Post-column Fluorescence Derivatization Research Articles

Co-Occurrence of Tetrodotoxin and Saxitoxins and Their Intra-Body Distribution in the Pufferfish Canthigaster valentini.

Pufferfish of the family Tetraodontidae possess tetrodotoxin (TTX) and/or saxitoxins (STXs), but the toxin ratio differs, depending on the genus or species. In the present study, to clarify the distribution profile of TTX and STXs in Tetraodontidae, we investigated the composition and intra-body distribution of the toxins in Canthigaster valentini. C. valentini specimens (four male and six female) were collected from Amami-Oshima Island, Kagoshima Prefecture, Japan, and the toxins were extracted from the muscle, liver, intestine, gallbladder, gonads, and skin. Analysis of the extracts for TTX by liquid chromatography tandem mass spectrometry and of STXs by high-performance liquid chromatography with post-column fluorescence derivatization revealed TTX, as well as a large amount of STXs, with neoSTX as the main component and dicarbamoylSTX and STX itself as minor components, in the skin and ovary. The toxins were also detected in the other tissues, but in much lower amounts than in the skin and ovary. The TTX/STX ratio varied greatly, depending on the tissue, but TTX was the major toxin component in the whole body, and STXs accounted for 25% and 13% of the total toxin amount in males and females, respectively. Like the marine pufferfish of the genus Arothron, C. valentini should be considered a pufferfish with considerable amounts of both TTX and STXs present simultaneously.

Development of Ultra-Performance Liquid Chromatography with Post-Column Fluorescent Derivatization for the Rapid Detection of Saxitoxin Analogues and Analysis of Bivalve Monitoring Samples.

Saxitoxin (STX) and its analogues produced by toxic dinoflagellates accumulate in bivalves, and routine monitoring of bivalves is important to prevent cases of human poisoning. In this study, we describe a rapid detection method for the analysis of STXs using ultra-performance liquid chromatography with post-column fluorescent detection and to investigate water depths and sampling points optimal for shellfish toxin monitoring. Cultured scallops (Mizuhopecten yessoensis) and mussels (Mytilus galloprovincialis) collected from various water depths and sampling points were used in this study. Irrespective of bivalve species, toxin concentrations in bivalves were lower at deeper water depths. The toxin concentrations of bivalves did not differ greatly when bivalves were collected from the same bay. Although the levels of contamination of bivalves with STXs can depend on various environmental and geographical factors, our findings are useful for formulating a sampling protocol for the prevention of harvesting contaminated shellfish.

Preconcentration and post-column fluorescent derivatization for the environmental water monitoring of an antihelmintic macrocyclic drug used in livestock

In this paper, a green analytical methodology based on fluorescence derivatization is proposed for the anti-helminthic drug monitoring ivermectin as environmental emergent contaminant. After sample clean-up, ivermectin was converted into a highly fluorescent derivative through a catalytic oxidation process followed by dehydration and tautomerization. Under optimal experimental conditions, a linear response was obtained for ivermectin within the range 0.38–600 μg L−1, with detection and quantification limits of 0.11 and 0.38 μg L−1, both values are lower than other previously reported. This method has been applied for ivermectin determination in environmental water samples at trace levels, showing its potential for contamination monitoring.

Column switching combined with hydrophilic interaction chromatography-tandem mass spectrometry for the analysis of saxitoxin analogues, and their biosynthetic intermediates in dinoflagellates

Hydrophilic-interaction chromatography (HILIC) is reportedly useful for the analysis of saxitoxin (STX) analogues, collectively known as paralytic shellfish toxins. Column switching and two-step gradient elution using HILIC combined with mass spectrometry enabled the simultaneous analysis of the 15 primary STX analogues and their biosynthetic intermediates, arginine, Int-A’, and Int-C’2, and the shunt product, Cyclic-C’. Crude extracts of toxin-producing dinoflagellates can be injected without any treatment except filtration. Enrichment of the compounds using this method was highly reproducible with respect to retention times (% RSD was under 1%) and highly sensitive (limits of detection (LODs) were in the range 0.9 (Int-C’2) - 116 (C3) μM) in terms of avoiding matrix effects associated with co-eluting substances. Validation studies demonstrated acceptable performance of this method for specificity, repeatability, linearity and recovery. A comparison of the quantitative results for STX analogues in Alexandrium tamarense using HPLC with post-column fluorescent derivatization and the column-switching HILIC–MS method revealed good agreement. The presence of Int-A’, Int-C’2, and Cyclic-C’ in toxic dinoflagellate species with different toxin profiles was confirmed using this method. Our data support the hypothesis that the early stages of the STX biosynthesis and shunt pathways are the same in dinoflagellates and cyanobacteria.

