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.     

Using sheath‐liquid reagents for capillary electrophoresis‐mass spectrometry: Application to the analysis of phenolic plant extracts

The combination of CE and MS is now a widely used tool that can provide a combination of high resolution separations with detailed structural information. Recently, we highlighted the benefits of an approach to add further functionality to this well‐established hyphenated technique, namely the possibility to perform chemical reactions within the sheath‐liquid of the CE‐MS interface 1 . Apart from using hydrogen/deuterium exchange for online determination of numbers of exchangeable protons, the addition of DPPH? (2,2‐diphenyl‐1‐picrylhydrazyl) to the sheath‐liquid can be used as a fast screening tool for studying antioxidant characteristics of individual components. Such a CE‐MS methodology allows rapid and information‐rich analysis with minimal reagent and sample consumption to be performed. In the present work, we demonstrate the applicability of this approach for the characterization of phenolic plant extracts from the Labiatae family, namely Rosmarinus officinalis and Melissa officinalis. Using the described approach, a wide range of compounds (15 and 13 phenolic compounds, respectively) could be confidently identified using a combination of high resolution CE‐MS separations with implementation of online deuterium exchange and DPPH? reactions. These compounds included polyphenols, phenolic acids, and triterpene acids. In conjunction with online MS/MS experiments, extensive structural information for aglyconic and glycosylated antioxidants present in the extracts could be obtained using simple experimental changes, which can be carried out prior to the purchasing of expensive chemical standards or the time‐consuming preparative isolation of individual compounds.     

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.     

Analysis of amino acids in latent fingerprint residue by capillary electrophoresis‐mass spectrometry

The analysis of the chemical composition of fingerprints is important for the development and improvement of existing fingerprint enhancement techniques. This study demonstrates the first analysis of a latent fingerprint sample, using an optimized CE‐MS method. In total 12 amino acids were detected in the fingerprint sample. MS/MS fragmentation was used to provide additional identity confirmation, for which eight of the twelve detected amino acids generated confirmatory product ions. Nine amino acids were quantified and their relative abundances were consistent with previous studies with serine and glycine being the most abundant. The successful detection of amino acids from latent fingerprints demonstrates that CE‐MS is a potential future technique for further study of such compounds in fingerprint samples.     

Rapid determination of 4‐aminobutyric acid and L‐glutamic acid in biological decarboxylation process by capillary electrophoresis‐mass spectrometry

4‐Aminobutylic acid (GABA) is a monomer of plastic polyamide 4. Bio‐based polyamide 4 can be produced by using GABA obtained from biomass. The production of L ‐glutamic acid (Glu) from biomass has been established. GABA is produced by decarboxylation of Glu in biological process. High‐performance liquid chromatography (HPLC) with derivatization is generally used to determine the concentration of GABA and Glu in reacted solution samples for the efficient production of GABA. In this study, we have investigated the rapid determination of GABA and Glu by capillary electrophoresis‐mass spectrometry (CE‐MS) without derivatization. The determination was achieved with the use of a shortened capillary, a new internal standard for GABA, and optimization of sheath liquid composition. Determined concentrations of GABA and Glu by CE‐MS were compared with those by pre‐column derivatization HPLC with phenylisothiocyanate. The determined values by CE‐MS were close to those by HPLC with pre‐column derivatization. These results suggest that the determination of GABA and Glu in reacted solution is rapid and simplified by the use of CE‐MS.     

Real‐time reaction monitoring by probe electrospray ionization mass spectrometry

Probe electrospray ionization (PESI) is a modified version of the electrospray ionization (ESI), where the capillary for sampling and spraying is replaced by a solid needle. High tolerance to salts and direct ambient sampling are major advantages of PESI compared with conventional ESI. In this study, PESI‐MS was used to monitor some biological and chemical reactions in real‐time, such as acid‐induced protein denaturation, hydrogen/deuterium exchange (HDX) of peptides, and Schiff base formation. By using PESI‐MS, time‐resolved mass spectra and ion chromatograms can be obtained reproducibly. Real‐time PESI‐MS monitoring can give direct and detailed information on each chemical species taking part in reactions, and this is valuable for a better understanding of the whole reaction process and for the optimization of reaction parameters. PESI‐MS can be considered as a potential tool for real‐time reaction monitoring due to its simplicity in instrumental setup, direct sampling with minimum sample preparation and low sample consumption. Copyright © 2010 John Wiley & Sons, Ltd.     

