A hydrodynamic injection approach for capillary electrophoresis using rotor–stator valves

Sample injection is a critical step in a capillary electrophoresis (CE) analysis. Electrokinetic injection is the simplest approach and is often selected for implementation in portable CE instruments. However, in order to minimize the effect of sample matrix upon the results of a CE analysis, hydrodynamic injection is preferred. Although portable CE instruments with hydrodynamic injection have been reported, injection has always been performed at the grounded end of the capillary. This simplifies fluidic handling but limits coupling with electrochemical detectors and electrospray ionization–mass spectrometry (ESI–MS). We demonstrated previously that injection at the high-voltage (HV) end of the capillary could be performed using an HV-compatible rotary injection valve (fixed-volume injection). However, the mismatch between the bore sizes of the channels on the rotor–stator valve and the separation capillary caused peak tailing and undesired mixing, impairing analytical performance. In this work, we present an HV-compatible hydrodynamic injection approach that overcomes the issues associated with the fixed-volume injection approach reported previously. The performance of the CE instrument was demonstrated by analyzing a mixture of 13 amino acids by CE coupled to laser-induced fluorescence, which showed relative standard deviations for peak area and migration time below 5% and 1%, respectively, for triplicate analysis. Additionally, replicate measurements of a mixture of amino acids, peptides, nucleobases, and nucleosides by CE coupled to electrospray ionization–mass spectrometry (CE–ESI–MS) were performed to evaluate peak tailing, and results were similar to those obtained with a commercial CE–ESI–MS setup.     

Highly sensitive and selective separation of intact parathyroid hormone and variants by sheathless CE‐ESI‐MS/MS

Parathyroid hormone (PTH) is a common clinical marker whose quantification relies on immunoassays, giving variable results as batch, brand, or target epitope changes. Sheathless CE‐ESI‐MS, combining CE resolution power and low‐flow ESI sensitivity, was applied to the analysis of PTH in its native conformation in the presence of related forms. Fused silica and neutral‐coated capillaries were investigated, as well as preconcentration methods such as transient isotachophoresis, field‐amplified sample injection (FASI), and electrokinetic supercharging (EKS). The method for the separation of PTH and its variants was first developed using fused‐silica capillary with UV detection. An acidic BGE was used to separate 1–84 PTH (full length), 7–84 PTH, and 1–34 PTH. Acetonitrile was added to the BGE to reduce peptide adsorption onto the capillary wall and transient isotachophoresis was used as analyte preconcentration method. The method was then transferred to a sheathless CE‐ESI‐MS instrument. When using a fused silica capillary, CE‐MS was limited to μg/mL levels. The use of a neutral coating combined with FASI or EKS allowed a significant increase in sensitivity. Under these conditions, 1–84 PTH, 7–84 PTH, and 1–34 PTH were detected at concentrations in the low ng/mL (FASI) or pg/mL (EKS) range.     

Optimization of capillary electrophoresis conditions for coupling to a mass spectrometer via a sheathless interface

When optimizing a capillary electrophoresis/electrospray ionization mass spectrometry (CE/ESI-MS) system, consideration has to be given not only to the separation but also to the electrospray stability. Methods developed for CE/UV analysis of drugs and peptides were considered and modified to be suitable for a CE/MS system with a robust sheathless interface. Different concentrations of the organic modifiers acetonitrile, methanol and 2-propanol were used in the separation buffer. The type and concentrations of these modifiers were also compared with reference to electrospray stability, sensitivity and time of analysis. In addition, different ionic strengths in the buffers were evaluated with reference to electrospray stability. The repeatability was used for the estimation of electrospray stability. The degree to which these parameters influenced the separation and the ESI stability was studied using a nine-peptide standard mixture and the antibiotic drugs bacampicillin and ampicillin as test substances. The analysis time and resolution were used as measures of the efficiency of the separation. A time-of-flight MS analyzer was used since it has the potential advantages of becoming a better fit for integration of CE with MS owing to the speed and sensitivity of this mass analyzer. The detection limit, i.e. 1 microM, for bacampicillin was comparable to what could be achieved with CE/MS on a quadrupole instrument using selected ion monitoring and sheath flow ESI.     

