Coupling of Capillary Electromigration Techniques to Mass Spectrometry

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COUPLING OF CAPILLARY ELECTROMIGRATION TECHNIQUES TO MASS SPECTROMETRY

12

Christian Neus€uß, Jennifer R€omer, Oliver H€ocker, Kevin Jooß Aalen University, Aalen, Germany

CHAPTER OUTLINE 12.1 Introduction .............................................................................................................................. 293 12.2 Coupling of Electromigration Techniques to Mass Spectrometry ................................................... 294 12.3 Interfaces for CE-ESI-MS Coupling ............................................................................................. 295 12.3.1 Sheath Liquid Interfaces ...................................................................................... 295 12.3.2 Sheathless Interfaces ........................................................................................... 297 12.4 Methods and Applications in CE-MS ........................................................................................... 299 12.4.1 Back Ground Electrolytes for CE-MS ..................................................................... 299 12.4.2 Capillary Coatings for CE-MS ................................................................................ 300 12.4.3 Parameters of Sheath Liquid in CE-MS .................................................................. 301 12.4.4 Important Applications of CE-MS .......................................................................... 302 12.5 Combining MS-Interfering CE Electrolytes With Mass Spectrometry .............................................. 303 12.5.1 Complete Exchange of the Background Electrolyte .................................................. 305 12.5.2 Compromise Between Separation and Ionization Efficiency ..................................... 305 12.5.3 Alternative CE-ESI-Interface ................................................................................. 306 12.5.4 Alternative Ionization Techniques .......................................................................... 306 12.5.5 Two-Dimensional Separation Techniques ............................................................... 307 12.6 Future Trends ............................................................................................................................ 309 References ........................................................................................................................................ 309

12.1 INTRODUCTION Mass spectrometry has revolutionized analytical chemistry in the last decades and is currently a routine tool in many analytical laboratories. The success of mass spectrometry is based on the high selectivity of mass determination. Selectivity and potentially also sensitivity can be further extended by dedicated Capillary Electromigration Separation Methods. https://doi.org/10.1016/B978-0-12-809375-7.00012-5 # 2018 Elsevier Inc. All rights reserved.

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fragmentation techniques and/or high-resolution mass analyzers leading to accurate mass determination. In this way, mass spectrometry often combined with chromatographic separation is a key technology in many scientific fields ranging from environmental analysis to bioanalytical chemistry. Capillary electrophoresis is a microfluidic technique and fits perfectly with mass spectrometry. This is due to small liquid volumes to be combined with the high vacuum requirement of MS as well as the fact that in both cases ions are separated; thus, theoretically no “additional ionization” is required in front of the mass analyzer. Initially, we discuss interfacing techniques for efficient and reproducible coupling of CE to MS. The small volumes in CE are on the one hand side often beneficial (low reagent costs, low consumption of resources, no dilution of small sample volumes); however, compared to LC-MS, the low concentration sensitivity limits the application area of CE-MS, since upscaling is not possible in a simple way. Anyway, the selectivity of CE for ionic molecules, potentially enabling separation of very similar compounds, makes CE-MS a valuable tool for the characterization of ionic analytes, often difficult to characterize by LC-MS. Thus, CE-MS represents an important tool to characterize intact proteins, peptides, metabolites, glycans, phenols, nucleotides, and many more compound types. In the second part of this chapter, various background electrolytes and coatings are presented along with their applications. In contrast to LC, many CE methods with optical detection (UV/Vis absorption, fluorescence) cannot be easily transferred to CE-MS methods. Though the main parameter for selectivity is the pH (compare Chapter 10), the separation power of CE often relies on the flexible use of electrolyte composition, including nonvolatile components. A majority of these electrolyte systems cannot be transferred easily to a system of volatile electrolytes usually required in CE-MS. Thus, this subject is one major focus of this chapter. Finally, we will try to envision future prospects of CE-MS, including combinations with other techniques and the extension to microchip devices. For a more detailed view on CE-MS, we refer to excellent reviews on coupling techniques [1,2], and applications [3] as well as to a recent compilation edited by de Jong [4].