Comparative metabolite profiling of carboxylic acids in rat urine by CE‐ESI MS/MS through positively pre‐charged and 2H‐coded derivatization

A new approach to the selective comparative metabolite profiling of carboxylic acids in rat urine was established using CE‐MS and a method for positively pre‐charged and ²H‐coded derivatization. Novel derivatizing reagents, N‐alkyl‐4‐aminomethyl‐pyridinum iodide (alkyl=butyl, butyl‐d9 or hexyl), containing quaternary amine and stable‐isotope atoms (deuterium), were introduced for the derivatization of carboxylic acids. CE separation in positive polarity showed high reproducibility (0.99–1.32% RSD of migration time) and eliminated problems with capillary coating known in CE‐MS anion analyses. Essentially complete ionization and increased hydrophobicity after the derivatization also enhanced MS detection sensitivity (e.g. formic acid was detected at 0.5 pg). Simultaneous derivatization of one sample using two structurally similar reagents, N‐butyl‐4‐aminomethyl‐pyridinum iodide (BAMP) and N‐hexyl‐4‐aminomethyl‐pyridinum iodide, provided additional information for recognizing a carboxylic acid in an unknown sample. Moreover, characteristic fragmentation acquired by online CE‐MS/MS allowed for identification and categorization of carboxylic acids. Applying this method on rat urine, we found 59 ions matching the characteristic patterns of carboxylic acids. From these 59, 32 ions were positively identified and confirmed with standards. For comparative analysis, 24 standard carboxylic acids were derivatized by chemically identical but isotopically distinct BAMP and N‐butyl‐d9‐4‐aminomethyl‐pyridinium iodide, and their derivatization limits and linearity ranges were determined. Comparative analysis was also performed on two individual urine samples derivatized with BAMP and N‐butyl‐d9‐4‐aminomethyl‐pyridinium iodide. The metabolite profiling variation between these two samples was clearly visualized.     

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.     

Identification of RNA sequence isomer by isotope labeling and LC–MS/MS

Recently, we developed a method for modified ribonucleic acid (RNA) analysis based on the comparative analysis of RNA digests (CARD). Within this CARD approach, sequence or modification differences between two samples are identified through differential isotopic labeling of two samples. Components present in both samples will each be labeled, yielding doublets in the CARD mass spectrum. Components unique to only one sample should be detected as singlets. A limitation of the prior singlet identification strategy occurs when the two samples contain components of unique sequence but identical base composition. At the first stage of mass spectrometry, these sequence isomers cannot be differentiated and would appear as doublets rather than singlets. However, underlying sequence differences should be detectable by collision‐induced dissociation tandem mass spectrometry (CID MS/MS), as y‐type product ions will retain the original enzymatically incorporated isotope label. Here, we determine appropriate instrumental conditions that enable CID MS/MS of isotopically labeled ribonuclease T1 (RNase T1) digestion products such that the original isotope label is maintained in the product ion mass spectrum. Next, we demonstrate how y‐type product ions can be used to differentiate singlets and doublets from isomer sequences. We were then able to extend the utility of this approach by using CID MS/MS for the confirmation of an expected RNase T1 digestion product within the CARD analysis of an Escherichia coli mutant strain even in the presence of interfering and overlapping digestion products from other transfer RNAs. Copyright © 2014 John Wiley & Sons, Ltd.     

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.     

Quantitative analysis of hindered amine light stabilizers by CZE with UV detection and quadrupole TOF mass spectrometric detection

The current work describes the development of a CZE method with quadrupole QTOF‐MS detection and UV detection for the quantitation of Cyasorb 3529, a common hindered amine light stabilizer (HALS), in polymer materials. Analysis of real polymer samples revealed that the oligomer composition of Cyasorb 3529 changes during processing, a fact hampering the development of a straightforward method for quantitation based on calibration with a Cyasorb 3529 standard. To overcome this obstacle in‐depth investigations of the oligomer composition of this HALS using QTOF‐MS and QTOF‐MS/MS had to be performed whereby 22 new oligomer structures, in addition to the ten structures already described, were identified. Finally, a CZE method for quantitative analysis of this HALS was developed starting with a comprehensive characterization of a Cyasorb 3529 standard using CZE‐QTOF‐MS, subsequently allowing the correct assignment of most Cyasorb 3529 oligomers in an electropherogram with UV detection. Employing the latter detection technique and hexamethyl‐melamine as internal standard, peak areas obtained for the melamine could be correlated with those from the triazine ring, the UV‐absorbing unit present in the HALS. This approach finally allowed proper quantitation of the single oligomers of Cyasorb 3529, an imperative for the quantitative assessment of this HALS in real polymer samples.     

Path-integral calculations of heavy atom kinetic isotope effects in condensed phase reactions using higher-order Trotter factorizations

A convenient approach to compute kinetic isotope effects (KIEs) in condensed phase chemical reactions is via path integrals (PIs). Usually, the primitive approximation is used in PI simulations, although such quantum simulations are computationally demanding. The efficiency of PI simulations may be greatly improved, if higher-order Trotter factorizations of the density matrix operator are used. In this study, we use a higher-order PI method, in conjunction with mass-perturbation, to compute heavy-atom KIE in the decarboxylation of orotic acid in explicit sulfolane solvent. The results are in good agreement with experiment and show that the mass-perturbation higher-order Trotter factorization provides a practical approach for computing condensed phase heavy-atom KIE.     

