A capillary electrophoresis and off-line capillary electrophoresis/electrospray ionization-quadrupole time of flight-tandem mass spectrometry approach for ganglioside analysis

A systematic study for the optimization and implementation of high-performance capillary electrophoresis (HPCE) in conjunction with negative ion electrospray ionization-quadrupole time of flight-tandem mass spectrometry (ESI-QTOF-MS/MS) for the analysis of complex glycolipids is described. The performance of the capillary electrophoresis (CE) and off-line CE/ESI-QTOF-MS approach has been explored for screening a complex ganglioside mixture from bovine brain. All instrumental and solution parameters demonstrated to require special adjustment and to have the most substantial effect on the CE separation, abundance of product ions produced in a low-energy collision-induced dissociation (CID) process and their detection by MS/MS, when attempting to identify and sequence single ganglioside molecular species from CE eluted fractions. Upon optimization of the experimental parameters, an efficient methodology emerged providing the general basic requirements for combined CE/ESI-MS analysis of this type of complex glycoconjugate.     

On-line sheathless capillary electrophoresis/nanoelectrospray ionization-tandem mass spectrometry for the analysis of glycosaminoglycan oligosaccharides

A novel approach in glycosaminoglycomics, based on sheathless on-line capillary electrophoresis/nanoelectrospray ionization-quadrupole time of flight-mass spectrometry (CE/nanoESI-QTOF-MS) and tandem MS of extended chondroitin sulfate/dermatan (CS/DS) oligosaccharide chains is described. The methodology required the construction of a new sheathless CE/nanoESI-QTOF-MS configuration, its implementation and optimization for the high sensitivity analysis of CS/DS oligosaccharide mixtures from conditioned culture medium of decorin transfected human embryonic kidney (HEK) 293 cells. Under newly established sheathless on-line CE/(-)nanoESI conditions for glycosaminoglycan (GAG) ionization and MS detection, single CS/DS oligosaccharide components of extended chain length and increased sulfation degree were identified. Molecular ions corresponding to species carrying 5 and 6 negative charges could be generated for large GAG oligosaccharide species in the negative ion nanoESI-MS. The optimized on-line conditions enabled the detection of molecular ions assigned to oversulfated tetradeca-, octadeca-, and eicosasaccharide CS/DS molecules, which represent the category of largest sulfated GAG-derived oligosaccharides evidenced by CE/ESI-MS. By on-line CE/ESI tandem MS in data-dependent acquisition mode the oversulfated eicosasaccharide species could be sequenced and the localization of the additional sulfate group along the chain could be determined.     

Off-line capillary electrophoresis/fully automated nanoelectrospray chip quadrupole time-of-flight mass spectrometry and tandem mass spectrometry for glycoconjugate analysis

Implementation and optimization of an off-line capillary electrophoresis (CE)/(−)nanoESIchip-quadrupole time-of-flight (QTOF) mass spectrometric (MS) and tandem MS system for compositional mapping and structural investigation of components in complex carbohydrate mixtures is described. The approach was developed for glycoscreening and applied to O-glycosylated peptides from urine of a patient suffering from α-N-acetylhexosaminidase deficiency, known as Schindler's disease. The fundamental issue of sensitivity, previously representing a serious drawback of the off-line CE/MS analysis, could be positively addressed by the off-line conjunction of CE with automated chip-based ESI-QTOF-MS to provide flexibility for CE/chip MS coupling and enhance structural elucidation of single components in heterogeneous mixtures. Copyright © 2004 John Wiley & Sons, Ltd.     

Capillary electrophoresis-mass spectrometry for glycoscreening in biomedical research