Analysis of process impurities in the basic drug SB-253149 using capillary electrophoresis and on-line mass spectrometric detection

Capillary electrophoresis with on-line electrospray ionisation mass spectrometry (CE/ESI-MS) has been used to identify process impurities in a batch of the anti-atherosclerotic drug, SB-253149. The impurities were separated from the main drug compound by capillary electrophoresis (CE) using an ammonium formate buffer at low pH in an untreated fused silica capillary. The CE method was initially developed using UV as the detection mode and then later structural elucidation work was achieved using an ion trap mass spectrometer. To maintain peak resolution and peak shape when the CE system was coupled to the mass spectrometer, a modified capillary cassette linked to a coaxial sheath flow electrospray ionisation (ESI) interface was used. By performing MS/MS experiments in conjunction with chemical knowledge of the reactivities of SB-253149, it was possible to propose molecular structures for impurities detected in the batch of SB-253149. The results from this study revealed that most of the process impurities in SB-253149 were dimeric derivatives of the parent molecule as well as trace levels of the starting material. This type of information was vital in process control and optimisation for the synthetic route for this drug.     

Development of a capillary zone electrophoresis-electrospray ionisation tandem mass spectrometry method for the analysis of fluoroquinolone antibiotics

The applicability of a capillary zone electrophoresis–electrospray ionisation tandem mass spectrometric (CZE–ESI-MS–MS) method for the separation of nine fluoroquinolones was investigated. Method optimisation involved systematic trouble-shooting starting with the type and duration of capillary pre-washing and conditioning, the choice of both the CE run buffer, MS sheath liquid, CE run potential, ESI spray voltage, sheath gas flow-rate, MS capillary voltage and CE capillary and MS capillary temperatures. Another extremely important factor was found to be the degree to which the CE capillary protrudes into the ESI chamber as well as whether or not sheath gas and spray voltage are employed during the CE injection or not. The importance of the latter has, to our knowledge, not been addressed elsewhere. Nine fluoroquinolones have been separated and detected in a single run by this technique.     

Analysis of synthetic chemical drugs in adulterated Chinese medicines by capillary electrophoresis/electrospray ionization mass spectrometry

Sixteen synthetic chemical drugs, often found in adulterated Chinese medicines, were studied by capillary electrophoresis/UV absorbance (CE/UV) and capillary electrophoresis/electrospray ionization mass spectrometry (CE/ESI-MS). Only nine peaks were detected with CZE/UV, but on-line CZE/MS provided clear identification for most compounds. For a real sample of a Chinese medicinal preparation, a few adulterants were identified by their migration times and protonated molecular ions. For coeluting compounds, more reliable identification was achieved by MS/MS in selected reaction monitoring mode. Micellar electrokinetic chromatography (MEKC) using sodium dodecyl sulfate (SDS) provided better separation than capillary zone electrophoresis (CZE), and, under optimal conditions, fourteen peaks were detected using UV detection. In ESI, the interference of SDS was less severe in positive ion mode than in negative ion mode. Up to 20 mM SDS could be used in direct coupling of MEKC with ESI-MS if the mass spectrometer was operated in positive ion mode. Because of better resolution in MEKC, adulterants can be identified without the use of MS/MS.     

