‐tetrahydrocannabinol
λ
phonon wavelength
Section 1Introduction
1Forty‐Year Evolution of High‐Throughput Mass Spectrometry: A Perspective
Thomas R. Covey
SCIEX, Concord, Ontario, Canada
1.1 Introduction
The field of drug discovery has been the primary driver behind the development of quantitative high‐throughput mass spectrometry (HT‐MS) over the past several decades. Hypothesis‐driven science guides the search for effective chemical interventions in disease increasingly supplemented with stochastic methods implemented to broaden the range of chemistries to be tested for efficacy. This later approach has generated the need to be able to make quantitative measurements on tens to hundreds of thousands of drug candidates per day in multiple in vitro and in vivo experimental scenarios. For any method of measurement such as mass spectrometry that is serial in nature, addressing daily numbers of that magnitude would require an analysis to be completed in approximately one second (1 Hz). It is fair to say that it has been only within the past few years that mass spectrometry‐based throughputs at this rate have been shown to be possible in a way that can be practically implemented into the drug discovery process. It is the purpose of this manuscript to attempt to explain how this came about. For a more thorough discussion of the role of high‐throughput experimentation (HTE) and the impact HT‐MS has on pharmaceutical R&D, see Chapter 14 of this book [1], review articles [2–7], and two earlier books on the topic of mass spectrometry in drug discovery [8, 9].
This chapter is not an exhaustive review of the literature but rather attempts to define what the trends in the field of HT‐MS were over the past 40 years using specific examples to illustrate its evolution. It is a personal perspective where many of the examples cited are technologies the author was in some way involved in either their development or early testing. This perspective attests to the importance of addressing as many of the bottlenecks in the overall HT‐MS workflow as possible, so a wide variety of technologies are shown to be contributors to the overall solution above and beyond just the speed of sample introduction. Historical context is provided for advances in all of the areas of consideration so that the gains in the field of HT‐MS since the first indications of its possibility nearly 40 years ago can be fully appreciated. Failures as well as successes are included in this perspective as they are shown to have provided to all working in the field valuable clues regarding what new directions to pursue eventually leading to where the industry is at today.
The areas to be covered, in more or less chronological order, are summarized in this chapter. Section 1.2 begins with a brief historical perspective on the development of ionization technologies and interfaces to mass spectrometry for LC/MS which laid the foundations for HT‐MS today. This culminated in the domination of atmospheric pressure ionization (API) for online LC/MS in the late 1980s utilizing both electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI). The later technique was the first to demonstrate the feasibility of HT‐MS in 1986 but ESI now serves as the basis for most systems today because it provides the broadest compound coverage, has been developed to operate reliably at high fluid linear velocities similar to APCI, and like APCI, has been demonstrated to be sufficiently resistant to contamination to sustain 24/7 operation under high sample loads.
Included in Section 1.2 are the origins of alternative sampling techniques that utilize ESI and APCI, popularly referred to as “ambient ionization,” as part of a mission to bypass chromatography to gain speed. Some are beginning to indicate possible utility for HT‐MS. Also in this section is the development of methods for the direct and indirect measurement of molecular affinities to biological targets which have begun to be incorporated into industrialized drug discovery programs with some of their earliest origins 20–30 years ago.
As described in Sections 1.3 and 1.4 of this chapter, approximately a decade after API took hold in the early 1990s, there was a proliferation of means to improve the speed of liquid chromatography and chromatographic systems which have evolved into the most widely used approach to HT‐MS today. These developments are more thoroughly covered in Chapters 2[10], 3[11], and 4[12] of this book.
Section 1.5 of this chapter describes how the 1 Hz sampling rate barrier was finally broken using matrix‐assisted laser desorption ionization (MALDI) around 2005 with high repetition rate lasers. This was in response to the realization that high performance liquid chromatography (HPLC) needed to be bypassed to achieve this goal. MALDI originated in the 1980s as a means to obtain molecular weight information on large biomolecules and has emerged recently as the HT‐MS method of choice for solid sample introduction thoroughly covered in Chapter 12 of this book [13].