Application of capillary electrophoresis (CE) in combination with mass spectrometry (MS) and tandem MS to glycoscreening in biomedical projects is highlighted. In the first part recent CE-MS experiments by sheath liquid CE and multiple stage MS are reported. Neutral and negatively charged N-glycan mixtures from ribonuclease B and fetuin, high-mannose type N-glycoforms, oligosaccharides from lipopolysaccharides (LPS) of Haemophilus influenzae, polysaccharides of Pseudomonas aeruginosa and Staphylococcus aureus were analyzed. A particular emphasis is devoted to the applicability of novel off- and on-line CE-MS and tandem MS methods for screening of proteoglycan-derived oligosaccharides, glycosaminoglycans (GAGs), such as hyaluronates from Streptococcus agalactiae, chondroitin/dermatan sulfates (CS/DS) from bovine aorta and human skin fibroblast decorin, and heparin/heparan sulfate (HS) from porcine and bovine mucosa. The performance of CE-MS/MS for identification of glycoforms in glycopeptides and glycoproteins is illustrated by experiments performed on complex mixtures from urine of patients suffering from a hereditary N-acetylhexosaminidase deficiency (Schindler's disease) and urine of patients suffering from cancer cachexia. For determination of glycosylation patterns in glycoproteins like enzymes and antibodies by CE/MS, both CE-matrix assisted laser desorption/ionization (MALDI) and CE-electrospray ionization (ESI)-MS were functional. Finally, the potential of CE-ESI-MS strategy in glycolipid analysis is demonstrated for gangliosides from bovine brain for which particular CE buffer conditions are required.     

Determination of sulfation pattern in brain glycosaminoglycans by chip-based electrospray ionization ion trap mass spectrometry

Chondroitin sulfate (CS) and dermatan sulfate (DS) glycosaminoglycans display variability of sulfation in their constituent disaccharide repeats during chain elongation. Since a large proportion of the extracellular matrix of the central nervous system (CNS) is composed of proteoglycans, CS/DS disaccharide degree and profile of sulfation play important roles in the functional diversity of neurons, brain development, and some of its pathological states. To investigate the sulfation pattern of CS/DS structures expressed in CNS, we introduced here a novel method based on an advanced system encompassing fully automated chip nanoelectrospray ionization (nanoESI) in the negative ion mode and high capacity ion trap multistage mass spectrometry (MS²–MS³) by collision-induced dissociation (CID). This method, introduced here for the first time in glycomics of brain glycosaminoglycans, was particularly applied to structural investigation of disaccharides obtained by β-elimination and digestion with chondroitin B and AC I lyase of hybrid CS/DS chains from wild-type mouse brain. Screening in the chip-MS mode of DS disaccharide fraction resulting after depolymerization with chondroitin B lyase revealed molecular ions assigned to monosulfated disaccharide species having a composition of 4,5-Δ-[IdoA-GalNAc]. By optimized CID MS²–MS³, fragment ions supporting the localization of sulfate ester group at C4 within GalNAc were produced. Chip ESI MS profiling of CS disaccharide fraction obtained by depolymerization of the same CS/DS chain using chondroitin AC I lyase indicated the occurrence of mono- and bisulfated 4,5-Δ-[GlcA-GalNAc]. The site of oversulfation was determined by MS²–MS³, which provided sequence patterns consistent with a rare GlcA-3-sulfate–GalNAc-6-sulfate structural motif.      

Improved workup for glycosaminoglycan disaccharide analysis using CE with LIF detection

This work describes improved workup and instrumental conditions to enable robust, sensitive glycosaminoglycan (GAG) disaccharide analysis from complex biological samples. In the process of applying CE with LIF to GAG disaccharide analysis in biological samples, we have made improvements to existing methods. These include (i) optimization of reductive amination conditions, (ii) improvement in sensitivity through the use of a cellulose cleanup procedure for the derivatization, and (iii) optimization of separation conditions for robustness and reproducibility. The improved method enables analysis of disaccharide quantities as low as 1 pmol prior to derivatization. Biological GAG samples were exhaustively digested using lyase enzymes, the disaccharide products and standards were derivatized with the fluorophore 2‐aminoacridone and subjected to reversed polarity CE‐LIF detection. These conditions resolved all known chondroitin sulfate (CS) disaccharides or 11 of 12 standard heparin/heparan sulfate disaccharides, using 50 mM phosphate buffer, pH 3.5, and reversed polarity at 30 kV with 0.3 psi pressure. Relative standard deviation in migration times of CS ranged from 0.1 to 2.0% over 60 days, and the relative standard deviations of peak areas were less than 3.2%, suggesting that the method is reproducible and precise. The CS disaccharide compositions are similar to those obtained by our group using tandem MS. The reversed polarity CE‐LIF disaccharide analysis protocol yields baseline resolution and quantification of heparin/heparan sulfate and CS/dermatan sulfate disaccharides from both standard preparations and biologically relevant proteoglycan samples. The improved CE‐LIF method enables disaccharide quantification of biologically relevant proteoglycans from small samples of intact tissue.     

