Table of Contents
Cover
Title Page
Copyright
List of Contributors
Chapter 1: Detection in Capillary Electrophoresis – An Introduction
1.1 UV Absorption
1.2 Fluorescence
1.3 Conductivity
1.4 Mass Spectrometry
References
Chapter 2: Electrospray Ionization Interface Development for Capillary Electrophoresis–Mass Spectrometry
2.1 A Brief Introduction to the Development of CE-MS
2.2 Fundamentals of ESI and Electrochemical Reactions in CE-MS
2.3 Interface Designs
2.4 Specific Interface Applications
2.5 Conclusion
Abbreviations
Acknowledgments
References
Chapter 3: Sheath Liquids in CE-MS: Role, Parameters, and Optimization
3.1 Introduction
3.2 Sheath-Liquid Functions and Sheath-Flow Interface Design
3.3 Sheath-Liquid-Related Parameters and their Selection
3.4 Sheath Liquids for Non-ESI CE-MS Interfaces
3.5 Sheath-Flow Chemistry
3.6 Conclusions
References
Chapter 4: Recent Developments of Microchip Capillary Electrophoresis Coupled with Mass Spectrometry
4.1 Introduction
4.2 Microchip Capillary Electrophoresis
4.3 Reviews on MCE and MCE-MS
4.4 Principal Requirements for MCE- MS
4.5 MCEMS by Direct Off-Chip Spraying
4.6 MCE- MS with Connected Sprayer
4.7 MCE-MS Devices with Integrated Sprayer
4.8 Multidimensional MCE-MS Devices
4.9 Conclusions and Perspectives
Appendix
References
Chapter 5: On-Line Electrophoretic, Electrochromatographic, and Chromatographic Sample Concentration in CE-MS
5.1 Introduction
5.2 Electrophoretic and Electrochromatographic Sample Concentration or Stacking
5.3 On-line/In-line SPE with CE-MS
5.4 Conclusion
Acknowledgment
References
Chapter 6: CE-MS in Drug Analysis and Bioanalysis
6.1 Introduction
6.2 CE-MS in Drug Analysis
6.3 CE-MS in Bioanalysis
6.4 CE-MS in Drug Metabolism Studies
6.5 Quantitative Aspects in CE-MS
6.6 Conclusions
Abbreviations
References
Chapter 7: CE-MS for the analysis of intact proteins
7.1 Introduction
7.2 CE of Intact Proteins
7.3 MS Detection of Intact Proteins
7.4 Applications of Intact Protein CE-MS
7.5 Conclusions
Abbreviations
References
Chapter 8: CE-MS in Food Analysis and Foodomics
8.1 Introduction: CE-MS, Food Analysis, and Foodomics
8.2 Concluding Remarks
Acknowledgments
References
Chapter 9: CE-MS in Forensic Sciences with Focus on Forensic Toxicology
9.1 Introduction
9.2 Sample Preparation of Forensically Relevant Matrices
9.3 Separation Modes and Analytical Conditions
9.4 Applications
9.5 Conclusions
References
Chapter 10: CE-MS in Metabolomics
10.1 Introduction
10.2 Sample Preparation and MS Systems
10.3 Application
10.4 Conclusions
Acknowledgments
References
Chapter 11: CE-MS for Clinical Proteomics and Metabolomics: Strategies and Applications
11.1 Introduction
11.2 Clinical Proteomics
11.3 Clinical Metabolomics
11.4 Conclusions and Perspectives
Abbreviations
Acknowledgments
References
Index
End User License Agreement
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Guide
Cover
Table of Contents
Begin Reading
List of Illustrations
Chapter 2: Electrospray Ionization Interface Development for Capillary Electrophoresis–Mass Spectrometry
Figure 2.1 Schematic of positive-mode electrospray ionization.
Figure 2.2 Common sheath-flow interface arrangements. (a) coaxial sheath-flow interface with sheath gas, (b) liquid junction interface, and (c) pressurized liquid junction interface.
Figure 2.3 Schematic illustration of the flow-through microvial interface apparatus, including a dissected view of needle tip with inserted capillary (inset).
Figure 2.4 Comparison of electrokinetically pumped nanospray CZE interfaces. (a) Overall design of the interface. The separation capillary is threaded through a PEEK cross (1.25 mm through hole) into a glass emitter. A tube (∼1 mm i.d.) connects a side arm of the cross to a sheath electrolyte reservoir, which is connected through a platinum electrode to a high-voltage power supply. The mass spectrometer inlet is held at ground potential. (b) Designs of three generations of interface. The original interface uses a flat separation capillary, which is able to approach within ∼1 mm of the emitter orifice; typical orifice diameters are 2–10 µm. The second generation uses a separation capillary with an etched tip, which approaches within ∼200 µm of the emitter orifice; typical orifice diameters are 2–10 µm. The third-generation interface also uses an etched separation capillary but with a much larger exit orifice. The etched tip approaches within a few micrometers of the orifice; typical orifice diameters are 15–35 µm.
Figure 2.5 Methods for creating electrical contact in sheathless interfaces. (a) Conductive coating applied to the emitter tip, (b) wire inserted at tip, (c) wire inserted through hole, (d) split-flow interface with a metal sheath, (e) porous, etched capillary walls in metal sleeve, (f) junction with metal sleeve, (g) microdialysis junction, and (h) junction with conductive emitter tip.
Figure 2.6 Principle of the HSPS sheathless interface (Beckman Coulter)/CESI 8000 (SCIEX).
