LC/MS LC/MS A Practical User’s Guide MARVIN C MCMASTER A JOHN WILEY & SONS, INC., PUBLICATION Copyright  2005 by John Wiley & Sons, Inc All rights reserved Published by John Wiley & Sons, Inc., Hoboken, New Jersey Published simultaneously in Canada No part of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, recording, scanning, or otherwise, except as permitted under Section 107 or 108 of the 1976 United States Copyright Act, without either the prior written permission of the Publisher, or authorization through payment of the appropriate per-copy fee to the Copyright Clearance Center, Inc., 222 Rosewood Drive, Danvers, MA 01923, (978) 750-8400, fax (978) 750-4470, or on the web at www.copyright.com Requests to the Publisher for permission should be addressed to the Permissions Department, John Wiley & Sons, Inc., 111 River Street, Hoboken, NJ 07030, (201) 748-6011, fax (201) 748-6008, or online at http://www.wiley.com/go/permission Limit of Liability/Disclaimer of Warranty: While the publisher and author have used their best efforts in preparing this book, they make no representations or warranties with respect to the accuracy or completeness of the contents of this book and specifically disclaim any implied warranties of merchantability or fitness for a particular purpose No warranty may be created or extended by sales representatives or written sales materials The advice and strategies contained herein may not be suitable for your situation You should consult with a professional where appropriate Neither the publisher nor author shall be liable for any loss of profit or any other commercial damages, including but not limited to special, incidental, consequential, or other damages For general information on our other products and services or for technical support, please contact our Customer Care Department within the United States at (800) 762-2974, outside the United States at (317) 572-3993 or fax (317) 572-4002 Wiley also publishes its books in a variety of electronic formats Some content that appears in print may not be available in electronic formats For more information about Wiley products, visit our web site at www.wiley.com Library of Congress Cataloging-in-Publication Data: McMaster, Marvin C LC/MS: a practical user’s guide / Marvin C McMaster p cm Includes bibliographical references and index ISBN-13 978-0-471-65531-2 (cloth) ISBN-10 0-471-65531-7 (cloth) Liquid chromatography—Handbooks, manuals, etc High performance liquid chromatography—Handbooks, manuals, etc Mass spectrometry—Handbooks, manuals, etc I Title QD79.C454M363 2005 543 84—dc22 2004063820 Printed in the United States of America 10 To the memory of my son, Chris McMaster, my writing partner and the artist on the first two books in this series Chris has passed on to bigger and better things painting sunrises and rainbows CONTENTS Preface xi Introduction to LC/MS 1.1 1.2 1.3 1.4 1.5 Why LC/MS?, Molecular Weights and Structure Studies, LC/MS Systems, System Costs, Competitive Systems, The HPLC System 2.1 2.2 2.3 2.4 HPLC System Components, Gradient versus Isocratic Systems, 14 Micro HPLC Systems, 16 HPLC Tubing and Fittings, 18 The HPLC Column and Separation Modes 3.1 3.2 3.3 3.4 3.5 3.6 3.7 21 Column Construction, 21 Column Packing Materials, 23 Normal-Phase Columns, 25 Other Bonded-Phase Silica Columns, 26 Optimizing Reverse-Phase Column Use, 28 Silica Ion-Exchange Columns, 30 Silica Size-Separation Columns, 31 vii viii CONTENTS 3.8 Zirconium Bonded-Phase Columns, 31 3.9 Polymer Reverse-Phase Columns, 32 HPLC and Column Maintenance 33 4.1 HPLC Maintenance, 33 4.2 Column Maintenance, 37 Sample Preparation and Separations Development 5.1 5.2 5.3 5.4 5.5 5.6 5.7 Mobile-Phase Preparation, 41 Mobile-Phase pH Control Using Buffers, 42 Sample Preparation, 44 Cartridge Column Cleanup, 44 On-Column Sample Concentration, 45 Isocratic and Gradient Methods Development, 46 Automated Methods Development, 49 LC/MS Interfaces 6.1 6.2 6.3 6.4 6.5 59 HPLC and the Ionization Source, 60 Vacuum Pumps, 61 Analyzer and Ion Detector Designs, 61 Data and Control Systems, 66 Peak Detection, ID, and Quantitation, 69 Mass Analyzers 8.1 8.2 8.3 8.4 8.5 8.6 51 Solvent Removal and Ionization, 51 Atmospheric-Pressure Interfaces, 52 Electrospray Interface, 53 Ion Spray Interface, 54 Secondary Detectors, 55 LC/MS Overview 7.1 7.2 7.3 7.4 7.5 41 71 Quadrupole Analyzer, 71 Ion Trap Analyzer, 74 Linear Ion Trap Analyzer, 78 Time-of-Flight Analyzer, 79 Fourier Transform Analyzer Design, 81 Magnetic Sector Analyzers, 83 Mass Spectrometer Maintenance 9.1 High-Vacuum Operation, 85 9.2 MS Hardware Maintenance, 88 9.3 System Electrical Grounding, 92 85 ix CONTENTS 10 Application Areas for LC/MS 10.1 10.2 10.3 10.4 10.5 10.6 10.7 10.8 Compound Discovery, 95 Identification of Complex Biological Compounds, 96 Analysis of Trace Impurities and Metabolites, 97 Arson Residue Investigation, 98 Industrial Water and Pesticide Analysis, 98 Toxicology and Drugs of Abuse, 98 Clinical Therapeutic Drug Screening, 99 Pesticide Manufacturing, 101 11 Trace Analysis and LC/MS/MS 11.1 11.2 11.3 11.4 103 LC/MS/MS Triple-Quadrupole System, 103 MS/MS Operating Modes, 104 Ion Trap MS/MS Operation, 106 Hybrid LC/MS/MS Systems, 108 12 Drug Discovery and Benchtop LC/MS 12.1 12.2 