Capillary electrophoresis-mass spectrometry

CHAPTER 4

Capillary electrophoresis-mass spectrometry: history, general principles, theoretical aspects, and state-of-the-art applications 2, 3 Chiara Fanali1, Giovanni D’Orazio2, Salvatore Fanali 1 2

Centro Integrato di Ricerca, Campus Bio-Medico University, Roma, Italy; Istituto per I Sistemi Biologici, Consiglio Nazionale delle Ricerche, Monterotondo, Italy; 3Teaching Committee of Ph.D. School in Natural Science and Engineering, University of Verona, Verona, Italy

4.1 Introduction Capillary electrophoresis (CE) is a modern analytical technique that can separate both charged and uncharged compounds. The analysis is carried out in capillaries with a narrow I.D. (10e300 mm), under a relatively high electric field (100e1000 V/cm). At the base of modern CE there is the work of Hjerten that first showed the possibility of separating various compounds in free zone electrophoresis, using rotating tubes with a 3 mm I.D. in absence of conventional supports. Theory, advantages, and disadvantages in using such an approach were reported in 1967 [1]. Later, based on the experience employing capillary isotachophoresis (ITP), Mikkers et al. demonstrated the possibility to achieve good free zone electrophoretic separations of some anionic compounds utilizing a polytetrafluorethylene (PTFE) narrow-bore tube with a 200 mm I.D. [2]. Although such works were the starting point for the development of modern CE, theory and new developments have been proposed by Jorgenson [3,4], who performed capillary zone electrophoresis (CZE) separations of some alkyl amines on 75 mm I.D. glass capillaries in a short time. The novel electrodriven technique offered, for the first time, the possibility to analyze both charged and uncharged compounds even in the same run. This was possible because, under the influence of the electric field, and due to the presence of charged/chargeable groups on the capillary wall, an electroosmotic flow (EOF) is generated. This driving force transports the mobile phase, or better the background electrolyte (BGE), to the detector. Obviously, the EOF is also transporting charged and uncharged Hyphenations of Capillary Chromatography with Mass Spectrometry ISBN 978-0-12-809638-3 https://doi.org/10.1016/B978-0-12-809638-3.00011-9

Copyright © 2020 Elsevier Inc. All rights reserved.

413

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Hyphenations of Capillary Chromatography with Mass Spectrometry

analytes to the detector. Different modes have been studied and developed in CE, namely CZE, capillary gel electrophoresis (CGE), micellar electrokinetic chromatography (MEKC), ITP, and capillary electrochromatography (CEC). Before 1989, the different groups working in the CE field had to use lab-made apparatuses even constructing high voltage power supplies. Injection was quite simple using either the hydrodynamic or electrokinetic mode. Detection was performed on column after removing a small part of the outside polyimide layer of the column and positioning the capillary in ultraviolet (UV) or fluorescence detectors. In 1990, some companies commercialized dedicated CE instrumentation, and since then a large number of research groups have been involved in studies and applications in different fields. Although basic instrumentation included UV detectors, other systems were developed. Among them conductivity [2,5], fluorescence and laser induced fluorescence (LIF) [6,7], and amperometric electrochemical devices [8] were the most applied. Finally mass spectrometry (MS), widely used in high performance liquid chromatography (HPLC), was coupled with CE. Clearly, it is simplistic to consider MS as only a detector. In fact, this technique offers a great information potential in relation to analyte structure and identification. This is useful especially when complex samples have to be analyzed. Electrospray ionization mode was the most popular approach, it being demonstrated for the first time by Smith’s group [9]. During this time CE was also coupled to continuous-flow fast atom bombardment [10], mainly reporting the separation of compounds with a high molecular weight. In literature, the hyphenation of CE with MS has been widely reported, after the availability of commercial interfaces, in various fields, e.g., proteomic, toxicology, pharmaceutical, environment, food, agrochemical, nutraceutical, etc. The aim of this chapter is to provide a short overview of CE coupled to MS focusing on historical achievements, principles, and some selected applications.

4.2 Brief history, theory, and general principles The development of modern CE techniques has introduced great improvements in compound analysis that had been demonstrated previously with classical electrodriven approaches. For the first time, positively charged, negatively charged, and uncharged compounds could be analyzed.

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The pioneering work carried out by Hjerten, reported in 1967 [1], has to be considered the start point of modern CE. Although the separations were obtained utilizing tubes with a 3 mm I.D., all problems related to the new technique were addressed and partly resolved. Among them were electroosmotic flow, detection, sample injection, cooling system, and constant I.D. of the tube used. The cooling system was based on water; it being also transparent to the UV beam was effective for the zone measurement. Detection was performed by scanning the whole capillary. Samples were injected as a sharp zone reducing dispersion. Quartz tubes were prepared and a rotary movement was introduced to avoid zone sedimentation [1]. Although excellent results were obtained, the instrumentation was quite large and difficult to handle. The presence of EOF was also considered. Given the scope of the work (accurate measurement of the electrophoretic mobility), the EOF was, at that time, a problem. Instead, later on, this parameter was considered as a resource. Jorgenson’s group presented an excellent study dealing with high-resolution analyte separations utilizing both electrophoresis and EOF [3,4]. Glass capillaries with a 75 mm I.D. were used. Analytes were negatively charged (due to derivatization) and moved to the cathode direction. In CZE, cations and/or anions migrate as zones with a certain velocity (n), under the application of an electric field (E). Separations by using CZE are carried out on open capillaries containing a selected background electrolyte. Cations and/or anions, after injection move under the effect of the applied E according to their own charge/mass in the cathodic or anodic compartments, respectively. In absence of EOF, their n can be calculated using the following equation: n ¼ mep

V L

(4.1)

where mep is the electrophoretic mobility, V the applied voltage, and L the capillary length. Analysis time (t) is calculated by: t¼

l lL ¼ n mep V

(4.2)

with l the length of the capillary at the detector window. Some considerations concerning zones broadening have to be made. After the injection, zones are separated along the capillary and they are subjected to a zone broadening effect responsible for a reduction of

