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.     

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.     

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.     

CE‐MS method development for peptides analysis,especially hepcidin,an iron metabolism marker

A method for the resolution of a peptides mixture including hepcidin‐25, an iron metabolism marker, was developed by CE‐ESI‐MS. Several strategies were tested to optimize peptide separation, such as the addition of cyclodextrins or organic solvents in the BGE or the use of coated capillaries. Best results in terms of resolution, symmetry and efficiency were obtained with a BGE made of 500 mM ammonium acetate pH 4.5/ACN 70:30 v/v. Using the methodology of experimental design, BGE concentration, sheath liquid composition and MS‐coupling parameters were then optimized in order to obtain the best signal intensity for hepcidin. Finally, a 225 mM BGE and a sheath liquid composed of isopropanol/water 80:20 v/v containing 0.5% v/v formic acid were selected as it constitutes the best compromise for selectivity, peak shape and sensitivity.     

Analysis of biologically active amines by CE

This paper provides an overview on the current status of the analysis of biogenic amines by CE. The basic CE separation and detection strategies for the analysis of biogenic amines are briefly described. CZE and MEKC that provide highly efficient and reproducible analysis of biogenic amines are particularly surveyed. With respect to the detection of biogenic amines, we focus on LIF, UV-visible absorption, electrochemiluminescence, and MS. Derivatization strategies, indirect methods, and on-line concentration techniques such as field-amplified sample stacking, sweeping, and use of polymer solution are described. To show the practicality of CE, we highlight currently developed techniques for the determinations of biogenic amines in biological samples, including foods, beverages, cerebrospinal fluids, urine, and single cells.     

Ion‐pairing high‐performance liquid chromatography‐mass spectrometry of impurities and reduction products of sulphonated azodyes

An HPLC separation method with triethylammonium acetate mobile phase additive developed for the analysis of impurities in polysulphonated azo dyes provides good separation selectivity and compatibility with electrospray ionisation (ESI) mass spectrometry. The negative‐ion ESI mass spectra containing only peaks of deprotonated molecules [M–H]^– for monosulphonic acids, [M–xH]^x–, and sodiated adducts [M–(x + y)H + yNa]^x– for polysulphonic acids allow easy molecular mass determination of unknown impurities. Based on the knowledge of the molecular masses and of the fragment ions in the MS/MS spectra, probable structures of trace impurities in commercial dye samples are proposed. To assist in the interpretation of the mass spectra of complex polysulphonated azodyes, additional information can be obtained after chemical reduction of azodyes to aromatic amines. The structures of the non‐sulphonated reduction products can be determined by reversed‐phase HPLC/MS with positive‐ion atmospheric pressure chemical ionisation and of the sulphonated products by ion‐pairing HPLC/MS with negative‐ion ESI.     

Metal displacement and stoichiometry of protein‐metal complexes under native conditions using capillary electrophoresis/mass spectrometry

Increases in the study of protein‐metal complexes, as well as in metal displacement in protein‐metal complexes under native conditions for optimum catalytic properties in drug research and catalyst design, demands a separation/detection technology that can accurately measure metal displacement and stoichiometry in protein‐metal complexes. Both nuclear magnetic resonance (NMR) and X‐ray diffraction techniques have been used for this purpose; however, these techniques lack sensitivity. Electrospray ionization mass spectrometry (ESI‐MS) using direct infusion offers higher sensitivity than the former techniques and provides molecular distribution of various protein‐metal complexes. However, since protein‐metal complexes under native conditions usually are dissolved in salt solutions, their direct ESI‐MS analysis requires off‐line sample clean‐up prior to MS analysis to avoid sample suppression during ESI. Moreover, direct infusion of the salty solution promotes non‐specific salt adduct formation by the protein‐metal complexes under ESI‐MS, which complicates the identification and stoichiometry measurements of the protein‐metal complexes. Because of the high mass of protein‐metal complexes and lack of sufficient resolution by most mass spectrometers to separate non‐specific from specific metal‐protein complexes, accurate protein‐metal stoichiometry measurements require some form of sample clean up prior to ESI‐MS analysis. In this study, we demonstrate that capillary electrophoresis/electrospray ionization in conjunction with a medium‐resolution (～10 000) mass spectrometer is an efficient and fast method for the measurement of the stoichiometry of the protein‐metal complexes under physiological conditions (pH ～7). The metal displacement of Co²⁺ to Cd²⁺, two metal ions necessary for activation in the monomeric AHL lactonase produced by B. thuringiensis, has been used as a proof of concept. Copyright © 2010 John Wiley & Sons, Ltd.     

