Negative-ion Electrospray Tandem Mass Spectrometry Research Articles

Development of a highly sensitive liquid chromatography–mass spectrometry method to quantify plasma leukotriene E4 and demonstrate pharmacological suppression of endogenous 5-LO pathway activity in man

Low basal endogenous concentrations (<20 pg/mL) of the 5-lipoxygenase (5-LO) pathway biomarker leukotriene E4 (LTE4) in human plasma present a significant analytical challenge. Analytical methods including liquid chromatography–mass spectrometry and enzyme linked immunosorbent assays have been used to quantify plasma LTE4 in the past but have not provided consistent data in the lower pg/mL-range. With our new method, a detection limit (<1 pg/mL plasma) significantly below basal levels of LTE4 was achieved by combining large volume sample purification and enrichment by anion-exchange mixed mode solid phase extraction (SPE) with large volume injection followed by chromatographic separation by ultra performance liquid chromatography (UPLC) and quantification by highly sensitive negative-ion electrospray tandem mass spectrometry (MS/MS). The method was reproducible, accurate and linear between 1 and 120 pg/mL plasma LTE4. The method was used to perform an analysis of plasma samples collected from healthy volunteers in a Phase 1 study with the FLAP (5-lipoxygenase activating protein) inhibitor AZD5718. Basal endogenous LTE4 levels of 5.1 ± 2.7 pg/mL were observed in healthy volunteers (n = 34). In subjects that had been administered a single oral dose of AZD5718, significant suppression (>80%) of plasma LTE4 level was observed, providing pharmacological evidence that endogenous 5-LO pathway activity could be assessed.

Profiling of Human Milk Oligosaccharides for Lewis Epitopes and Secretor Status by Electrostatic Repulsion Hydrophilic Interaction Chromatography Coupled with Negative-Ion Electrospray Tandem Mass Spectrometry.

Human milk oligosaccharides (HMOs) are one of the most abundant ingredients in breast milk, and they play a beneficial role for newborns and are important for infant health. The peripheral fucosylated sequences of HMOs, such as the histo-blood group ABH(O) and Lewis a, b, x, and y antigens, are determined by the expression of the secretor (Se) and Lewis (Le) genes in the mammary gland, and are often the recognition motifs and serve as decoy receptors for microbes. In this work, we developed a method for determination of secretor status and Lewis blood phenotype and assignment of Lewis blood-group epitopes. The method was based on electrostatic repulsion/hydrophilic interaction chromatography coupled with tandem mass spectrometry (ERLIC-MS/MS). A specifically designed stationary phase, aspartic acid-bonded silica (ABS), was used to separate the acidic and neutral HMOs by electrostatic repulsion followed by HILIC. Negative-ion electrospray MS/MS was then used for analysis of secretor status and Lewis blood phenotypes and assignment of important epitopes of HMOs from the lactating mothers by selecting a specific set of unique fragment ions.

Assignment by Negative-Ion Electrospray Tandem Mass Spectrometry of the Tetrasaccharide Backbones of Monosialylated Glycans Released from Bovine Brain Gangliosides

Gangliosides, as plasma membrane-associated sialylated glycolipids, are antigenic structures and they serve as ligands for adhesion proteins of pathogens, for toxins of bacteria, and for endogenous proteins of the host. The detectability by carbohydrate-binding proteins of glycan antigens and ligands on glycolipids can be influenced by the differing lipid moieties. To investigate glycan sequences of gangliosides as recognition structures, we have underway a program of work to develop a “gangliome” microarray consisting of isolated natural gangliosides and neoglycolipids (NGLs) derived from glycans released from them, and each linked to the same lipid molecule for arraying and comparative microarray binding analyses. Here, in the first phase of our studies, we describe a strategy for high-sensitivity assignment of the tetrasaccharide backbones and application to identification of eight of monosialylated glycans released from bovine brain gangliosides. This approach is based on negative-ion electrospray mass spectrometry with collision-induced dissociation (ESI-CID-MS/MS) of the desialylated glycans. Using this strategy, we have the data on backbone regions of four minor components among the monosialo-ganglioside-derived glycans; these are of the ganglio-, lacto-, and neolacto-series.Graphical abstract

A UPLC/MS/MS method for determination of protosappanin B in rat plasma and its application of a pharmacokinetic and bioavailability study.