Determination of UV filters in river water samples by in‐line SPE‐CE‐MS

The use of SPE coupled in‐line to CE using electrospray MS detection (in‐line SPE‐CE‐ESI‐MS) was investigated for the preconcentration and separation of four UV filters: benzophenone‐3, 2,2‐dihydroxy‐4‐methoxybenzophenone, 2,4‐dihydroxybenzophenone and 2‐phenylbenzimidazole‐5‐sulphonic acid. First, a CE‐ESI‐MS method was developed and validated using standard samples, obtaining LODs between 0.06 μg/mL and 0.40 μg/mL. For the in‐line SPE‐CE‐ESI‐MS method, three different sorbents were evaluated and compared: Oasis HLB, Oasis MCX, and Oasis MAX. For each sorbent, the main parameters affecting the preconcentration performance, such as sample pH, volume, and composition of the elution plug, and sample injection time were studied. The Oasis MCX sorbent showed the best performance and was used to validate the in‐line SPE‐CE‐ESI‐MS methodology. The LODs reached for standard samples were in the range between 0.01 and 0.05 ng/mL with good reproducibility and the developed strategy provided sensitivity enhancement factors between 3400‐fold and 34 000‐fold. The applicability of the developed methodology was demonstrated by the analysis of UV filters in river water samples.     

Recent advances in capillary electrophoresis‐mass spectrometry: Instrumentation,methodology and applications

Capillary electrophoresis (CE) offers fast and high‐resolution separation of charged analytes from small injection volumes. Coupled to mass spectrometry (MS), it represents a powerful analytical technique providing (exact) mass information and enables molecular characterization based on fragmentation. Although hyphenation of CE and MS is not straightforward, much emphasis has been placed on enabling efficient ionization and user‐friendly coupling. Though several interfaces are now commercially available, research on more efficient and robust interfacing with nano‐electrospray ionization (ESI), matrix‐assisted laser desorption/ionization (MALDI) and inductively coupled plasma mass spectrometry (ICP) continues with considerable results. At the same time, CE‐MS has been used in many fields, predominantly for the analysis of proteins, peptides and metabolites. This review belongs to a series of regularly published articles, summarizing 248 articles covering the time between June 2016 and May 2018. Latest developments on hyphenation of CE with MS as well as instrumental developments such as two‐dimensional separation systems with MS detection are mentioned. Furthermore, applications of various CE‐modes including capillary zone electrophoresis (CZE), nonaqueous capillary electrophoresis (NACE), capillary gel electrophoresis (CGE) and capillary isoelectric focusing (CIEF) coupled to MS in biological, pharmaceutical and environmental research are summarized.     

Enantiomeric Separation of Omeperazole Enantiomers by Aqueous CE Using UV and MS Detection

This paper details the analysis of the enantiomers of omeprazole, using aqueous CE coupled with MS detection. Following our previously published work: where a non-aqueous CE–UV method was developed for omeprazole and 5-hydroxy-omeprazole; coupling to electro-spray ionization (ESI) MS detection has now been investigated, using a sheath-flow interface for introduction. An aqueous CE method was developed and designed to afford increased compatibility with ESI–MS detection, employing an ammonium acetate buffer system (pH 5.8). Common partial filling methods could not be utilized to avoid the entrance of cyclodextrin into the MS, and therefore a modified method of non-continuous-flow CE–MS was applied, with the CE separation carried out without applied ESI voltage, before reapplying and allowing flow into the MS for data collection. A chiral CE separation of omeprazole and 5-hydroxyomeprazole was achieved, and chiral CE resolution of omeprazole has been demonstrated using MS detection.     

Optimization of capillary electrophoretic-electrospray ionization-mass spectrometric analysis of catecholamines

The capillary electrophoretic-mass spectrometric analysis (CE-MS) of catecholamines was optimized with coaxial sheath flow interface and electrospray ionization (ESI). The parameters studied included the sheath liquid composition and its flow rate, separation conditions in ammonium acetate buffer together with the ESI and cone voltages as mass spectrometric parameters. In addition, the effect of ESI voltage on injection as well as the siphoning effect were considered. The optimized conditions were a sheath liquid composition of methanol-water (80:20 v/v) with 0.5% acetic acid, with a flow rate of 6 microL/min. The capillary electrophoretic separation parameters were optimized with 50 mM ammonium acetate buffer, pH 4.0, to +25 kV separation voltage together with a pressure of 0.1 psi. The most intensive signals were obtained with an ESI voltage of +4.0 kV and a cone voltage of +20 V. The nonactive ESI voltage during injection as well as avoidance of the siphoning effect increased the sensitivity of the MS detection considerably. The use of ammonium hydroxide as the CE capillary conditioning solution instead of sodium hydroxide did not affect the CE-MS performance, but allowed the conditioning of the capillary between analyses to be performed in the MS without contaminating the ion source.     