A systematic approach toward comparing electrospray ionization efficiencies of derivatized and non‐derivatized amino acids and biogenic amines

Ionization efficiency (IE) in mass spectrometry (MS) has been studied for many different compounds, and different IE scales have been constructed in order to quantitatively characterize IE. In the case of MS, derivatization has been used to increase the sensitivity of the method and to lower the limits of detection. However, the influence of derivatization on IE across different compounds and different derivatization reagents has not been thoroughly researched, so that practitioners do not have information on the IE‐enhancing abilities of different derivatization reagents. Moreover, measuring IE via direct infusion of compounds cannot be considered fully adequate. Since derivatized compounds are in complex mixtures, a chromatographic method is needed to separate these compounds to minimize potential matrix effects. In this work, an IE measurement system with a chromatographic column was developed for mainly amino acids and some biogenic amines. IE measurements with liquid chromatography electrospray ionization mass spectrometry (LC/ESI/MS) were carried out, and IE scales were constructed with a calibration curve for compounds with and without derivatization reagent diethyl ethoxymethylenemalonate. Additionally, eluent composition effects on ionization were investigated. Results showed that derivatization increases IE for most of the compounds (by average 0.9 and up to 2‐2.5 logIE units) and derivatized compounds have more similar logIE values than without derivatization. Mobile phase composition effects on ionization efficiencies were negligible. It was also noted that the use of chromatographic separation instead of flow injection mode slightly increases IE. In this work, for the first time, IE enhancement of derivatization reagents was quantified under real LC/ESI/MS conditions and obtained logIE values of derivatized compounds were linked with the existing scale.     

Isothiocyanates as derivatization reagents for amines in liquid chromatography/electrospray ionization–tandem mass spectrometry

The applicability of 3‐pyridyl isothiocyanate, p‐(dimethylamino)phenyl isothiocyanate and m‐nitrophenyl isothiocyanate as the derivatization reagents for amines in high‐performance liquid chromatography/electrospray ionization–tandem mass spectrometry (LC/ESI‐MS/MS) was examined. The generated derivatives of amines with these reagents were favorably separated on the reversed‐phase column and detected by ESI‐MS/MS. The C–N bond of the generated thiourea structure was efficiently cleaved by collision‐induced dissociation and gave the single and intense product ion. Among the three reagents, 3‐pyridyl isothiocyanate was the most suitable as the derivatization reagent with regard to the reactivity to amines and the detection sensitivity. Copyright © 2009 John Wiley & Sons, Ltd.     

Independent highly sensitive characterization of asparagine deamidation and aspartic acid isomerization by sheathless CZE‐ESI‐MS/MS

Amino acids residues are commonly submitted to various physicochemical modifications occurring at physiological pH and temperature. Post‐translational modifications (PTMs) require comprehensive characterization because of their major influence on protein structure and involvement in numerous in vivo process or signaling. Mass spectrometry (MS) has gradually become an analytical tool of choice to characterize PTMs; however, some modifications are still challenging because of sample faint modification levels or difficulty to separate an intact peptide from modified counterparts before their transfer to the ionization source. Here, we report the implementation of capillary zone electrophoresis coupled to electrospray ionization tandem mass spectrometry (CZE‐ESI‐MS/MS) by the intermediate of a sheathless interfacing for independent and highly sensitive characterization of asparagine deamidation (deaN) and aspartic acid isomerization (isoD). CZE selectivity regarding deaN and isoD was studied extensively using different sets of synthetic peptides based on actual tryptic peptides. Results demonstrated CZE ability to separate the unmodified peptide from modified homologous exhibiting deaN, isoD or both independently with a resolution systematically superior to 1.29. Developed CZE‐ESI‐MS/MS method was applied for the characterization of monoclonal antibodies and complex protein mixture. Conserved CZE selectivity could be demonstrated even for complex samples, and foremost results obtained showed that CZE selectivity is similar regardless of the composition of the peptide. Separation of modified peptides prior to the MS analysis allowed to characterize and estimate modification levels of the sample independently for deaN and isoD even for peptides affected by both modifications and, as a consequence, enables to distinguish the formation of l ‐aspartic acid or d ‐aspartic acid generated from deaN. Separation based on peptide modification allowed, as supported by the ESI efficiency provided by CZE‐ESI‐MS/MS properties, and enabled to characterize and estimate studied PTMs with an unprecedented sensitivity and proved the relevance of implementing an electrophoretic driven separation for MS‐based peptide analysis. Copyright © 2016 John Wiley & Sons, Ltd.     