416

Hyphenations of Capillary Chromatography with Mass Spectrometry

efficiency and resolution. This effect depends on some parameters such as longitudinal diffusion, convection, and temperature. The use of capillaries with narrow diameters minimizes the convection and temperature effects; therefore capillaries with small diameters (10e100 mm I.D.) are usually used in CE [4]. Supposing that only diffusion occurs during CZE experiments, the spatial variance (s2) can be calculated as follows: s2 ¼ 2Dt ¼

2DlL mep V

(4.3)

where D is the analyte diffusion coefficient. The number of theoretical plates N can be defined as: N¼

lL s2

(4.4)

Combining Eqs. (4.3) and (4.4) allows the following calculation of N: N¼

mep V 2D

(4.5)

Some considerations related to the above-reported equations can help to understand the powerfulness of CZE techniques. In fact, from Eqs. (4.2) and (4.5) ways can be derived on how to achieve a fast separation and high efficiency, respectively. The increase of the applied voltage at 30 kV can offer both decrease of analysis time and increase of efficiency. Analytes moving with higher mobility show shorter analysis time and higher efficiency. A similar effect can also be observed reducing l and/or L. Clearly, these parameters cannot be modified indefinitely because other consequences could occur, e.g., hardware problems and efficient dispersion of the heat generated by the Joule effect. As previously mentioned analyte velocity can also be influenced by the EOF. As reported by Pretorius in 1974 [11], and later on pointed out by Jorgenson [4], the flow velocity of the EOF can be calculated by:   εz neof ¼ E (4.6) 4ph with ε, z, and h are the dielectric constant, zeta potential, and viscosity, respectively. Eq. (4.6) can be applied to both CZE and CEC. The flow profile of the EOF is flat, which is the contrary of that present in pressure-driven techniques (parabolic). Considering Eq. (4.6), it can be

Capillary electrophoresis-mass spectrometry

417

concluded that the EOF velocity is not influenced by the geometry and size of the channels. Therefore, higher efficiency can be obtained. This was observed by several authors especially in CEC [4,12e16]. The EOF can be modified by controlling some experimental parameters, e.g., buffer concentration and type, organic solvent. These parameters, for example, modify z, the thickness of the double layer formed on the surface of the capillary and/or particles and the viscosity of the BGE, respectively. In general the use of acetonitrile instead of methanol and a lower concentration of the buffer increase the EOF. The direction of the EOF is usually toward the cathode, e.g., in untreated capillaries due to the presence of silanol groups on the surface. However, when the wall is positively charged (because modified) the EOF moves in the opposite direction (anode). This “new” driving force, the EOF, was utilized by Terabe et al. in 1984 in his studies concerning MEKC [17]. In this work, the authors showed, for the first time, the separation of uncharged compounds (e.g., phenol and its derivatives) utilizing a buffer at high pH containing micelles (sodium dodecyl sulfate). The proposed mechanism consisted in generating a high EOF applying 25 kV. The driving force moved both uncharged analytes and micelles to the cathode while the movement of the micelles, negatively charged, was also influenced by the electric field. Therefore, their velocity was decreased. Analytes were selectively complexed by the micelles and therefore were detected at a different time. Such pioneering work was fundamental in developing this methodology for practical applications in various fields such as pharmaceutical [18e27], agrochemical [28], chiral [29e31], biomedicine [32,33], etc. Fig. 4.1 shows the analysis of atrazine in water samples by using MEKC. The CEC approach is another CE mode where an essential role is played by the EOF. After the fundamental work performed by Pretorius [11] and Jorgenson [4], several groups have been involved in studies dealing with theory, instrumentation, hyphenation with MS and applications. Several review papers have been reported in the literature [34e47]. In CEC, analytes are separated on capillaries containing selected stationary phases and mobile phases. Therefore, this technique results as a combination of CE and HPLC and thus offering high efficiency and high selectivity, respectively [48]. As mentioned before, the EOF is an important parameter to be controlled. This is achieved by selecting the appropriate experimental conditions, e.g., type of stationary phase (SP), buffer in the mobile phase (MP), pH, ionic strength, content and type of organic solvent,

418

Hyphenations of Capillary Chromatography with Mass Spectrometry

Figure 4.1 The separation of atrazine and simazine (A), and the analysis of herbicides (B) in water by using MEKC. Peak identity: (1) atrazine, (2) internal standardd hydroquinone monobenzyl ether. Capillary, 50 cm  75 mm I.D.; BGE, 10 mM phosphate buffer pH 8 and 25 mM of sodium dodecyl sulfate; applied voltage, 10 kV. (Reproduced from C. Desiderio, S. Fanali, Atrazine and simazine determination in river water samples by micellar electrokinetic capillary chromatography, Electrophoresis 13, 1992, 698e700 with permission of John Wiley and Sons.)

etc. The SP is present in the capillary usually as modified silica particles (packed), or monolithic material, or bonded/adsorbed on the wall [49]. Capillary isotachophoresis is a relatively old miniaturized CE technique where analytes zones are separated utilizing discontinuous electrolytes. Leading and terminating electrolytes (with the highest and lowest electrophoretic mobilities, respectively) are employed, while analytes must have mobilities in between them. Analytes (cations or anions) are separated in contiguous sharp zones according to their mobility, moving with the same velocity. The main feature of ITP stands in its concentrating capability and in the large sample volume that can be injected without sacrificing efficiency and resolution. These effects were demonstrated by Gysler et al. using a single capillary for ITP-CZE-MS [50]. Recombinant human interleukin-6 after acidic degradation and other proteins were analyzed. Lower sample concentrations than in LC-MS were used in ITP-MS.

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419

An ITP-MS approach, using a sheathless (SLess) interface with a porous tip, was applied to the analysis of IgG antibodies. The capillary was filled with a relatively high volume of sample (about 37% of the length) obtaining higher sensitivity than with nano-LC. This was due to the concentrating capacity of ITP. The method resulted useful for the analysis of IgG1 compounds present at a concentration lower than the limit of detection (LOD) observed in nano-LC-MS [51].