Section 1.6 describes the understanding that some form of high‐speed chemically based separation to replace HPLC would remain a requirement to keep the versatility of HT‐MS on par with LC/MS. This initiated more concerted efforts to find a high‐speed substitute in ion mobility technology considered in detail in Chapter 6 of this book [14]. In 2008, differential ion mobility spectrometry (DMS) was adapted to the mass spectrometer (MS) for HT‐MS because of its unique separation mechanism that, like HPLC, separates based on the chemical properties of molecules. Further discussion of DMS is provided in Chapter 7 of this book [15] as well as Section 1.6 of this chapter.
By the second decade of the twenty‐first century, the cumulative gains in API MS sensitivity over the past 40 years approached one million‐fold and are described in Section 1.7 of this chapter. This development has played a major role in the evolution of HT‐MS as it has made possible the reduction in the volume of sample required to achieve biologically relevant limits of quantitation (LOQ) by a similar amount, approximately 6 orders of magnitude. This relaxes the requirements for sample preparation, reduces system contamination, and enables the use of high‐speed low‐volume dispensers in the picoliter to nanoliter range.
The final topic in Section 1.8 describes the emergence of low‐volume sample introduction techniques into the MS representing an important culmination of the past 40 years of HT‐MS evolution. These approaches have the potential to streamline several bottlenecks in the high‐throughput workflow such as the elimination or minimization of sample preparation, the elimination of the time wasting and ambiguous results generated from sample cross contamination, and simplification of automation by leveraging the microtiter plate format. Chapter 5 of this book [16] also elaborates on some applications of this approach to many aspects of the drug discovery process in addition to Section 1.8 of this chapter.
As throughputs increased over this time, new software and robotic systems designed to improve sample handling, management, data acquisition, and data analysis advanced in lock step. The role they play is vital to enable these HT‐MS developments to reach their throughput potentials. This aspect is conspicuously omitted from this chapter for no other reason than its scope is of sufficient magnitude to be the topic of another book. Initial access to information regarding these aspects is provided in references [17–21].
1.2 Ionization Foundations of High‐Throughput Mass Spectrometry
During the late 1960s through the 1980s, two different approaches to ionization emerged in the field of mass spectrometry that established the foundation for today's HT‐MS technologies, API [22–27], and MALDI [28]. The efforts leading to MALDI were primarily driven by the quest to find a means to create intact gas‐phase ions from biopolymers. Until then, obtaining molecular ions from molecules with molecular weights above 2000 amu was considered a heroic effort, a single mass spectrum the centerpiece of an entire PhD thesis. API became popular primarily from the efforts to find a viable means to interface the HPLC to the MS although its capacity for high mass measurement contributed to its success as well [29, 30].
In the 1980s, the vast majority of commercial instruments were based on ionization inside the MS vacuum system [31]. The difficulties faced interfacing HPLC and MS prior to API were expressed in a famous icon created by Patrick Arpino in 1982 shown in the insert in Figure 1.1. Titled “A Difficult Courtship,” it is referred to the fundamental incompatibility between the vacuum‐based gas‐phase world of MS, the bird, and the liquid‐phase world of LC, the fish [32]. A line from the Broadway play “Fiddler on the Roof” summarizes the difficulties of such a relationship when Tevye says to his daughter intent on marrying against his wishes: “a bird may love a fish, but where would they build a home together?” The home would eventually reside in an atmospheric pressure ion source when it was finally realized that spraying liquids into vacuum systems or through vacuum locks and stages was not such a good idea after all.
Figure 1.1 Histomap of LC/MS interfaces from 1967 to present and some emerging HT‐MS technologies superimposed. The width of the region (x‐axis) that each technique occupies illustrates its growth from initial invention and early publications (thin line) to an approximation of its commercial proliferation and incorporation into industrialized processes relative to the other techniques during that period of time. The map is bifurcated down the middle into two halves. On the left is shown atmospheric pressure ionization LC/MS interfaces and on the right are those based on ionization under vacuum conditions. The famous 1980s bird/fish icon represents the incompatibility of the gas‐phase world of MS and liquid‐phase world of LC as viewed at that time.
Source: Reproduced with permission from Arpino [32].
All acronyms are defined in the chapter text. Ion mobility is included in the Histomap even though it is not an LC/MS interface or sample introduction system because of its potential as a high‐speed substitute for some functions of HPLC in emerging HT‐MS systems. LAP MALDI and MALDESI are included in the electrospray area because they both utilize the Ion Evaporation ionization mechanism of ESI at atmospheric pressure. This Histomap was adapted from a similar Histomap in references [27, 33, 34]. The Histomap concept was developed by John Sparks and published by Rand McNally to track the course of world cultural history since the dawn of civilization in a 6‐ft long Histomap [35].