Derivatization of chiral carboxylic acids with (S)‐anabasine for increasing detectability and enantiomeric separation in LC/ESI‐MS/MS

A simple and practical derivatization procedure for increasing the detectability and enantiomeric separation of chiral carboxylic acids in LC/ESI‐MS/MS has been developed. (S)‐Anabasine (ANA) was used as the derivatization reagent and rapidly reacted with carboxylic acids [3‐hydroxypalmitic acid (3‐OH‐PA), 2‐(β‐carboxyethyl)‐6‐hydroxy‐2,7,8‐trimethylchroman (γ‐CEHC), and etodolac] in the presence of 4‐(4,6‐dimethoxy‐1,3,5‐triazin‐2‐yl)‐4‐methylmorpholium chloride. The resulting ANA‐derivatives were highly responsive in ESI‐MS operating in the positive‐ion mode and gave characteristic product ions during MS/MS, which enabled sensitive detection using selected reaction monitoring; the detection responses of the ANA‐derivatives were increased by 20–160‐fold over those of the intact carboxylic acids and the limits of detection were in the low femtomole range (1.8–11 fmol on the column). The ANA‐derivatization was also effective for the enatiomeric separation of the chiral carboxylic acids; the resolution was 1.92, 1.75, and 2.03 for 3‐OH‐PA, γ‐CHEC, and etodolac, respectively. The derivatization procedure was successfully applied to a biological sample analysis; the derivatization followed by LC/ESI‐MS/MS enabled the separation and detection of trace amounts of 3‐OH‐PA in neonatal dried blood spot and γ‐CEHC in human saliva with a simple pretreatment and small sample volume.     

Analysis of biogenic amines by solid-phase microextraction and high-performance liquid chromatography with electrochemical detection

We investigated the new solid-phase microextraction method by high-performance liquid chromatography with electrochemical detection for the analysis of biogenic amines. The Carbowax–Templated Resin 50 μm (purple) fibre coating offers good performances for dopamine and serotonin separation, i.e., good selectivity and high sensibility (0.1 μg l⁻¹). We also tested this fibre for biogenic amines quantification of rat striatum. The coating seems to be selective towards the amines and has low affinity for the metabolites, allowing a good separation and preventing drawbacks from the biological matrix. These first results obtained using this original separation method offer large perspectives of application to many biological samples.     

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.     

Precolumn derivatization reagents for high‐speed analysis of amines and amino acids in biological fluid using liquid chromatography/electrospray ionization tandem mass spectrometry

A rapid analytical method for amines and amino acids was developed, involving derivatization with the novel reagent 3‐aminopyridyl‐N‐hydroxysuccinimidyl carbamate (APDS), followed by reversed‐phase high‐performance liquid chromatography and electrospray ionization tandem mass spectrometry (HPLC/ESI‐MS/MS). More than 100 different analytes with amino groups, including amino acids in biological fluids such as mammalian plasma, could be measured within 10 min. The analytes were easily derivatized with APDS under the mild conditions. Selective reaction monitoring of ESI‐MS/MS in positive mode was carried out to include the transitions of all of the protonated molecular ions of analytes derivatized with APDS to the common fragment at m/z 121, which was derived from the amino pyridyl moiety of the reagent. We evaluated the retention time precision, the quantification limits, the linearity, the intra‐ and inter‐day precisions and the accuracy of 22 typical amino acids found in biological fluids, by analyzing a standard amino acid mixture and rat plasma. The intra‐day relative standard deviations (RSDs) of the retention times of the 22 amino acids and their internal standards were within 0.9% and the inter‐day RSDs were less than 1.1%, except for asparagines, with an RSD of 1.9%. The intra‐day and inter‐day RSDs of amino acid analyses in rat plasma were within 8.0% and 4.5%, respectively. The method, which facilitates the amino acid analysis of more than 100 samples in a day, represents an alternative to traditional amino acid analysis techniques, such as chromatography using postcolumn derivatization by ninhydrin. Copyright © 2009 John Wiley & Sons, Ltd.     