Highly sensitive detection of five typical fluoroquinolones in low‐fat milk by field‐enhanced sample injection‐based CE in bubble cell capillary

Fluoroquinolones are a group of synthetic antibiotics with a broad activity spectrum against mycoplasma, Gram‐positive, and Gram‐negative bacteria. Due to the extensive use of fluoroquinolones in farming and veterinary science, there is a constant need in the analytical methods able to efficiently monitor their residues in food products of animal origin, regulated by Commission Regulation (European Union) no. 37/2010. Herein, field‐enhanced sample injection for sample stacking prior the CZE separation was developed inside a bubble cell capillary for highly sensitive detection of five typical fluoroquinolones in bovine milk. Ethylenediamine was proposed as the main component of BGE for the antibiotics separation. The effect of BGE composition, injection parameters, and water plug length on the field‐enhanced sample injection‐based CE with UV detection was investigated. Under the optimized conditions, described field‐enhanced sample injection‐based CE‐UV analysis of fluoroquinolones provides LODs varying from 0.4 to 1.3 ng/mL. These LOD values are much lower (from 460 to 1500 times) than those obtained by a conventional CE in a standard capillary without bubble cell. The developed method was finally applied for the analysis of fluoroquinolones in low‐fat milk from a Swiss supermarket. Sample recovery values from 93.6 to 106.0% for different fluoroquinolones, and LODs from 0.7 to 2.5 μg/kg, were achieved. Moreover, the proposed ethylenediamine‐based BGE as volatile and compatible with MS system, enabled the coupling of the field‐enhanced sample injection‐based CE with a recently introduced electrostatic spray ionization MS via an iontophoretic fraction collection interface for qualitative fluoroquinolones identification.     

Coupling CE with atmospheric pressure photoionization MS for pharmaceutical basic compounds: optimization of operating parameters

The use of CE coupled with MS (CE-MS) has evolved as a useful tool to analyze charged species in small sample volumes. Because of its sensitivity, versatility and ease of implementation, the ESI interface is currently the method of choice to hyphenate CE to MS. An alternative can be the atmospheric pressure photoionization (APPI) source, however, numerous parameters must be optimized for its coupling to CE. After evaluation of the sheath liquid composition and the CE capillary outlet position, an experimental design methodology was assessed for optimizing other ionization source parameters, such as sheath liquid flow rate, drying gas flow rate and temperature, nebulizing gas pressure, vaporizer temperature, and capillary voltage. For this purpose, a fractional factorial design (FFD) was selected as a screening procedure to identify factors which significantly influence sensitivity and efficiency. A face-centered central composite design (CCD) was then used to predict and optimize sensitivity, taking into account the most relevant variables. Sensitivity was finally evaluated with the optimized conditions and height-to-noise ratios (H/N) around 10 were achieved for an injection of 200 ng/mL of each analyte.     

The Development of a Sheathless Interface for Capillary Electrophoresis Electrospray Ionization Mass Spectrometry Using a Cellulose Acetate Cast Capillary

The fabrication of a novel sheathless interface for capillary electrophoresis–electrospray–mass spectrometry (CE–ESI–MS) is described. A programmable CO₂ laser was used to ablate small channels in the walls of a polyimide capillary near the terminus. Subsequent exposure of the channel region to a cellulose acetate solution followed by drying resulted in the formation of an electrically conductive semi-permeable membrane. Application of an appropriate voltage to the reservoir resulted in the simultaneous establishment of an electrical connection for CE and ESI. Interface viability was demonstrated by conducting a CE separation of a peptide mixture, with detection accomplished via positive ion mode ESI–MS. For the peptide Val-Tyr-Val, a limit of detection of 0.1 femtomole (S/N 3) was achieved using single reaction monitoring. Attributes of the interface include structural robustness, ease of fabrication, minimal interface dead volume, and the ability to alter post-separation analyte ionization status by use of appropriate buffers in the interface reservoir.     