12.3 12.4 95 111 Activity Screening, 111 Standardized LC/MS Screening, 113 Molecular Fragmentation for Structural Determination, 115 Process Monitoring, 116 13 Proteomics: LC/MALDI/TOF and MS/MS Libraries 119 13.1 Protein Molecular-Weight Determination by LC/MS, 120 13.2 De Novo Protein Purification, 121 13.3 Protein Analysis by Two-Dimensional GEP and LC/TOFMS, 122 13.4 LC/MS/MS Identification of Peptide Structures, 122 13.5 Tracer Labeling for Peptide ID, 124 13.6 Posttranslational Modified Protein, 124 13.7 Transient Peptides and Accumulation Proteins, 124 14 The Future of LC/MS 14.1 14.2 14.3 14.4 127 Instrumentation Improvements, 127 Affordable Benchtop LC/LITMS, 129 User-Customized Data Libraries, 129 Nucleomics and Restriction Fragment Analysis, 130 Appendix A LC/MS Frequently Asked Questions 131 Appendix B 139 Solvents and Volatile Buffers for LC/MS Appendix C Guide to Structure Interpretation 143 GLOSSARY OF LC/MS TERMS 151 allows plotting of intensity versus m/z spectra with very high sensitivity at each chromatographic point The sample is not destroyed, so the signal can be sampled repeatedly to produce a very accurate sample measurement Gas chromatography A separation technique in which the volatile analyte is swept by a carrier gas down a column packed with packing coated with an absorbing liquid Differential partition between the two phases by sample components leads to band separation and elution into a detector Often combined with a mass spectrometer to create a GC/MS system Hybrid MS/MS A combination of two or more mass spectrometer analyzers of different types Used like a triple quadrupole system to separate and then fragment compounds in the LC effluent to aid in identifying a compound’s structure from its fragmentation ions Injector A device used to move a sample in undiluted form onto the head of the HPLC column See also Autosampler Internal standard A compound added to all analyzed samples in equal concentration during the last dilution before sample injection Its purpose is to correct for variations in sample injection size It can also be used to correct for variations in peak retention times Ion-exchange chromatography Partition chromatography in which the column has a bonded phase with a permanent or inducible charge opposite to that which attracts the target molecules Compounds are eluted either with a counterion, salt, or pH gradients Ion spray interface An atmospheric-pressure ionizing interface between the LC and MS modules Column effluent is nebulized or entrained with an inert gas in a capillary tube and sprayed across a coronal discharge needle operating at high voltage to evaporate solvent and volatile buffer and ionize the sample It is the interface of choice for neutral and nonpolar samples Ion trap detector (ITD) A desktop three-dimensional spherical-segment ion trap MS that ionizes and holds the ionized sample within a circular electromagnet until swept with a dc/RF frequency signal that releases the ionized sample into the ion detector Linear ion trap (LIT) A linear ion trap mass spectrometer is based on a quadrupole analyzer with end electrodes for trapping or releasing ions It has a much higher loading capacity than that of a three-dimensional spherical ion trap, which translates to more accurate measurements for trace analysis Mobile phase The liquid phase being pumped through an HPLC column to act as a carrier for the sample, to establish equilibration with the stationary phase, and eventually to elute the separated compound peaks Molecular weight Summation of the weights of all the elements in a molecule, expressed in amu or dalton In mass spectral analysis, the weight of the molecular ion produced in a soft chemical ionization such as ion spray or electrospray MS/MS system A tandem, triple quad, or hybrid dual mass spectrometer system used to study MS fragmentation mechanisms The second analyzer is 152 GLOSSARY OF LC/MS TERMS used to further fragment ions separated in the first analyzer Separation of the fragments can be done by scanning the trapping analyzer or in a third analyzer m/z Mass divided by charge measured in amu or dalton The x-axis for a mass spectrum, indicating that MS spectra are dependent on both the ion’s mass and the charge on the ion Neutral loss experiment A MS/MS experiment in which the first analyzer measures the precursor ion fragments while the second analyzer looks for product ions that have a constant mass difference, the mass of the neutral loss product molecule formed during the fragmentation Only precursor ions that form this neutral compound will show in the product ion scan that is produced Normal-phase chromatography Partition separation on unbounded polar silica columns Mobile phase is usually a nonpolar solvent but can be an acidic polar solvent Pacification Washing a system with N nitric acid after removing the HPLC column Cleans the pump heads, check valves, and the injection loop and protects them against salt corrosion for a period of time Plunger and seal Solvent flow in an HPLC pump is created by a sapphire plunger driven by a reciprocating cam-and-spring mechanism through a Teflon seal into a pumping chamber Check valves in the pump’s inlet and outlet fitting prevent reverse solvent flow and allow pressure