Figure 2.7 Schematic of CITP/CZE-nanoESI-QQQ MS setup used in this study. The lower part shows a detailed view of the sheathless interface design (not to scale).
Figure 2.8 (a) Schematic diagram of automated cIEF-MS. The flow-through microvial provides the electrical contact and chemical environment for cIEF. The CE-MS interface improves the ionization efficiency of the mobilized effluent without significant dilution. (b) Total ion chromatogram for a protein mixture separated in an N–CHO coated capillary.
Figure 2.9 (a) A base peak chromatogram of total IgG tryptic digest from human serum, (b) an extracted ion chromatogram of IgG1 glycopeptides of high concentration with assignments, (c) an extracted ion chromatogram of IgG1 glycopeptides of low concentration with assignments. The blue square is N -acetylglucosamine, the red triangle is fucose, the green circle is mannose, the yellow circle is galactose, and the purple diamond is N -acetylneuraminic acid.
Chapter 3: Sheath Liquids in CE-MS: Role, Parameters, and Optimization
Figure 3.1 Different types of sheath-flow interfaces. Coaxial sheath-flow interface (a), miniaturized coaxial sheath-flow interface (b, reproduced from Liu et al . [12] with permission of Wiley), and miniaturized liquid junction interface (c, reproduced from Fanali et al . [13] with permission of Wiley).
Figure 3.2 Effect of hydrostatic pressure applied to the sheath-liquid reservoir in the miniaturized liquid junction interface (see Figure 3.1c) on migration times and resolution of four β-blockers.
Figure 3.3 Expanded view of the electrospray tip with an ionic boundary propagating into the CE capillary. The empty and filled circles correspond to the background electrolyte (BGE) and liquid sheath (LS) counterions, respectively.
Figure 3.4 Influence of liquid-sheath effect on selectivity. CE-UV electropherograms at 215 nm of a 1 mg ml−1 SLV308 sample solution containing four impurities. Methanol/water 50 : 50% (v/v) containing 0.2% formic acid was used as sheath liquid (SL). Conditions: (a) 100 mM TRIS adjusted to pH 2.5 with phosphoric acid, inlet vial–outlet vial: BGE-BGE; (b) 100 mM TRIS adjusted to pH 2.5 with phosphoric acid, inlet vial–outlet vial: BGE-SL; (c) 100 mM TRIS adjusted to pH 3.0 with formic acid, inlet vial–outlet vial: BGE-GE; (d) 100 mM TRIS adjusted to pH 3.0 with formic acid, inlet vial–outlet vial: BGE-SL.
Figure 3.5 Effect of methanol and iso -propanol content in SL on charge state distribution of Bet v 1a. BGE: 10 mmol l−1 NH4 HCO3 , pH 7.50; SL composition: 0.1% v/v FA in ultrapure water with (a) 25%, (b) 50%, (c) 75% v/v methanol, and (d) 50% v/v iso -propanol.
Figure 3.6 Influence of nebulizer pressure and SL flow rate on the peak height of malondialdehyde (MDA) obtained from the third set of experiments (tested ranges reduced to 1–10 µl min−1 for SL flow rate and 20–30 psi for nebulizer pressure for finding the optimum with higher precision) employed for optimizing the CE-MS interface parameters.
Figure 3.7 Sheath-liquid composition optimization. Experimental conditions: FS capillary 65 cm × 50 µm i.d., injection 500 mbar s, CE voltage 30 kV, sheath-liquid composition iso -propanol/water (50 : 50 v/v) and flow rate 25 µl min−1 . Influence of sensitivity on peak height/noise (H/N) of (a) % toluene, (b) % acetone, and (c) % formic acid.
Figure 3.8 Effect of the MS capillary voltage on the signal intensity obtained for phenacetin using a sheath liquid with 5% acetone as dopant ( ) and without dopant ( ).
Figure 3.9 Mass spectrum for melamine from a CZE-ESI-Q-TOFMS run using a deuterium oxide/mono-deuterated MeOH (80 : 20), 0.1% formic acid sheath liquid. Numbers in parentheses refer to mass error in ppm.
Figure 3.10 Effect of online DPPH• reaction experiments for selected components: (a) rosmarinic acid found in Rosmarinus officinalis extract and (b) caffeic acid found in Melissa officinalis .
Figure 3.11 Charge state distribution of human growth hormone with the respective electropherogram obtained in scan mode (m /z = 850–2000) with an acidic BGE (75 mM ammonium formate pH = 2.5 + ACN 20%, v/v) and a sheath liquid composed of (a) isopropanol–water–formic acid (49.5 : 49.5 : 1, v/v), (b) m-NBA 1.0% (w/v) in isopropanol–water–formic acid (49.5 : 49.5 : 1, v/v), and (c) sulfolane 0.5% (w/v) in isopropanol–water–formic acid (49.5 : 49.5 : 1, v/v).
Chapter 4: Recent Developments of Microchip Capillary Electrophoresis Coupled with Mass Spectrometry
Figure 4.1 Principle layout of a chip for capillary electrophoresis; a, buffer reservoir; b, sample reservoir; c, sample waste reservoir; d, outlet reservoir; e, point of detection. (a) Design for pinched sample injection. (b) Design for gated sample injection.
Figure 4.2 (a) Photograph of the Agilent LabChip™. (b) Channel detail of LabChip™.
Figure 4.3 Simplified fabrication process for fast prototyping of microfluidic chips on soda-lime glass substrates.
Figure 4.4 MS coupling of microfluidic chips; (a) different approaches to generate the nanospray; (b) microfluidic device for HPLC or CE separation.
