Table of Contents

Preface XI

The Structure of HPLC-MS for Practitioners XIII

List of Contributors XV

Part I Overview, Pitfalls, Hardware-Requirements 1

1 State of the Art in the LC/MS 3
O. Schmitz

1.1 Introduction 3

1.2 Ionization Methods at Atmospheric Pressure 5

1.2.1 Overview of API Methods 6

1.2.2 ESI 6

1.2.3 APCI 8

1.2.4 APPI 9

1.2.5 APLI 10

1.2.6 Determination of Ion Suppression 11

1.2.7 Best Ionization for Each Question 11

1.3 Mass Analyzer 12

1.4 Future Developments 13

1.5 What Should You Look forWhen Buying a Mass Spectrometer? 14

References 15

2 Technical Aspects and Pitfalls of LC/MS Hyphenation 19
M.M. Martin

2.1 Instrumental Requirements for LC/MS Analysis – Configuring the Right System for Your Analytical Challenge 20

2.1.1 (U)HPLC andMass Spectrometry – Not Just a Mere Front-End 20

2.1.2 UHPLC System Optimization – Gradient Delay and Extra-column Volumes 21

2.1.3 Does YourMass Spectrometer Fit Your Purpose? 33

2.1.4 Data Rates and Cycle Times ofModernMass Spectrometers 38

2.1.5 Complementary Information by Additional Detectors or Mass SpectrometryWon’t Save theWorld 39

2.2 LC/MS Method Development and HPLC Method Adaptation – How toMakeMy LC Fit forMS? 43

2.2.1 Method Development LC/MS – LC Fits theMS Purposes 44

2.2.2 Converting Classical HPLC Methods into LC/MS 53

2.3 Pitfalls and Error Sources – Sometimes Things Do GoWrong 54

2.3.1 No Signal at All 54

2.3.2 Inappropriate Ion Source Settings and Their Impact on the Chromatogram 56

2.3.3 Ion Suppression 58

2.3.4 Unknown Mass Signals in the Mass Spectrum 59

2.3.5 Instrumental Reasons for the Misinterpretation ofMass Spectra 65

2.4 Conclusion 68

2.5 Abbreviations 69

References 70

3 Aspects of the Development of Methods in LC/MS Coupling 73
T. Teutenberg, T. Hetzel, C. Portner, S. Wiese, C. vom Eyser, andJ. Tuerk

3.1 Introduction 73

3.2 From Target to Screening Analysis 74

3.2.1 Target Analysis 74

3.2.2 Suspected-Target Screening 74

3.2.3 Non-target Screening 74

3.2.4 Comparable Overview of the Different Acquisition Modes 75

3.3 The Optimization of Parameters in Chromatography andMass Spectrometry 75

3.3.1 Requirements and Recommendations for HPLC/MS Analysis Taking DIN 38407-47 as an Example 75

3.3.2 The Definition of Critical Peak Pairs in the Context of HPLC/MS Coupling 77

3.3.3 The Separation of Polar Components fromthe Column Void Time 79

3.3.4 Determining the HPLC Method Parameters Using the Example of the Separation of Selected Pharmaceuticals 80

3.3.5 Carrying out Screening Experiments 84

3.3.6 Evaluation of the Data and Discussion of the Influencing Parameters 86

3.3.7 Using Simulation Software for Fine Optimization 98

3.3.8 Choosing the Stationary Phase Support 99

3.3.9 The Influence of the Inner Column Diameter and theMobile Phase Flow Rate 103

3.3.10 The Influence of the Injection Volume 104

3.3.11 Establishing theMass Spectrometric Parameters 115

3.3.12 Optimization of theMass Spectrometric Parameters 117

3.3.13 Quantification Using LC/MS 122

3.3.14 Screening Using LC/MS 128

3.3.15 Miniaturization – LC/MS Quo Vadis? 132

References 135

Part II Tips, Examples, Trends 139

4 LC/MS for Everybody/for Everything? – LC/MS Tips 141
F. Mandel

4.1 Introduction 141

4.2 Tip Number 1 142

4.2.1 Choosing the Right LC/MS Interface 142

4.3 Tip Number 2 148

4.3.1 Which Mobile Phases Are Compatible with LC/MS? 148

4.4 Tip Number 3 149

4.4.1 Phosphate Buffer – The Exception 149

4.5 Tip Number 4 150

4.5.1 Paired Ions 150

4.5.2 Which “Antidote” Is Available? 151

4.5.3 Summary 152

4.6 Tip Number 5 152

4.6.1 Using Additives to Enhance Electrospray Ionization 152

4.6.2 Additives for APCI 153

4.6.3 Summary 154

4.7 Tip Number 6 154

4.7.1 How Can I Enhance Sensitivity of Detection? 154

4.8 Tip Number 7 155

4.8.1 No Linear Response and Poor Dynamic Range? 155

4.8.2 The Reasons 156

4.8.3 Possible Solutions 156

4.8.4 Summary 157

4.9 Tip Number 8 157

4.9.1 HowMuch MSn Do I Need? 157

4.9.2 Solutions 158

4.9.3 Summary 158

4.10 Need More Help? 166

References 167

Part III User Reports 169

5 LC Coupled to MS – a User Report 171
A.Muller and A. Hofmann

References 176

6 ProblemSolvingwithHPLC/MS– aPracticalViewfromPractitioners 177
E. Fleischer

6.1 Introduction and Scope 177

6.2 Case Example 1 181

6.2.1 Investigation ofMethohexital Impurities and Decomposition Products 181

6.2.2 Sample Preparation 181

6.3 Case Example 2 183

6.3.1 Separation of Oligomers from Caprolactam, Multicomponent Separation of Impurities on a Gram Scale 183

6.4 Case Example 3 184

6.4.1 Preparation and Isolation of bis-Nalbuphine from Nalbuphine 184

6.5 Case Example 4 186

6.5.1 Isolation and Elucidation of Dopamine Impurities 186

7 LC/MS from the Perspective of a Maintenance Engineer 189
O.Muller

7.1 Introduction and Historical Summary 189

7.2 Spray Techniques 190

7.3 Passage Through the Ion Path 191

7.4 The Analyzer 191

7.5 Maintenance 193

References 198

Part IV Vendor’s Reports 201

8 LC/MS – the Past, Present, and Future 203
T.L. Sheehan and F. Mandel

9 Vendor’s Report – SCIEX 207
D. Schleuder

10 Manufacturer Report – Thermo Fisher Scientific 213
M.M. Martin

10.1 Liquid Chromatography for LC/MS 214

10.2 Mass Spectrometry for LC/MS 215

10.3 Integrated LC/MS Solutions 217

10.4 Software 217

References 219

About the Authors 221

Index 227

About the Author

Stavros Kromidas studied biology and chemistry at the University of Saarbrücken, where he obtained his Ph.D. degree on the development of new chiral stationary phases for HPLC in 1983. After working for Waters GmbH for five years, he founded NOVIA GmbH, a provider of professional training and consulting in analytical chemistry, serving as the CEO until 2001. Since 2001 he works as an independent consultant for analytical chemistry, based in Saarbrücken (Germany). For more than 20 years he has regularly held lectures and training courses on HPLC, and has authored numerous articles and several books on various aspects of chromatography.