Caesalpinia sappan L. is a traditional medicinal plant which is used for promoting blood circulation and cerebral apoplexy therapy in China. Previous reports showed that the extracts of Caesalpinia sappan L. could exert vasorelaxant activity and anti-inflammation activity. Protosappanin B is a major constituent of C. sappan L., and showed several important bioactivities. The separation was achieved by an Acquity UPLC BEH Symmetry Shield RP18 column (1.7 μm, 2.1 × 100 mm) column with the gradient mobile phase consisting of 5 mm ammonium acetate aqueous solution and acetonitrile. Detection was carried out by using negative-ion electrospray tandem mass spectrometry via multiple reaction monitoring. Plasma samples were preprocessed by an extraction with ethyl acetate, and apigenin was used as internal standard. The current UPLC-MS/MS assay was validated for linearity, accuracy, intraday and interday precisions, stability, matrix effects and extraction recovery. After oral and intravenous administration, the main pharmacokinetic parameters were as follows: peak concentrations, 83.5 ± 46.2 and 1329.6 ± 343.6 ng/mL; areas under the concentration-time curve, 161.9 ± 69.7 and 264.9 ± 56.3 μg h/L; and half-lives, 3.4 ± 0.9 and 0.3 ± 0.1 h, respectively. The absolute bioavailability in rats of protosappanin B was 12.2%. The method has been successfully applied to a pharmacokinetic and bioavailability study of protosappanin B in rats.

Sequence analysis of the pyruvylated galactan sulfate-derived oligosaccharides by negative-ion electrospray tandem mass spectrometry

Five sulfated oligosaccharide fragments, F1–F5, were prepared from a pyruvylated galactan sulfate from the green alga Codium divaricatum, by partial depolymerization using mild acid hydrolysis and purification with gel-permeation chromatography. Negative-ion electrospray tandem mass spectrometry with collision-induced dissociation (ES-CID-MS/MS) is attempted for sequence determination of the sulfated oligosaccharides. The sequence of F1 with homogeneous disaccharide composition was first characterized to be Galp-(4SO4)-(1 → 3)-Galp by detailed nuclear magnetic resonance spectroscopic analyses. The fragmentation pattern of F1 in the product ion spectra was established on the basis of negative-ion ES-CID MS/MS, which was then applied to sequence analysis of other sulfated oligosaccharides. The sequences of F2 and F3 were deduced to be Galp-(4SO4)-(1 → 3)-Galp-(1 → 3)-Galp-(1 → 3)-Galp and 3,4-O-(1-carboxyethylidene)-Galp-(6SO4)-(1 → 3)-Galp, respectively. The sequences of major fragments in F4 and F5 were also deduced. The investigation demonstrated that negative-ion ES-CID-MS/MS was an efficient method for the sequence analysis of the pyruvylated galactan sulfate-derived oligosaccharides which revealed the patterns of substitution and glycosidic linkages. The pyruvylated galactan sulfate-derived oligosaccharides were novel sulfated oligosaccharides different from other algal polysaccharide-derived oligosaccharides.

Negative-Ion Electrospray Tandem Mass Spectrometry and Microarray Analyses of Developmentally Regulated Antigens Based on Type 1 and Type 2 Backbone Sequences.

Type 1 (Galβ1-3GlcNAc) and type 2 (Galβ1-4GlcNAc) sequences are constituents of the backbones of a large family of glycans of glycoproteins and glycolipids whose branching and peripheral substitutions are developmentally regulated. It is highly desirable to have microsequencing methods that can be used to precisely identify and monitor these oligosaccharide sequences with high sensitivity. Negative-ion electrospray tandem mass spectrometry with collision-induced dissociation has been used for characterization of branching points, peripheral substitutions, and partial assignment of linkages in reducing oligosaccharides. We now extend this method to characterizing entire sequences of linear type 1 and type 2 chain-based glycans, focusing on the type 1 and type 2 units in the internal regions including the linkages connecting type 1 and type 2 disaccharide units. We apply the principles to sequence analysis of closely related isomeric oligosaccharides and demonstrate by microarray analyses distinct binding activities of antibodies and a lectin toward various combinations of type 1 and 2 units joined by 1,3- and 1,6-linkages. These sequence-specific carbohydrate-binding proteins are in turn valuable tools for detecting and distinguishing the type 1 and type 2-based developmentally regulated glycan sequences.