Figure 4.5 Schematic representation of the chip-CE configuration using (a) a disposable nanoelectrospray emitter or (b) a sheath-flow ESMS interface.
Figure 4.6 Glass chip–based CE/MS apparatus and the expanded view of the coupled microsprayer.
Figure 4.7 Diagram of the MCE device with a subatmospheric ES interface. The expanded view shows the coupling of the ESI tip with the separation channel in the liquid junction.
Figure 4.8 Separation of bradykinin, angiotensin I, angiotensin II, [Sar1, Ala8]angiotensin II, and [Val4]angiotensin III five ions). (a) Short chip and (b) long chip. Separation buffer, 50 mM acetic acid–ammonium acetate buffer (pH 5.7) containing 50% (v/v) acetonitrile.
Figure 4.9 Schematic drawing of the MCE device with integrated nanospray emitter. SO, sample outlet; SI, sample inlet; BI, buffer inlet; MS, mass spectrometer.
Figure 4.10 Separation and mass spectrometric characterization of 4 LMW test solutes in <100 s at low-micromolar level.
Figure 4.11 Schematic diagrams of the short-channel (a) and the long-channel (b) CE-ESI-MS chips. The length of the separation channel (measured from the injection cross to the outlet) was 4.7 cm for the short-channel chip and 20.5 cm for the long-channel chip. For both chips, the channels were all 75 µm wide at full width and 10 µm deep. The reservoirs are labeled S (sample), B (buffer), SW (sample waste), and SC (side channel). The direction of electro-osmotic fluid flow is indicated by the arrows in (a).
Figure 4.12 Image of the electrospray plume generated from the corner of a CE-ESI-MS chip acquired with a CCD camera. The liquid being sprayed was 50/50 (v/v) methanol/water with 0.2% acetic acid. The voltages applied to the microchip reservoirs raised the potential at the spray tip to 3.5 kV above that of the mass spectrometer inlet and caused the liquid to be pumped out of the chip at a flow rate of ∼40 nl min−1 .
Figure 4.13 Schematic views of the SU-8 microchips used for direct coupling of cIEF to on-chip ESI/MS (a) and for multiplex cIEF-tITP separation coupled to on-chip ESI/MS (b).
Figure 4.14 Schematic diagram of the polymer microchip. Dimensions of the channel are 50 µm of width, 20 m of depth, and 30 mm of length. The width of the outlet channel is tapered from 50 to 10 µm. (Designations of the sample reservoir and buffer reservoir are incorrect in the Figure The buffer reservoir is at the beginning.)
Figure 4.15 Schematic for the microchip-based LC--CE-MS system. The blue squares on the microchip denote the location of weirs that were used to retain the packed particles. Valve 1 (V1) was used to perform LC injections, and valve 2 (V2) was used to open and close the vent line. Valves are shown in the “sample loading” configuration. Electrospray was performed from the lower- right corner of the microchip.
Figure 4.16 Electropherogram of glycans released from plasma glycoproteins using standard CE equipment with 35 cm separation length and MCE with a separation distance of 14 mm. Upper and lower axes correspond to the time (seconds) of the CE and microchip electrophoresis separation, respectively. Reproduced from Smejkal et al . [105] with permission of Elsevier.
Chapter 5: On-Line Electrophoretic, Electrochromatographic, and Chromatographic Sample Concentration in CE-MS
Figure 5.1 Simplified schematic of t-ITP stacking in capillary electrophoresis. (a) The leading electrolyte (LE) is injected before the sample solution (S). The terminating electrolyte (TE) is injected. The electrophoretic mobility of the leading ion is faster than the sample and terminating ions, all having the same sign of charge (+ or −). (b) Voltage is applied and the sample ions are stacked behind the leading ion. This ITP state is transient and dissipates with the continued application of voltage. (c), The t-ITP stacked sample ions separate by electrophoresis.
Figure 5.2 t-ITP CE-MS of cationic peptides. Conditions: Bare fused-silica capillary 104 cm × 50 mm; Separation voltage, 25.0 kV; LE, 50 mM ammonium acetate (pH 4.8); Separation electrolyte, 50 mM acetic acid (pH 3.1); Sample, the three peptides mixture in 25 mM acetic acid; Sample injection, 28 kV for 40 s; (a) Without t-ITP; (b) t-ITP with H+ as terminating ion where 50 mM ammonium acetate (pH 4.8) was injected (50 mbar for 60 s) prior to sample injection; (c) t-ITP with β-alanine as terminating ion where 50 mM ammonium acetate (pH 4.8) was injected (60 s at 50 mbar) prior to sample injection, 40 mM β-alanine (pH 3.6 with acetic acid) was then injected (50 mbar for 20 s).
Figure 5.3 Simplified schematic of field-enhanced or -amplified stacking in capillary electrophoresis with a fused-silica capillary and cathodic electro-osmotic flow (EOF). (a), The sample solution (S) is injected after conditioning the capillary with background solution (BGS) or electrolyte. If a voltage is applied, the field strength in the S > BGS and thus the electrophoretic velocity of the analyte (v ep ) will be faster in the S than in the BGS. (b) The cations (+) migrated quickly into the right or cathodic boundary between the matrix and BGS. The velocity decreased in the boundary and caused the stacking of the analytes. The same process occurred with the anions (−) but at the anodic boundary.
Figure 5.4 FASS-CE-MS/MS analysis of 10 ppb HAAs in DI water. Separation electrolyte: 50% MeOH containing 2.5% ammonium acetate buffer at pH 3.5. Peak designation: 1, DCAA; 2, TCAA; 3, DBAA; 4, MCAA; 5, MBAA.