buildup to overcome flow resistance in the column Precursor ion The target ion submitted for fragmentation studies in a MS/MS system A precursor scan is a mass scan of the fragmentation ions produced by thermal collision with a heavy gas molecule Quadrupole analyzer Mass spectrometer analyzer based on four circular rods held in a hyperbolic configuration and swept with a variable-frequency dc/RF signal, allowing selection of individual mass fragments Resolution equation A measure of a column’s separating power It combines retention, separation, and efficiency factors into a single equation and shows their interactions Retention factor An HPLC column resolution factor measuring how separation is affected by residence time on the column Retention is controlled by mobile-phase polarity and to a certain degree by column temperature Retention time The length of time a compound stays on the column under a given set of experimental conditions Reverse-phase chromatography Partition chromatography in which the column has a nonpolar bonded phase and the mobile phase uses polar solvents mixed with water The majority of reverse-phase separations are made on C18 bonded phase on silica columns Ring electrode The central electrode of a three-dimensional ion trap, used to hold ion fragments in circular orbits until time to elute them into the detector Roughing pump The first pump in a vacuum system, used to reduce pressure initially from atmospheric pressure to a low pressure that can serve as a starting point for the high-performance vacuum pump Today’s roughing pump is usually a mechanical rotary vane oil pump GLOSSARY OF LC/MS TERMS 153 Scan A mass spectrometer operational mode in which the amount of each mass unit is measured by continuously changing the dc/RF frequency on a quadrupole Mass can be scanned low to high or high to low, the latter leading to less intermass tailing and more accurate relative height measurements Secondary detector A detector, usually a UV detector, connected to an effluent diverter in the ionizing interface to take the excess effluent from the MS measurement and provide a second measurement of the chromatography peaks by an alternative form of detection Separation factor A column resolution factor controlled by the column’s chemistry and by temperature Changes in this factor result in shifting of relative peak positions See also Alpha factor and Resolution equation SIC (single-ion chromatogram) A chromatogram produced by displaying the ion current produced versus time for a given mass (m/z) It can be produced by operating in single-ion mode or extracted from a scanned fragment database SIM (single-ion monitoring) The mass spectrometer measures one or only a few specific m/z points Since fewer mass measurements are made than in scan, they are made more often, with a proportional increase in sensitivity Also an acronym for selective ion monitoring Size-separation chromatography Chromatography in which separation is made on the size of the molecules on controlled-pore-size columns Also referred to as gel filtration Spectra Bar plots of signal intensity in volts versus ion fragment mass/charge, measured in amu, for a given MS scan or range of scans The data are usually summed around unit mass and presented as a bar graph of intensities relative to the base peak SPE Sample filtration and extraction cartridge columns used for preinjection filtration and chromatographic cleanup of a sample SPE columns are available for all separation modes used in LC/MS Supercritical fluid chromatography (SCF) A column separation technique using pressure and temperature control to convert a gas into a fluid that is used as the mobile phase for liquid/solid chromatography Sample recovery is made by releasing the pressure to turn the mobile phase back into a gas Surrogate A standard compound added in known amounts to all processed samples Its purpose is to detect and correct for sample loss due to extractions and handling errors Usually, it is a deuterated or other isotopic-labeled derivative of an analyzed compound not normally found in nature Target compound quantitation Quantitation based on identifying a compound from its precursor scan by locating target and qualifier ion fragments Once identified, the target ion signal strength is compared to known amounts of standards to determine the amount present Target ion One of a compound’s MS ion fragments chosen for identifying and quantitating the amounts of the compound present in mixtures of standards and unknowns 154 GLOSSARY OF LC/MS TERMS Temperature ramp A gradual controlled increase of temperature with time It is used in combination with holds and other ramps in building a column heater temperature program for resolving compounds on a zirconium column TIC (total ion chromatogram) A chromatogram produced by measuring the total ion current from a mass spectrometer versus time A TIC data point represents a summation of all mass fragments present at a given time Time-of-flight LC/MS (LC TOF/MS) A chromatographic technique in which an MS detector analyzes effluent by ionizing it with pulse laser energy bursts and identifying mass fragments by using the time they take to travel a flight tube and reach a detector LC/TOFMS is becoming popular in