CE‐ESI‐MS coupled with dynamic pH junction online concentration for analysis of peptides in human urine samples

In this article, an approach has been developed for the analysis of some small peptides with similar pI values by CE‐ESI‐MS based on the online concentration strategy of dynamic pH junction. The factors affected on the separation, detection and online enrichment, such as BGE, injection pressure, sheath flow liquid and separation voltage have been investigated in detail. Under the optimum conditions, i.e. using 0.5 mol/L formic acid (pH 2.15) as the BGE, preparing the sample in 50 mM ammonium acetate solution (pH 7.5), 50 mbar of injection pressure for 300 s, using 7.5 mM of acetic acid in methanol–water (80% v/v) solution as the sheath flow liquid and 20 kV as the separation voltage, four peptides with similar pI values, such as L ‐Ala‐L ‐Ala (pI=5.57), L ‐Leu‐D ‐Leu (pI=5.52), Gly‐D ‐Phe (pI=5.52) and Gly‐Gly‐L ‐Leu (pI=5.52) achieved baseline separation within 18.3 min with detection limits in the range of 0.2–2.0 nmol/L. RSDs of peak migration time and peak area were in the range of 1.45–3.57 and 4.93–6.32%, respectively. This method has been applied to the analysis of the four peptides in the spiked urine sample with satisfactory results.     

CE‐ESI‐TOF/MS for human growth hormone analysis

CE is a powerful analytical tool used to separate intact biomolecules such as proteins. The coupling of CE with TOF/MS produces a very promising method that can be used to detect and identify proteins in different matrices. This paper describes an efficient, rapid, and simple CE‐ESI‐TOF/MS procedure for the analysis of endogenous human growth hormone and recombinant human growth hormone without sample preparation. Operational factors were optimized using an experimental design, and the method was successfully applied to distinguish human growth hormone and recombinant human growth hormone in unknown samples.     

Structural analysis of glycosphingolipid analogues obtained by the saccharide primer method using CE‐ESI‐MS

A glycosphingolipid analogue (12‐azidododecyl β‐lactoside) as a saccharide primer has been shown to be useful for the synthesis of oligosaccharide libraries by mammalian cells. In the present study, CE‐ESI‐MS was employed to elucidate the structure of glycosphingolipid analogues derived from 12‐azidododecyl β‐lactoside (Lac‐C12N3) by mammalian cells. MDCK cells and COLO201 cells were cultured with Lac‐C12N3, and the glycosylated products secreted into the medium were collected and separated into acidic and neutral products by column chromatography. The acidic products could be directly analyzed by CE‐ESI‐MS, while the neutral products were converted to anionic derivatives via a reaction with propiolic acid. With this method, it was possible to analyze both acidic and neutral products glycosylated by MDCK cells and COLO201 cells at high sensitivity.     

CE‐MS with electrokinetic supercharging and application to determination of neurotransmitters

Electrokinetic supercharging (EKS) is known as one of the most effective online electrophoretic preconcentration techniques, though pairing with it with mass spectrometry has presented challenges. Here, EKS is successfully paired with ESI‐MS/MS to provide a sensitive and robust method for analysis of biogenic amines in biological samples. Injection parameters including electric field strength and the buffer compositions used for the separation and focusing were investigated to achieve suitable resolution, high sensitivity, and compatibility with ESI‐MS. Using EKS, the sensitivity of the method was improved 5000‐fold compared to a conventional hydrodynamic injection with CZE. The separation allowed for baseline resolution of several neurotransmitters within 16 min with LODs down to 10 pM. This method was applied to targeted analysis of seven biogenic amines from rat brain stem and whole Drosophila tissue. This is the first method to use EKS with CE‐ESI‐MS/MS to analyze biological samples.     

Development of a CE‐MS2 method for the enantiomeric separation of L/D‐carnitine: Application to the analysis of infant formulas

A new chiral analytical method based on CE‐MS is proposed for the identification and simultaneous quantification of D /L ‐carnitine in infant formulas. Previous derivatization of carnitine with FMOC enabled the optimization of the chiral separation using CE with UV detection. An optimization of electrospray‐MS parameters using a partial filling of the non‐volatile chiral selector (succinyl‐γ‐CD) was performed. A selective fragmentation using MS² experiments with an ion trap analyser was carried out to confirm the identity of D /L ‐carnitine according to the current legislation. Satisfactory results were obtained in terms of linearity, precision, and accuracy. Interestingly, the CE‐MS² method developed allowed a sensitivity enhancement with respect to UV detection of 100‐fold, obtaining an LOD of 100 ng/g for D ‐carnitine. The determination of L ‐carnitine and its enantiomeric purity in 14 infant formulas supplemented with carnitine was successfully achieved, sample preparation only requiring an ultrafiltration with centrifugal filter devices to retain the components with the highest molecular weights.     