4.3 Hyphenation with mass spectrometry The hyphenation of CE with MS has been commonly performed with the electrospray ionization (ESI) interface, either laboratory-made or commercially available. Such a combination is necessary not only to utilize a more sensitive detector but also to advantageously determine the mass and identify the analyzed compounds. This is particularly useful when complex samples are considered. Coupling CE with MS is not an easy task because, for the electrophoretic separation, a high electric field has to be applied. Therefore, an electric connection between the CE instrument and the interface has to be made. The interfaces studied and applied until now are: sheathless, coaxial sheath-flow, liquid-junction, and pressurized liquid-junction ones. The sheathless interface was firstly proposed by Olivares et al. [52], demonstrating the possibility to perform CE-MS analysis of some quaternary ammonium ions with efficiencies ranging between 35,000 and 140,000 theoretical plates. The capillary used for the CE separation was inserted into a stainless steel capillary with a double function, (i) cathodic end for CZE and (ii) electrospray needle. This approach exhibits some drawbacks, e.g., spray and CE run stability and careful selection of the electrolyte solution used for the electrophoretic separation. Moini studied a new sheathless interface [53], where the electric contact was obtained through a porous tip at the end of the capillary. The pores were prepared by treating this part of the capillary with hydrofluoric acid. The tip was inserted into the ESI needle that was filled with the BGE. This approach offered some advantages, e.g., absence of dead volumes, high voltage applied outside the capillary, and higher stability. This type of hyphenation was also employed for the fast separation of some enantiomers of pharmaceutical interest (separations were performed in about 1 min) [54]. Based on such studies, the SLess interface was proposed by Beckman Coulter, as a commercially available product. Fig. 4.2 reports schemes of different

420

Hyphenations of Capillary Chromatography with Mass Spectrometry

Figure 4.2 Sheathless interfaces: (A) Conductive coating applied to the emitter tip, (B) wire inserted at the tip, (C) wire inserted through a hole, (D) split-flow interface with a metal sheath, (E) porous, etched capillary walls in a metal sleeve, (F) junction with metal sleeve, (G) microdialysis junction, and (H) junction with a conductive emitter tip. (Reproduced from E.J. Maxwell, D.D.Y. Chen, Twenty years of interface development for capillary electrophoresiseelectrospray ionizationemass spectrometry, Anal. Chim. Acta 627, 2008, 25e33 with permission of Elsevier.)

sheathless interfaces studied for CE-MS hyphenation. As can be observed, the voltage is applied at the end of the capillary or tips either by insertion of a metal connection or coated outside. The system offered a higher sensitivity in comparison to the sheath flow tool. However, some stability problems and limitations to the selection of the spray liquid are present. In fact, since the selected mobile phase also acts as ion-spray liquid, often it is not easy to have only one solution useful for both processes (electrophoresis and spray formation). Fig. 4.3 reports the scheme of other interfaces used in CE, namely coaxial sheath-flow, liquid-junction, and pressurized liquid-junction. The sheath liquid (SL) interface has been introduced and commercialized by some companies. As can be observed in Fig. 4.3, the end of the separation capillary is inserted into a coaxial capillary tube allowing the sheath liquid to elute through the outside capillary wall. The flow is usually 200e800 nL/min and 2e6 mL/min in the column and in the sheath liquid, respectively. Although good results have been obtained, this type of interface presented also some drawbacks especially due to the relatively high flow of the sheath liquid. In fact, turbulence causing reduction of efficiency and resolution of the separated zones were observed. In addition zone dilution affected the sensitivity. On the other hand this approach offered

Capillary electrophoresis-mass spectrometry

421

Figure 4.3 Most common interfaces used for CE-MS (A) coaxial sheath-flow, (B) liquidjunction and (C) pressurized liquid-junction. (Reproduced from E.J. Maxwell and D.D.Y. Chen, Twenty years of interface development for capillary electrophoresiseelectrospray ionizationemass spectrometry, Anal. Chim. Acta 627, 2008, 25e33 with permission of Elsevier.)

higher spray stability and a better selection of the mobile phase could be made. Later, the SL interface, either laboratory-made or commercially available, was used for CZE-MS separations [55e58]. A detailed scheme of an SL interface can be observed in Fig. 4.4. Alternatively, a liquid-junction (LJ) interface has been studied, it being characterized by good spray and CE separation stability, reduced turbulence, and higher sensitivity. The capillary column and a tip are positioned into a small electrode compartment, at a very low distance (about 100 mm), allowing the transfer of the separated zone to the MS instrument. Karger’s group developed an LJ subatmospheric electrospray interface coupling both CZE and nano-LC with MS [59]. The CZE analysis of some proteins was carried out successfully. The capillary column was connected to a tip by the LJ interface that was pressurized at different pressures to obtain the optimum flow rate. Additionally, the same group also studied a microdevice with an integrated LJ interface for proteins and peptides analysis employing CZE-MS [60,61].

422

Hyphenations of Capillary Chromatography with Mass Spectrometry

Figure 4.4 Scheme of a sheath-liquid ESI interface currently used in CE-MS.

Recently, Foret’s group presented a newly developed “interface-free CE-nano-spray/MS” system. It consisted of a single 15 mm I.D. capillary used for both CZE separation and as nanospray emitter. The flow of the BGE was estimated to be 30 nL/min. In order to stabilize the spray, the end tip was outside-coated with hydrophobic material (water dispersion of Teflon and UV curable polish) eliminating the wetting properties of the capillary surface. The method was optimized, validated, and applied to the analysis of serum samples. The CE analysis time was three time shorter than a reported HPLC-MS one, and only a 1 nL volume of sample was injected [62]. An LJ interface for CZE-MS applications was also studied [63]. Later, the interface was also considered for CEC-MS separations. Even though good results were achieved, the LJ interface showed some drawbacks, e.g., perfect alignment of the capillary and tip and need of a microscope to verify their distance for the optimum transfer of the separated zone to the MS [64]. Bearing in mind such problems, an implemented LJ interface was prepared utilizing a multiport fittings tee made of ULTEMÔ Resin (https://www.sabic.com/en/products/specialties/ultem-resins/ultem-resin) (amorphous thermoplastic polyetherimide), with a zero dead-volume. The use of the tee avoided the inspection of the capillary-tip connection. In addition, nitrogen was used to pressurize the electrode compartment. In this study the performance of the interface, analyzing the same compounds, was compared with the previous developed interface [65]. Fig. 4.5A and B show the scheme of the LJ nanospray interface.