Storage and source of polycyclic aromatic hydrocarbons in sediments downstream of a major coal district in France

During the 20th century, the local economy of the Upper Loire Basin (ULB) was essentially based on industrial coal mining extraction. One of the major French coal districts with associated urban/industrial activities and numerous coking/gas plants were developed in the Ondaine-Furan subbasins, two tributaries of the upper Loire main stream. To determine the compositional assemblage, the level and the potential sources of contamination, the historical sedimentary chronicle of the 16 US EPA priority polycyclic aromatic hydrocarbons (PAHs) has been investigated. PAH concentrations were determined using gas chromatography/mass spectrometry (GC/MS) in a dated core, sampled in the Villerest flood-control reservoir located downstream of the Ondaine-Furan corridor (OFC). The most contaminated sediments were deposited prior to 1983 (Σ16PAHs ca. 4429–13,348 ng/g) and during flood events (Σ16PAHs ca. 6380 ng/g – 1996 flood; 5360 ng/g – 2003 flood; 6075 ng/g – 2008 flood), especially in medium and high molecular weight PAHs. Among them, typical pyrogenic PAHs such as FLT, PYR, BbF and BaP were prevalent in most of the core samples. In addition, some PAHs last decade data is available from the Loire Bretagne Water Agency and were analyzed using high-performance liquid chromatography with postcolumn fluorescence derivatization (HPLC/FLD). These results confirm that the most highly contaminated sediments were found downstream of OFC (Σ16PAHs ca. 2264–7460 ng/g). According to the observed molecular distribution, PAHs are originated largely from high-temperature pyrolytic processes. Major sources of pyrogenic PAHs have been emphasized by calculation of specific ratios and by comparison to reported data. Atmospheric deposition of urban and industrial areas, wood combustion and degraded coal tar derived from former factories of coking/gas plants seem to be the major pyrogenic sources. Specifically, particular solid transport conditions that can occur during major flood events lead us to emphasize weathering of former contamination sources, such as more preserved coal tar.

The presence of 12β-deoxydecarbamoylsaxitoxin in the Japanese toxic dinoflagellate Alexandrium determined by simultaneous analysis for paralytic shellfish toxins using HILIC-LC–MS/MS

A differential screening study using high-resolution (HR)-hydrophilic interaction chromatography (HILIC)-electrospray ionization (ESI)–quadrupole time-of-flight mass spectrometry (Q-TOF MS) was conducted to identify saxitoxin (STX) analogues in the marine dinoflagellate toxic sub-clone Alexandrium tamarense Axat-2 and the non-toxic sub-clone UAT-014-009 derived from the same Japanese isolate. One unknown compound was identified only in the toxic sub-clone and was found to have the molecular formula C9H16N6O2. This structure differed from that of decarbamoyl STX (dcSTX; C9H16N6O3) by the loss of a single oxygen. A 12-deoxy-dcSTX standard (a mixture of 12α- and β-deoxy-dcSTX) was chemically prepared from dcSTX by reduction with sodium borohydride. The unknown compound in the toxic strain of A. tamarense was identified as 12β-deoxy-dcSTX by comparison of its HR-HILIC-LC–MS retention time and HR–MS/MS spectrum with those of the chemically prepared standard, and the identification was confirmed by high-sensitivity HPLC analysis with post-column fluorescent derivatization. Moreover, two Japanese isolates of A. catenella showing toxin profiles different from that of A. tamarense were also found to contain 12β-deoxy-dcSTX. Previously, 12β-deoxy-dcSTX was isolated from the freshwater cyanobacterium Lyngbya wollei, which produces a unique set of STX analogues. This study is the first evidence of the presence of 12β-deoxy-dcSTX in marine dinoflagellates.