Revealing the potential of capillary electrophoresis/mass spectrometry: the tipping point

The hyphenation of capillary electrophoresis and mass spectrometry (CE/MS) remains a minor technique compared with liquid chromatography/mass spectrometry (LC/MS), which represents nowadays the standard instrumentation, regardless of its introduction thirty years ago. However, from a theoretical point of view, CE coupling should be quite favorable especially with electrospray ionization mass spectrometry (ESI‐MS). At the time, the sensitivity provided by CE/MS was often limited, due to hyphenation requirements, which at some point appeared to disqualify CE/MS from benefiting from the performance gain driving the evolution of MS instruments. However, this context has been significantly modified in a matter of a few years. The development of innovative CE/MS interfacing systems has enabled an important improvement regarding sensitivity and reinforced robustness in order to provide an instrumentation accessible to the largest scientific community. Because of the unique selectivity delivered by the electrophoretic separation, CE/MS has proved to be particularly relevant for the analysis of biological molecules. The conjunction of these aspects is motivating the interest in CE/MS analysis and shows that CE/MS is mature enough to enrich the toolbox of analytical techniques for the analysis of complex biological samples. Here we discuss the characteristics of the major types of high‐sensitivity CE/ESI‐MS instrumentation and emphasize the late evolution and future positioning of CE/MS analysis for the characterization of biological molecules like peptides and proteins, through some pertinent applications.     

Staggered multistep elution solid-phase extraction capillary electrophoresis/tandem mass spectrometry: a high-throughput approach in protein analysis

An approach based on staggered multistep elution solid-phase extraction (SPE) capillary electrophoresis/tandem mass spectrometry (CE/MS/MS) was developed in the analysis of digested protein mixtures. On-line coupling of SPE with CE/MS was achieved using a two-leveled two-cross polydimethylsiloxane (PDMS)-based interface. Multistep elution SPE was used prior to CE to provide an additional dimension of separation, thus extending the separation capacity for the peptide mixture analysis. By decreasing in the number of co-eluting peptides, problems stemming from ionization suppression and finite MS/MS duty cycle were reduced. As a result, sequence coverage increased significantly using multistep elution SPE-CE/MS/MS compared to one-step elution SPE-CE/MS/MS in the analysis of a single protein tryptic digest (49% vs. 18%) and a six protein tryptic digest (22-71% vs. 10-44%). A staggered CE method was incorporated to increase the throughput. The electropherograms of consecutive CE runs were partially overlapped by injecting the sample plug at a fixed time interval. With the use of a 5 min injection interval, slightly poor results were obtained in comparison with the sequential CE method while the total analysis time was reduced to 28%.     

Investigation of commercial sorbents for the analysis of opioid peptides in human plasma by on‐line SPE‐CE

In this study, we investigated the performance of several commercial sorbents (Sep‐pack^® C18, ^tC18, C8 and ^tC2, Oasis^® HLB, Isolute^® ENV+, Strata^?‐X and Oasis^® MCX) for the determination of opioid peptides by solid‐phase extraction coupled on‐line to capillary electrophoresis (SPE‐CE). First, standard solutions were analyzed in order to achieve the lowest LOD and the best electrophoretic separations using UV detection. The best results were obtained using C18, C8 and ^tC2 sorbents, which were examined for the analysis of spiked human plasma samples. A double‐step sample clean‐up pretreatment, which consisted of precipitation with acetonitrile and filtration, was needed to prevent saturation of the on‐line SPE microcartridge. The filtration step was critical to obtain optimum analyte recovery and to clean up the sample matrix. A range of centrifugal filters and filtration conditions were tested and the recoveries of the sample pretreatment were evaluated by CE‐ESI‐MS. The LODs attained through SPE‐CE‐UV were approximately ten‐fold better with C18 than with C8 and ^tC2. The 0.1 μg/mL LODs achieved by C18‐SPE‐CE‐UV were further improved until we could detect 1 ng/mL concentrations of opioid peptides in plasma samples by C18‐SPE‐CE‐ESI‐MS, due to the outstanding selectivity of the MS detection.     