Figure 5.5 Overlaid extracted ion electropherograms of 50 mg kg−1 butyl 1-(pyridinyl-4yl) piperidine 4-carboxylate derivative test mixture of potentially genotoxic alkyl halides (G, K, T, and M) using FESI at 10 kV for 150 s (a) of 500 mg kg−1 BPPC derivative test mix using typical hydrodynamic injection (b).
Figure 5.6 Effect of sample matrix concentration on focusing using dynamic pH junction and t-ITP. Conditions: bare fused-silica capillary, 90 cm × 50 mm; BGS, 0.5 M formic acid at pH 2.15; sample matrix, ammonium acetate at pH 7.5 with concentration varied (a) 10 mM, (b) 25 mM, (c) 50 mM, and (d) 75 mM; injection, 30 s of a mixture containing 1 mmol l−1 of each peptide; sample i.d., (1) l -Ala-l -Ala, (2) l -Leu-d -Leu, (3) Gly-d -Phe, (4) Gly-Gly-l -Leu.
Figure 5.7 Simplified schematic of analyte focusing by micelle collapse or AFMC in PF-EKC with a cathodic EOF. (a) The S of neutral analytes prepared in a micellar solution is injected after conditioning the capillary with BGS (nonmicellar) and injection of micellar solution for separation. The symbol * represents a low-conductivity electrolyte or organic-solvent-rich solution for micelle collapse. (b) Application of voltage caused the analytes to accumulate at the sb. (c) A final AFMC zone is formed when all the analytes are transported and released into the sb. (d) The micelles from the micellar solution penetrated the AFMC formed zone and caused them to separate by EKC principles. Note that the EOF is to the cathode where the detector is also located.
Figure 5.8 Extracted base peak electropherograms for pindolol (m /z = 249) and metoprolol (m /z = 268) analyzed by CZE-ESI-MS (a) and MSS-CZE-ESI-MS (b). BGS = 30 mM ammonium acetate, pH 5 with 20% acetonitrile; sample matrix = BGS (a) and 10 mM SDS with 13 mM ammonium acetate, pH 5 (b); S = 1.3 µg ml−1 (a) and 0.13 µg ml−1 (b) of pindolol and metoprolol; injection scheme: 2.6 mm (6 s at 50 mbar) of sample solution (a) and 0.6 cm (15 s at 50 mbar) of sample matrix followed by 2.6 cm (60 s at 50 mbar) of sample solution (b).
Figure 5.9 Schematic of on-line SPE CE-MS.
Figure 5.10 Electropherograms of CE-MS and SPE-CE-MS analysis of a peptide mixture. (a) CE-MS analysis of a peptide mixture at a concentration of 0.25 mg ml−1 . (b) SPE-CE-MS analysis of a peptide mixture at a concentration of 5 ng ml−1 , loading sample volume 50 µl. The CE condition: 10 mM, pH 3.80 ammonium formate as the running buffer; the separation capillary: 50 cm, 50 µm i.d. A 400 V cm−1 electric field was applied for SPE-CE-MS and CE-MS experiments.
Figure 5.11 Construction of a fritless microcartridge for in-line SPE CE-MS.
Figure 5.12 Extracted ion electropherograms obtained by SPE-CEMS of (a) blank CSF and (b) CSF spiked with the opioid peptides (5 ng ml−1 each). Experimental conditions: sample loading, 10 min at 930 mbar; elution, 63 s at 50 mbar; separation voltage, 25 kV; extracted ions: 434.7 (DynA), 611.2 (End1), and 574.2 (Met).
Chapter 6: CE-MS in Drug Analysis and Bioanalysis
Figure 6.1 Pharmaceutical and biomedical analysis: this chapter covers the fields of CE-MS in “drug analysis” and “bioanalysis.”
Figure 6.2 CE-MS electropherograms in negative ESI obtained for selected NSAIDs at 1 µg ml−1 . (a) Conventional CZE mode with a sheath-liquid interface; BGE: ammonium acetate 50 mM, pH 8.5, and (b) NACE mode with a sheathless interface; BGE, ammonium acetate 5 mM in ACN–MeOH 80 : 20 (v/v). (Reproduced from [24] with permission from Elsevier.)
Figure 6.3 CE-MS2 electropherogram of 0.02 µg ml−1 R -duloxetine in the presence of 100 µg ml−1 S -duloxetine achieved by the PFT countercurrent approach. (Reproduced from [56] with permission from Elsevier.)
Figure 6.4 Correlation between pK a values obtained for 50 drugs by CE-MS and (a) literature data and (b) values predicted by ACD/Labs. (Reproduced from [63] with permission from Wiley.)
Figure 6.5 CE-MS two-step workflow used in bioanalysis. (a) Screening with CE-TOF/MS and (b) Quantitation by CE-MS/MS of two urine samples containing cocaine and methadone, respectively. (Reproduced from [72] with permission from Elsevier.)
Figure 6.6 Qualitative evaluation of matrix effects by CE-MS. (a) Schematic representation of the coaxial sheath-flow interface used as a postcapillary infusion device and (b) Typical ESI-MS response measured.
Figure 6.7 Analysis of insulin by CE-UV-TOF/MS. (a) MS detection, total ion electropherogram (TIE); (b) MS detection, extracted mass spectrum; (c) MS detection, extracted ion electropherogram (XIE); and (d) UV detection. a. and e. insulin (calibration standard) from the first injection, b. and f. insulin (sample to be identified and quantified) from the second injection, c. procaine (UV-IS) from the first injection, d. procaine (UV-IS) from the second injection, * neutral excipients from the first injection, and ** neutral excipients from the second injection. (Reproduced from [46] with permission from Elsevier.)