the analysis of charged biochemicals, proteins, and DNA restriction fragments with multiple charges Triple-quad LC/MS/MS A tandem quadrupole system in which an LC feeds a mass detector with three quadrupole units in series The second quadrupole acts as a holding and collision cell in which fragments separated in Q1 can interact with a heavy gas such as xenon and fragment for separation in Q3 Used primarily for studying fragmentation mechanisms to aid in precursor compound structure identification Turbomechanical pump A high-vacuum pump that uses a series of vanes, mounted on a shaft, that rotate rapidly between stator plate entraining air molecules, dragging them out of the volume evacuated A turbo pump operates like a jet engine to evacuate a mass spectrometer to the high vacuum (10−5 to 10−7 torr) needed for operation Void A channel inside the packing of an HPLC column that ruins the separation Voids can open spontaneously when a column is shocked or flow is reversed They can also be caused by fines breaking off irregular packing or by silica packing dissolving at pH above 8.0 Void volume A measure of the total solvent volume inside a particle-packed HPLC column This value is used in the resolution equation to normalize retention values to make them independent of column length Also used to define washout volume for column regeneration APPENDIX E LC/MS SELECTIVE READING LIST JOURNALS American Journal of Mass Spectrometry American Laboratory Analyst Analytical Biochemistry Analytical Chemistry Environmental Science and Technology Journal of Pharmaceutical and Biomedical Analysis LC/GC Rapid Communications in Mass Spectrometry BOOKS Ardrey, Bob, Liquid Chromatography–Mass Spectrometry: An Introduction, Wiley, Chichester, West Sussex, England, 2003, 276 pp Barcello, Damia, ed., Applications of LC-MS in Environmental Chemistry, Elsevier Science, New York, 1996 Lee, Mike S., LC/MS Applications in Drug Development, Wiley-Interscience, New York, 2002, 243 pp McLafferty, Fred W., and Turecek Frantisek, Interpretation of Mass Spectra, 4th ed., University Science Books, Mill Valley, CA, 1993, 377 pp LC/MS: A Practical User’s Guide, by Marvin C McMaster Copyright  2005 John Wiley & Sons, Inc 155 156 LC/MS SELECTIVE READING LIST McMaster, M C., HPLC: A Practical User’s Guide, VCH Publishers, New York, 1994, 211 pp McMaster, Marvin, and Christopher McMaster, GC/MS: A Practical User’s Guide, Wiley-VCH, New York, 1998, 167 pp Snyder, L R., and J J Kirkland, Introduction to Modern Liquid Chromatography, Wiley, New York, 1979, 863 pp Synder, L L., et al., Practical HPLC Methods Development, Wiley, New York, 1997, 299 pp Watson, J Throck, Introduction to Mass Spectrometry, 2nd ed., Raven Press, New York, 1985, 351 pp Willoughby, Ross, et al., A Global View of LC/MS, Global View Publishing, Pittsburg, 2002 PAPERS Allen, Mark, and Bob Shushan, “Atmospheric Pressure Ionization–Mass Spectrometry Detection for Liquid Chromatography and Capillary Electrophoresis,” LC/GC, 11(2), 112–126, 1993 Amirav, Aviv and Ori Granot, “LC-MS with Supersonic Molecular Beams,” J Am Soc Mass Spectrom., 11, 587–591, 2000; www.tau.ac.il/chemistry/ amirav/lcms.shtml Chalk, Rod, and Urs Matter, “Analysis of High-Mass Biopolymers: Applications and Advantages of Cryodetector Mass Spectrometry,” Am Biotech Lab., April 2004, 26–28 Duncan, W.P., and Perkins, P.D “LC-MS With Simultaneous Electrospray and Atmospheric Pressure Chemical Ionization,” Am Lab., March 2005, 28–33 Henry, Richard A., “Highly Selective Zirconia-Based Phases for HPLC Applications,” Am Lab., November 2002, 18–25 Henion, Jack, et al., “High-flow Ion Spray Liquid Chromatography/Mass Spectrometry.” Anal Chem., 65, 439–446, 1993 Hopfgartner, G., et al., “High-Flow Ion Spray Liquid Chromatography/Mass Spectrometry,” Anal Chem., 65, 439–446, 1993 Sandra, Pat, et al., “Consideration on Column Selection and Operating Conditions for LC-MS, Interfaces for LC-MS, Mass Analysers for LC-MS,” LC/GC Europe, December 2001; www.lcgceurope.com Smith, Richard D., “Trends in Mass Spectrometry Instrumentation for Proteomics,” Trends Biochem., 20(12), 2002, S3–S7; www.trends.com Tiller, P R., et al., “Qualitative Assessment of Leachables Using Data-Dependent Liquid Chromatography/Mass Spectrometry and Liquid Chromatography/Tandem Mass Spectrometry,” Rapid Commun Mass Spectrom., 11, 1570–1574, 1997 Zahleen, K., et al., “Screening Drugs of Abuse by LC/MS,” Agilent Technologies, publication 5889-1267EN, 2004, pp.; www.agilent.com/chem INDEX A + element, 146 A + element, 146 Accelerating voltage, 84 Accumulator Q1 increased sensitivity, 81, 108 Accurate mass, 83, 84, 108 Acetone spiked gradients, 46 Activity screens, 95, 111 A/D conversion, 68, 73 Adducts, 71 Affinity chromatography, 149 Affinity column, 27,124 Affinity tag reagent, 124 Air leak check, 88, 92 Alpha factor, 47, 149 Alzheimer plaque protein, 125 Amine column oxidation, 38 Amine ion pairing, 43 AMU offset lens, 73, 137 Analog creation, 112 Analyzers for MS, 61, 64,136 venting, 89 Analyzer source assembly, 89 Animal testing, 111 Antibody purification, 28 APCI, 149 Application areas for LC/MS, 95 Arson investigation, 98 Atmospheric TOF interfaces, 81 Automated methods development, 49, 132 Atmospheric pressure ionization, Automated