Capillary electrophoresis-mass spectrometry

423

Figure 4.5A Scheme of the instrumentation used for CEC-MS separations, employing a nano-liquid-junction interface. (Reproduced from G. D’Orazio, S. Fanali, Pressurized nano-liquid-junction interface for coupling capillary electrochromatography and nanoliquid chromatography with mass spectrometry, J. Chromatogr. A 1317, 2013, 67e76 with permission of Elsevier.)

Figure 4.5B Nano-liquid-junction interface for coupling CEC with MS. (Reproduced from G. D’Orazio, S. Fanali, Pressurized nano-liquid-junction interface for coupling capillary electrochromatography and nano-liquid chromatography with mass spectrometry, J. Chromatogr. A 1317, 2013, 67e76 with permission of Elsevier.)

424

Hyphenations of Capillary Chromatography with Mass Spectrometry

4.4 Two-dimensional separation methods Multidimensional separations have been widely studied in separation science. In chromatography a high peak capacity and resolution power can be obtained, and therefore the method can be helpful especially when complex samples have to be analyzed. On the other hand, these advantages have been previously shown in conventional electrophoresis [66]. A multidimensional separation system has been proposed by Ramsey’s group. Capillary LC was coupled with a microchip to achieve both chromatographic and CE separations. The chip and MS device were also coupled for detection and characterization of peptides (digested protein). A peak capacity of 1400 in less than 40 min was obtained [67]. Twodimensional MEKC-CE with microchip instrumentation was successfully used for the analysis of labeled peptides and proteins digest detecting the separated compounds with LIF [68]. A microchip for two-dimensional (2D) ITP-CZE was also used by Wu et al. for the analysis of milk proteins [69]. An interesting 2D CE separation has been previously reported analyzing single cell proteins utilizing capillary sieving electrophoresis (CSE) in the first dimension, and MEKC in the second [70]. On the other hand, CZE and ITP were previously coupled for analyte separation and pretreatment using the column-coupling CE equipment. This system was particularly helpful because it allowed the separation of compounds at trace levels and the treatment of matrices at high ionic strengths. Preconcentration is a feature of ITP: ions are concentrated, separated, and detected increasing the sensitivity 103e104 times. Such characteristics have been reported by Fanali et al. for the analysis of tryptophan enantiomers in urine [71]. The amino acids, such as 2,4dinitrophenyl derivatives, were separated by ITP in the first capillary. Afterward, the amino acids zone was cut and transferred onto the CZE column and separated in presence of a BGE supplemented with a- or b-cyclodextrin for enantiomeric separation. The same apparatus type was used for coupling CZE and CZE for highly sensitive enantiomer separation. In the first capillary, some hydroxyacid enantiomers were separated in the presence of chiral selectors with high UV absorbance, and transferred onto the second capillary for the CZE separation in absence of the chiral selector, enhancing sensitivity [72]. Mikus et al. demonstrated the possibility to perform a 3D electrodriven separation utilizing a commercial apparatus with three capillaries. In the first dimension, a preconcentration step was carried out with ITP and analytical separation in the second and third

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425

capillaries by ITP and CZE, respectively. Phthalic acid in urine samples was analyzed achieving good results related to repeatability and sensitivity [73]. An interesting approach involving the combination of capillary isoelectricfocusing (IEF) with CZE in a single capillary was proposed by Liu et al. [74]. The polyacrylamide-coated capillary was treated in a small portion with hydrogen fluoride to prepare an etched interface to allow the conductivity. The interface was easily prepared and no dead volumes were present. The tool was applied to the separation of some proteins in absence of EOF. A 2D IEF-CZE approach was proposed by Wang et al. for protein separation [75]. The two capillaries were connected to each other by an etched porous interface. The first capillary contained a monolithic immobilized gradient for protein immobilization, while the second one a BGE (20 mM glutamic acid). Proteins were transferred onto the second capillary by a syringe pump. Capillary zone electrophoresis and CEC have also been coupled with nuclear magnetic resonance. A model mixture of lysine/histamine and five alkyl benzoates were separated by CZE and CEC, respectively. Nuclear magnetic resonance was on-line connected and a stop-flow applied. The 2D separation method could be of great potential for the characterization of separated compounds. However their applications, at the moment, are rare [76]. In a later 2D method, CE was combined with inductively coupled plasmaMS (ICP-MS) and was applied to the analysis of Mn in serum and brain samples. The metal ion was found to be bonded to citrate or transferrin and albumin. Manganese in the studied samples could be a useful marker for neurological disorders in people in contact with this compound [77]. Heart-cut 2D-CE was proposed by Kohl et al. for the separation of bovine serum albumin (BSA) tryptic digest [78]. The system made use of an isolated mechanical valve switching separated zone in the first dimension (containing phosphate buffer), to the second column containing a BGE more compatible with the MS instrument. Therefore, the system could be used when a nonvolatile electrolyte had to be used in the first dimension. Fig. 4.6 shows a scheme of the setup used for the 2D-CE separation, characterized by an electrically-isolated mechanical valve.

4.5 Selected examples of CE-MS applications In this section some selected applications of CE-MS, discussing the advantages in using these techniques over others will be proposed, mainly focusing on problems and solutions encountered in the coupled approach (Table 4.1).

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Hyphenations of Capillary Chromatography with Mass Spectrometry

Figure 4.6 Schematic setup of the 2D CE-MS system utilizing a four-port valve. Position A and B, load and injection, respectively. Capillaries 1A, 1B, 2A, and 2B are connected to the valve (S, W, P, and C). (Reproduced from F.J. Kohl, C. Montealegre, C. Neusüß, On-line two-dimensional capillary electrophoresis with mass spectrometric detection using a fully electric isolated mechanical valve, Electrophoresis 37, 2016, 954e958 with permission of John Wiley and Sons.)