Identification of a highly sulfated fucoidan from sea cucumber Pearsonothuria graeffei with well-repeated tetrasaccharides units

Sea cucumber fucoidan is a major bioactive component of sea cucumber. The structures of fucoidans have significant influences on their biological activities. The present study clarified the delicate structure of a fucoidan from Pearsonothuria graeffei. Fucoidan was obtained after papain digestion and purified by ion chromatography. The carbohydrate sequence of fucoidan was firstly determined by negative-ion electrospray tandem mass spectrometry (ES–MS) with collision-induced dissociation of the oligosaccharide fragments, which were obtained by mild acid hydrolysis, and completed by NMR for assignment of the anomeric conformation. It was unambiguously identified as a tetrasaccharide repeating unit with a backbone of [→3Fuc (2S, 4S) α1→3Fucα1→3Fuc (4S) α1→3Fuc#7×10#]n. The glycosidic bonds between the non-sulfated and 2,4-O-disulfated fucose residues were selectively cleaved, and highly ordered oligosaccharide fragments with a tetrasaccharide repeating unit were obtained. The highly 4-O- and 2, 4-di-O-sulfated polysaccharide deserves further developments for Pharmacia use.

Unravelling Glucan Recognition Systems by Glycome Microarrays Using the Designer Approach and Mass Spectrometry

Glucans are polymers of d-glucose with differing linkages in linear or branched sequences. They are constituents of microbial and plant cell-walls and involved in important bio-recognition processes, including immunomodulation, anticancer activities, pathogen virulence, and plant cell-wall biodegradation. Translational possibilities for these activities in medicine and biotechnology are considerable. High-throughput micro-methods are needed to screen proteins for recognition of specific glucan sequences as a lead to structure–function studies and their exploitation. We describe construction of a “glucome” microarray, the first sequence-defined glycome-scale microarray, using a “designer” approach from targeted ligand-bearing glucans in conjunction with a novel high-sensitivity mass spectrometric sequencing method, as a screening tool to assign glucan recognition motifs. The glucome microarray comprises 153 oligosaccharide probes with high purity, representing major sequences in glucans. Negative-ion electrospray tandem mass spectrometry with collision-induced dissociation was used for complete linkage analysis of gluco-oligosaccharides in linear “homo” and “hetero” and branched sequences. The system is validated using antibodies and carbohydrate-binding modules known to target α- or β-glucans in different biological contexts, extending knowledge on their specificities, and applied to reveal new information on glucan recognition by two signaling molecules of the immune system against pathogens: Dectin-1 and DC-SIGN. The sequencing of the glucan oligosaccharides by the MS method and their interrogation on the microarrays provides detailed information on linkage, sequence and chain length requirements of glucan-recognizing proteins, and are a sensitive means of revealing unsuspected sequences in the polysaccharides.

Fatty acid profiling of the seed oils of some varieties of field peas (Pisum sativum) by RP-LC/ESI-MS/MS: Towards the development of an oilseed pea

Reversed-phase liquid chromatography coupled to negative-ion electrospray tandem mass spectrometry (RP-LC/ESI-MS/MS) was used to study the fatty acid profile from the oil of harvested field pea (Pisum sativum) varieties as part of a research project to develop this legume as a commercial oilseed for Canada. The seed oils from pea samples contained palmitic and stearic acids as major saturated fatty acids. Oleic, linoleic and linolenic acids were the major unsaturated fatty acids found. Small percentages of other long chain fatty acids were also detected. This profile suggests that the species of field pea investigated might have the potential to be used as raw materials to develop a future new oilseed crop for the food industry. Fatty acid extracts did not require further manipulation before final analysis by RP-LC/ESI-MS/MS, indicating the utility and relative simplicity of this technique for future screening studies.

Preparation of Hybrid Fucoidan-derived Oligosaccharides and Their Structural Analysis by Negative-ion Electrospray Tandem Mass Spectrometry

Acomplex sulfated fucoidan(SPF) was obtained by hot water extraction from Sargassum pallidum.A series of hybrid oligosaccharides were acquired by mild acid hydrolysis with low pressure gel permeation chromatography(LPGPC).Based on the monosaccharides composition,methylation and electrospray collision-induced dissociation mass spectrometry(ES-CID-MS/MS) analysis,21 kinds of oligosaccharides were sulfated hybrid fucoidan-derived oligosaccharides.The resulted oligosaccharides were mainly composed of α1→3 linked Fuc,β1→4 linked Xyl and β1→6 linked Gal.The sulfation occurred at C4 or C2 of fucose,C2 of xylose and C4 of galactose residue,respectively.Most of fucose residues were present in nonreducing terminal of oligosaccharides.The technique of ES-CID-MS/MS was suitable for the sequences analysis of other hybrid oligosaccharides of various fucoidans.More importantly,all these diverse sulfated oligosaccharides can be printed onto the glycol-chip,and studying their interactions with proteins.