spotter plate system, 79 Autosampler, 13, 132, 149 Autotune, 89, 92, 129, 137 Axial modulation, 77 Background contamination, 139 Backpressure column blank, 34 sources, 132 Bacterial effluent system, 101 Base peak, 149 Batch identifiers, 98 Bioassay evaluation, 113 screening activity, 115 Biological catalyst, 79 fate, 96 large molecules, 79 proteins, 121 Biotin reporter group, 124 Blade shaft vanes, 87 Blood levels, 99 Bonded-phase column, 3, 27 Bound dipeptides, 113 Breakdown products, 97, 105 LC/MS: A Practical User’s Guide, by Marvin C McMaster Copyright  2005 John Wiley & Sons, Inc 157 158 Buffer, 28, 133 pH control, 29, 43 precipitation, 132 volatile, 2, 16, 29, 43 washout, 29 Butterfly valve, 87 By-products of drugs, 112 C18 silica column, 149 Calibrate and tune, 89, 137 Calibration curve, 69 Calibration jumping, 92 Calibration standards, 137, 149 Candidate compounds, 111 Capillary zone, electrophoresis (CZE), 137 Charged aerosol detector, 57, 135 Cartridge column cleanup, 44, 98 c-DNA expressed protein, 121 Certified methods, 130 Chain of distribution, 99 Characteristic mass loss, 148 Check valve, 10, 150 blockage, 34–35 Chelating agent, 31, 43 Chemical linker, 113 Chromatophore, 64, 79, 122 Chromatography changes, 134 Cleaning MS source, 89 Cleavage fragments, 97 Clinical screening, 111, 115 Coeluting compound, 99 Collision cell, 64, 108 Collision fragments, 64, 103, 115 Collision gas in ion trap, 78 Column, 21–22, 150 affinity, 27 aging, 48 amino–Si, 27 backpressure, 16 blank or bridge, 19, 34, 42 C18, 23 cyanopropyl–Si, 27 desalting GPC, 49 diameter, 23 diol, 27 enantiomeric, 28 end cap, 22 equilibration, 29 flushing, 42 hybrid silica, 25 ion exchange, 30, 37 maintenance, 29, 37 normal phase, 25 INDEX octyl–Si, 26 organic washing, 29 over-pressurize, 32 oxidation protection, 38 phenyl–Si, 27, 48 polymeric, 25, 32, 39 protein separation, 37 SCX-Si, 30 separation model, silica, 25 silica pH limits, 25 size separation, 31 SPE, 44–45 standard, 23 storage, 29 void volume, 30, 44 washing, 20, 29, 37, 48 zirconium, 25, 31, 39 Zr washing, 31 Column bridge, 30, 42, 150 Column cleaning, 28, 134 Column efficiency, 46 Column switching, 133 Combinational library, 113 Combinational synthesis, 113 Common daughter ion, 104 Competitive systems, Complex Biologicals ID, 96 Compound discovery, 78, 95 purification, 115 Compression fitting, 19 Computer control and acquisition, 14 searchable database, 114 system, 68, 74 Containment vessel seating, 92 Contaminated water, 33 Conversion completion, 96 Core molecule, 113 C-term amino acid attachment, 113 Curtain gas, 64 Cutting agents, 99 Cyclotonic frequency, 82 CZE/MS model system, Database library searches, 78 Data/control system, 66, 150 Data libraries, user customized, 129 Data systems, 64, 66, 129 Daughter ion, 1, 52, 60, 101 fragments, 79, 129 scans, 106, 115, 122 159 INDEX Daughter mode, 64 SCANQ1, SIMQ2, 104 dc/RF field, 68, 72 signal, 6, 62, 67–68, 72 scanning, 69, 72, 85 voltage, 106 Dead volume, 18 Deconvolution software, 53, 121 Definitive identification, 98 Definitive structure, 95 Degradation products, 111, 112 Deluant ID, 98 Denatured protein MW, 120 Denaturing buffer, 122 De novo protein purification, 121 Destabilizing frequency, 106 Detector, 14, 56–57, 150 charged aerosol, 57, 135 diode array UV, 14, 80 dynode, 62, 73, electron multiplier, 73, 85 electrooptical array, 83 fluorescence (FL), 56 FTMS plates, 82 horn, 73, 85 mass, 85 microflow cell, 18 refractive index (RI), 57 secondary, 55–57 UV, 36, 56, 135 Diagnostics gradient, 46 MS problems, 88 Differential pumping, 150 Digital converter, 68, 73 Diode array impact detector, 64 limitations, 95 Disease agent, 96 Diverter valve, 29 DNA restriction fragments MS size limits, 130 structure studies, 96 Double-bond calculation, 148 Double-sector MS, 84 Draw out lens, 72 Drive cam, 12 Drug by-products, 112 candidates, 111 discovery, 111–114 metabolite structure, 115 packing artifacts, 118 Drug development, 111 Drugs of abuse screening, 99 Dynode mass size limits, 130 Edman degradation, 113 EDTPA chelator, 31, 39 Efficiency factor, 48, 150 for column aging, 48 EI and CI, 149–150 Ejection voltage (LIT), 79 Electrical continuity check, 92 Electromagnet, 83 Electron cascade, 73 Electron gate, 75–76 Electrophoresis, 97 Electrospray (ES), 52, 53, 150 Elemental composition, 145–149 Elevated temperature, 128 EM voltage, 75, 91 Entrained gases, 37 Entrance lens cleaning, 89 Environmental soil and water, 101 Enzymatic activity, 96, 119 cleavage, 97, 122 EPA methods, 7, 98 ESI-LC/MS system protein mol wt., 120 Evacuation of MS system, 92 Excitation voltage, 79 Explosive mazing, 122 Explosive residues, 98 FAQs, 131–138 FDA approval, 111 registration, 115 Filament switching, 128 Filter, 41, 132 degas solvents, 128 fritted column, 17, 23 microporous, 44 solvent, 42 Final analyzer (Q3), 104 Fitting column inlet, 21 Flight tube, 80 folding, 81 TOF, 79 Flow cell volume, 14 Flow rate check, 34 Focus lens, 61 Fore pump, 17, 59, 85 Formulations, 97, 101, 117 Fossil proteins, 125 160 Fourier transform, 60, 81 “chirp” signal, 82 LC/MS, 150 sensitivity, 83 software, 82 Four solvent gradient, 16, 104 Four solvent gradient HPLC, 101 Fragmentation analysis, 103 cleavage, 103 heavy inert gas, 75, 113 library ID, 99, 143 