Real time monitoring of accelerated chemical reactions by ultrasonication‐assisted spray ionization mass spectrometry

Ultrasonication has been used to accelerate chemical reactions. It would be ideal if ultrasonication‐assisted chemical reactions could be monitored by suitable detection tools such as mass spectrometry in real time. It would be helpful to clarify reaction intermediates/products and to have a better understanding of reaction mechanism. In this work, we developed a system for ultrasonication‐assisted spray ionization mass spectrometry (UASI–MS) with an ~1.7 MHz ultrasonic transducer to monitor chemical reactions in real time. We demonstrated that simply depositing a sample solution on the MHz‐based ultrasonic transducer, which was placed in front of the orifice of a mass spectrometer, the analyte signals can be readily detected by the mass spectrometer. Singly and multiply charged ions from small and large molecules, respectively, can be observed in the UASI mass spectra. Furthermore, the ultrasonic transducer used in the UASI setup accelerates the chemical reactions while being monitored via UASI–MS. The feasibility of using this approach for real‐time acceleration/monitoring of chemical reactions was demonstrated. The reactions of Girard T reagent and hydroxylamine with steroids were used as the model reactions. Upon the deposition of reactant solutions on the ultrasonic transducer, the intermediate/product ions are readily generated and instantaneously monitored using MS within 1 s. Additionally, we also showed the possibility of using this reactive UASI–MS approach to assist the confirmation of trace steroids from complex urine samples by monitoring the generation of the product ions. Copyright © 2014 John Wiley & Sons, Ltd.     

Partially bio‐based aromatic polyimides derived from 2,5‐furandicarboxylic acid with high thermal and mechanical properties

Two novel bio‐based diamines are synthesized through introduction of renewable 2,5‐furandicarboxylic acid (2,5‐FDCA), and the corresponding aromatic polyimides (PIs) are then prepared by these diamines with commercially available aromatic dianhydrides via two‐step polycondensation. The partially bio‐based PIs possess high glass transition temperatures (T_gs) in the range from 266 to 364 °C, high thermal stability of 5% weight loss temperatures (T_5%s) over 420 °C in nitrogen and outstanding mechanical properties with tensile strengths of 79–138 MPa, tensile moduli of 2.5–5.4 GPa, and elongations at break of 3.0–12.3%. Some colorless PI films (PI‐1‐b and PI‐1‐c) with the transmittances at 450 nm over 85% are prepared. The overall properties of 2,5‐FDCA‐based PIs are comparable with petroleum‐based PI derived from isophthalic acid, displaying the potential for development of innovative bio‐based materials. © 2018 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2018 , 56, 1058–1066     

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.     

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.     

Interaction energies of ZnO in acrylic polymer lattices

Water‐based acrylic polymers are frequently used as binders in ceramic materials that contain ZnO as a major component. Thin flexible ceramic films used in semiconductor elements are prepared from ceramic powder, polymer binder, dispergant and plasticizer. In the present work, the chemical reaction of acrylic acid, a part of the polymer, and ceramic powder (ZnO) has been studied by semi‐empirical calculations and Fourier transform infrared spectroscopy (FTIR). Systematic evaluation of model structures using varying contents of acrylic acid showed that ZnO adhesion increases as the carboxylate content in the polymer system increases. Interaction energy surfaces provide a quantitative insight into the spatial distribution of ZnO particles in various model structures. These results are also in good agreement with our infrared spectroscopy measurements.     

Synthesis and characteristics of photopolymerized benzophenone

Photopolymerization is extensively used in today's industrial field due to its advantages of rapid reaction, environmental friendly, energy saving and economical. Benzophenone is a most common photoinitiator (PI) using in photopolymerization because of its superior ability to initiate acrylate monomers. However, the intrinsic nature of initiator molecules is that they migrate out of polymer network, which limits its application, especially in the domain of food packaging materials. A polymerizable PI 4‐methylbenzophenone acrylate (MBPAc) was synthesized by a facile procedure and characterized by ¹H NMR, ¹³C NMR, and MS analyses. A systematic study of the photopolymerization kinetics of MBPAc was explored by the Real‐Time Fourier Transform Infrared Spectrometer. The results show that the final conversion and photopolymerization rate of acrylate monomers are closely related to the factors of their chemical structure, viscosity, functionality and light intensity, which means MBPAc is an efficient PI. Ultraviolet‐visible Spectrophotometer and vitro cytotoxicity measurement results indicate that the noncytotoxic MBPAc shows significantly lower migration than its analogue. © 2016 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2017 , 55, 313–320     

Rapid screening and determination of 4‐chloroamphetamine in saliva by paper spray‐mass spectrometry and capillary electrophoresis‐mass spectrometry

A novel drug‐screening system, consisting of paper spray‐MS (PS‐MS) and a CE‐ESI‐MS method was developed. This system can be easily switched either to PS‐MS for rapidly screening samples or to the traditional CE‐ESI‐MS method for separation and to obtain detailed mass spectral information, while sharing the same mass spectrometer. In the former case, when a sharp (15°‐tip) chromatography paper was used, the optimized distance from the paper tip to the mass inlet was 7.7 mm, whereas the optimized distance for the CE‐ESI tip was ～13.5 mm. Using 4‐chloroamphetamine as a model compound, the LODs for PS‐MS and CE‐ESI‐MS were determined to ～0.1 and 0.25 ppm, respectively. Comparisons of results obtained using PS‐MS and CE‐ESI‐MS and the experimental conditions are described.     

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.