Chapter 7: CE-MS for the analysis of intact proteins
Figure 7.1 CE-UV electropherogram of insulin obtained with (a) alkaline BGE and (b) acidic BGE. Upper trace: BGE containing 10% ACN. Lower trace: BGE without the addition of ACN.
Figure 7.2 Total ion electropherograms obtained using CZE-ESI-MS of a mixture of the basic proteins lysozyme (1), cytochrome c (2), and ribonuclease A (3) using a BGE of ammonium acetate buffer at pH 5.5 and (a) a bare fused-silica capillary or (b) an coated capillary. (c) Mass spectra of the main peaks obtained with the coated capillary.
Figure 7.3 Schematic representation for the most commonly used interfaces for (a) CE-MALDI-MS and (b) CE-ICP-MS.
Figure 7.4 (a) CZE-ESI-MS analysis of stressed hGH. The blue trace represents the EIE of hGH (1), deamidated hGH (2), and dideamidated hGH (3). The brown trace represents the EIE of peaks 1–3 after spontaneous elimination of PhePro. (B) Simulated isotope pattern of intact rhGH (I) and charge deconvoluted mass spectra of peaks 1 (II), 2 (III), and 3 (IV) of stressed rhGH, respectively. The blue line is to clarify the shift of the maximum of the isotopic distribution by 1 Da at a time.
Figure 7.5 CZE-ESI-MS of recombinant human erythropoietin employing a sheathless interface in combination with a neutrally coated capillary. (a) Base peak electropherogram; (b1) contour plot with zooms of (b2) the 14+ charge state of the glycoforms and (b3) the SiA13 sialoforms of the 14+ glycoforms.
Figure 7.6 CZE analysis with off-line MS detection of partially digested antibody. (a) CZE-UV electropherogram showing the charge variants of the Fc/2 (peak 1–3) and F(ab′)2 (peaks between 30 and 45 min) fragments. (b) MALDI-TOF-MS and (c) ESI-TOF-MS mass spectra obtained for the three separated and off-line collected Fc/2 charge variants. Identified glycoforms are annotated in panel (c).
Figure 7.7 CZE-ESI-MS analysis of trypsinogen sample using a positively charged coated capillary and a BGE of 25 mM ammonium acetate (pH 8.0) containing no (a) or 25 μM (b) aprotinin. Extracted-ion electropherograms of trypsinogen (2398 m /z ), trypsinogen variants (2397–2399, 1814.1, and 1798.5 m /z ) and cortisone (EOF marker) (371.2 m /z ) are depicted. (c) Mass spectra obtained for peak 4 during CZE-ESI-MS of trypsinogen using a BGE of 25 mM ammonium acetate (pH 8.0) containing no (I), 2 (II), 15 (III), 50 (IV), or 150 μM (V) aprotinin. Arrows indicate trypsinogen–aprotinin complex ions.
Figure 7.8 CZE-ICP-MS analysis of a rabbit liver MT sample using a 2-acrylamido-2-methyl-1-propanesulfonic acid coated capillary. The proteins are detected by their cadmium, copper, and zinc content as monitored with ICP-MS. Numbers indicate the MT isoforms that were identified.
Figure 7.9 CZE-ESI-MS analysis of four standard proteins. (a) Base peak electropherogram. Colored MS spectra correspond to migration profiles of the designated proteins. (b) ETD and HCD fragmentation spectra of cytochrome c with the identified fragment ions. (c) Sequence maps show combined fragmentation patterns of ETD and HCD for the four standard proteins. *Number of fragmentation sites: total fragmentation sites/overlapped fragmentation sites.
Figure 7.10 CZE-ESI-MS analysis of cytochrome c incubated with Bcl-XL peptide, showing both the oxidized (1) and reduced cytochrome c (2). Insets show the ESI mass spectra of the peaks.
Figure 7.11 CIEF-ESI-MS analysis of a mixture of the noncovalent protein complexes GADH (1) and CPK (2). (a) Mass spectra of monomeric, dissociated species are obtained upon addition of an acidic sheath flow and high ESI voltages. (b) Mass spectra of GAPDH tetrameric and CPK dimeric complexes are obtained with a medium-pH sheath liquid and lower ESI voltages.
Chapter 8: CE-MS in Food Analysis and Foodomics
Figure 8.1 Separation of PST mixture using four different CE methods. (a) Electropherogram of CZE-UV using 30 mM phosphate buffer pH 2.5, sample injected at 5 kV for 10 s. (b) Extracted ion electropherogram) of CZE-MS at selected m /z using 35 mM morpholine pH 5.0, sample injected at 5 kV for 10 s. (c) Electropherogram of CZE-C4D using 25 mM sodium acetate buffer pH 4.22, sample injected at 5 kV for 10 s. (d) Electropherogram of MEKC-FLD using 30 mM phosphate buffer at pH 8.5 containing 80 mM SDS, sample injected at 0.7 psi for 10 s.
Figure 8.2 CE-MS electropherograms of arsenic standards and rice extracts with postextraction addition of the internal standard o -arsanilic acid (o -ASA). DMA, dimethylarsinic acid; As III, arsenite; MMA, monomethylarsonic acid; As V, arsenate. CE conditions: sodium carbonate at pH 11 as separation buffer. Injection: 15 mbar for 8 s. Running voltage: +30 kV.