Microanalysis and preliminary pharmacokinetic studies of a sulfated polysaccharide from Laminaria japonica

A rapid, sensitive and reproducible high performance liquid chromatography (HPLC) method with post-column fluorescence derivatization has been developed to determine the amount of low-molecular-weight sulfated polysaccharide (GFS) in vivo. The metabolism of GFS has been shown to fit a two component model following its administration by intravenous injection, and its pharmacokinetic parameters were determined to be as follows: half-time of distribution phase (t 1/2α)=11.24±2.93 min, half-time of elimination phase (t 1/2β)=98.20±25.78 min, maximum concentration (C max)=110.53 μg/mL and peak time (T max)=5 min. The pharmacokinetic behavior of GFS was also investigated following intragastric administration. However, the concentration of GFS found in serum was too low for detection, and GFS could only be detected for up to 2 h after intragastric administration (200 mg/kg body weight). Thus, the bioavailability of GFS was low following intragastric administration because of the metabolism of GFS. In conclusion, HPLC with postcolumn derivatization could be used for quantitative microanalysis and pharmacokinetic studies to determine the presence of polysaccharides in the serum following intravenous injection.

Mycoflora and mycotoxin contamination of Roundup Ready soybean harvested in the Pampean Region, Argentina

A total of 89 freshly harvested soybean seed samples (Roundup Ready [transgenic] soybean cultivars) from the 2010/2011 crop season were collected from five locations in the Northern Pampean Region II, Argentina. These samples were analyzed for internal mycoflora, toxin production of isolated fungi, and for a range of mycotoxins. Mycotoxin analysis of aflatoxins (AFs), zearalenone (ZEA), fumonisins (FBs) and ochratoxin A (OTA) was done by HPLC-FLD (high performance liquid chromatography with postcolumn fluorescence derivatization), alternariol and alternariol monomethyl ether with HPLC-UV (HPLC with UV detection), trichothecenes (deoxynivalenol, nivalenol, T-2 toxin, HT-2 toxin, fusarenon X, 3-acetyldeoxynivalenol and 15-acetyldeoxynivalenol were analyzed by GC-ECD (gas chromatography with electron capture detector). Fungal colonization was more frequently found for samples from América, Saladillo and Trenque Lauquen than for samples from General Villegas and Trenel; a total of 1,401 fungal isolates were obtained from the soybean seeds. The most commonly identified fungal genera were Alternaria, Sclerotinia, Chaetomium, Cladosporium, Aspergillus, Penicillium, Phomopsis and Fusarium. Alternaria alternata, A.tenuissima, Aspergillus flavus, Penicillium citrinum, Fusarium verticillioides and F.semitectum were the predominant toxigenic fungal species. Mycotoxin production was confirmed for several isolates of toxigenic species, including Aspergillus flavus, A. parasiticus, Alternaria alternata, A.tenuissima, Fusarium graminearum, F semitectum and F. verticillioides. In particular, the percentage of mycotoxigenic Alternaria alternata (100%), A.tenuissima (95%) and aflatoxigenic strains of A. flavus (57%) were remarkably high. Although none of the mycotoxins, AFs, ZEA, FBs, trichothecenes and OTA, were directly detected in samples of soybean seeds, the frequent presence of toxigenic fungal species indicates the risk of multiple mycotoxin contamination.

Multiresidue confirmatory method for determination of quinolones in milk by HPLC: method development and validation according to the criteria of Commission Decision 2002/657/EC

Veterinary drugs have become an integral part of the livestock production and play an important role in maintaining animal welfare. The use of veterinary medicines may be cause of the presence of drug residues in animal food products if appropriate withdrawal periods are not respected or if contaminated feeds are used. This work presents the development of an high performance liquid chromatography with postcolumn fluorescence derivatization (HPLC-FLD) method for the quantitative detection of eight quinolones – norfloxacin, ciprofloxacin, danofloxacin, enrofloxacin, difloxacin, oxolinic acid, nalidixic acid, and flumequine – in bovine milk. After deproteination and extraction with a metaphosphoric acid 1% w/v/methanol/acetonitrile (60/20/20 v/v/v) solution, the sample is partially evaporated and cleaned up on a reversed phase solid phase extraction (SPE) cartridge. The extract is analyzed using an HPLC-FLD. Mean recovery ranged between 65-88%. The method is validated as a confirmatory method according to Decision 2002/657/EC. All the verified parameters (linearity, selectivity/specificity, trueness, precision, CC, ruggedness and stability) were satisfactory and the method is able to quantify all the analytes in milk in the concentration range 15-60 μg/Kg for danofloxacin and 25-150 μg/Kg for the other quinolones.