Combining size-exclusion chromatography and fully automated chip-based nanoelectrospray quadrupole time-of-flight tandem mass spectrometry for structural analysis of chondroitin/dermatan sulfate in human decorin

Chondroitin/dermatan sulfate (CS/DS) chain of decorin (DCN) from human skin fibroblasts (HSk) was released by reductive β-elimination reaction and digested with chondroitin AC I lyase. Enzymatic hydrolysis mixture of CS/DS chains was separated by size-exclusion chromatography (SEC). Collected octasaccharide fraction was subjected to fully automated chip-based nanoelectrospray (nanoESI) quadrupole time-of-flight (QTOF) MS and tandem MS (MS/MS). MS of human skin fibroblasts DCN CS/DS displayed a high complexity due to the large variety of glycoforms, which under chip-nanoESI MS readily ionized to form multiply charged ions. Except for the regularly tetrasulfated octasaccharide, the investigated fraction contained four additional octasaccharides of atypical sulfation status. Two new oversulfated glycoforms and two undersulfated species were identified. Remarkably, the series of decasaccharides discovered in the same SEC pool was found to encompass a trisulfated and a novel hexasulfated [4,5-Δ-GlcAGalNAc(IdoAGalNAc)?] species. MS/MS by collision-induced dissociation (CID) on the [M-4H]? ion corresponding to the previously not reported [4,5-Δ-GlcAGalNAc(IdoAGalNAc)?](5S) corroborated for a novel motif in which three N-acetylgalactosamine (GalNAc) moieties are monosulfated, 4,5-Δ-GlcA and the first IdoA from the non-reducing end bear one sulfate group each, while the second N-acetylgalactosamine from the reducing end is unsulfated.     

Copper-coated microsprayer interface for on-line sheathless capillary electrophoresis electrospray mass spectrometry of carbohydrates

A sturdy home-built sheathless CE/ESI-QTOF-MS system was developed and optimized for carbohydrate analysis. The interface and employed methodology provided a simple analytical solution to laborious CE/MS interfacing methods and to problems in characterization of complex carbohydrate mixtures that require high-resolution separation of the components. The CE/ESI interface, feasible in any MS laboratory, consists of a one-piece CE column having the CE terminus in-laboratory shaped as a microsprayer and coated with copper. The CE microsprayer was inserted into an in-house made stainless steel clenching device and the whole assembly was mounted onto a quadrupole TOF mass spectrometer. The analytical potential of the interface in terms of suitability, microsprayer performance, copper coat durability, ionization efficiency, spray stability, and sensitivity was tested first on a simple mixture of standard saccharides, which were separated, resolved, and detected with high separation efficiency. The approach was next assessed for the screening of a biological sample, a complex mixture of O-glycosylated sialylated amino acids from urine of a patient suffering from Schindler disease. Preliminary data allow this method to be considered as one of general applicability in structural glycobiology and glycomics and easy to be implemented for proteomic surveys as well.     

Identification of hyaluronic acid oligosaccharides by direct coupling of capillary electrophoresis with electrospray ion trap mass spectrometry

A new method for the identification of oligosaccharides obtained by enzymatic digestion of hyaluronic acid (HA) with bacterial hyaluronidase (HA lyase, E.C. 4.2.2.1, from Streptococcus agalactiae) using online capillary electrophoresis/electrospray mass spectrometry (CE/ESI-MS) is presented. A fused-silica capillary coated with polyacrylamide was used with a 40 mM ammonium acetate buffer at pH 9.0 and a separation voltage of +30 kV applied to the inlet. Separation was achieved for oligosaccharides containing 4-16 monomers. The migration behavior follows the chain length of the oligomers, regardless of charge state. However, no linear relationship was found for the relation between mobility and chain length. Using an ion trap mass analyzer, complementary structural information was obtained by MS/MS and MS(n) experiments.     

Quantification of Fumaria officinalis isoquinoline alkaloids by nonaqueous capillary electrophoresis-electrospray ion trap mass spectrometry

A capillary electrophoresis (CE) method using non-aqueous (NA) separation solutions combined with an ion trap mass spectrometer (MS and MS/MS) as detection device is presented for the separation, identification and quantification of isoquinoline alkaloids from Fumaria officinalis. The best results were obtained with a mixture of acetonitrile-methanol (9:1, v/v) containing 60mM ammonium acetate and 2.2M acetic acid as running electrolyte and an applied voltage of 30 kV. Electrospray MS measurements were performed in the positive ionization mode with isopropanol-water (1:1, v/v) as sheath liquid at a flow rate of 3 microl/min. Alkaloids were detected as [M+H](+)-ions and showed typical fragmentation patterns in MS/MS experiments. The developed assay was used for the quantification of seven isoquinoline alkaloids representing different structural subtypes in Fumariae herba extracts and F. herba containing phytopharmaceuticals.     