Figure 8.3 (a) CE-MS extracted ion electropherograms of detected proteins in a cheese extract. Mass spectra from (b) α-lactoalbumin, (c) β-lactoglobulin, and (d) lysozyme. (e) CE-MS extracted ion electropherograms of low-molecular-mass compounds. Capillary coated with poly-(TEDETAMA-co-HPMA) 50 : 50 copolymer. CE conditions: 35 mM ammonium acetate at pH 4.8 as separation buffer. Injection: 0.5 psi for 10 s. Running voltage: −30 kV.
Figure 8.4 CE-TOF MS extracted ion electropherograms (EIEs) of (a) the 14 most abundant sulfur-containing compounds and (b) methionine and cysteine from inactive dry yeast (n-IDY) and GSH-enriched inactive dry yeast (g-IDY). A continuous line for g-IDY permeate and a dotted line for n-IDY permeate are used. CE conditions: 3 M formic acid as separation buffer. Running voltage: +25 kV. Injection: 0.5 psi for 80 s.
Figure 8.5 Total ion electropherograms (TIE) of glycerophospholipid profile and EIEs for each phospholipid from Arbequina olive oil samples analyzed. Conditions were as shown in Figure 8.2. Peaks: lyso-PA, lysophosphatidic acid; PC, phosphatidylcholine; lyso-PE, lysophosphatidylethanolamine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PA, phosphatidic acid; PG, phosphatidylglycerol. CE conditions: 100 mM ammonium acetate in 60 : 40 (v/v) methanol/ACN with 0.5% acetic acid as separation buffer. Running voltage: +25 kV. Injection: 50 mbar during 5 s.
Figure 8.6 Global Foodomics strategy used by Ibáñez et al . [85] to investigate the activity of rosemary polyphenols against colon-cancer HT29 cells at molecular level. Contribution of CE-MS on this workflow is highlighted.
Chapter 9: CE-MS in Forensic Sciences with Focus on Forensic Toxicology
Figure 9.1 Incorporation in and loss of compounds from hair.
Figure 9.2 CE-ESI-MS electropherogram of (6)-etilefrine plotted in the single ion track (m /z 5181.7–182.7) mode with 3 mg ml−1 CM-beta-CD as a chiral selector (pH 4.3).
Figure 9.3 Scheme of two-step forensic toxicology analysis.
Figure 9.4 CE-ESI-MS enantioseparation of ketamine, prilocaine, and mepivacaine. CE conditions, running buffer, 20 mM ammonium formate at pH 3 in the presence of S -beta-CD (a) ketamine and (b) top: reconstructed ion electropherogram, middle: ion 221, bottom: ion 247, prilocaine and mepivacaine.
Figure 9.5 Chiral analysis of amphetamine, ephedrine, norephedrine, methamphetamine, MDA, MDMA, and MDEA enantiomers with low HS-gamma-CD concentration and MS detection. (a) Standard solution. (b) Spiked plasma after LLE extraction.
Figure 9.6 MRM pherograms of (a) a standard mixture of THC-COOH (0.1 µg ml−1 ), THC-COOH-glu (0.1 µg ml−1 ), and IS (0.5 µg ml−1 ), (b) a control urine sample, (c) a control urine sample spiked with THC-COOH and THC-COOH-glu (each 0.5 µg ml−1 in urine), (d) sample (e) diluted fourfold with methanol.
Figure 9.7 Extracted ion electrochromatograms of (a) blank urine sample and (b) blank urine sample spiked with the drugs of abuse mixture, after SPE procedure. Samples were electrokinetically injected (12 kV, 10 s).
Figure 9.8 CE-ESI-ion trap MS analysis of a urine sample from a subject under therapeutic treatment with GHB. GHB concentration: 47 µg ml−1 . Inset: expanded view of electropherogram at the migration time of GHB.
Figure 9.9 Schematic representation of the clinically relevant Tf isoforms. Asn, asparagine residue; hexagon, N -acetylglucosamine; pentagon, mannose; diamond galactose; and triangle, sialic acid. Trisialo-Tf contains one biantennary di-sialylated N -glycan and one biantennary mono-sialylated N -glycan.
Figure 9.10 Mass traces for EtG and EtS together with the chemical structures (on the left) and extracted MS spectra (upper graphs on the right) and MS2 spectra (lower graphs on the right) obtained with a standard containing 17.3 µg ml−1 EtG and 15.4 µg ml−1 EtS dissolved in 10-fold diluted running buffer.
Figure 9.11 CE-ESI/MS/MS electropherograms for seven androgen glucuronides in the negative ionization mode.
Figure 9.12 CE-MS data obtained for the SPE extract prepared from 2.5 ml of urine sample derived from a patient under therapy. (a) Mass trace, (b) mass spectrum, (c) SRM mass trace 2, and (d) MS mass spectrum.
Figure 9.13 CE-MS-MS analysis of alkyl methylphosphonic acids (5 mg l−1 ).
Figure 9.14 Separate incubation of dGMP (deoxyguanosine monophosphate) and dAMP (deoxyadenosine monophosphate) with cisplatin. (a) Incubated solution of dGMP. (b) Incubated solution of dAMP. (c) Electropherogram after coinjection of both solutions.
Chapter 10: CE-MS in Metabolomics
Figure 10.1 (a) Three-phase electroextraction setup, (b) analytical workflow.