An HPLC Method for Microanalysis and Pharmacokinetics of Marine Sulfated Polysaccharide PSS-Loaded Poly Lactic-co-Glycolic Acid (PLGA) Nanoparticles in Rat Plasma

This study was aimed at developing a sensitive and selective HPLC method with postcolumn fluorescence derivatization for the detection of propylene glycol alginate sodium sulfate (PSS) in rat plasma. Plasma samples were prepared by a simple and fast ultrafiltration method. PSS was extracted from rat plasma with d-glucuronic acid as internal standard. Isocratic chromatographic separation was performed on a TSKgel G2500 PWxL column with the mobile phase of 0.1 M sodium sulfate at a flow rate of 0.5 mL/min. Analyte detection was achieved by fluorescence detection (FLD) at 250 nm (excitation) and 435 nm (emission) using guanidine hydrochloride as postcolumn derivatizing reagent in an alkaline medium at 120 °C. The calibration curve was linear over a concentration range of 1–500 μg/mL, and the lower limit of detection (LLOD) was found to be 250 ng/mL. This validated method was applied successfully to the pharmacokinetic study of PSS and PSS-loaded poly lactic-co-glycolic acid (PLGA) nanoparticles (PSS-NP) in rat plasma after a single intravenous (PSS only) and oral administration (PSS and PSS-NP). Significant differences in the main pharmacokinetic parameters of PSS and PSS-NP were observed. The relative bioavailability of PSS-NP was 190.10% compared with PSS which shows that PSS-NP can improve oral bioavailability.

Single-cell analysis of paralytic shellfish toxins in Alexandrium tamarense by HPLC with post-column fluorescent derivatization

We developed a methodology for analyzing the C-toxin (C2) content in single Alexandrium tamarense cells; this method was based on high performance liquid chromatography (HPLC). C2 is the main paralytic shellfish toxin (PST) detected in a clonal culture of A. tamarense, which is a common causative organism in cases of paralytic shellfish poisoning in Japan. This HPLC method employs post-column fluorescent derivatization (FL). Mobile phase, column size, flow rate, reagent concentrations, and lamp type for the fluorescent detector were all optimized for the detection of C2. With this improved methodology, we could measure 1 fmol of C2 with a signal to noise ratio (S/N)=2. Clonal heterogeneity within the toxic strain, which was maintained for 13 years after re-isolation from the original clonal culture, ranged from <1fmol to 700fmolcell−1. This report is the first to demonstrate definitively that PST content varies on a cell-by-cell basis in a clonal culture of a dinoflagellate that causes paralytic shellfish poisoning.

Co-occurrence and statistical correlations between mycotoxins in feedstuffs collected in the Asia–Oceania in 2010

In this study, a total of 1468 feed samples collected in the Asian-Oceania region during the year 2010 were analysed for the presence of aflatoxins B1, B2, G1 and G2, fumonisin B1 and B2, zearalenone, ochratoxin A and deoxynivalenol. The samples tested were diverse, ranging from cereals such as corn, wheat and rice to processing by-products, namely soybean meal, corn gluten meal, dried distillers grains with solubles and other fodder, including straw, silage and finished feed. The samples were analysed by using high liquid chromatography with UV detector, postcolumn fluorescence derivatization and mass spectrometer after prior clean-up. The positive correlation between the occurrence of aflatoxins B1 and B2 and fumonisins B1 and B2 was very high, as was to be expected. Both correlation between the Fusarium mycotoxins deoxynivalenol and zearalenone and the storage mycotoxins ochratoxin A and aflatoxins proved to be low. The correlations between individual mycotoxins depended strongly on the commodity itself. These data indicate that the contamination of feedstuffs with only one mycotoxin is rare and that mycotoxins occur more frequently together, representing a risk for domestic livestock.