12.2 COUPLING OF ELECTROMIGRATION TECHNIQUES TO MASS SPECTROMETRY In general, the coupling of separation techniques to mass spectrometry can be performed either online or offline. Due to the small volumes inherent to capillary electrophoresis fraction collection is rarely applied. The only offline coupling to mass spectrometry is to be found in some studies of the coupling to MALDI MS. Typically, a sheath liquid is applied in this context for both electrical contact at the outlet and for a sufficient flow for droplet formation on a reasonable time scale. In most cases, online coupling to mass spectrometry is utilized with capillary electromigration techniques. The benefits of on-line coupling consist mainly of the high time resolution for immediately derived results, including both mass spectra and electropherograms. Fast separations by CE are retained in CE-MS using short capillaries [5]; however, the need to reach the mass spectrometer outside of the CE instrument requires longer capillaries (>50 cm) than in CE with optical detection, when commercial instrumentation is used. CE is frequently coupled to ICP-MS for elemental analysis. For the analysis of organic compounds, ESI is by far the most important ionization technique for CE-MS. This is due to the fact that in CE usually ionic compounds are separated and CE is a microfluidic technique

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easily combined with the generation of small and highly charged droplets for efficient ionization in ESI-MS. However, alternative ionization techniques have been developed often within the context of applying non-ESI-compatible background electrolytes (BGE) for separations (Section 12.5) [6].

12.3 INTERFACES FOR CE-ESI-MS COUPLING The coupling of capillary electrophoresis with electrospray ionization mass spectrometry is challenging due to the requirement of two individual electrical circuits, varying flow rates in the separation capillary, and direction of the electroosmotic flow. Two categories of interfaces can be distinguished for the hyphenation: sheath liquid interfaces with an additional make up flow and sheathless liquid interfaces where the background electrolyte is sprayed directly. Sheath liquid interfaces with flow rates in the μL/min range are still the most frequently used for most applications due to their ease of use and versatility. Many different nanospray interfaces have been developed in recent years, offering improved sensitivity due to lower flow rates (nL/min range).

12.3.1 SHEATH LIQUID INTERFACES In these interfaces, an additional liquid (“sheath liquid” or “make-up liquid”) is used to provide electrical contact at the end of the separation capillary by adding an additional liquid flow to the effluent of the CE capillary. The sheath liquid either mixes with the CE effluent after separation and is sprayed directly or mixes in a narrow gap between the capillary outlet and the separate electrospray emitter. The latter is known as a liquid junction interface. Dilution of the analyte by the additional liquid flow reduces the signal intensity as electrospray is a concentration-dependent process. On the other hand, sheath liquid composition can be chosen to optimize the ionization efficiency. Organic solvents such as isopropanol and methanol increase ionization efficiency and are used with water in 1:1 to 5:1 ratio. High organic solvent content on the other hand reduces conductivity, potentially limiting the CE current. Also, the make-up liquid allows sheath liquid chemistry with reactions like H/D exchange [7] and postcolumn chemistry [8].

12.3.1.1 Coaxial sheath liquid interface The coaxial sheath liquid interface is the most widely used interface for CE-MS coupling. In this interface, as developed by Smith and coworkers [9], an additional sheath liquid, coaxially delivered by a metal needle surrounding the capillary, provides both electrical contact and a constant flow, mixing with the capillary effluent at the sprayer tip. Such a “triple tube” design commercialized by Agilent Technologies is shown in Fig. 12.1. For sheath liquid flow rates of 1–10 μL/min, typical for standard 360 μm O.D. capillaries, an additional nebulizer gas is required to obtain a stable spray. The position of the CE capillary with respect to the surrounding metal needle needs to be carefully adjusted to enable electrical contact between the background electrolyte and sheath liquid for grounding while avoiding additional dead volume, so that the capillary needle slightly protrudes the metal needle (around 100 μm). Positioning can influence signal intensity and separation efficiency and was evaluated, for example, by Geiser and coworkers [10]. Nebulizer gas flow rate should be as low as possible to reduce the suction effect leading to peak broadening caused by the pressure difference between capillary inlet and outlet.

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FIG. 12.1 Coaxial sheath liquid interface.