mass intervals, 148 pattern matching, 6, 145 peptides, 123 scans, 105 structure studies, 115, 143 Frequency “chirp” signals, 82 Full scan mode, 104 Future of LC/MS, 127 Gas chromatography, 151 confirmation, 101 LC/MS conversion, 7, 137 limiting factors, model system, Gauges, vacuum pressure, 87–88 Gel filtration, 120 Gel permeation (GPC), 31 desalting, 30 mixture clean up, 97 molecular weights, 120 Generic LC/MS, 2, 114 Glass cathode surface, 78 Glyburide, 106 Glycosylated groups, 124 Gradient HPLC, 14–15 two-pump, 15, 36 isocratic development, 46 mixing valve, 15, 36 performance, 36, 48 Grounding strap, 69 Guard column, 44 Hazardous materials, 101 Hazmat studies, 101 Heated filament electrode, 75 Heavy inert gases, 104, 106 Helium gas, 131 solvent purging, 42 trap ion stabilization, 76 Herbicide production, 108 High sensitivity, 106 FTMS, 108 INDEX impurity detection, 97 linear ion trap, 108 High vacuum, 51, 59, 85, 135 History of sample, 145 Hormones, 124 Hot cathode gauge, 88 Housekeeping proteins, 119 HPLC, compression fittings, 18 data model, detectors, 56–57 dual-pump gradient, 15, 46 FAQs, 131 gradient system, 2, inert, 22 injector, 13 isocratic system, 2, 14 grade solvents, 41 gradient, 14, 131 limiting factors, maintenance, 33 micro syringe pump, 16 microflow, 16 mixing-valve gradient, 15 peak sharpening, 35, 131–133 pump, 10–11 pump head, 11 piston and seal, 10 scouting gradient, 114 separation speed, 128 system washing, 35 tubing and fittings, 35 ultra-fast systems, 128 water purity check, 37 HPLC column, 6, 9, 21 aminopropyl-, 27 C18, 23 cleaning, 39, 134 cyanopropyl, 27 high resolution, 16 killers, 133 lifetime, 28 model, 22 packing, 22 separation modes, 22 versatility, 132 Hybrid columns, 23, 24, 26 Hybrid MS/MS, 4, 83, 108, 150 Hydrophobic interaction, 30 Hyperbolic ion trap orbits, 76 Immiscible solvents, 39 Immunology screens, 99 Impactor plate, 54 161 INDEX Industrial waste dump, 101 Injector, 13, 151 loop and valve diagram, 13 loop capacity, 44 loop washing, 14, 35 microvolume, 18 plugging, 35 Interface, 51 APCI, 52 electrospray (ESI), 52, 134 FAQs, 134 ion spray(IS), 52, 55–6, 134 multimode source, 54 nanospray, 54, 134 robotic spotter plate, 52 switching, 62 Interfering compounds, 99 Intermediate compounds, 112 Internal standard, 151 deuterated, 102 In vitro matrix, 111 Ion accumulator, 108 Ion cyclotron resonance, 82 Ion detector, 5, 62–3, 73, 135 diode array, 80 dynode, 75 replacement, 91–2 Ion ejection from trap, 78 Ion exchange, 38, 151 weak, 30 Ion gate, 75–6 Ionization, 51 filament, 128 fly-through, 128 Ion monitoring(SIM), 101 Ion pairing reagent, 2, 43, 133 Ion spray (IS), 52, 54, 151 alternating filament, 128 interface, 3, Ion trap, 59, 106, 135, 151 three-dimensional, 75 analyzers, 74 detector (ITD), 74 ion detector, 78 linear, 78–9 MS/MS, 74, 106 sensitivity, 76 systems, 63 Isocratic system, 10 Isotope-coded reagents, 124 Isotopic abundance, 146 Isotopic patterns, 103, 146 Large biochemicals, 119, 130, 135 Laser beam bombarding, 52, 79–80 LC advantages, LC/LC/MS/MS systems, 128 LC/MS, 4, 59 basic (isocratic) system, buffers, 140 data model, FAQs, 136 generic methods, 11, 114 interfaces, 51, 60 ion trap, 59 overview, 59–60, 127 protein MW, 120 proteomics, 95 purification guide, 96 quadrupole, 59 sales, separations database, 114 solvents, 140 startup, 92 system costs, system model, ultra-fast, 128 use problems, LC/MS Applications in Drug Development, 118 LC/MS/MS, 5, 79, 103, 137 fragmentation, 97 hybrid systems, 97 operating modes, 66, 105 structural studies, 115 system model, triple quad or tandem, 65 LC/QFTMS system, 108 LC/Qtrap system, 108 Libraries, 104 database searching, 4, 143 precursor ID, 120 spectral, 129 user customized, 130 Light scattering detectors, 120 Linear ion trap (LIT), 135, 151 analyzer, 78–79 capacity, 79 MS/MS scanning, 78–79, 106 trace sensitivity, 108 Linked scan mode, 66 Logical fragmentation interval, 148 Magnetic sector analyzers, 83 Maintenance, 30, 85 MALDI/TOF, 64, 79, 122 flight tube, 80 162 MALDI/TOF (continued ) lab cost, spotter plate source, 52, 122 protein structure, 112, 136 MALDI/TOF/TOF system, 109 Mandated testing, 98 Manufacturing control, 117 Manufacturing QC, 101 Mass analyzer, 71 Mass/charge ratios, 52 Mass intervals, 148 Mass range scanning, 104, 137 Mass spectral database customizing, 130 searching, 104, 143 Mass spectrometer, 16 analyzer, analyzer vacuum, 57 cleaning, 89, 135 differing mass linearity, 83 hybrid MS/MS, 64 interface, 51 ion trap, 63–64 laser source, 80 lens, 72 LIT, 135 MALDI/TOF, 64, 109 quadrupole, 63 tandem, 64 tuning, 88 Mass spectrum, 67 Measurement precision, 108 Mechanical oil pump, rotary vane, 86 Merrifield synthesis, 113 Metabolic enhancement, 111 Metabolites drug, 116 structure determination, 97 Metal capillary, 53 Metal chelators, 30, 38 Methods development automated, 49 gradient, 46 isocratic, 46 Microflow columns, 134 Middle quad, Q2, 104, 108 Mixed mode columns, 28, 31 Mobile phase, 9, 41, 151 filtering and degassing, 42 Molecular ion adducts, 145 molecular weight, 145 protein envelope, 120 INDEX Molecular weight, 6, 145, 151 calculation, 120 determination, 60 effect of adducts, 145 information, 111 peak labeling, 136 proteins, 120 MRM mode, 64, 106 SIMQ1, SIMQ3, 104, 106 MS detector