Microanalysis of oligosaccharide HS203 in beagle dog plasma by postcolumn fluorescence derivatization method

A rapid and sensitive postcolumn fluorescence derivatization method was developed for microanalysis of antidiabetic oligosaccharide HS203 in beagle dog plasma. After plasma protein was removed by a simple and fast ultrafiltration method, chromatographic separation was performed on an Asahipak GS-320 HQ column with a mobile phase of 50mmol/L phosphate buffer (pH 6.7) and acetonitrile (83/17, v/v). The column effluent was monitored by fluorescence detection at 249nm (excitation) and 435nm (emission) using guanidine hydrochloride as a postcolumn derivatizing reagent. A satisfactory resolution of the analyte was achieved and the limit of detection was found to be 4ng (more sensitive than silver staining of HS203 in polyacrylamide gel electrophoresis). The method described above was successfully applied to a pharmacokinetic study of HS203 and to monitor blood glucose level simultaneously in beagle dog. It is also possible to be applied for microanalysis of other oligosaccharides in biological samples.

Determination of Idebenone in Plasma by HPLC with Post-Column Fluorescence Derivatization Using 2-Cyanoacetamide

Idebenone is a benzoquinone analog that is used in the treatment of several neurological disorders including Friedreich's ataxia. It was found that the reaction of idebenone with 2-cyanoacetamide under alkaline conditions generates fluorescent products, and the reaction was considered to proceed via Craven's reaction. The reaction mixture from idebenone gave fluorescence with excitation and emission maximum wavelengths at 358 nm and 409 nm, respectively. It was adopted for HPLC with post-column fluorescence derivatization of idebenone. Idebenone in the plasma showed a linear response in the range of 0.5-32 ng (25-1600 ng/mL), and the quantitation limit (S/N=10) was 12.5 ng/mL. The detection limit (S/N=3) of the standard solution of idebenone was 0.1 ng. The HPLC system was applied to the human blood plasma obtained by finger prick. The plasma sample obtained by finger prick gave a similar chromatogram to that of venous blood obtained by venipuncture.

Catecholamine analysis with strong cation exchange column liquid chromatography–peroxyoxalate chemiluminescence reaction detection

A liquid chromatography-chemiluminescence detection method was developed and validated for the determination of catecholamines (norepinephrine, epinephrine, and dopamine) in mouse brains. Chromatography was performed on a strong cation exchange column (150 × 2.0-mm id) using an isocratic mobile phase of 65 mM potassium acetate/75 mM potassium phosphate (95:5, pH 3.5) at a flow rate of 0.2 mL/min following post-column fluorescence derivatization of catecholamines with ethylenediamine and peroxyoxalate chemiluminescence reaction detection. The recovery of catecholamines added to mouse brain samples was more than 95.0%, while intra- and inter-day precision of the assay were <4.8%. The validated method was used to determine norepinephrine and dopamine concentrations in mouse brains without prior sample purification.

Determination of Ubiquinone in Blood by High-Performance Liquid Chromatography with Post-Column Fluorescence Derivatization Using 2-Cyanoacetamide

It was shown that ubiquinone (CoQ(10)) and ubiquinol (CoQ(10)H(2)) produce fluorescence products under alkaline conditions when reacted with 2-cyanoacetamide. The reaction mixture from CoQ(10) gave fluorescence with excitation and emission maximum wavelengths at 442 nm and 549 nm, respectively. This reaction was considered to proceed via Craven's reaction. Moreover, 2-cyanoacetamide was shown to be a useful reagent for high-performance liquid chromatography (HPLC) with post-column fluorescence derivatization of CoQ(10) and CoQ(10)H(2) in blood. CoQ(10) showed a linear response in the range of 0.32-1276 ng, and the detection limit (S/N = 3) was 0.16 ng. Moreover, the sample pretreatment by deproteinization and extraction of CoQ(10) and CoQ(10)H(2) from plasma using 1-propanol with potassium formate was effective for excellent separation of CoQ(10) and CoQ(10)H(2) from other fluorescent substances in the blood. This simple and rapid pretreatment was considered to minimize the oxidation of CoQ(10)H(2). On the other hand, CoQ(10) and CoQ(10)H(2) in plasma samples obtained by finger prick were detected, as in venous blood obtained by venipuncture. Our method involving the simple and rapid collection of plasma by finger prick and sample pretreatment is thought to be applicable for the determination of CoQ(10)H(2)/total CoQ(10) ratio as a biomarker of oxidative stress.