Table 4.1 Selected CE-MS applications. Capillary

Ammonium (tetramethyl, trimethylphenyl, tetraethyl, tetrapropyl, tetrabutyl) ions Paralytic shellfish poisons

CZE-ESI-MS

100 mm I.D.  100 cm

CZE-IS-MS Ion spray

50 mm I.D.  90 cm

Saxitoxin and neosaxitoxin, antibacterial drugs Peptides (bradykinin, neurotensin derivatives and angiotensin II derivatives)

CZE-IS-MS Ion spray

50 mm I.D.  90 cm

MEKC-ESIMS

50 mm I.D.  67 e100 cm

Pentachlorophenols

CZE-ESI-MS

50 mm I.D.  100 cm

Triazines (simazine, atrazine, propazine, simetryn, ametryn, prometon, prometryn, terbutryn)

MEKC-ESIMS

50 mm I.D.  80 cm

Experimental conditions

Interface

References

BGE: (50/50) watermethanol with 104 M KCl

SLess

[52]

BGE: 20 mM sodium citrate buffer (pH 2) or 0.1 M acetic acid (pH 2.9) BGE: trisma buffer (pH 7.2)

SL: 0.1 M acetic acid

[89]

SL or LJ: aqueous formic acid (0.2%)

[90]

BGE: 10 mM formate buffer pH 3.0 with100 mM sucrose monododecanoate; partial filling BGE: 2-[NCyclohexylamino methanesulfonic acid] (CHES buffer pH 10) BGE: 50 mM ammonium acetate and 0.7 mM CTAB at pH 3.85

SL: water-organic solvent-formic acid (50:50:1)

[82]

SL: water-2propanol (20:80) containing 0.5% ammonia SL: methanol-water (80:20) with 0.1% trifluoroacetic acid (FA)

[55]

[56]

427

CE mode

Capillary electrophoresis-mass spectrometry

Analytes

Continued

Table 4.1 Selected CE-MS applications.dcont'd Capillary

Enantiomersarylpropionic acids (etodolac and metabolites, ibuprofen and metabolites, carprofen, flurbiprofen, ketoprofen, naproxen) Proteins (lysozyme, cytochrome c) recombinant cytokine fragments

CZE-ESI-MS

50 mm I.D.  44 cm coated with polyacrylamide;

ITP-CZE-ESIMS

75 mm I.D.  80 cm polyacrylamide coated

Peptides (cytochrome c tryptic digest; angiotensin)

CZE-ESI-MS ITP-ESI-MS

Chip

Coptisine, berberine, and palmatine in Chinese medicine

CZE-ESI-MS

50 mm I.D.  70 cm

Experimental conditions

Interface

References

BGE: 50 mM acetic acid/ammonium acetate pH 4.8 and 5 mM vancomycin; counterflow mode

SL: methanol ewatereammonia (50:48:2)

[57]

Leading electrolyte: 20 mM ammonium acetate pH 4.2; terminating electrolyte: 10 mM acetic acid; BGE: as leading electrolyte BGE: 100 mM ammonium acetate or 1% (v/v) formic acid in water or 20 mM 6aminocaproic acid/ acetic acid, pH 4.4 BGE: 50 mM ammonium acetate pH 3.8

SL: methanol/acetic acid (80:20, v/v)

[50]

SL: 1% (v/v) formic acid in 50% (v/v) methanol/water or 0.8% (v/v) acetic acid pH 4.4 in 50% methanol

[61]

SL: methanol-water (80:20) with 1% acetic acid

[86]

Hyphenations of Capillary Chromatography with Mass Spectrometry

CE mode

428

Analytes

75 mm I.D.  40 cm coated with polyvinyl alcohol (PVA) 50 mm I.D.  87 cm coated

BGE: 20 mM ε aminocaproic acid/ acetic acid pH 4.4 BGE: 100 mM ammonium acetate buffer at pH 6 with 5 mM b-CD

SL: 1% (v/v) acetic acid in 50% methanol SL: methanol-water (1:1 v/v) with 25% BGE without b-CD

[59]

CZE-ESI-MS

75 mm I.D.  64 cm coated with polyvinyl alcohol

BGE: 30 mM ammonium acetate at pH 5.5 or 100 mM ammonium formate pH 2.8

LJ: spray liquid, 1% v/v acetic acid in 50% v/v methanol. pressurized

[63]

CZE-ESI-MS

75 mm I.D.  50 cm (coated)

BGE: 1% aqueous solution of formic acid

LJ: spray liquid, 50% acetonitrile in 1% formic acid, pressurized, 100 kPa

[92]

CZE-ESI-MS

20 mm I.D.  60 e120 cm

BGE: 0.1% polybrene in 0.1% acetic acid

SLess: ESI needle was filled with 1 M formic acid

[53]

CZE-ESI-MS

Dansyl (DNS) and fluorescein isothiocyanate (FITC) aminoacid enantiomers in food b-blocker drugs (pindolol, oxprenolol, atenolol, nadolol) and peptides (bradykinin, angiotensinI, neurotensin, leuenkephalin or tryptic digest) Peptides and protein (angiotensin I, bradykinin, neurotensin, cytochome C tryptic) Amino acids, peptides, and intact protein

CZE-ESI-MS

[91]

Capillary electrophoresis-mass spectrometry

Peptides (angiotensins)

429

Continued

Table 4.1 Selected CE-MS applications.dcont'd Capillary

Opioid peptides in plasma

CZE-ESI-MS

75 mm I.D.  72 cm

Proteins (insulin, carbonic anhydrase II, ribonuclease A, Lysozyme) Organophosphorus pesticides (dialifos, fensulfothion, fenamiphos, isofenphos, methamidophos, profenofos) and acebutolol, alprenolol, atenolol, clenbuterol, fluoxetine, metoprolol, mianserin, mirtazapine, nadolol, nor-fluoxetine, oxprenolol, propranolol, and venlafaxine enantiomers (pesticides in drinking water)