A convenient and sensitive method for haloacetic acid analysis in tap water by on‐line field‐amplified sample‐stacking CE–ESI‐MS

In this study, we propose a simple strategy based on flow injection and field‐amplified sample‐stacking CE–ESI‐MS/MS to analyze haloacetic acids (HAAs) in tap water. Tap water was passed through a desalination cartridge before field‐amplified sample‐stacking CE–ESI‐MS/MS analysis to reduce sample salinity. With this treatment, the signals of the HAAs increased 300‐ to 1400‐fold. The LODs for tap water analysis were in the range of 10 to 100 ng/L, except for the LOD of monochloroacetic acid (1 μg/L in selected‐ion monitoring mode detection). The proposed method is fast, convenient, and sensitive enough to perform on‐line analysis of five HAAs in the tap water of Taipei City. Four HAAs, including trichloroacetic acid, dichloroacetic acid, dibromoacetic acid, and monobromoacetic acid, were detected at concentrations of approximately 1.74, 1.15, 0.16, and 0.15 ppb, respectively.     

CE‐MS analysis of heroin and its basic impurities using a charged polymer‐protected gold nanoparticle‐coated capillary

The first application of charged polymer‐protected gold nanoparticles (Au NPs) as semi‐permanent capillary coating in CE‐MS was presented. Poly(diallyldimethylammonium chloride) (PDDA) was the only reducing and stabilizing agent for Au NPs preparation. Stable and repeatable coating with good tolerance to 0.1 M HCl, methanol, and ACN was obtained via a simple rinsing procedure. Au NPs enhanced the coating stability toward flushing by methanol, improved the run‐to‐run and capillary‐to‐capillary repeatabilities, and improved the separation efficiency of heroin and its basic impurities for tracing geographical origins of illicit samples. Baseline resolution of eight heroin‐related alkaloids was achieved on the PDDA‐protected Au NPs‐coated capillary under the optimum conditions: 120 mM ammonium acetate (pH 5.2) with addition of 13% methanol, separation temperature 20°C, applied voltage ?20 kV, and capillary effective length 60.0 cm. CE‐MS analysis with run‐to‐run RSDs (n=5) of migration time in the range of 0.43–0.62% and RSDs (n=5) of peak area in the range of 1.49–4.68% was obtained. The established CE‐MS method would offer sensitive detection and confident identification of heroin and related compounds and provide an alternative to LC‐MS and GC‐MS for illicit drug control.     

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.     

CE–MS fingerprinting of Laurencia complex algae (Rhodophyta)

The use of CE–ESI‐MS has been considered as a new chemical strategy for the possible discernment of genera and species of the Laurencia complex. After the selection of the CE–MS and the extraction conditions, a total of 28 specimens of the complex, including different species of four genera (Laurencia, Laurenciella, Palisada, and Osmundea) collected from five intertidal locations on the Island of Tenerife (Canary Islands, Spain) were analyzed. CE–MS fingerprints revealed that CE–MS can be used as a useful tool for these studies in order to assess similarities and differences between them and that it constitutes an important starting point for further studies in the field.     

Potential of prodendronic polyamines with modulated segmental charge density as novel coating for fast and efficient analysis of peptides and basic proteins by CE and CE‐MS

In this work, the suitability of a new polymer family has been investigated as capillary coatings for the analysis of peptides and basic proteins by CE. This polymer family has been designed to minimize or completely prevent protein–capillary wall interactions and to modify the EOF. These coating materials are linear polymeric chains bearing as side cationizable moiety a dentronic triamine derived from N,N,N’,N’‐tetraethyldiethylenetriamine (TEDETA), which is linked to the backbone through a spacer (unit labeled as TEDETAMA). Four different polymers have been prepared and evaluated: a homopolymer which comprised only of those cationizable repetitive units of TEDETAMA, and three copolymers that randomly incorporate TEDETAMA together with neutral hydrosoluble units of N‐(2‐hydroxypropyl) methacrylamide (HPMA) at different molar percentages (25:75, 50:50 and 75:25). It has been demonstrated that the composition of the copolymers influences the EOF and therefore the separation of the investigated biopolymers. Among the novel polymers studied, poly‐(TEDETAMA‐co‐HPMA) 50:50 copolymer was successfully applied as coating material of the inner capillary surface in CE‐UV and CE‐MS, providing EOF reversing together with fast and efficient baseline separation of peptides and basic proteins. Finally, the feasibility of the polymer‐coated capillary was shown through the analysis of lysozyme in a cheese sample.     