1.2.1 Historical Context of the Development of LC/MS. Ionization in Vacuum or at Atmospheric Pressure?
The Histomap in Figure 1.1 provides a perspective on the extent to which the LC/MS interfacing problem was being addressed in research groups worldwide and the diversity of approaches that were developed, commercialized, and proliferated. From its earliest beginnings in the 1960s, the LC/MS field was divided into two camps, those generating ions created at atmospheric pressure initiated by Malcom Dole in Chicago [36] (ESI on a mobility analyzer at atmospheric pressure), and those creating ions under vacuum conditions initiated by Victor Talroze in Moscow [37] (low‐volume liquids introduced into an electron impact ionization source in a vacuum). The atmosphere versus vacuum ionization divide would extend for two more decades and as seen in the Histomap, the vacuum‐based techniques for LC/MS interfacing overwhelmingly dominating the commercial landscape through the 1980s. This was largely because commercial API systems initially did not exist and when one emerged in 1978, it was designed primarily for direct air pollution monitoring only relegating it to the commercial fringes of the analytical mass spectrometry field. Modifications to main stream commercial mass spectrometers for API operation were extensive whereby it was seemingly more pragmatic to develop LC interfaces that maintained vacuum‐based ionization. After all mass spectrometers were designed to analyze ions created in a vacuum since the earliest beginnings of its use for chemical analysis in 1912 [38] and there had been little thought to do otherwise for much of the twentieth century.
LC/MS interfaces with ionization in a vacuum followed two paths. The route proposed by McLafferty [39], Henion [40, 41], and Vestal [42, 43] involved chemical ionization using the vaporized LC mobile phase as the reagent gas with either the direct liquid introduction or Thermospray interfaces.
The other route championed by McFadden [44], Games [45], and Willoughby [46] upheld the importance of maintaining the option of electron impact ionization capability with the moving belt and particle beam interfaces. These interfaces removed all of the mobile phase using various types of vacuum locks and stages so only dried sample material would enter vacuum on either a Kapton belt or as a dried gas‐phase particle. Thermal desorption of the dried sample inside vacuum enabled electron impact ionization or chemical ionization when reagent gas was piped in.
Kelsey Cook was exploring an early form of ESI under vacuum called Electrohydrodynamic Ionization (EHDI) [47]. With this approach, low flows of liquid charged by a high voltage on the capillary transporting it were sprayed directly inside the vacuum system. With the exception of the work of Thomson discussed later in this Section 1.2.1, the attributes of atmospheric ESI were not yet realized, its extraordinarily mild ionization and multiple charging properties. These capabilities were not observed with EHDI because of the poor evaporation rates of droplets in a vacuum due to inefficient thermal energy transfer at these pressures. The droplets could not evaporate to the Rayleigh limit and undergo the coulomb explosions required to achieve ion emission diameters. Also, the creation of energetic electrons by the Townsend discharge process occurring at these pressures using the voltages required to form a spray caused fragmentation of whatever molecules made it to the gas phase. Had the sprayer been moved from inside the vacuum system to atmosphere, requiring substantial modifications to the MS as mentioned earlier, history may have been rewritten. For the reasons described above, it was not commercialized as seen in its dead ended trace in the Histomap.
During this time, attempts to ionize higher molecular weight compounds were done under vacuum with the high‐energy particle impact techniques of Fast Atom Bombardment [48] (FAB) and Californium Plasma Desorption [49], both of which were replaced by MALDI which could produce intact gas‐phase molecular ions, as could ESI, from much larger molecules.
References to the initial publications of these ionization and LC/MS interfacing techniques are provided in Figure 1.1 with a more detailed description of the various interfaces displayed provided in earlier published versions of this Histomap [27, 34, 50].
In 1978, an important event occurred that provided a base upon which to develop HT‐MS systems in the future. As noted in the Histomap, an API MS was commercialized as a component of a van‐based mobile laboratory for environmental and regulatory applications as described in more detail later in this chapter. Although samples were introduced as gasses or solids by a variety of techniques, not in the liquid form, this technology drew the attention of a few experts in the LC/MS field as a potential solution to the LC/MS interfacing problem particularly because some earlier liquid introduction work at atmospheric pressure by the Horning's with APCI [51, 52] and Thomson with Ion Evaporation [53–55] provided proof‐of‐principle.