Sequence analysis of the sulfated rhamno-oligosaccharides derived from a sulfated rhamnan

Three sulfated rhamno-oligosaccharides, designated O1, O2 and O3, were obtained by mild acid hydrolysis of the sulfated rhamnan and purified by gel-permeation chromatography. On the basis of one- and two-dimensional nuclear magnetic resonance (1D, 2D NMR) spectroscopic analyses, the oligosaccharide O1 was characterized to be α-l-Rhap-(2SO4)-(1→3)-α-l-Rhap. The fragmentation pattern of the homogeneous disaccharide in the product ion spectra was recognized by negative-ion electrospray tandem mass spectrometry with collision-induced dissociation (ES-CID MS/MS). With the principles established, the sequences of the oligosaccharides O2 and O3 were deduced to be α-l-Rhap-(2SO4)-(1→3)-α-l-Rhap-(1→3)-α-l-Rhap, and α-l-Rhap-(2SO4)-(1→3)-α-l-Rhap-(1→3)-α-l-Rhap-(1→3)-α-l-Rhap (2SO4), respectively. The investigation demonstrated that the sulfated rhamnan-derived oligosaccharides were novel sulfated oligosaccharides different from those of other polysaccharides-degraded from algae, and it could be possible to determine the sequence of the sulfated rhamno-oligosaccharides directly from the glycosidic cleavage fragmentation in the product ion spectra.

Sequence determination and anticoagulant and antithrombotic activities of a novel sulfated fucan isolated from the sea cucumber Isostichopus badionotus

BackgroundThe aim is to analyze the structure, anticoagulant and antithrombotic activities of a sulfated fucan isolated from sea cucumber Isostichopus badionotus (fucan-Ib). MethodsFucan-Ib was hydrolyzed under mild acid conditions. The oligosaccharide fragments were fractionated by gel-filtration chromatography and the structures were determined by negative-ion electrospray tandem mass spectrometry with collision-induced dissociation and two-dimensional NMR. Anticoagulant activities were measured by activated partial thromboplastin, thrombin and prothrombin times, and by in vitro inhibition experiments with factors IIa and Xa. Antithrombotic activities were determined in vitro by measuring the length and weight of the thrombus generated. ResultThe linear polysaccharide sequence of fucan-Ib was deduced from the structures of its oligosaccharide fragments produced by acid hydrolysis. Under mild conditions, the glycosidic bonds between the non-sulfated and 2,4-O-disulfated fucose residues were selectively cleaved and highly ordered oligosaccharide fragments with a tetrasaccharide repeating unit [→3Fuc(2S,4S)α1→3Fuc(2S)α1→3Fuc(2S)α1→3Fucα1→]n were obtained. In in vitro assays fucan-Ib showed good anticoagulant and antithrombotic activities compared with heparin and the fucosylated chondroitin sulfate isolated from the same source (fCS-Ib). The two polysaccharides, fucan-Ib and fCS-Ib, differ in the mechanism of action; the former exhibited activity mainly by potentiation of antithrombin acted on thrombin and factor Xa whereas the latter mainly through heparin cofactor II. ConclusionFucan-Ib has a well defined structure with tetrasaccharide tandem repeats and good anticoagulant and antithrombotic activities. General importanceFucan-Ib has a well defined structure and can be readily quality-controlled, and therefore has potential therapeutic value as an affective antithrombotic drug with low risk of bleeding.

Development and validation of a liquid chromatography–tandem mass spectrometry method for the simultaneous determination of nine corticosteroid residues in bovine liver samples

A liquid chromatography tandem mass spectrometry (LC–MS/MS) confirmatory method for the simultaneous determination of nine corticosteroids in liver, including the four MRL compounds listed in Council Regulation 37/2010, was developed. After an enzymatic deconjugation and a solvent extraction of the liver tissue, the resulting solution was cleaned up through an SPE Oasis HLB cartridge. The analytes were then detected by liquid chromatography–negative-ion electrospray tandem mass spectrometry, using deuterium-labelled internal standards. The procedure was validated as a quantitative confirmatory method according to the Commission Decision 2002/657/EC criteria. The results showed that the method was suitable for statutory residue testing regarding the following performance characteristics: instrumental linearity, specificity, precision (repeatability and intra-laboratory reproducibility), recovery, decision limit (CCα), detection capability (CCβ) and ruggedness. All the corticosteroids can be detected at a concentration around 1 μg kg −1; the recoveries were above 62% for all the analytes. Repeatability and reproducibility (within-laboratory reproducibility) for all the analytes were below 7.65% and 15.5%, respectively.