Analysis of monoclonal antibody by a novel CE‐UV/MALDI‐MS interface

mAbs are highly complex proteins that present a wide range of microheterogeneity that requires multiple analytical methods for full structure assessment and quality control. As a consequence, the characterization of mAbs on different levels is particularly product‐ and time‐consuming. CE‐MS couplings, especially to MALDI, appear really attractive methods for the characterization of biological samples. In this work, we report the last instrumental development and performance of the first totally automated off‐line CE‐UV/MALDI‐MS/MS. This interface is based on the removal of the original UV cell of the CE apparatus, modification of the spotting device geometry, and creation of an integrated delivery matrix system. The performance of the method was evaluated with separation of five intact proteins and a tryptic digest mixture of nine proteins. Intact protein application shows the acquisition of electropherograms with high resolution and high repeatability. In the peptide mapping approach, a total number of 154 unique identified peptides were characterized using MS/MS spectra corresponding to average sequence coverage of 64.1%. Comparison with NanoLC/MALDI‐MS/MS showed complementarity at the peptide level with an increase of 42% when using CE/MALDI‐MS coupling. Finally, this work represents the first analysis of intact mAb charge variants by CZE using an MS detection. Moreover, using a peptide mapping approach CE‐UV/MALDI‐MS/MS fragmentation allowed 100% sequence coverage of the light chain and 92% of the heavy chain, and the separation of four major glycosylated peptides and their structural characterization.     

CE‐SDS method development,validation, and best practice—An overview

CE‐SDS has been implemented in the biopharmaceutical industry and is being used for the characterization of therapeutic proteins in most Biological License Applications currently submitted. An overview is presented on the separation mechanism, methodology, and good working practices/best practices. The CE‐SDS platform method development and validation are discussed and typical scientifically and regulatory issues and troubleshooting situations are highlighted.     

Improved sample preparation method for glycan analysis of glycoproteins by CE‐LIF and CE‐MS

CE is a high‐resolution separation technique broadly used in the biotechnology industry for carbohydrate analysis. The standard sample preparation protocol for CE analysis of glycans released from glycoproteins generally requires derivatization times of overnight at 37°C, using ≥100 fold excess of fluorophore reagent, 8‐aminopyrene‐1,3,6‐trisulfonic‐acid, if the sample is unknown, or it is a regulated biotherapeutic product, possibly containing terminal sialic acid(s). In this paper, we report on significant improvements for the standard CE sample preparation method of glycan analysis. By replacing the conventionally used acetic acid catalyst with citric acid, as low as 1:10 glycan to fluorophore molar ratio (versus the typical 1:≥100 ratio) maintained the >95% derivatization yield at 55°C with only 50 min reaction time. Terminal sialic acid loss was negligible at 55°C during the derivatization process, and indicating that the kinetics of labeling at 55°C was faster than the loss of sialic acid from the glycan. The reduced relative level of 8‐aminopyrene‐1,3,6‐trisulfonic‐acid simplified the removal of excess reagent, important in both CE‐LIF (electrokinetic injection bias) and CE‐MS (ion suppression). Coupling CE‐ ESI‐MS confirmed that the individual peaks separated by CE corresponded to single glycans and increased the confidence of structural assignment based on glucose unit values.     

Comparative analysis of CE‐SSCP to standard RFLP‐CE‐FLA method in quantification of known viral variants within an RNA virus quasispecies

RNA viruses display the highest replication error rate in our biosphere, leading to highly diverse viral populations termed quasispecies. The gold standard method for detection and quantification of variants in a quasispecies is cloning and sequencing, but it is expensive, laborious and time consuming. Therefore, other mutation detection approaches, including SSCP, are often used. In this study, we demonstrate development and the usage of a CE‐SSCP method for quantification of two nearly identical viral variants in heterogenic population of a mumps virus strain and its comparison to RFLP‐CE‐fragment length analysis (RFLP‐CE‐FLA). Analyzed PCR fragments were of the same size (245 bp) with one difference in their nucleotide sequence. The limit of detection of both methods was at 5% of the minor variant. When PCR amplicons of the two variants were pooled, methods' results were very similar. On the contrary, the quantification results of samples in which variants were mixed prior to PCR showed substantial difference between the two methods. Our results indicate that although both methods can be used for detection and monitoring of a specific mutation within a viral population, caution should be taken when quantitative analysis of complex samples is based solely on results of one method.     