CZE-ESI-MS

30 mm I.D.  30 cm

CEC-ESI-MS

100 mm I.D.  26 or 34 cm Packed with silica C18 or vancomycin

Experimental conditions

Interface

References

BGE: 50 mM of acetic acid and 50 mM of formic acid at pH 3.5 with ammonia BGE: 100 mM acetic acid (pH 3.1)

SL: isopropanol:water (60:40) v/v with a 0.05% (v/v) formic acid SLess

[93]

Mobile phase: 100 mM NH4 formate pH w 2.5/ acetonitrile, 1:9 (v/v) for pesticides and 100 mM NH4Ac pH w 6/MeOH/ACN, 1:9 (v/v) for enantiomers separation

SL: 1% formic acid (v/v in MeOH/ H2O, 70:30 (v/v) under hydrostatic pressure of w2.5 kPa or 0.5% (v/v) AcH in MeOH/H2O 80:20 (v/v) w2.5 kPa

[64]

[94]

Hyphenations of Capillary Chromatography with Mass Spectrometry

CE mode

430

Analytes

MEKC-ESIMS

50 mm I.D.  70.5 cm

Twenty aminoacids in urine (Ala, Pro, Gly, Val, Ile, Leu, Tyr, Gln, Trp, His, Met, Ser, Thr, Phe, Asn, Lys, Cys, Glu, Asp, Arg) Methamidophos, fensulfothion, fenamiphos, isophenphos, profenofos, sulprophos Or acidic drugs IgG1 glycosylation antibodies

MEKC-ESIMS

50 mm I.D.  90 cm

CEC-ESI-MS

ITP-MS

BGE: 75 mM perfluorooctanic acid adjusted to pH 9.0 with ammonium hydroxide 14.2 M BGE: 150 mM APFO adjusted to pH 9.0 with 14.2 M ammonium hydroxide

SL: 2propanol:formic acid (99.9:0.1, v/v)

[95]

SL: isopropanol/ water/formic acid (90:10:1 v/v/v)

[85]

100 mm I.D.  34 cm Packed with silica C18 particles

BGE: ACN/H2O, 10 mM NH4Ac pH 4.5, 90/10 (v/v) or ACN/H2O, 15 mM NH4Fo pH 2.5, 75/ 25 (v/v)

LJ: 0.1% formic acid (v/v) in MeOH/ H2O, 80:20 (v/v) 0.1% NH3 (v/v) in MeOH/H2O, 80:20 (v/v)

[65]



Leading electrolyte: 100 mM ammonium acetate pH 4; terminating electrolyte: acetic acid

SLess: porous tip

[51]

Capillary electrophoresis-mass spectrometry

17 Nmethylcarbamates

Continued

431

Table 4.1 Selected CE-MS applications.dcont'd Capillary

FMOC-amino acid enantiomers (DLalanine (DL-Ala), Lhistidine (L-His), DLHis, Llysine (L-Lys), DL-Lys, L-valine (LVal), DL-Val, Lmethionine (L-Met), DL-Met, L-leucine (LLeu), D-Leu, Ltyrosine (L-Tyr), DTyr, DL-threonine (DL-Thr), L-arginine (L-Arg), DL-Arg, Lasparagine (L-Asn), L-serine (L-Ser), DLSer, L-proline (LPro), L-tryptophan (L-Trp), D-Trp, Lglutamic acid (LGlu), L-Phe, Lcysteine (L-Cys), DLCys, D-glutamine (D-Gln), DL-Orn, DOrn, DL-citrulline (DL-Cit), D-Cit, Lpipecolic acid (L-Pip), DPip)

CZE-ESI-MS

50 mm I.D.  60 cm (coated)

Experimental conditions

BGE: 0.5 mM vancomycin in 50 mM ammonium formate pH 7.0 with 10% methanol

Interface

References

SL: e methanol and 50 mM ammonium formate (pH 7.0) (50:50, v/v)

[96]

Hyphenations of Capillary Chromatography with Mass Spectrometry

CE mode

432

Analytes

CZE-ESI-MS

50 mm I.D.  72 cm

Pheniramine (PHM), phenylephrine (PHE), paracetamol (PCM) in urine

ITP-CZE-MS

ITP column: polytetrafluorethylene (PTFE) 800 mm I.D.  9 cm. CZE: 300 mm I.D.  16 cm

Varenicline and its targeted metabolite, 2-hydroxyvarenicline, in urine

ITP-CZE-MS

ITP column: polytetrafluorethylene (PTFE) 800 mm I.D.  9 cm. CZE: 300 mm I.D.  16 cm

BGE: 50 mM acetic acid and 50 mM formic acid, adjusted to pH 3.5 with ammonium hydroxide. Leading electrolyte ¼ 10 mM NH4Ac þ 20 mM HAc (pH 4.5); terminating electrolyte ¼ 10 mM HAc (pH 3.1) in ITP, and BGE ¼ 10 mM HAc (pH 3.1). Leading electrolyte ¼ 10 mM NH4Ac þ 20 mM HAc (pH 4.5); terminating electrolyte ¼ 10 mM HAc (pH 3.1) in ITP, and BGE ¼ 10 mM HAc (pH 3.1).

SL: propan-2-olwater (60:40 v/v) with formic acid (0.05% v/v)

[97]

SL: methanol/water (50/50, v/v) with 0.1% (v/v) HAc.

[98]

SL: methanol/water (50/50, v/v) with 0.1% (v/v) HAc.