Identification of reaction products from reactions of free chlorine with the lipid-regulator gemfibrozil

High global consumption rates have led to the occurrence of pharmaceutically active compounds (PhACs) in wastewater. The use of chlorine to disinfect wastewater prior to release into the environment may convert PhACs into uncharacterized chlorinated by-products. In this investigation, chlorination of a common pharmaceutical, the antihyperlipidemic agent gemfibrozil, was documented. Gemfibrozil (2,2-dimethyl-5-(2,5-dimethylphenoxy)pentanoic acid) was reacted with sodium hypochlorite and product formation was monitored by gas chromatography-mass spectrometry (GC-MS). The incorporation of one, two or three chlorine atoms into the aromatic region of gemfibrozil was demonstrated using negative-ion electrospray ionization mass spectrometry (ESI-MS) and tandem mass spectrometry (ESI-MS/MS). Further analysis using 1H nuclear magnetic resonance (NMR) spectroscopy identified the reaction products as 4′-ClGem (5-(4-chloro-2,5-dimethylphenoxy)-2,2-dimethylpentanoic acid) 4′,6′-diClGem (5-(4,6-dichloro-2,5-dimethylphenoxy)-2,2-dimethylpentanoic acid), and 3′,4′,6′-triClGem (5-(3,4,6-trichloro-2,5-dimethylphenoxy)-2,2-dimethylpentanoic acid), products consistent with electrophilic aromatic substitution reactions. The rapid reaction of gemfibrozil with free chlorine at pH conditions relevant to water treatment indicates that a mixture of chlorinated gemfibrozils is likely to be found in wastewater disinfected with chlorine.

Structural analysis and differentiation of reducing and nonreducing neutral model starch oligosaccharides by negative-ion electrospray ionization ion-trap mass spectrometry

Negative-ion electrospray tandem mass spectrometry with an ion-trap provided a sensitive tool for sequence determination of oligosaccharides derived from starch. Fragmentations of [M−H] − as well [M+Cl] − ions allow an unambiguous differentiation between α-(1 → 4) and α-(1 → 6) linkage based on the presence of diagnostic [M−H−90] − and [M−H−78] − ions. For nonreducing saccharides with terminal sucrose groups, the fragmentation starts with elimination of the fructose unit, followed by cross-ring cleavages of the remaining rings. The specific fragmentation features of reducing and nonreducing saccharides can be used for their differentiation.

Determination of the Phenoxyacid Herbicides MCPA, Mecoprop and 2,4-D in Kidney Tissue Using Liquid Chromatography with Electrospray Tandem Mass Spectrometry

An analytical method was developed to determine the phenoxyacid herbicides 2,4-D, MCPA and mecoprop in kidney tissue from animals where poisoning is suspected. Samples were Soxhlet extracted using diethyl ether and the extracts cleaned-up using anion exchange solid phase extraction cartridges. Analysis was performed using liquid chromatography with negative-ion electrospray tandem mass spectrometry (LC-MS/MS). The method was evaluated by analysing control kidney samples fortified at 1 and 5 mg/kg. Mean recoveries ranged from 82 to 93% with relative standard deviations from 3.2 to 19%. The limit of detection was estimated to be 0.02 mg/kg.

Confirmatory method for the determination of resorcylic acid lactones in urine sample using immunoaffinity cleanup and liquid chromatography–tandem mass spectrometry

The presence of zeranol (α-zearalanol) in urine samples due to natural contamination or illegal treatment is under debate within the European Union. The simultaneous determination of zeranol, its epimer taleranol (β-zearalanol), zearalanone and the structurally related mycotoxin zearalenone with the corresponding α- and β-zearalenol metabolites appears to be critical in deciding whether an illegal use has occurred. The aim of this study is to develop and validate a simple analytical procedure applicable to bovine and swine urine samples for the determination of all six resorcylic acid lactones. After an enzymatic deconjugation, the urine was subjected to a one-step cleanup on a commercially available immunoaffinity chromatography cartridge. The analytes were detected by liquid chromatography–negative-ion electrospray tandem mass spectrometry using deuterium-labelled internal standards. The method was validated as a quantitative confirmatory method according to European Commission Decision 2002/657/EC. The evaluated parameters were: linearity, specificity, precision (repeatability and intra-laboratory reproducibility), recovery, decision limit, detection capability and ruggedness. The decision limits (CCα) obtained, were between 0.56 and 0.68 μg L −1; recovery above 66% for all the analytes. Repeatability was between 1.4 and 5.3% and within-laboratory reproducibility between 1.9 and 16.1% for the six resorcylic acid lactones.