12.3.1.2 Nanoflow sheath liquid interfaces To address the reduced sensitivity caused by the dilution effect, several approaches to reduce the outer diameter of the CE capillary and the surrounding needle to lower the flow of sheath liquid to the nL/min range have been developed. Nanoelectrospray produces smaller initial droplets due to the sharper tips and lower flow rates with more efficient ionization and higher tolerance of ES-interfering components in the sprayed liquid [11–13]. The spray tip can be positioned closer to the MS inlet enhancing the sampling efficiency. In most cases, nebulizing gas can be omitted because of the low liquid flow rate. Narrow tapered needles enable operation without additional nebulizer gas [14]. The flow-through microvial interface with a stainless steel needle and beveled tip is one example, where standard capillaries can be used. The high electrical field on the sharpest point of the asymmetrical tip allows modifier liquid flow rates of 100–400 nL/min. The inside of the emitter acts as an outlet vial and mixing chamber for the capillary effluent and modifier liquid [15]. This interface was applied to cIEF-MS [16]. Another nanoflow interface of a similar type is a low sheath flow interface with a blunt [17]) or tapered stainless steel needle ([18] with flow rates of 0.7 and 0.3 μL/min, respectively). A nanoflow sheath liquid interface, where the separation capillary is introduced into a glass emitter, was reported by Hsieh et al. [19] and further developed by the Dovichi group [20,21] (Fig. 12.2). The sheath liquid inside the emitter is consumed in the ES process without additional pressure applied. The influence of different orifice diameters (8–30 μm) on the ionization efficiency was tested and the capillary tip O.D. reduced by etching to minimize dead volume inside the emitter tip. In the most recent design, orifice diameters of around 30 μm are used, since they are more robust toward clogging. This interface was used in several application areas, including proteomics and peptide analysis with

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FIG. 12.2 Nanoflow sheath liquid interface with the separation capillary threaded through a PEEK T-union into a glass emitter. The side arm connects to a sheath liquid reservoir, open to ambient pressure with a platinum electrode where an electrospray voltage or grounding can be applied.

sensitivities in the picomolar range [22]. CMP Scientific commercialized this interface under the name EMASS-II. Nanospray can also be achieved by a liquid junction arrangement. Liquid junction interfaces combine the benefits of sheath liquid and sheathless interfaces by applying a liquid junction between the CE capillary outlet and a separate interface spray tip to perform separations independent of the electrospray process. The low μm gap allows both electrical contact and the addition of a spray liquid. Separation electrolyte and spray liquid mix in the gap between the separation capillary and spray emitter. The flow rate is typically in the low nL/min range and can be controlled by applying pressure to the make-up liquid [23] or a slight vacuum to the needle tip and is independent of the CE capillary flow rate [24]. Flow rate adjustments and alignment of the CE capillary with the spray tip to achieve reproducible gaps and dead volumes are difficult. Krenkova et al. developed a system, where the capillaries are selfaligned in a microfluidic chip by a defined angle, which significantly increases the system robustness and enables flow rates of 100 nL/min to be used [25]. The typical set up for a liquid junction interface is shown in Fig. 12.3.

12.3.2 SHEATHLESS INTERFACES Sheathless interfaces utilize only the background electrolyte for spray generation. Therefore, the EOF, pH, and capillary coating influence the flow rate and spray performance. An additional pressure may be required, however, should be as low as possible to avoid peak broadening. To facilitate grounding, the interface electrical contact can be provided in several ways (Fig. 12.3). An electrically conducting coating applied to the capillary tips is the most straightforward solution, but critical regarding life time. The coating can be applied by sputtering, vapor deposition, and electroplating of metals [26] or emitter tips can be fabricated from conductive polymers or carbon can be applied [27]. Inserting metal wires through small holes close to the end of the capillary [28] or a submicrometer fracture is used to allow

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FIG. 12.3 Typical set up for a liquid junction interface. The sheath liquid flow can be delivered by external pressure or vacuum from the mass spectrometer inlet.

electrical contact of the background electrolyte with surrounding electrolyte [29]. Different variations for contact closure in sheathless interfacing are shown in Fig. 12.4. An interface by etching the capillary wall at the outlet over a length of several cm to a wall thickness of 5 μm, where the glass becomes porous and conductive, was published by Moini [30] and is commercially available as CESI8000 by Sciex (Fig. 12.5). The capillary end is etched by hydrofluoric acid to allow electrical contact with the surrounding metal cylinder filled with BGE. The porous tip protrudes from the liquid and acts as an emitter. Clogging is minimized compared to internally tapered emitters, HV

(A)

HV

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(F) HV

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FIG. 12.4 Methods for contact closure in sheathless CE-MS interfacing. (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. Reproduced from Maxwell and Chen with permission of Elsevier.