Capillary electrophoresis of complex natural polysaccharides

Complex natural polysaccharides, glycosaminoglycans (GAGs), are a class of ubiquitous macromolecules that exhibit a wide range of biological functions and participate and regulate multiple cellular events and (patho)physiological processes. They are generally present either as free chains (hyaluronic acid and bacterial acidic polysaccharides) or as side chains of proteoglycans (PGs; chondroitin/dermatan sulfate, heparin/heparan sulfate, and keratan sulfate) and are most often found in cell membranes and in the extracellular matrix. The recent emergence of modern analytical tools for their study has produced a virtual explosion in the field of glycomics. CE, due to its high resolving power and sensitivity, has been useful in the analysis of intact GAGs and GAG-derived oligosaccharides and disaccharides affording concentration and structural characterization data essential for understanding the biological functions of GAGs. In this review, novel off-line and on-line CE-MS and MS/MS methods for screening of GAG-derived oligosaccharides and disaccharides will be discussed.     

Capillary electrophoresis/mass spectrometry: a promising tool for the control of some physiologically hazardous compounds. I-derivatives of 3-quinuclidinol

A method based on the coupling of capillary electrophoresis with mass spectrometry (CE/MS) was developed for the monitoring of 3-quinuclidinol and its four N-alkyl derivatives (methyl, ethyl, propyl and isopropyl derivatives). A fragmentation study (collision-induced dissociation of ions in an ion trap) and optimization of the ion optics set-up for CE/MS experiments using direct infusion of a methanolic solution of the standards into the mass spectrometer were carried out in advance. Molecular ions of all quaternary compounds and the quasi-molecular ion [M + H]+ of free 3-quinuclidinol prevail in the mass spectra. In the MS/MS of propyl and isopropyl derivatives, the elimination of the alkyl chain dominates, leading to the ion at m/z 128. The fragmentation of the other compounds is more complex. Previous CE separation of the mixture of isobaric propyl and isopropyl derivatives is necessary for their unambiguous identification. A 10 mM ammonium acetate buffer (pH 4.0) is the optimum running electrolyte, allowing the CE separation of methyl, ethyl, propyl and isopropyl derivatives. A 0.5% (v/v) solution of acetic acid in methanol provides sufficient detection sensitivity when used as the sheath liquid. Limits of detection of 0.1 ppm for 3-quinuclidinol and 0.05 ppm for quaternary derivatives were achieved under the optimum conditions. The optimized method was applied to the determination of 3-quinuclidinol and related quaternary derivatives spiked into a sample of pond water. The experimental set-up for CE/MS/MS was investigated, which strongly increases the identification capability of the technique.     

Identification of oligomeric domains within dermatan sulfate chains using differential enzymic treatments, derivatization with 2-aminoacridone and capillary electrophoresis

Galactosaminoglycans, i.e. dermatan sulfate (DS) and chondroitin sulfate, are linear heteropolysaccharides consisting of repeating disaccharide units of L-iduronic acid (L-IdoA) or D-glucuronic acid (D-GlcA) residues linked to N-acetyl-galactosamine. High-performance capillary electrophoresis (HPCE or CE) has been successfully used for determining the disaccharide composition of glycosaminoglycans. However, only limited information is available on how to identify oligomeric domains rich in D-GlcA or L-IdoA. The aim of this study was therefore to develop a rapid and accurate CE procedure by which such oligosaccharides can be determined together with the variously sulfated disaccharides. Isolated dermatan sulfates of human origin were separately digested with chondroitinases ABC, AC and B and the enzymic products were derivatized with 2-aminoacridone. CE analysis of these products was performed using a phosphate buffer, pH 3.0, and reversed polarity at 30 kV. The derivatization enabled their detection with laser-induced fluorescence (LIF) and UV at 260 nm at much higher sensitivity than the detection of nonderivatized delta-saccharides at 232 nm and therefore components undetectable at 232 nm were nicely detected after derivatization. Except for delta-disaccharides, altogether five distinct oligosaccharides with differences in charge density were identified. Depending on the lyase that produced these oligomers, information on the presence of L-IdoA- or D-GlcA-containing domains within the DS chain and the sulfation pattern of these oligomeric domains was obtained. This CE method could also be useful in studying the functional oligomeric domains in galactosaminoglycan chains.     