Sequence determination of a non-sulfated glycosaminoglycan-like polysaccharide from melanin-free ink of the squid Ommastrephes bartrami by negative-ion electrospray tandem mass spectrometry and NMR spectroscopy

A non-sulfated polysaccharide was isolated from the ink sac of squid Ommastrephes bartrami after removal of the melanin granules. The carbohydrate sequence of this polysaccharide was assigned by negative-ion electrospray tandem mass spectrometry with collision-induced dissociation of the oligosaccharide fractions produced by partial acid hydrolysis of the polysaccharide. The structural determination was completed by NMR for assignment of anomeric configuration and confirmation of linkage information and it was unambiguously identified as a glycosaminoglycan-like polysaccharide containing a glucuronic acid-fucose (GlcA-Fuc) disaccharide repeat in the main chain and a N-acetylgalactosamine (GalNAc) branch at Fuc position 3: -[3GlcAbeta1-4(GalNAcalpha1-3)Fucalpha1](n)-. Partial hydrolysis of the polysaccharide to obtain several oligosaccharide fractions with different numbers of the repeating unit assisted the assignment. In the negative-ion tandem mass spectrometric analysis, the unique (0,2)A type fragmentation was important to establish the presence of a 4-linked fucose in the main polysaccharide chain and a GalNAc branch at the Fuc position-3 of the disaccharide repeat.

Sequence Determination of Sulfated Carrageenan-Derived Oligosaccharides by High-Sensitivity Negative-Ion Electrospray Tandem Mass Spectrometry

Negative-ion electrospray tandem mass spectrometry with collision-induced dissociation is assessed for sequence determination of multiply sulfated oligosaccharide fragments of carrageenan obtained from partial depolymerization of the polysaccharides by either enzymatic digestion or mild acid hydrolysis. Carrageenan oligosaccharides with homogeneous disaccharide compositions were used to establish their fragmentation pattern, which was then applied to sequence determination of unusual oligosaccharides with either "hybrid" biose compositions or odd-numbered residues. As sulfate groups are labile, sulfate loss during collision-induced association was prevented by sodium adduction. The product ion spectra of [M - Na]- (where M represents the sodium salt of oligosaccharides) feature an extensive series of B- and C-type glycosidic cleavages, whereas the Y-type cleavage occurs mainly at the sulfated residues. The assignment of reducing or nonreducing terminal fragments was assisted by oligosaccharide reduction and the product ion spectra of the derived alditols. Due to the anionic nature of the sulfate present, high-sensitivity detection (1-5 pmol, using a hexasaccharide as an example) was obtained.

Sequence Analysis of Alginate-Derived Oligosaccharides by Negative-Ion Electrospray Tandem Mass Spectrometry

Negative-ion electrospray tandem mass spectrometry (ES-MS/MS) with collision-induced dissociation (CID) is attempted for sequence determination of alginate oligosaccharides, derived from polyanionic alginic acid, polymannuronate, and polyguluronate by partial depolymerization using either alginate lyase or mild acid hydrolysis. Sixteen homo- and hetero-oligomeric fragments were obtained after fractionation by gel-filtration and strong anion exchange high performance liquid chromatography. The product-ion spectra of these alginate oligosaccharides were dominated by intense B-, C-, Y-, and Z-type ions together with (0,2)A- and (2,5)A-ions of lower intensities. Internal mannuronate residues (M) produce weak but specific decarboxylated Z(int)-ions (Z(int) - 44 Da; int: denotes internal), which can be used for distinction of M and a guluronate residue (G) at an internal position. A reducing terminal M or G, although neither gives rise to a specific ion, can be identified by differences in the intensity ratio of fragment ions of the reducing terminal residue [(2,5)A(red)]/[(0,4)A(red)] (red: denotes reducing terminal).