Optimization of micro‐HPLC peak focusing for the detection and quantification of low hepcidin concentrations

Micro‐high‐performance liquid chromatography is a miniaturized, economic and ecological chromatographic system allowing the use of reduced size chromatographic columns. Coupled with electrospray ionization tandem mass spectrometry, this technique can be used to detect and quantify low concentrations of peptides. In this study, hepcidin was used as the model compound and analysed using octadecylsilica stationary phase by means of a gradient elution mode at a flow rate of 4 μL/min. Several parameters were studied to optimize peak focusing. Using the methodology of experimental design, the mobile‐phase gradient conditions and the sample composition were optimized in order to maximize the sensitivity and minimize retention time. Stability of the target peptide in solution was also demonstrated.     

Automated sample preparation for CE‐SDS

Traditionally, CE with SDS (CE‐SDS) places many restrictions on sample composition. Requirements include low salt content, known initial sample concentration, and a narrow window of final sample concentration. As these restrictions require buffer exchange for many sample types, sample preparation is often tedious and yields poor sample recoveries. To improve capacity and streamline sample preparation, an automated robotic platform was developed using the PhyNexus Micro‐Extractor Automated Instrument (MEA) for both the reduced and nonreduced CE‐SDS assays. This automated sample preparation normalizes sample concentration, removes salts and other contaminants, and adds the required CE‐SDS reagents, essentially eliminating manual steps during sample preparation. Fc‐fusion proteins and monoclonal antibodies were used in this work to demonstrate benefits of this approach when compared to the manual method. With optimized conditions, this application has demonstrated decreased analyst “hands on” time and reduced total assay time. Sample recovery greater than 90% can be achieved, regardless of initial composition and concentration of analyte.     

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.     

Coupling of CE‐MS for protein and peptide analysis

The review is focused on the latest developments in the analysis of proteins and peptides by capillary electrophoresis techniques coupled to mass spectrometry. First, the methodology and instrumentation are overviewed. In this section, recent progress in capillary electrophoresis with mass spectrometry interfaces and capillary electrophoresis with matrix‐assisted laser desorption/ionization is mentioned, as well as separation tasks. The second part is devoted to applications—mainly bottom‐up and top‐down proteomics. It is obvious that capillary electrophoresis with mass spectrometry methods are well suited for peptide and protein analysis (proteomic research) and it is described how these techniques are complementary and not competitive with the often used liquid chromatography with mass spectrometry methods.     

An update for human blood plasma pretreatment for optimized recovery of low‐molecular‐mass peptides prior to CE–MS and SPE–CE–MS

Protein precipitation and centrifugal filtration are well‐established methods for concentrating and purifying peptides with a low relative molecular mass (M_r) from human blood plasma before proteomic and peptidomic studies using high‐performance separation techniques, but there is little information on peptide recoveries. Here, we evaluate acetonitrile precipitation followed by a range of centrifugal filtration conditions for the analysis of low M_r peptides in human blood plasma before CE–MS and SPE coupled online to CE–MS. Three opioid peptides were used as model compounds, that is, dynorphin A 1–7, endomorphin 1, and methionine enkephalin and 3, 10, and 30 K M_r cut‐off cellulose acetate filters (Amicon® Ultra‐0.5) and 10 K M_r cut‐off polyethersulfone filters (Vivaspin® 500) were studied. Unexpectedly, recoveries and repeatability were only optimum after passivating the 10 K M_r cut‐off cellulose acetate filters with PEG to avoid peptide adsorption on the inner walls of the plastic sample reservoir.     

A 2‐D‐HPLC‐CE platform coupled to ESI‐TOF‐MS to characterize the phenolic fraction in olive oil

A 2‐D‐HPLC/CE method was developed to separate and characterize more in depth the phenolic fraction of olive oil samples. The method involves the use of semi‐preparative HPLC (C18 column 250×10 mm, 5 μm) as a first dimension of separation to isolate phenolic fractions from commercial extra‐virgin olive oils and CE coupled to TOF‐MS (CE‐TOF‐MS) as a second dimension, to analyze the composition of the isolated fractions. Using this method, a large number of compounds were tentatively identified, some of them by first time, based on the information concerning high mass accuracy and the isotopic pattern provided by TOF‐MS analyzer together with the chemical knowledge and the behavior of the compounds in HPLC and CE. From these results it can be concluded that 2‐D‐HPLC‐CE‐MS provides enough resolving power to separate hundreds of compounds from highly complex samples, such as olive oil. Furthermore, in this paper, the isolated phenolic fractions have been used for two specific applications: quantification of some components of extra‐virgin olive oil samples in terms of pure fractions, and in vitro studies of its anti‐carcinogenic capacity.