replacement, 57 MS/MS, 60, 151 analyzers, 64 capabilities, 108 experiments, 66, 135 hybrid, 60, 66 libraries, 119 operating modes, 66, 105 standard conditions, 114 system, 5, 104 MS secondary analyzer, 104 MSn type systems, 108 Multiple protein ions, 53, 120 m/z ratio, 6, 52, 67, 71, 80, 152 Nanoflow systems, 17, 52 Nanospray, 54 Nebulizer gases, 54, 137 Neutral loss, 104, 105, 152 linked SCANQ1, SCANQ3, 105 precursor, 106 NIST spectral library, 129, 137 Nitric acid washing, 20, 35 Nitrogen rule, 144 Nonpolar impurity, 37 Nonvolatile buffer, 2, 18 Normal phase, 25, 26, 152 N-terminal amino acid, 113 Octyldecyl-, 26 Off-spec production, 101 Oil changing, 88 Oil diffusion pump, 86, 135 shut down, 87, 89 On-column concentration, 45–46 Optical isomers, 28 Optimized methods, 114 Organic acids pKa , 43 Outlet fitting pores, 17, 21 Overpressure pump setting, 131 Oxalate column washout, 30, 38 Oxidized amino columns, 30 Oxymyoprotein, 125 163 INDEX Pacification, 20, 4, 35, 134, 152 Packaging artifacts, 111 Parallel synthesis, 113 Parent mode, 64 SIMQ1, SCANQ3, 66, 104 Partial amide formation, 113 Particle diameters, 23 Pascal (PA), 57 Peak detection, 69 labeling, 116, 129 matching, 84 sharpening, 22 Peptide, 120, 124 hormones, 124 structures, 120 Pesticides, 98, 101 pH control, 2, 42 Phospholipids, 119 Phosphorylated groups, 124 Photodiode arrays, 64, 135 Pinhole entrance, 61, 74, 90 Pirani gauges, 87 Piston and seal, 11, 152 broken pump, 34 pKa , 43, 133 Polyacrylamide plates, 122 Polymeric columns, 133 bonded phase, 32 pressure protection, 39 Polysaccharide ID, 97 Polystyrene immobiling beads, 113 Porous frit, 21 Preclinical testing, 111 Precolumn cleanup, 45, 98 Precursor ion, 152 QA purity, 101, 106 scanning, 104 Preparative purification, 114 Pressure checking, 25, 32–33 differential, 61 sensitivity, 38 polymer columns, 25 transducer, 12 Primary fragments, 104 Priority pollutants testing, 98 Process QC monitoring, 116 Protein ESI-QTOF/TOF, 109 molecular weight, 53, 120 multicharged ions, 53, 120 structure determination, 122 types, 119 Proteomics, 95, 119 PTM proteins, 119, 124 Pulse dampener, 12 Pulsed laser source, 52, 122 Pumps finish, 59 head wetting, 42 maintenance, 33, 48 plunger, 11 roughing, 59, 85 seal, 12 syringe, 17 QC laboratories, 101 QqQ triple quadrupole, 108 QTOF MS/MS, 108 Qtrap system, 7, 108 costs, Quadrupole, 62, 71, 135, 152 maintenance, 91 Q1, 104 rods, 63, 72, 91 Quadrupole-based ion trap, 108 Quantitate, 69 Quaternary solvent system, 16, 101 Rapid response bioassay, 111 Rational synthesis, 112 Reaction impurities, 115 Regulator agency, 98 Reluctance, 83 Repeller, 61, 75 Resolution equation, 47, 152 Restriction fragments, 97, 130 Retention factor, 47, 152 Reverse phase, 3, 28, 152 RF signal, 62 Ring electrode, 74, 76, 152 gas inlet port, 75 Robotic workstations, 52, 131 Rough pump, 17, 152 rotary vane, 59 hoses, 59 oil change, 59 Sample preparation, 41, 44, 132 Sampling rate, 69 SCAN mode, 152 four-segment, 75–76, 106 quadrupole, 71, 104 range, voltage, 62–63 164 SCAN, SCAN mode, 104, 105 SCAN, SIM mode, 104 SCF, 8, 153 model system, SCF/MS model system, Scouting gradient, 114 SDS, 122 Searchable spectral library, 104 peptides, 120 Secondary detectors, 55–56, 75, 134, 153 Sensitivity requirements, 103 Separation factor, 47, 153 Separation mixed modes, 28, 31 Sequencing proteins, 120 Serendipitous synthesis, 96 SIC, 153 Signal amplification, 73 Silica, 25 columns, 38 gel permeation, 31, 120 column hydration, 26 nonpolar washout, 38 pressure resistance, 37 problems, 25, 133 surface charge states, 26 SIM mode, 101, 104, 153 SIM/SCAN 104, 105, SIM/SIM, 104, 106 Six column volume flush, 42 Size separation, 31, 120, 133, 153 Solvent, 41, 132 air bubbles, 42 analyzer rinsing, 91 contamination, 88 gradients, 36, 48 degassing, 10, 42 reservoir, 10, 42 volatilization, 51 Source body, 89 cleaning, 88–91 contamination, 89 disassembly, 89 reassembly, 91 Spatial array detector, 83 SPE cartridge, 45, 132, 141, 153 cleaning water, 43 sample preparation, 45 windowing mixtures, 45 Spectra, 67, 85, 145, 153 FTMS, 82 Spherical particles, 23 Splitter, 18, 51 Spotter plate, 79 INDEX Stable circular path, 77 Standard LC/MS screening, 113 Startup HPLC, 42, 136 LC/MS, 92, 136 Stator plates, 87 Stepping motor drive, 12 Stokes radius, 120 Storage voltage, 76 Street drugs, 99 Structure ID, 123 interpretation, 5, 98, 143 false assignment, 143 protein mapping, 123 Superconducting detector, 130 Supersonic molecular beams, 128 Surge protector grounding, 92 Surrogate, 153 Switch-valve failure diagnostic, 36 System electrical grounding, 92 reassembly, 92 shutdown, 89 startup, 92 Tandem mass spectrometer, 64 Target compound, 52, 97, 114, 153 Target spectra tuning, 143 Temperature, 153 changes, 48 elevated in LC/MS, 128 ramps, 25 with zirconium, 32, 133 Therapeutic drug screens, 101, 111 Thermal collision, 104 Thermoconductivity gauge, 87 Three-dimensional folding, 119 Threshold for autotune, 89 TIC peak labeling, 116, 129 Time-of-flight LC/MS, 64, 135, 154 analyzer, 59, 79 flight tube, 80–81 large mass, 108, 130 Time slice window, 80 Torr, 51, 87 Total ion chromatogram (TIC), 67, 118, 154 Toxicology, 98 Trace analysis, 103 contaminates, 95 impurities, 97, 103 Tracer labeling, 124 165 INDEX Transmitter