12.4 METHODS AND APPLICATIONS IN CE-MS

299

FIG. 12.5 Principle of the sheathless porous tip interface with a porous tip segment for contact closure and spray tip.

since the internal diameter is constant over the whole capillary. In the commercially available interface, 30 μm I.D. capillaries are used, a whole capillary cooling can be applied and connection of CE to MS is user friendly. Flow rates are reported down to 5–20 nL/min, allowing low ion suppression and high sensitivity [31]. A porous tip interface with detachable tip was developed by Wang et al. [32], where a piece of capillary with smaller O.D. and a porous etched tip is glued into the separation capillary. The larger bore separation capillary combines high sample loadability with the fine spray tip of a small I.D. capillary. An “interface-free” interface was reported, where the inlet separation voltage is used to create an electrospray and no additional grounding is needed [33]. To match the separation and electrospray currents, only capillaries with an I.D. of <20 μm and low concentrated buffer solutions are suitable for this application. The outlet of the separation capillary acts as an emitter and is sharpened by grinding to an angle of 5 degree. Sheathless interfaces often require a high EOF created by coated capillaries in many cases. This limits the application for anion separations with high pH electrolytes. Most separations of peptides, proteins and other cations are performed at low EOF and low pH conditions or with coated capillaries suppressing the EOF to achieve the required separations. Pressurization can be used to enhance flow, however, leading to lower separation efficiency.

12.4 METHODS AND APPLICATIONS IN CE-MS 12.4.1 BACK GROUND ELECTROLYTES FOR CE-MS For the hyphenation of CE and MS, some requirements need to be fulfilled. First of all, the background electrolyte (BGE) should be compatible with efficient electrospray ionization. Thus, a volatile BGE is beneficial to avoid interference from salts in the ionization process. Most commonly used BGEs for CE-MS applications are formic acid (HFA) and acetic acid (HAc) for low pH ranges and their respective ammonium salts for acidic to neutral pH ranges. Alternatively, ammonium carbonate or ammonium hydroxide can be used for the high pH range. An overview of the most common BGEs and their buffering range is given in Fig. 12.6.

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FIG. 12.6 Most common volatile BGEs and their respective pH buffer range. Reprinted and adapted from Pantu˚ckova´ P, Gebauer P, Bocek P, Kriva´nkova´ L. Electrolyte systems for on-line CE-MS. Electrophoresis 2009;30(1):203–14 with permission from John Wiley and Son.

Despite limited buffer capacity, the total pH range is covered by these volatile electrolytes. However, beside the pH, the selectivity in CE is also influenced by the type of electrolyte and potential additives. Thus, the use of volatile electrolytes limits the selectivity for the separation of certain analytes. For further discussion and possible solutions, see Section 12.5. Besides volatility, the ionic strength of the BGE and the resulting current is limited for mass spectrometric detection. Here, not only the Joule heating needs to be considered, but also the interfacing with MS restricts the applicable current. As a result of the limited current, either the ionic strength of the BGE must be adjusted or a lower voltage applied. A lower voltage should be avoided if it is possible to assure optimum separation efficiency and fast separations.

12.4.2 CAPILLARY COATINGS FOR CE-MS Fused silica capillaries are commonly used due to their convenient handling and inertness. In addition, they are available in various diameters and are relatively inexpensive. However, measurements with a bare fused silica capillary are associated with some difficulties such as the adsorption of the analyte on the capillary wall. The use of a BGE of extreme pH can help avoid these interactions. At low pH, silanol groups are mostly protonated and uncharged. Thus, the ionic interactions with positively charged analytes are reduced. If a BGE of high pH is used, some analytes like proteins can have a negative net charge and are repulsed from the negatively charged silanol groups. Measuring negatively charged analytes by negative electrospray ionization can result in lower signal intensities compared with the same analytes in positive ESI mode. Depending on the noise, this can lead to higher limits of detection and loss of sensitivity. Furthermore, many analytes, such as proteins, still tend to adsorb on the wall of fused silica capillaries. Therefore, it is common to choose a suitable capillary coating to reduce analyte adsorption and to enhance the stability and performance of the separation. This can be achieved by using a neutral or a charged coating. Neutral coatings prevent analyte adsorption via shielding the silanol groups of the capillary wall. Charged capillary coatings, mainly positively charged, can prevent analyte adsorption via electrostatic repulsion.