Figure 10.2 Electropherogram of a standard mixture of γ-glutamyl peptides by CE-MS/MS. Peak identification: 1, γ-Glu-Gly; 2, γ-Glu-Ala; 3, γ-Glu-Ser; 4, γ-Glu-Val; 5, γ-Glu-Thr; 6, γ-Glu-Ile; 7, γ-Glu-Leu; 8, γ-Glu-Gly-Gly; 9, γ-Glu-Asn; 10, γ-Glu-Ornithine; 11, γ-Glu-Asp; 12, γ-Glu-Homocysteine; 13, γ-Glu-Lys; 14, γ-Glu-Gln; 15, γ-Glu-Ala-Gly; 16, γ-Glu-Glu; 17, γ-Glu-Met; 18, γ-Glu-His; 19, Ophthalmate; 20, γ-Glu-Ser-Gly; 21, γ-Glu-Phe; 22, γ-Glu-Val-Gly; 23, γ-Glu-Norvaline-Gly; 24, γ-Glu-Arg; 25, γ-Glu-Citrulline; 26, γ-Glu-Homoserine-Gly; 27, γ-Glu-Thr-Gly; 28, GSSG; 29, GSH; 30, γ-Glu-Tyr; 31, γ-Glu-Ile-Gly; 32, γ-Glu-Leu-Gly; 33, γ-Glu-Asn-Gly; 34, γ-Glu-Asp-Gly; 35, γ-Glu-Homocysteine-Gly; 36, γ-Glu-Gln-Gly; 37, γ-Glu-Glu-Gly; 38, γ-Glu-Trp; 39, γ-Glu-Tyr-Gly. Experimental conditions: concentration of each peptide, 20 µmol l−1 . The numbers on the right are m /z for analyte ions of Q1 (protonated precursor ion) and Q3 (product ion) in multiple reaction monitoring mode.
Figure 10.3 Selected CE-TOFMS electropherograms for intermediate metabolites of glycolysis, pentose phosphate, and the TCA pathways extracted from mouse liver.
Figure 10.4 Supervised hierarchical clustered heat map of 25 metabolites identified by one-way ANOVA. Each column shows the metabolic pattern of individual animals in the control, food-restricted, and fatigued groups. The quantity of each metabolite in the individual samples is expressed as a relative value obtained by the autoscaling method and is represented by a color scheme with red and green for high and low concentrations of metabolites, respectively.
Figure 10.5 Receiver-operating characteristic curve analysis of the ability of γ-glutamyl peptides alone or in combination with aspartate transaminase (AST), alanine transaminase (ALT), and methionine sulfoxide to discriminate each group from all other liver diseases and healthy controls. The solid and dashed curves represent the ROC curves for the training and validation cohorts, respectively. AUCt and AUCv in each panel indicate the area under the curve values in the training and validation cohorts, respectively. The group label indicates the group discriminated from all the other groups by a multiple logistic regression model.
Figure 10.6 Data obtained from the following four distinct cell types: (1) ventral basal thalamocortical neuron, (2) nonburst-firing thalamic reticular nucleus neuron, (3) bursting thalamic reticular nucleus neuron, and (4) astrocyte. (a) Electrophysiological recordings of the individual cells (1–4) shown in the (b) photomicrographs (scale bar = 20 µm). (c) Extracted ion chromatograms corresponding to the cytoplasm sampled from the neurons and glial cells are shown. Peaks correspond to ornithine (dark red), GABA (light red), glycine (yellow), serine (gold), tryptophan (rainbow), glutamine (light green), glutamic acid (light blue), tyrosine (dark blue), and proline (indigo).
Figure 10.7 Quantification of metabolites involved in glutathione biosynthesis and related pathways. The columns represent average concentrations (nmol g−1 tissue), and the error bars indicate the standard deviation. *p < 0.05; **p < 0.01; ***p < 0.001; and N.D., not detected.
Figure 10.8 Changes in the level of representative primary metabolites after aphid introduction. The data points are mean values and the error bars indicate the standard error. Asterisk denotes statistical significance with p < 0.01.
Chapter 11: CE-MS for Clinical Proteomics and Metabolomics: Strategies and Applications
Figure 11.1 Scheme of the two-dimensional CE system containing a replaceable enzymatic microreactor for on-line protein digestion. The magnets hold the trypsin-modified magnetic beads, forming the replaceable microreactor. High-voltage connections are shown as dashed curves.
Figure 11.2 Base peak electropherograms of rat testis H1 histones digested with endoproteinase Arg-C obtained with sheathless CE-MS using positively charged capillaries. (a) PEI-coated capillary, separation voltage of −25 kV. (b) M7C4I-coated capillary, separation voltage of −25 kV. (c) M7C4I-coated capillary, separation voltage of −12.5 kV. BGE: (pH 2.7)0.1% (v/v) formic acid. Sample amount: 6.15 ng (300 fmol). Capillary length: 100 cm with porous tip. Inner diameter 30 µm, outer diameter 150 µm.
Figure 11.3 Urinary peptide profiles distinguishing patients with chronic kidney disease (CKD) from healthy subjects. Compiled data sets of urine samples from 230 patients with CKD (a) and 379 healthy control subjects (b) are shown. Normalized molecular mass (y -axis) is plotted against normalized CE migration time (x -axis). The mean signal intensity is represented in three-dimensional depiction. CE-MS analysis was performed at low pH (1% acetic acid, pH ∼2) using a 90 cm (50 µm ID) capillary, coupled to TOF MS via a sheath-liquid interface (flow rates in the range of 200 nl min−1 , without nebulizer gas) in the mass region 350–3000 m/z . The sample was injected for 99 s using 1 psi for injection (circa 60 nl).