FTMS plates, 82 Trapping cell, 64 electrodes, 78 frequency, 106 FTMS plates, 82 LIT volume, 106 voltage, 74 Triple quad LC/MS/MS, 64–65, 104, 154 Troubleshooting LC/MS, 117 Trypsin, protein cleavage, 124 Tubing, 18, 35 Turbomolecular pump, 154 three-dimensional ion trap, 75 linear ion trap, 87 service, 88 shutdown, 87 trade in, 88, 135 venting, 87, 89 Turnkey quantitation, 127 Two-dimensional gel electrophoresis, 97, 122 Two-dimensional LC, 128 Ultrafast LC/MS, 128 Ultrasonication, 42, 44 Ultraviolet detector, 118 Union, 18 Urine and plasma samples, 101 Vacuum, 61 high, 59 measurement, 87 pumping system, 59 rotary rough pump, 59, 85 seating, 92 turbo pump, 61 two stage, 59, 85 Valve, solvent diversion, 62 Very fast analyzer, 109 Voids, 45, 154 Void volume, 47, 154 Volatile buffer, 29 Volatiles and extractables, 98 Volumes, extra-column, 22 Washing out metals, 30, 38 Water, 36, 132 HPLC grade, 36 purification, 37, 41 Windowing with SPE, 45 Zero–dead volume, 18 Zirconium columns, 25, 31, 133 Bronstead acids, 39, 43 charge states, 26 chelating compounds, 31 cleaning, 32, 39 elevated temperature, 133 pH stability, 43 [...]... compounds in a mixture The final oven temperature necessary to remove a large compound from a column can degrade many thermally labile compounds In the last two years, LC/ MS sales have nearly equaled GC /MS sales because of the additional compounds that can be analyzed by LC/ MS and the greater range of separation variables that can be utilized in HPLC separation The editors of Analytical Instrumentation Industry... column can separate almost any mixture that can be dissolved A mass spectrometer can ionize the separated peak solution and provide a molecular weight for each peak component An LC/ MS/ MS system can fragment the parent ion into a distinctive fragmentation pattern and can separate the daughter ions for identification and quantitation The characteristic fragmentation pattern from each parent ion can be identified... industrial laboratories as well as other areas of LC/ MS application that are anticipated when regulated methods become available Special emphasis is placed on drug discovery and development, protein analysis, impurities, and metabolite determinations These areas have fueled the rapid growth of LC/ MS sales in the last few years The needs of these labs go beyond the desire to provide separation and molecular... 5971 MSD mass spectrometer from a GC /MS that had been purchased originally for $86,000 with an $18,000 three-solvent gradient HPLC and a $12,000 ion spray interface Getting started in LC/ MS is not a casual adventure 1.5 COMPETITIVE SYSTEMS HPLC is not the only separation system being used as a front end for mass spectral analysis Applications using GC /MS preceded LC/ MS by a number of years and are very... cleaning contaminated analyzers are explained The basic theory for controlling analyzer and detector sensitivity and scanning ranges is discussed Two of the great advances in interpretation of mass spectral data have been the introduction of accurate mass-molecular-weight determination and computer scanning of library databases of known fragmentation patterns to aid compound identification These have... data systems for GC /MS equipment I added consulting and teaching in this specialty to my portfolio and designed a book, GC /MS: A Practical User’s Guide, to provide a teaching tool Again, I added a final chapter on the growing art of LC/ MS I feel another book and course are needed now that commercial sales of LC/ MS systems has nearly equaled those of GC /MS systems This tool combines my expertise and interests... mixtures Fragmentation studies using LC/ MS, LC/ MS/ MS, and mixed analyzer systems to supplement LC/ MS comprise a rapidly growing technology It is important to understand the changes in system costs, hardware configurations, applications, and techniques that seem to be driving these changes 1.3 LC/ MS SYSTEMS Basically, an LC/ MS system is an HPLC pumping system, injector, and column married to a mass spectrometer... evacuated mass spectrometer Electrical lenses focus the charged beam of ions and carry them into the mass analyzer They are swept down the analyzer by a scanning direct-current/radio-frequency (dc/RF) signal that selects ions of a particular mass/charge (m/z) value to strike the Voltage signal strength (V) Spectral data plane z m/ µ) (am Run time (min) FIGURE 1.4 LC/ MS data model Chromatographic data... look at the components that make up the various analyzers used in mass spectrometry We compare the advantages and areas of specific applications of quadrupole, ion trap, Fourier transform, and time-of-flight (TOF) analyzer configurations A variety of systems for generating the high vacuum used in analyzer operations are described and evaluated Techniques for maintain a system under operating conditions and... diode-array UV detectors can detect compounds that absorb UV light anywhere within wavelengths available to the array The final hardware component in an LC/ MS system is a computer, used for data acquisition and processing This can be used simply as a stripchart recorder or an integrator, or it can be used as a computer-based system for controlling all system components and acquiring peak data, quantifying