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Apart from analyte wall interactions, capillary coatings influence the electroosmotic flow (EOF). The direction and magnitude of the EOF are crucial for a stable and successful separation. If the EOF is directed to the capillary inlet, sheath liquid and even air can enter the capillary. This leads to an unstable current or even a total current breakdown. For this reason, the EOF needs to be directed toward the mass spectrometer. In case of a weak EOF, it is possible that sheath liquid counterions enter the capillary changing the separation conditions, for example, pH, with a loss of resolution. To avoid this problem, either a strong EOF is needed or the sheath liquid counterions should have the same pKa values and electrophoretic mobility. If sheathless liquid ionization is applied, the Taylor cone and following electrospray is only generated by the BGE. Therefore, the EOF needs to be directed to the MS to obtain a stable spray. Thus, charged capillary coatings are more common with this kind of interface. Coating techniques can be classified as either dynamic or static. Dynamic coating agents are incorporated into the BGE. The interactions between analytes and silanol groups of the column wall are reduced by competition with the dynamic coating compounds. The coating compounds enter the MS, which can lead to ionization suppression, additional background signals, and contamination of the ion source. In contrast, static coatings are anchored to the capillary wall prior to separation. This can be performed by either chemical bonding (static-covalent) or by adsorption (static-adsorbed). These coatings offer the advantage that almost no coating material enters the MS during the separation. Statically adsorbed coatings such as the cationic polybrene (PB) or neutral acrylamide-based Ultratrol (LN) coatings have the advantage that the coating is performed using simple rinsing steps. After a few runs, these capillary coatings need to be renewed. In general, these adsorbed precoatings are not suitable for use at extreme pH. A comparison of the separation of erythropoietin (EPO) measured with a neutral and a cationic coating is shown in Fig. 12.7. Static-covalent coatings are used to permanently modify the capillary wall and are stable over a wide pH range. Poly(vinylalcohol) (PVA) can be permanently fixed on the capillary wall by thermal immobilization and for higher stability crosslinked with glutaraldehyde. The coating procedure, including the polymerization by heating, is more extensive than generating a statically adsorbed coating by flushing. The resulting coating suppresses the EOF and is stable over a wide pH range. In conclusion, dynamic coatings are rarely applied, whereas both types of static coatings are used in CE-MS. Static-covalent coatings are more common and more stable compared with statically adsorbed coatings. Further details for capillary coatings in CE-MS can be found in the review of Huhn et al. [34].

12.4.3 PARAMETERS OF SHEATH LIQUID IN CE-MS The standard interface is a coaxial interface using a sheath liquid and a nebulizer gas. The choice of the sheath liquid is important to obtain optimum analyte ionization efficiency. In general, mixtures of water and organic solvents such as methanol, isopropanol, and acetonitrile are common. Depending on the functional groups of the analyte, additives including volatile acids and bases in the sheath liquid can be beneficial for ionization. As mentioned before, the choice of additives can influence the separation if a low EOF is present. Here, the added ions should be identical to the ions present in the BGE in order to avoid moving boundaries of different ions and pH regions. If a strong EOF is present, the additive should be selected to obtain the best ionization efficiency for the analyte to improve signal intensity.

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13 SiA 12 SiA

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FIG. 12.7 CE-MS of human recombinant erythropoietin using a neutral coating (A) and a cationic coating (B).

12.4.4 IMPORTANT APPLICATIONS OF CE-MS CE-MS is often utilized as a complementary technique to LC-MS for the analysis of charged and polar metabolites in different fields. A main goal of metabolomics is the identification of biomarkers for diseases. For the analysis of biomarkers, body fluids such as blood, urine, and cerebrospinal fluid (CSF) are often used. With the help of chemometric methods, CE-MS is also promising for highthroughput separations of metabolite profiles. This can be useful in diagnostics and screening for metabolite changes in patients. A representative example is the discovery of schizophrenia biomarkers in plasma by Koike et al. [35]. Compared to LC-MS metabolomic studies, the CE-MS methods allow the

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identification and measurement of ionized and low-molecular-weight metabolites. Five metabolomes showed a significant difference between healthy patients, patients with first-episode schizophrenia, and patients with autism spectrum disorders. Here, only 100 μL of plasma from each patient was required for the analysis. Due to its selectivity, an important application of CE-MS is the analysis of intact proteins, especially in a biopharmaceutical context. The analysis of posttranslational modifications (PTM) is of interest due to their influence on the activity and function of proteins. One example of PTMs is glycosylation. The analysis of intact glycosylated proteins with a large variation of glycosylation patterns is challenging. Here, selectivity with regard to charge and size is essential. Neus€uss et al. [36] analyzed human recombined EPO and separated 44 glycosylated forms according to their number of negatively charged sialic acid residues and glycan size. In addition, taking acetylation into account, about 135 isoforms could be identified. This illustrates the impressive separation efficiency of CE combined with the advantages of accurate mass determination. CE-MS is also often used for the analysis and control of drugs and food products due to its fast separation time, high separation performance, and resolution. As a marker for food quality and freshness 5-hydroxymethylfurfural (HMF) was analyzed by CE-MS in addition to HPLC-UV by Bignardi et al. [37]. The goal was to develop a comparable method to HPLC-UV for the quantification of HMF and simultaneously reduce the cost of reagents and the separation time. The method was applied to selected food products. Quantitative data for HMF were in the same range as those obtained by HPLC-UV and the separation time was reduced for CE-MS. Marakova et al. [38] developed a CE-MS method for vitamin B in pharmaceuticals and dietary supplements. Here, the enhancement of sensitivity and selectivity with CE-MS compared to CE-UV was significant. Compared with LC-MS the CE-MS method afforded lower cost, simplicity, flexibility, and was environment friendly. The method was suitable for routine analysis in multidrug quality control laboratories. An overview of additional applications is given in the review of Ty´cˇova´ et al. [1].