A promising capillary electrophoresis–electrospray ionization–mass spectrometry method for carbohydrate analysis

A method for adapting widely used CE conditions for the separation of fluorescently labeled carbohydrates to permit online ESI‐MS detection is presented. Reverse polarity separations were performed in bare fused‐silica capillaries with an acidic BGE. Under these conditions, negatively charged 8‐aminopyrene 1,3,6‐trisulfonate‐labeled carbohydrates migrate forward against the EOF, which is towards the capillary inlet. Therefore, the CE‐MS interface must simultaneously back‐fill the capillary, in order to maintain the CE circuit, and provide a stable forward flow at the sprayer tip to support the electrospray process. This was achieved using a junction‐at‐the‐tip interface, which provides a flow of solution to the junction formed by the capillary terminus and the inner wall of the emitter needle tip. Because the flow rate required for this arrangement is much less than in conventional sheath flow interfaces, dilution of the analytes is minimized. Optimized separation conditions permit baseline resolution of glucose oligomers containing up to 15 glucose units, while longer oligomers, up to 33 glucose units, were observed as resolved peaks in the negative ion mode mass spectrum.     

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.     

High‐throughput polymer tip‐electrospray ionization mass spectrometry for enhanced detection of neopterin and biopterin in clinical urine samples

Urinary biopterin (Bio) and neopterin (Neo) are important markers for clinical diagnosis of hyperphenylalaninemia. Herein, we developed a high‐throughput analysis method based on electrospray ionization mass spectrometry (ESI‐MS) with polymer tips for the rapid quantitative detection of Bio and Neo in clinical urine samples. Different polymer tips were investigated. It is found that the best detection sensitivity was achieved with hydrophobic polymer tip, ie, polyethylene tips. The high‐throughput polymer tip‐ESI‐MS method allowed a rapid analysis speed at ~40 seconds per sample. The limits of quantification (LOQ) (S/N ≥ 10) for the detection of Bio and Neo were improved to be 5.0 ng/mL. Acceptable relative standard deviation (RSD) values for Neo and Bio were measured to be 12.2% and 13.4% for direct measurement of Bio and Neo in raw urine samples, respectively. Furthermore, Bio and Neo were directly quantified from 18 clinical urine samples by presented method. The ratios of urinary Bio‐to‐Neo were analyzed for diagnosis of hyperphenylalaninemia. The results demonstrated that the present polymer tip‐ESI‐MS method is a promising strategy for the rapid analysis of clinical samples.     

高效液相色谱-激光诱导荧光法分析水样中的胺类物质

通过色谱条件和衍生条件的优化,建立了微量胺类物质的高效液相色谱-激光诱导荧光检测分析方法。该方法灵敏度高,在优化的条件下分析亚精胺、腐胺和组胺,检出限达到10^-10 mol/L数量级,且稳定性好。连续进样5次,3种生物胺保留时间的RSD(n=5)小于0.3%,峰面积的RSD(n=5)小于3%,平均加标回收率为94.99%~104.7%。将该方法应用于实际水样中3种生物胺的检测及7种茶叶茶水中胺类物质的分析,取得了良好的结果。该方法灵敏度高,稳定性好,可用于水样中微量胺类物质的分析。     

Pressurized CEC coupled with QTOF‐MS for urinary metabolomics

Pressurized CEC (pCEC) coupled with ESI‐QTOF‐MS using a sheathless interface was applied for metabolomics to develop an alternative analytical method for metabolic profiling of complex biofluid samples such as urine. The hyphenated system was investigated with mixed standards and pooled urine samples to evaluate its precision, repeatability, linearity, sensitivity, and selectivity. The applied voltage, mobile phase, and gradient elution were optimized and applied for the analysis of urinary metabolites. Multivariate data analysis was subsequently performed and used to distinguish lung cancer patients from healthy controls successfully. High separation efficiency has been achieved in pCEC due to the EOF. For metabolite identification, the pCEC‐MS separation mechnism was helpful for discriminating the fragment ions of glutamine conjugates from co‐eluted metabolites. Three glutamine conjugates, including phenylacetylglutamine, acylglutamine C8:1, and acylglutamine C6:1 were identified among 16 differential urinary metabolites of lung cancer. Receiver‐operating‐characteristic analysis of acylglutamine C8:1 resulted in an area‐under‐curve value of 0.882. Overall, this work suggests that this pCEC‐ESI‐QTOF‐MS method may provide a novel and useful platform for metabolomic studies due to its superior separation and identification.     

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.