Analysis of phospholipase A2 glycosylation patterns from venom of individual bees by capillary electrophoresis/electrospray ionization mass spectrometry using an ion trap mass spectrometer

A method based on tryptic digestion, ultrafiltration and capillary electrophoresis/mass spectrometry (CE/MS) has been developed for the analysis of the glycosylation pattern in the phospholipase A2 (PLA) of individual honeybees. Without reducing the disulfide bonds, PLA was digested with trypsin and filtered with a 3 kDa molecular weight (MW) cut-off membrane. With this procedure, the glycopeptides could be isolated from the nonglycosylated peptides. After tryptic digestion and ultrafiltration, the disulfide bonds were reduced before analysis by CE. To reduce the adsorption, CE separation was performed on successive multiple ionic-polymer (SMIL) polybrene (PB) coated capillary columns. The SMIL-PB columns allowed partial separation of the glycopeptides and eight glycopeptides were identified by on-line coupling of CE with electrospray ionization (ESI) mass spectrometry. The analysis of phospholipase A2 from the venom of individual bees indicated that the variation and relative abundances of different glycopeptides were similar between the younger and the older bees.     

Analysis of dissolution test sample solutions by high speed capillary electrophoresis

Summary The quantitative use of high speed capillary electrophoresis (HSCE) is examined by applying high voltages across short capillaries. Acceptable performance in terms of injection precision and migration times were achieved within 1–2 minute analysis times. HSCE was used for the novel CE application of dissolution test sample solution analysis. The results generated by HSCE compared well with those generated using validated on-line UV absorbance measurements. It is concluded that HSCE is a viable alternative and supplement to standard analytical methods employed in dissolution test analysis.     

Analysis of dissolution test sample solutions by high speed capillary electrophoresis

Summary The quantitative use of high speed capillary electrophoresis (HSCE) is examined by applying high voltages across short capillaries. Acceptable performance in terms of injection precision and migration times were achieved within 1–2 minute analysis times. HSCE was used for the novel CE application of dissolution test sample solution analysis. The results generated by HSCE compared well with those generated using validated on-line UV absorbance measurements. It is concluded that HSCE is a viable alternative and supplement to standard analytical methods employed in dissolution test analysis.     

A simple fabrication method for tapered capillary tip and its applications in high‐speed CE and ESI‐MS

Fabrication of capillaries with tapered tips is an important technique that is required in many analytical chemistry areas, such as ESI‐MS, CE, electrochemical analysis, and microinjection. This paper describes a simple and effective grinding‐based fabrication method for capillaries with tapered tips. A novel grinding mode utilizing the combination of rotation and precession of an elastic capillary was developed, which significantly improved the controllability to the grinding process as well as the capillary tip shape. The capillary was fabricated by fixing it in an electric drill installed perpendicularly, and grind the capillary tip rotated around its own axis as well as the drill axis on sandpapers. Compared with conventional fabrication techniques for capillary tips, the present method is easy to control the capillary tip shape in routine laboratories without the requirement of expensive equipments or poisonous reagent (e.g. hydrofluoric acid (HF) solution). Various capillaries with different tip diameters and tip taper angles could be fabricated using the present method with good controllability and reproducibility. These capillaries were applied in high‐speed CE and ESI‐MS analysis to demonstrate the feasibility and potential of this fabrication method.