12.5 COMBINING MS-INTERFERING CE ELECTROLYTES WITH MASS SPECTROMETRY Besides the technical challenges related to CE-MS coupling, the influence of the BGE on the electrospray ionization process plays a major role for successful measurements. Generally, volatile BGEs with low ionic strength are selected for CE-MS to prevent ion suppression, contamination, or potential damage to the MS instrument. Ion suppression can be explained by interfering buffer components that capture the generated electrospray charge, salt deposits on the source, and ion pairing with the analyte. Based on these characteristics, the most widely applied BGEs for CE-MS are acetic acid (HAc), formic acid (HFA), and their respective ammonium salts. However, low ionic strength BGEs are linked to zone broadening during the migration of ions mainly due to electrodispersion. In CZE, common nonvolatile BGEs include borate, phosphate, citrate tris(hydroxymethyl)-aminomethane (TRIS), phthalate, and 2-(cyclohexylamino)ethanesulfonic acid (CHES). These compounds are often used to improve the separation selectivity and efficiency, which is exemplarily displayed for a TRIS-based BGE in Fig. 12.8 [39]. In this work, the influence of TRIS on the impurity profile of a potential new drug is shown. Based on a better “mobility match” between the coion and the sample ions as well as a higher ionic strength, electrodispersion is less prominent in BGE (A) compared to BGE (C). Despite BGE (C) being the most volatile and thus, the most MS compatible electrolyte system, baseline separation could only be achieved for BGE (A) indicating the importance of appropriate coions such as TRIS.

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FIG. 12.8 Effect of concentrations of TRIS on peak shape for the impurity profiling of a potential new drug. CE electropherogram (UV 215 nm) of a 1 mg/mL sample at full scale (left) and detail (right). (A) 100 mM TRIS adjusted to pH 3.0 using HFA, (B) mixture of BGE A/C 20/80% (v/v), that is, 20 mM TRIS and (C) 50 mM ammonium formate. Reprinted from van Wijk AM, Muijselaar PG, Stegman K, de Jong GJ. Capillary electrophoresis-mass spectrometry for impurity profiling of basic pharmaceuticals using non-volatile background electrolytes. J Chromatogr A 2007;1159(1–2):175–84 with permission from Elsevier.

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(A)

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The limited choice of electrolytes for CE-MS not only restricts the versatility of the CE separation itself but also affects the potential use of sample stacking by isotachophoresis (ITP)-CZE combinations. In addition, certain modes of CE often require ESI-interfering electrolyte components such as chiral CZE (cyclodextrines, etc.), CIEF (ampholytes), and MEKC (surfactants). On the other hand, nonaqueous capillary zone electrophoresis (NACE) systems often provide different selectivities than classical aqueous CE and are ideal for coupling with MS due to the high volatility and low surface tension of the organic solvents. Still, NACE is associated with challenges in understanding migration, interaction, and dissociation processes in organic solvents. In general, there are five different approaches to tackle the challenges mentioned: (I) Complete exchange of ESI-interfering electrolyte components or additives, ideally retaining the selectivity and separation efficiency; (II) Use of lower concentrated BGEs to achieve a compromise between separation efficiency and ESI interference; (III) Use of an alternative CE-ESI interface than the standard sheath-liquid interface; (IV) Application of a different type of ionization; (V) Implementation of multidimensional methods, off- or on-line, using the ESI-interfering BGE as the first dimension and an MS-friendly separation technique as second dimension coupled to MS. Sometimes more than one strategy is combined to obtain satisfactory results.