[99]

Capillary electrophoresis-mass spectrometry

Opioid peptides in cerebrospinal fluid

433

Continued

CE mode

Capillary

Cathinone derivatives enantiomers

CZE-ESI-MS (partial filling)

20 mm I.D.  90 cm

Estrogens in milk or yogurt

MEKC-ESIMS

50 mm I.D.  60 cm

Phenolic plant extracts

CZE-ESI-MS

50 mm I.D.  73 cm

5-Nitroimidazole antibiotics (metronidazole, secnidazole, and ternidazole) in urine

CEC-ESI-MS

75 mm I.D.  25 cm packed with RP18 and silica (3:1, w/w)

Experimental conditions

BGE: 55% of the capillary is filled with 0.125% HS-g-CD in 15 mM (þ)-18-C-6TCA, while the BGE was 15 mM (þ)-18-C-6-TCA. BGE: 45 mM of ammonium perfluorooctanoate (APFO) at pH 9.0 containing 10% v/v of MeOH BGE: 50 mM ammonium acetate (pH 9.1) ACN/MeOH/water (45:10:45, v/v/v) containing 5 mM of ammonium acetate buffer (pH 5) BGE: acetonitrile/

Interface

References

SLess-porous tip

[83]

SL: 2-propanol/ water (96:4, v/v) flow rate 1.7 mL/ min

[84]

SL: 2,2- diphenyl1-picrylhydrazyl (DPPH) in methanol/ammonia LJ: isopropanol/ water (50:50, v/v) and formic acid (0.05%, v/v)

[100]

[101]

Hyphenations of Capillary Chromatography with Mass Spectrometry

Analytes

434

Table 4.1 Selected CE-MS applications.dcont'd

methanol/water with ammonium acetate (5 mM pH 5) (45:10:45 v/v/v) CZE-ESI-MS ITP-ESI-MS

15 mm I.D.  60 cm

BGE: 1.5% formic acid (v/v; pH ¼ 1.9)

SLess

[62]

CZE-ESI-MS

15 mm I.D.  85 e100 cm or 5e10 mm I.D.  24e25 cm

BGE: 0.5% formic acid with (18-crown6)-2,3,11,12tetracarboxylic acid

SLess-porous tip

[54]

CEC-ESI-MS

Packed with silicaPinnacleTM II phenyl 75 mm I.D.  25.0 cm

Mobile phase: 5 mM NH4Ac at pH 4.5 in 60/40 ACN/ H2O v/v; separation voltage, þ15 kV

[102]

FMOC-amino acids enantiomers in cerebrospinal fluid

CZE-ESI-MS

50 mm I.D.  80 cm

BGE: 0.05 M ammonium bicarbonate (pH 8) isopropanol (85:15,

LJ: spray liquid, MeOH/H2O, 60:40 (v/v) with 0.1% (v/ v) NH4OH delivered at a pressure of 4.0 kPa SL: isopropanolwater-1 M ammonium bicarbonate (50:50:1, v/v/v)

[103]

Capillary electrophoresis-mass spectrometry

Drugs in serum (dexrazoxane and its hydrolyzed form ADR-925) Amphetamine, cathinone, normephedrone and pregabalin enantiomers Estrogens in water

Continued

435

CE mode

Capillary

Experimental conditions

Interface

References

SL

[78]

v/v) and 10 mM bcyclodextrin BSA tryptic digest

Heart-cut 2DCE-MS

1st dimension:50 mm I.D.  38.2 cm 2nd dimension: 50 mm I.D.  55.1 cm

BGE: 1st dimension,10 mM phosphoric acid (pH 2.5) 2nd dimension, 10%, v/v acetic acid

SL, Sheath Liquid; LJ, Liquid Junction; SLess, Sheathless; 18-C-6-TCA, 18-crown-6 ether-tetracarboxylic acid.

Hyphenations of Capillary Chromatography with Mass Spectrometry

Analytes

436

Table 4.1 Selected CE-MS applications.dcont'd

Capillary electrophoresis-mass spectrometry

437

It is known that, in order to avoid MS contamination and ion signal suppression, volatile solvents have to be used. In CZE and in MEKC some additives have to be added to the MP in order to increase the selectivity, e.g., charged complexing agents and ion forming micelles, respectively. Often such additives decrease sensitivity [79], and surfactant related spectra can be detected [80]. Therefore, frequently, they generate drawbacks that must be solved. An interesting approach was proposed by Lamoree et al. [81], who employed a 2D heart-cutting approach with two capillaries. Compounds were separated on the first capillary containing micelles and transferred to the second capillary for the CZE separation. The two columns were connected through an LJ interface. Alternatively to this method, Terabe’s group proposed a partial-filling approach, it being applied to the separation of neurotensin and angiotensin analogs (as cations at pH 3) by MEKC in presence of an uncharged surfactant such as sucrose monodecanoate and detected with MS [82]. The surfactant occupied only a small part of the capillary, while peptides reached the detector at higher velocity. Therefore, analytes were detected in absence of the surfactant with good sensitivity. A similar method was also used by Moini and Rollman for the CZE-MS chiral separation of cathinone derivatives. A negatively-charged highly sulfated cyclodextrin was employed as the chiral selector added to the BGE [83]. In an alternative to the partial filling method, Fanali et al. used a polyacrylamide coated capillary of 50 mm I.D. to minimize the EOF for the enantioresolution of some nonsteroidal antiinflammatory drugs by CZEMS [57]. The acetate buffer at pH 4.8 supplemented with vancomycin filled the whole capillary; however, being the chiral selector positively charged at this pH, it migrated in the opposite direction of the analytes (negatively charged). Carprofen, flurbiprofen, ketoprofen, and naproxen enantiomers were resolved and detected with good sensitivity in absence of the chiral selector. Fig. 4.7A and B show a scheme of the counterflow CZE separation and the electropherograms of the studied enantiomers, respectively. The chiral separation was obtained by carefully selecting experimental conditions, e.g., buffer pH, vancomycin concentration, applied voltage, and capillary coating. The optimized method was also applied to the chiral separation of (i) ibuprofen and its metabolites (2-hydroxyibuprofen and carboxyibuprofen); (ii) etodolac and its metabolites in urine, injected

438

Hyphenations of Capillary Chromatography with Mass Spectrometry

Figure 4.7A Scheme of the counter flow electrophoretic separation of the anionic analytes using vancomycin as the chiral selector. (Reproduced from S. Fanali, C. Desiderio, G. Schulte, S. Heitmeier, D. Strickmann, B. Chankvetadze, G. Blascke, Chiral capillary electrophoresis-electrospray mass spectrometry coupling using vancomycin as chiral selector, J. Chromatogr. A 800, 1998, 69e76 with permission of Elsevier.)