Gold nanomaterials based pseudostationary phases in capillary electrophoresis: A brand‐new attempt at chondroitin sulfate isomers separation

In this work, a CE method with bare gold nanorods (GNRs) based pseudostationary phase was developed and applied for the separation of chondroitin sulfate (CS) isomers, CS, and dermatan sulfate (DS). The separation efficiency was investigated by varying the experimental parameters such as concentration and pH of the BGE, separation voltage, internal diameter of capillary, different size, and morphology of gold nanomaterials. Results showed that different size and morphology of gold nanomaterials had different effects on the separation of CS and DS. The best separation of CS and DS was achieved in the BGE composed of aqueous 150 mmol/L (mM) ethylenediamine + 20 mM sodium dihydrogen phosphate + 30% v/v GNRs, pH 4.5, at the separation voltage of ?10 kV. Capillary was 59.2 cm in length (effective length 49 cm), 50 μm id capillary thermostated at 25°C. CE with bare GNRs used as pseudostationary phase was shown to be a suitable technique for the separation of CS and DS mixtures with wider peaks. RSD of migration time and peak area of CS and DS were 0.13, 0.14 and 0.86, 1.07%, respectively.     

Structural analysis of bikunin glycosaminoglycan

The structure of an intact glycosaminoglycan (GAG) chain of the bikunin proteoglycan (PG) was analyzed using a combined top-down and bottom-up sequencing strategy. PGs are proteins with one or more linear, high-molecular weight, sulfated GAG polysaccharides O-linked to serine or threonine residues. GAGs are often responsible for the biological functions of PGs, and subtle variations in the GAG structure have pronounced physiological effects. Bikunin is a serine protease inhibitor found in human amniotic fluid, plasma, and urine. Bikunin is posttranslationally modified with a chondroitin sulfate (CS) chain, O-linked to a serine residue of the core protein. Recent studies have shown that the CS chain of bikunin plays an important role in the physiological and pathological functions of this PG. While no PG or GAG has yet been sequenced, bikunin, the least complex PG, offers a compelling target. Electrospray ionization Fourier transform-ion cyclotron resonance mass spectrometry (ESI FTICR-MS) permitted the identification of several major components in the GAG mixture having molecular masses in a range of 5505-7102 Da. This is the first report of a mass spectrum of an intact GAG component of a PG. FTICR-MS analysis of a size-uniform fraction of bikunin GAG mixture obtained by preparative polyacrylamide gel electrophoresis, allowed the determination of chain length and number of sulfo groups in the intact GAGs.     

Multistage Tandem Mass Spectrometry of Chondroitin Sulfate and Dermatan Sulfate

Chondroitin/dermatan sulfate (CS/DS) is a glycosaminoglycan (GAG) found in abundance in extracellular matrices. In connective tissue, CS/DS proteoglycans play structural roles in maintaining viscoelasticity through the large number of immobilized sulfate groups on CS/DS chains. CS/DS chains also bind protein families including growth factors and growth factor receptors. Through such interactions, CS/DS chains play important roles in neurobiochemical processes, connective tissue homeostasis, coagulation, and cell growth regulation. Expression of DS has been observed to increase in cancerous tissue relative to controls. In earlier studies, MS(2) was used to compare the types of CS/DS isomers present in biological samples. The results demonstrated that product ion abundances reflect the types of CS/DS repeats present and can be used quantitatively. It was not clear, however, to which of the CS/DS repeats the product ions abundances were sensitive. The present work explores the utility of MS(3) for structural characterization of CS/DS oligosaccharides. The data show that MS(3) product ion abundances correlate with the presence of DS-like repeats in specific positions on the oligosaccharide chains.     