12.5.1 COMPLETE EXCHANGE OF THE BACKGROUND ELECTROLYTE At a first glance, the simple exchange of ESI-interfering BGE components seems to be the most straightforward approach. Nevertheless, it is often difficult to retain the original separation performance. Exemplarily, the analysis of proteinogenic glycans is conducted by capillary sieving electrophoresis coupled with laser-induced fluorescence (CSE-LIF) detection using 8-aminopyrene-1,3, 6-trisulfonic acid (APTS) as glycan label. However, a clear identification of glycans with optical detection is almost impossible and coupling CSE to MS is not feasible due to the nonvolatile nature of the pseudo-gel. For this reason, two CZE-TOF-MS methods (acidic and alkaline BGE) for glycan identification have been developed [40]. The system based on the acidic BGE (40 mM ε-aminocaproic acid, 131 mM HAc, pH 4) delivered an analogue migration profile compared to routine CSE-LIF systems allowing the unequivocal assignment of quantitative CSE-LIF to qualitative CZE-TOF-MS data for a medium complex glycan mixture released from a fusion protein, Fig. 12.9. Nevertheless, the mobility of sialylated glycans was slightly shifted relatively to nonsialylated glycans compared with common CSE-LIF systems. Thus, the transfer of structural assignments from CZE-MS to CSE-LIF was challenging in the absence of knowledge of the expected glycans or if very complex samples are analyzed.

12.5.2 COMPROMISE BETWEEN SEPARATION AND IONIZATION EFFICIENCY In a variety of applications, the ESI-interfering BGE components are mandatory for resolution, separation efficiency, and stability of the CE methods. Therefore, a compromise between MS-friendly conditions and separation performance is often required. The most common strategy for coupling CIEF with ESI-MS involves direct coupling using low ampholyte concentrations. The first online CIEF-ESIMS coupling was realized by placing the catholyte vial inside the ESI source during the focusing step [41]. A relatively low ampholyte concentration of 0.5% was considered the best compromise between CIEF resolution and MS sensitivity. Several other approaches are described in a recent review [42]. For the quality control of pharmaceuticals, chiral CE is commonly used employing nonvolatile chiral selectors such as cyclodextrins (CDs). In latter years, different methods have been developed for chiral

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FIG. 12.9 Separation of a medium complex glycan mixture by CZE-TOF-MS (BGE: 40 mM ε-aminocaproic acid, 131 mM HAc, pH 4) and CSE-LIF (Beckman glycan separation buffer). Assignment of respective peaks demonstrating the congruent separation. € C. Capillary electrophoresis/mass spectrometry of APTS-labeled glycans for the Reprinted and adapted from Bunz S-C, Rapp E, Neususs identification of unknown glycan species in capillary electrophoresis/laser-induced fluorescence systems. Anal Chem 2013;85 (21):10218–24 with permission from John Wiley and Son.

CE-MS, including counter migration of charged CDs and/or partial filling techniques using neutral CDs. Nevertheless, these methods represent a compromise between separation and ionization efficiency. A more detailed discussion can be found elsewhere [43]. Considering MEKC, similar attempts have been pursued to overcome the ion suppression caused by surfactants, for example, sodium dodecyl sulfate (SDS), including partial filling techniques or replacement of ESI-interfering surfactants by lowinterfering BGE components [44]. It is worth highlighting the application of polymer surfactants for MEKC-MS, since they have a close to zero critical micelle concentration and thus, cause less interference with the ESI process in contrast to traditional surfactants. Still, comparable issues are faced as described for chiral CE-MS.

12.5.3 ALTERNATIVE CE-ESI-INTERFACE In recent years, several new CE-ESI interfaces have been developed. Interfaces based on nanoESI processes compared with traditional ESI are generally more tolerant of ESI-interfering components and provide higher sensitivity (Section 12.3). However, there are few studies of semivolatile/nonvolatile BGEs in combination with nanospray interfaces. This field is rather new and in need of further investigation.

12.5.4 ALTERNATIVE IONIZATION TECHNIQUES Although ESI is the most frequently used ionization technique for CE-MS, alternative ion sources represent a potential solution to BGE-related MS interference. Soft ionization techniques, which are

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known to be less affected by nonvolatile BGE constituents, include matrix-assisted laser desorption/ ionization (MALDI), atmospheric pressure chemical ionization (APCI), atmospheric pressure photoionization (APPI), and inductively coupled plasma (ICP). Each technique has its own benefits and drawbacks. Although ion suppression does not affect CE-ICP-MS, it is limited to the detection of metal-containing analytes and some other elements such as phosphorous or selenium. APCI and APPI are generally more suited for the ionization of small, low-polarity compounds, which is contradictory to a majority of compounds analyzed by capillary electromigration separation techniques. Still both APCI and APPI were successfully applied for the coupling of MEKC to MS. In addition, there have been attempts to couple CIEF offline to MALDI-MS, although MALDI is known to be less sensitive to additives or salts. Still, a significant decrease of signal intensity was observed with traditional CIEF setups.