directly without any treatment. Her’s group utilized CZE-MS with an SL interface for the separation of a series of triazines. Compounds were analyzed in normal EOF mode, with acetate buffer at pH 4.5, achieving only partial resolution. By reversing the EOF and adding cetyltrimethylammonium bromide (CTAB) to the mobile phase, baseline resolution was obtained for the eight analytes. The MEKC-MS process can be carried out with good sensitivity by using volatile surfactants such as ammonium perfluorooctanoate (APFO) added to the BGE, as shown by D’Orazio et al. who employed an SL interface [84]. Estrogens were separated and analyzed in milk and yogurt samples. The same surfactant was also used by another group for the analysis of 17 N-methylcarbamates [85]. Fig. 4.8 reports the separation by MEKC-MS of some estrogens in yogurt samples using a volatile surfactant added to the BGE.

Capillary electrophoresis-mass spectrometry

439

Figure 4.7B CEeESI-MS electropherograms of racemic nonsteroidal antiinflammatory drugs: (A) (þ/)-carprofen, (B) (þ/)-flurbiprofen, (C) (þ/)-ketoprofen and (D) (þ/)-naproxen in the presence of 5 mM of vancomycin in the BGE. (Reproduced from S. Fanali, C. Desiderio, G. Schulte, S. Heitmeier, D. Strickmann, B. Chankvetadze, G. Blascke, Chiral capillary electrophoresis-electrospray mass spectrometry coupling using vancomycin as chiral selector, J. Chromatogr. A 800, 1998, 69e76 with permission of Elsevier.)

As can be seen in Table 4.1, the SL interface has been used for CZE-MS applications in various fields such as drug analysis [86,87], agrochemical, food, biomedicine, enantiomers. A new LJ interface was applied to the separation of some b-blockers, peptides, and a tryptic digest mixture by using CZE-MS [63]. Ammonium acetate at pH 3.5, or ammonium formate at pH 2.8, were effective for analyte separation. The method was optimized studying several parameters such as type of spray liquid, pressure applied at the interface and at the inlet capillary. Good sensitivity (LODs of 10 ng/mL and 200 ng/mL were

440

Hyphenations of Capillary Chromatography with Mass Spectrometry

Figure 4.8 MEKC-MS separation of estrogens in yogurt samples. Analyzed estrogens: E3 (1,3,5(10)-estratriene-3,16a,17b-triol), b-ZAL (2,4-dihydroxy-6-(6b,10-dihydroxy undecyl]benzoic acid m-lactone), a-ZAL (2,4-dihydroxy-6-(6a,10-dihydroxyundecyl) benzoic acid m-lactone), a-ZEL (2,4-dihydroxy-6-(6a,10-dihydroxy-trans-1-undecenyl) benzoic acid m-lactone), b-ZEL (2,4-dihydroxy-6-(6b,10-dihydroxy-trans-1-undece nyl)benzoic acid m-lactone), ZEN ((3S,11E)-14,16-dihydroxy-3-methyl-3,4,5,6,9, 10-hexahydro-1H-2-benzoxacyclotetradecine-1,7(8H)-dione), 17a-E2 (1,3,5(10)-estratriene-3,17a-diol), 17b-E2 (1,3,5-estratriene-3,17b-diol), EE2 ((17a-ethynyl-1,3,5(10)estratriene-3,17b-diol), 2-MeOE2 (1,3,5(10)-estratriene-2,3,17-triol 2-methyl ether), E1 (1,3,5(10)-estratriene-3-ol-17-one). Experimental conditions: separation capillary, 60 cm  50 mm I.D.; BGE, 45 mM of ammonium perfluorooctanoate (PFOA) at pH 9.0 (with 7 mM ammonium hydroxide) containing 10% (v/v) of MeOH. (Reproduced from G. D’Orazio, M. Asensio-Ramos, J. Hernández-Borges, M.A. Rodríguez-Delgado, S. Fanali, Evaluation of the combination of a dispersive liquid-liquid microextraction method with micellar electrokinetic chromatography coupled to mass spectrometry for the determination of estrogenic compounds in milk and yogurt, Electrophoresis 36, 2015, 615e625 with permission of John Wiley and Sons.)

Capillary electrophoresis-mass spectrometry

441

measured for b-blockers and peptides, respectively) and repeatability for both CZE runs and MS detection were obtained. Later, the same implemented interface was also applied to a CEC-MS study for the separation of some organophosphorus pesticides and drug enantiomers utilizing packed capillaries with modified silica (C18 or vancomycin) [64]. Several experimental parameters such as liquid ion-spray, mobile phase, and hydrostatic pressure at the electrode end compartment were studied and optimized. The method was validated and applied to the analysis of some pesticides in drinking water samples.

4.6 Conclusions and perspectives Modern electrodriven separation techniques have become very popular in the field of separation science and commercial instrumentation is now available. The different CE modes, ITP, CZE, MEKC, and CEC offer the possibility to analyze a large number of compounds, offering the desired selectivity and very high efficiency. Although several detectors have been used mainly based on UV, hyphenation with MS can be performed in order to determine the mass and characterize analytes. This is particularly useful when complex samples are analyzed. The coupling of CE with MS can be done with different interfaces, namely sheathless, sheath-liquid, and liquid junction. Although some drawbacks have been observed in coupling CEC with MS, some solutions have been proposed, e.g., using liquid-junction interface. Some examples of 2D separations have been reported showing the possibility to increase the number of analyzed compounds. This approach is only at the beginning and therefore additional work is needed. Examples of CE separations realized with chips have been reported. Interesting results have been obtained especially considering the short analysis time and the high efficiency. However, its use is quite limited due to the required costs. Finally, further studies are necessary, e.g., in CEC concerning the synthesis of new stationary phases offering high selectivity. This research could be performed also considering the needs of other techniques such as HPLC or nano-LC.

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