Reverse Homogeneous Liquid–Liquid Extraction as a Miniaturized Method for Extraction of Aflatoxins from Pistachio and Wheat and LC Post-column Derivatization-Fluorescence Detection

A rapid and simple quantitative method for the simultaneous determination of the four aflatoxins (AFs) B1, B2, G1 and G2 was developed using reverse homogeneous liquid–liquid extraction (RHLEE) and HPLC post-column derivatization-fluorescence (HPLC-FL) detection. The method based on the rapid extraction of AFs from a methanolic sample into chloroform after addition of water containing KBr, with a method we called reverse homogeneous liquid–liquid extraction. Recoveries were in the range of 88–125% and the limits of detection were between 0.001–0.042 ng g−1 for different AFs. Wheat and pistachio were chosen for the analysis of real samples and the method was also successfully applied to a FAPAS® TEST MATERIAL (T04151).

Determination of 9, 10-phenanthrenequinone in airborne particulates by high-performance liquid chromatography with post-column fluorescence derivatization using 2-aminothiophenol

9, 10-Phenanthrenequinone (PQ) is regarded as a harmful environmental pollutant and its presence has been reported in atmospheric environment. The measurement of PQ in environment should be necessary to evaluate the influence of PQ on human health. We found that PQ reacted with 2-aminothiophenol under acidic condition to form fluorescent derivative which emits green fluorescence at 510 nm. Based on this reaction, a simple and rapid determination method for PQ was developed by HPLC with post-column derivatization and fluorescence detection. By the proposed HPLC system, PQ was detected at 24 min and the detection limit was 67 fmol/injection (S/N = 3). The proposed method was able to determine the atmospheric PQ concentrations by the direct injection of extract from airborne particulates.

Improved separation and characterization of lipopolysaccharide related compounds by reverse phase ion pairing-HPLC/electrospray ionization-quadrupole-mass spectrometry (RPIP-HPLC/ESI-Q-MS)

A new approach for the separation and inline characterization of lipopolysaccharide (LPS) related compounds has been developed. The separation was based on the difference in the number of charged phosphate and ethanolamine groups, as non-stoichiometric substituents, on the polysaccharide backbone, and was achieved with reverse phase ion-paring chromatography (RPIP-HPLC). Tributylamine was used as an ion-pair reagent. In the conditions used in this study, tributylammonium then binds to the LPS related compounds through the negatively charged phosphate groups. This changes the hydrophobicity of the analytes at different positions and allows for separation based on both the number and position of the substituents on the analyte. The RPIP-HPLC was found to be effective for the separation of the O, N-deacylated derivative (deON) and polysaccharide portion (PS) from the LPS of Escherichia coli C strain. Post-column fluorescence derivatization (FLD), using sodium periodate and taurine, was used to detect the separated LPS related species. On the other hand, the separated species were also detected by direct infusion into the ESI-Q-MS using a volatile ammonium acetate buffer rather than the more traditional potassium phosphate buffer. The signal to noise ratio (S/N ratio) was low for the total ion chromatogram, however, high S/N ratios as well as good resolution were attained by selected ion monitoring (SIM) using m/ z numbers corresponding to species with different numbers of non-stoichiometric substituents. Five species for deON and ten species for PS were clearly identified on the SIM chromatogram on the RPIP-HPLC/ESI-Q-MS. Accordingly, the present method allows for the effective separation and inline identification of the species corresponding to the diverse non-stoichiometric substitutions in LPS related compounds.

Evolutionary Differences in Glycosaminoglycan Fine Structure Detected by Quantitative Glycan Reductive Isotope Labeling

To facilitate qualitative and quantitative analysis of glycosaminoglycans, we tagged the reducing end of lyase-generated disaccharides with aniline-containing stable isotopes (12C6 and 13C6). Because different isotope tags have no effect on chromatographic retention times but can be discriminated by a mass detector, differentially isotope-tagged samples can be compared simultaneously by liquid chromatography/mass spectrometry and quantified by admixture with known amounts of standards. The technique is adaptable to all types of glycosaminoglycans, and its sensitivity is only limited by the type of mass spectrometer available. We validated the method using commercial heparin and keratan sulfate as well as heparan sulfate isolated from mutant and wild-type Chinese hamster ovary cells, and select tissues from mutant and wild-type mice. This new method provides more robust, reliable, and sensitive means of quantitative evaluation of glycosaminoglycan disaccharide compositions than existing techniques allowing us to compare the chondroitin and heparan sulfate compositions of Hydra vulgaris, Drosophila melanogaster, Caenorhabditis elegans, and mammalian cells. Our results demonstrate significant differences in glycosaminoglycan structure among these organisms that might represent evolutionarily distinct functional motifs.