The Thomson work in the late 1970s described the theory behind the ESI mechanism which remains as the primary explanation of the electrospray process today, referred to as the Ion Evaporation Theory for ion production. He also showed its unique ability to produce multiply charged ions when interfaced to a MS [55]. By the mid‐1980s, John Fenn brought attention to electrospray to a broader audience in the West [56, 57] while Gall independently developed it in the Soviet Union at the same time [58, 59]. A means to couple ESI with conventional flow rate liquid chromatography followed in 1986 borrowing elements of the Thomson Ion Evaporation and the Fenn/Gall Electrospray, given the moniker Ion Spray [60], because it was the combination of the two which made it practical. From ion evaporation was borrowed the formation of droplets by high‐velocity gas shear forces. The ion evaporation interface charged the droplets remotely with an induction electrode. From electrospray was borrowed direct electrical charging of the liquid to replace the inductive charging of the ion evaporation interface which increased the charge density of the droplets and improved sensitivity. Ion Spray greatly increased the sensitivity of the Ion Evaporation interface and greatly increased the practicality for coupling to liquid chromatography Fenn and Gall's Electrospray. Figure 1.2a–d shows the first published drawings and photographs of these early developments and how they evolved into the most commonly used interface today.
Figure 1.2 The evolution of electrospray LC/MS interfaces. (a) The ion evaporation interface of Thomson showing pneumatic nebulization and inductive charging of the droplets. (b) Depiction of the Gall electrospray device showing direct electrical charging of the fluid.
Source: Adapted from Alexandrov et al. [59].
(c) Depiction of the Fenn electrospray device showing direct electrical charging of the fluid.
Source: Adapted from Whitehouse et al. [57].
(d) The ion spray interface of bruins which combined the ion evaporation nebulization with the direct electrical charging of electrospray.
Source: Reproduced with permission from Bruins et al. [60]/American Chemical Society.
The earliest compelling publication of the potential of API for HT‐MS was not with ESI but rather APCI. Based on the earlier APCI LC/MS work of the Horning's [52] and the APCI LC/MS/MS work of Henion and Thomson [61], the heated nebulizer APCI LC/MS interface was redesigned to operate at higher flow rates by increasing the desolvation chamber power and prototyped in the mid‐1980s and is shown in Figure 1.3c, d. Demonstrated in 1986 was a successful quantitative pharmacokinetic study by LC/MS/MS at the rate of one sample per minute, monitoring a drug and three of its metabolites for extended periods of time as shown in Figure 1.3a, b [62]. It is important to put this data in the context of the time when the successful completion of 1–10 LC/MS chromatograms per day before a catastrophic system failure occurred was considered an accomplishment. A common ritual, upon entering a laboratory exploring the utility of the various LC/MS interfaces in the 1980s, was to Spray N′ Pray.
Increasing amounts of ESI data from labs with early pre‐commercialization versions manifested in the testing of a large swath of chemical space from different application areas. A rapid realization was emerging from this data that the ionization process was uniquely mild among all other forms of ionization considered to that date. Clues that this would be true were present in the earlier data of Thomson who showed a multiply charged mass spectrum of the highly labile adenosine triphosphate in 1982 [55]. This indicated ESI was a much more versatile form of ionization, later shown to be capable of molecular ion generation from high molecular weight proteins [29] and oligonucleotides [30] and opening the door for the routine sequencing of proteins from the uniquely simple to interpret collision induced dissociation (CID) spectra of their doubly charged tryptic peptides [63–70]. Equally important was gaining access to a mass spectrometry‐based approach to determine the structures of very labile small molecules such as drugs, their metabolites, and conjugates. Previously intractable problems could be readily solved, for example unknown steroids in clinically important samples were readily identified [71]. In the late 1980s, the entire profile of over 20 metabolites of an administered drug was established in two sample runs, one to identify all the molecular ions and their retention times followed by a second to acquire CID spectra on all of them, published later in the early 1990s [72], a problem that would otherwise have taken years to solve, and would become even more straightforward when ESI was adapted to routine tandem high‐resolution accurate mass systems like QqTOF's [73] and Orbitraps. Previously unresolved chemical mysteries, that presented life or death consequences, could suddenly be solved in a matter of minutes or hours [74, 75].