Separation and identification of isomeric acidic degradation products of organophosphorus chemical warfare agents by capillary electrophoresis-ion trap mass spectrometry

Capillary electrophoresis (CE) coupled to ion trap mass spectrometry (MS) was evaluated for the separation and identification of chemical warfare agent degradation products (alkylphosphonic acids and alkyl alkylphosphonic acids). Different analytical parameters were optimized in negative ionization mode such as electrolyte composition (15 mM CH(3)COONH(4), pH 8.8), sheath liquid composition (MeOH/H(2)O/NH(3), 75:25:2, v/v/v), nebulization and ion trapping conditions. A standard mixture of five alkylphosphonic (di)acids and five alkyl alkylphosphonic (mono)acids containing isomeric compounds was used in order to evaluate CE selectivity and MS identification capability. The obtained electropherograms revealed that CE selectivity was very limited in the case of alkyl alkylphosphonic acid positional isomers, whereas isomeric isopropylphosphonic and propylphosphonic acids were baseline-separated. CE-MS-MS experiments provided an unambiguous identification of each isomeric co-migrating alkyl alkylphosphonic acids thanks to the presence of specific fragment ions. On the other hand, CE separation was mandatory for the identification of isomeric alkylphosphonic acids, which led to the same fragment ion and could not be differentiated by MS-MS. The developed method was applied to the analysis of soil extracts spiked with the analytes (before or after extraction treatment) and appeared to be very promising since resolution and sensitivity were similar to those observed in deionized water. Especially, analytes were detected and identified in soil extract spiked at 5 microg mL(-1) with each compound before extraction treatment.     

Energetic heterogeneity of the surface of a molecularly imprinted polymer studied by high-performance liquid chromatography

A sequential combination of reversed-phase liquid chromatography–mass spectrometry (LC–MS) and capillary electrophoresis (CE) has been explored in order to perform separation and characterization of a multicomponent peptide mixture from the synthesis of leuprolide. The mixture was first analyzed and fractionated by LC–MS, and the collected fractions were subsequently separated by CE. Unambiguous identification of the electrophoretic peaks was achieved by injecting the collected fractions separately and spiking the leuprolide crude mixture. Furthermore, structural information about the components of the mixture provided by several semi-empirical migration models has been used to check the accuracy of the structures previously proposed by LC–MS. Combination of the two orthogonal techniques results in an enhancement of their individual selectivity characteristics.     

Quantitative determination of the glycosaminoglycan Delta-disaccharide composition of serum, platelets and granulocytes by reversed-phase high-performance liquid chromatography

Seven Delta-disaccharide standards from heparan sulfate/heparin (HS/H) and nine Delta-disaccharide standards from chondroitin/dermatan sulfate (CS/DS) and hyaluronic acid (HA) were derivatized with the fluorophore 2-aminoacridone (AMAC) and separated in two runs each by reversed-phase HPLC with baseline separation and very short run times. This novel method facilitates the separation of the largest number of Delta-disaccharides from both CS/DS/HA and HS/H with one column and buffer system after fluorophore labeling in two runs at present. For the first time nine glycosaminoglycan (GAG) Delta-disaccharides from CS/DS/HA were separated after fluorophore labeling in one run. The limits of quantification (LOQs) were below 0.2 pmol for CS/DS/HA and HS/H Delta-disaccharides. We demonstrated applicability of our method for biological samples. Furthermore, normal ranges of the GAG Delta-disaccharide compositions from platelets and granulocytes were determined for the first time.     

Fraction collection in capillary electrophoresis for various stand-alone mass spectrometers

A procedure for collecting fractions during capillary electrophoresis for their analysis using various stand-alone instruments is described. The results of a systematic study of the optimization and application of capillary electrophoresis (CE) in conjunction with a reverse-phase high-performance liquid chromatography electrospray ionization quadrupole time of flight-tandem mass spectrometry (RP-HPLC-ESI-Q-TOF-MS/MS) and inductively-coupled mass spectrometry (ICP-MS) to the analysis of the seed extract of the Japanese Pagoda Tree (Sophora japonica) are presented. The off-line coupling of CE to the matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS) for the proteins mixture was applied. The cathode end of the capillary was placed inside a stainless steel needle using a coaxial liquid-sheath-flow configuration. The optimization of experimental parameters resulted in an efficient methodology for MS analysis of fractions. Several components contained in the extract of S. japonica were identified, some not previously known. It was demonstrated that low sensitivity, which is a real problem in off-line CE–MS analysis, could be tolerated because of a more flexible optimization of the CE separation conditions and the choice of independent stand-alone instruments for analysis of separated fractions. The estimated limit of detection for CE-RP-HPLC-ESI-Q-TOF-MS was 50 μM of polyphenols and for CE-ICP-MS, 1–100 μg/l.