Figure 11.4 Mass distribution of histone H1 peptides identified from perchloric acid extracted from rat testis digest using CE-MS and nano-RPLC-MS. CE conditions: M7C4I-coated capillary (length, 100 cm with porous tip; i.d., 30 µm); BGE, 0.1% (v/v) formic acid; separation voltage, −25 kV; nano-RPLC-MS was performed with a homemade fritless column: packed 10 cm with 3 µm reversed-phase C18 (Reprosil). The gradient (solvent A, 0.1% formic acid; solvent B, 0.1% formic acid in 85% acetonitrile) started at 4% B. The concentration of solvent B was increased linearly from 4% to 50% during 50 min and from 50% to 100% during 5 min. A flow rate of 250 nl min−1 was applied.
Figure 11.5 Peptide (a) and protein (c) identification sensitivity comparison of micro-SPE-CE-MS/MS (blue), direct injection CE-MS/MS (green), and nano-LC-MS/MS (red) of Pyrococcus furiosus (Pfu) tryptic digests run in duplicate. Venn comparisons of identified peptides (b) and proteins (d) from combined duplicate runs of 5 and 100 ng of Pfu digests using micro-SPE-tITP-CE and nano-LC.
Figure 11.6 (a) Base peak electropherogram (m/z 50–450) of human urine obtained with sheathless CE-MS using a porous tip sprayer. Conditions: BGE, 10% acetic acid (pH 2.2); sample injection, 2.0 psi for 30 s (1% of capillary volume). (b) Base peak electropherogram (m/z 50–450) of human urine obtained with CE-MS using a sheath-liquid interface. Conditions: BGE, 10% acetic acid (pH 2.2); sample injection, 0.5 psi for 30 s (1% of capillary volume).
Figure 11.7 (a) Representative electropherograms of pooled 24 h urine samples as quality controls analyzed intermittently over 29 days to demonstrate method robustness for population-based iodine nutrition assessment for large-scale epidemiological studies. (b) Control chart summarizing intermediate precision performance for CE method based on measurement of urinary iodide concentration (μM) and relative migration time (RMT) with an overall variance of 11% and 0.75% (n = 87), respectively. (c) Reliable quantitative performance of CE also demonstrated by external calibration curves derived from analyzing iodide calibrants with an internal standard over 5 weeks with consistent sensitivity relative to original calibration curve performed at the start of the study.
Figure 11.8 (a) Multiplexed separation based on serial injection of seven discrete sample segments within a single capillary by MSI-CE-MS; (b) ions migrate as a series of zones in free solution prior to ionization; (c) the procedure enables reliable quantification of polar metabolites and their isomers in different samples as ionization occurs within a short-time interval (≈2–6 min) under steady-state conditions with fast data acquisition by TOF-MS.
Figure 11.9 An accelerated workflow in metabolomics for biomarker discovery that takes advantage of multiplexed separations by MSI-CE-MS, such as a seven-segment format captures dynamic metabolomic responses to strenuous exercise for individual subjects, as well as their adaptive responses to exercise training. Unlike conventional MS-based data workflows, a single dilution trend filter of a pooled QC sample is used as a primary screen to certify reproducible plasma-derived molecular features while rejecting signal artifacts based on signal pattern recognition. This allows for targeted analysis of authentic metabolites from individual samples with high data fidelity that avoids data overfitting and false discoveries when using multivariate statistical methods. Unambiguous identification and quantification of lead plasma markers (HyX) and their associated metabolic pathway (purine degradation) provide deeper insight into exercise responsiveness that differ between subjects.
Figure 11.10 Venn diagram showing the number of identified molecular features detected by RPLC-MS, GC-MS, and CE-MS in a pooled extract of a mouse lung. For experimental details see [86].
List of Tables
Chapter 2: Electrospray Ionization Interface Development for Capillary Electrophoresis–Mass Spectrometry
Table 2.1 Areas of study and application have been listed for each interface described in Sections 2.3.1 and 2.3.2, along with CE separation mode, BGE, mass spectrometer used, and some analytical characteristics reported in each reference
Chapter 4: Recent Developments of Microchip Capillary Electrophoresis Coupled with Mass Spectrometry
Table 4.1 Review articles on microfluidic device coupling with MS
Table 4.2 Properties of capillary electrophoresis and microchip capillary electrophoresis
Table A.1 Overview of the most important relevant sources of microfluidic chips for capillary electrophoresis
Chapter 5: On-Line Electrophoretic, Electrochromatographic, and Chromatographic Sample Concentration in CE-MS
Table 5.1 Ionic species suiTable for CE-MS and their pK a values and ionic mobilities
Chapter 6: CE-MS in Drug Analysis and Bioanalysis
Table 6.1 Main characteristics of conventional approaches versus CE-based methods to assess acid/base properties (pK a ), lipophilicity (log P ), and protein binding (PPB) of a new chemical entity (NCE)
Chapter 7: CE-MS for the analysis of intact proteins
Table 7.1 Overview of CE-MS methods for the analysis of (glycosylated) biopharmaceuticals
Chapter 9: CE-MS in Forensic Sciences with Focus on Forensic Toxicology
Table 9.1 Summary of the cases studied
Table 9.2 Caffeine and its metabolites
Table 9.3 Name of some phosphonic acids
Table 9.4 Phosphonic acids and CID product ion spectra (M-H)−
Edited by
Gerhardus de Jong
Capillary Electrophoresis-Mass Spectrometry (CE-MS)
Principles and Applications
Editor
Prof. Gerhardus de Jong
Utrecht University
Department of Pharmaceutical Sciences
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