12.5.5 TWO-DIMENSIONAL SEPARATION TECHNIQUES There are two different purposes for the application of two-dimensional (2D) separation techniques. In the majority of cases, 2D approaches are used to obtain higher peak capacities by combining two separation techniques with different selectivity (e.g., a combination of CE and LC methods). These setups can also be used to enable mass spectrometric detection of analytes separated in highly ESI-interfering electrolyte systems as the first dimension. Hence, the remaining interfering buffer components of the first dimension are either completely removed from the sample or separated from the analytes of interest in the second dimension prior to MS detection. If an ESI-interfering CE method is applied as first dimension, it is beneficial to use CZE as the second dimension, due to matching of capillary geometries and the availability of MS-friendly BGE systems. On the other hand, cRPLC-MS was applied as a second dimension in some applications. In general, the 2D coupling techniques involving CE as the first dimension can be divided into offline and online approaches. In offline 2D methodologies, fractions of the first dimension separation are transferred individually to a second dimension separation such as CE-MS. This approach is rather straightforward and frequently preferred if the sample needs to be treated in-between (washing, derivatization, digestion, etc.). Still, the collection of CE effluent is generally associated with sample dilution since nL fractions are typically collected in at least a few μL of solvent. In principle, this limitation can be overcome by the integration of an additional sample enrichment step, such as solid-phase extraction, before the separation in the second dimension takes place. However, these approaches further complicate the whole system set-up. In addition, offline 2D methods are usually time and labor intensive, and in the majority of cases, cannot be automated easily. Online 2D approaches involving at least one capillary electromigration separation technique can be classified into two groups: (i) application of two different CE modes in one single capillary and (ii) the hyphenation of two individual separation systems via an interface. However, considering singlecapillary approaches the coupling to MS or other external detectors is often impossible due to the need of a (pressurized) outlet vial. This limitation does not apply to interfaces for 2D coupling. Over the last few years, several different interfaces have been developed with some allowing coupling to MS, for example, dialysis, flow gating, and mechanical-valve-based interfaces. The characteristics of each interface are discussed in recent reviews [45,46]. As an example, the results for the analysis of degradation products of acetylsalicylic acid (ASA)/ascorbic acid (AA) formulations are shown using CE-CE-MS and a mechanical 4-port-nL-valve interface, Fig. 12.10, where a highly ESI-interfering BGE, 100 mM tricine, was used as the first dimension [47].

FIG. 12.10 Electropherogram of a degraded ASA + AA effervescent tablet at two different wavelengths: 220 nm (blue line, dashed line) and 270 nm (red line, solid line) (A). Separation was performed using heart-cut CE-CE-MS using a highly ESI-interfering BGE as first dimension (100 mM tricine, pH 8.8). Interference-free mass spectra of peak 1 (B), peak 2 (C), peak 3 (F), degradation product #1 (D), and #2 (E). Due to the determination of accurate masses, the peaks were assigned to AA (peak 1), ASA (peak 2), SA (peak 3), diacetylated AA (#1), and monoacetylated AA (#2). € C. Quantification of ascorbic acid and acetylsalicylic acid in effervescent tablets Reprinted from Neuberger S, Jooß K, Ressel C, Neusuß by CZE-UV and identification of related degradation products by heart-cut CZE-CZE-MS. Anal Bioanal Chem 2016;408(30):8701–12 with permission from Springer Science + Business Media S.A.

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Still, the majority of the 2D applications are focused on enhancing separation efficiency and selectivity rather than to enable MS detection with ESI-interfering systems. Nevertheless, these techniques are quite promising for both approaches.

12.6 FUTURE TRENDS Due to its beneficial selectivity, small sample and reagent consumption and efficient separation CE-MS will continue to develop new application areas. The increasing interest and development of proteinbased pharmaceuticals will give rise to further applications and technological progress of CE-MS. On the one hand, the focus will be set to the development of robust and sensitive nanospray interfaces. On the other hand, two-dimensional separations will play a major role in the future. Both aspects will expand the field of applications for CE-MS due to an increase in sensitivity and separation capabilities. Additionally, CE will benefit from further developments in microchip technology as it allows higher throughput, integration with sample purification, and combination with other separation techniques.

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