Post-column High-performance Liquid Chromatography Research Articles

Determination of glyphosate and aminomethylphosphonic acid residues in Finnish soils by ultra‐high performance liquid chromatography–tandem mass spectrometry

Glyphosate [N-(phosphonomethyl) glycine] (GLY) adsorbs strongly in Finnish soils. A new method for GLY and its main degradation product, aminomethylphosphonic acid (AMPA) residues in clay soils (Protovertic Luvisol) was developed and validated. A new method was necessary because the previous one required laborious cleaning pre-treatments, and its recovery was quite poor (<40%–70%). In the new method, the earlier method's extraction solvent, 0.1 M potassium hydroxide (KOH), was replaced by more effective 0.6 M KOH. The old post-column high-performance liquid chromatography and fluorescence (HPLC-FLD) method was replaced by the ultra-high performance liquid chromatography–tandem mass spectrometry (UHPLC-MS/MS) method. Compounds were identified as their fluorenyl methyl chloroformate (FMOC) derivatives by a multiple reaction monitoring (MRM) technique and quantified by an internal standard method utilising multipoint matrix-matched calibration. Glufosinate-ammonium (GLUF) was used to monitor the effectiveness of extraction with good recovery (80–119%). All calibration curves were found to be linear (R2 ≥ 0.98) in the studied calibration range (0.01–3.31 mg kg−1 in fresh soil). The repeatability and reproducibility were 25% and 28% for GLY, and 20% and 24% for AMPA in real research soil samples. The method was effective throughout the calibration range in all the studied Finnish agricultural soils.•An improved method was created to analyse glyphosate (GLY) and AMPA in Finnish clay soil.•The challenge caused by strong GLY adsorption on soil was solved by using multipoint matrix-matched calibration curve samples which were prepared identically with the research samples.•The method performed well in all tested clay, loam and sandy loam soils.

Effect of Pretreatment Processes on Biogenic Amines Content and Some Bioactive Compounds in Hericium erinaceus Extract.

Hericium erinaceus is reported as a source of several nutritional contents and bioactive compounds, especially β-glucan. However, various uncontrolled processes lead to the formation of byproducts that can affect human health, including biogenic amines. These amines are concerning, because their presence is an important indicator of the process of hygiene and food spoilage or quality. A better understanding of various pretreatment processes can control the content of biogenic amines. In this work, we studied the effect of pretreatment processes, i.e., sample size (whole, ripping, and chopping); heating process (non-heating, blanching, and boiling); and drying method (nondrying, hot air drying, and freeze-drying) on biogenic amine contents in H. erinaceus extract. A method of the post-column high-performance liquid chromatography (HPLC) technique was used for the analysis of putrescine (PUT) and spermidine (SPD) in H. erinaceus extract following the acceptable guidelines. In this study, treatment 20 (chopping/non-heating/hot air drying) was suggested as a good choice for the pretreatment process, because low levels of PUT and SPD were shown in the extract while high levels of the bioactive compounds β-glucan and antioxidant activity were presented. This treatment process can be applied to the industry because of its easy operation and cost-saving.

A Comparison Study of Phenolic Contents and &amp;lt;i&amp;gt;in Vitro&amp;lt;/i&amp;gt; Antioxidant Activities of Australian Grown Faba Beans (&amp;lt;i&amp;gt;Vicia faba&amp;lt;/i&amp;gt; L.) Varying in Seed Coat Colours as Affected by Extraction Solvents

Twelve Australian grown faba bean genotypes with seed colour ranging from white, different shades of buff/beige, green, red and purple, were extracted using 80% methanol (v/v) or 70% acetone (v/v). The findings showed that commonly consumed buff-coloured genotypes have comparable phenolic contents and antioxidant activities to those with exotic seed coat colour (red, green and purple). In general, the extraction yield for methanol and acetone extracts of faba bean genotypes were similar. However, the acetone extracts of faba bean varieties with coloured seed coats exhibited higher antioxidant activities than their methanol extracts counterparts. Phenolic compounds and antioxidant activities of the genotypes were compared in an array of chemical-based assays and profiled using an on-line high-performance liquid chromatography-post column derivatization (HPLC-PCD) system. The total phenolic content (TPC) and total flavonoid content (TFC) of acetone extracts were approximately twice those of methanol extracts. The acetone extracts contained six times higher levels of di(phenyl)-(2,4,6-trinitrophenyl) imi- noazanium radical scavenging activity (DPPH), six times higher Total Equivalent Antioxidant Capacity (TEAC) and two times higher Ferric Reducing Antioxidant Power (FRAP) than those of methanol extracts. In general, the methanol extracts of white-coloured faba bean genotype exhibited comparable phenolic contents and antioxidant activities to varieties with coloured seed coats. However, the TPC, TFC, DPPH, TEAC and FRAP of acetone extracts from white-coloured genotype were 2 - 4, 1 - 2, 5 - 9, 2 - 3 and 1 - 2 times lower than those of faba bean varieties with coloured seed coats. HPLC-PCD analyses showed substantial antioxidant responses, represented as a dense “hump” of peaks in the HPLC chromatograms of acetone extracts from coloured-genotypes. This “hump” was not detected in the chromatograms of white-genotype acetone extracts, or in chromatograms of methanol extracts regardless of genotype or seed coat colour. All coloured-beans had higher phenolic contents and antioxidant activities than the white-genotype. Hydroxybenzoic acids/flavanols, hydroxycinnamic acids and flavonols were dominant in coloured beans. The findings suggest potentials in selecting commonly consumed buff-coloured faba bean genotypes within the breeding programs for enhanced levels of phenolic compounds and antioxidant activities, potentially increasing their health-promoting properties. And, there might also be potentials in developing faba bean extracts for pharmaceutical or natural medicines.

Isolation and Identification of Three γ-Glutamyl Tripeptides and Their Putative Production Mechanism in Aged Garlic Extract.

We analyzed aged garlic extract (AGE) to understand its complex sulfur chemistry using post-column high-performance liquid chromatography with an iodoplatinate reagent and liquid chromatography high resolution mass spectrometry (LC-MS). We observed unidentified peaks of putative sulfur compounds. Three compounds were isolated and identified as γ-glutamyl-γ-glutamyl- S-methylcysteine, γ-glutamyl-γ-glutamyl- S-allylcysteine (GGSAC) and γ-glutamyl-γ-glutamyl- S-1-propenyl-cysteine (GGS1PC) by nuclear magnetic resonance and LC-MS analysis based on comparisons with chemically synthesized reference compounds. GGSAC and GGS1PC were novel compounds. Trace amounts of these compounds were detected in raw garlic, but the contents of these compounds increased during the aging process. Production of these compounds was inhibited using a γ-glutamyl transpeptidase (GGT) inhibitor in the model reaction mixtures. These findings suggest that γ-glutamyl tripeptides in AGE are produced by GGT during the aging process.

Development of an Analytic Method for Sulfur Compounds in Aged Garlic Extract with the Use of a Postcolumn High Performance Liquid Chromatography Method with Sulfur-Specific Detection

Background: Garlic and its processed preparations contain numerous sulfur compounds that are difficult to analyze in a single run using HPLC.Objective: The aim of this study was to develop a rapid and convenient sulfur-specific HPLC method to analyze sulfur compounds in aged garlic extract (AGE).Methods: We modified a conventional postcolumn HPLC method by employing a hexaiodoplatinate reagent. Identification and structural analysis of sulfur compounds were conducted by LC-mass spectrometry (LC-MS) and nuclear magnetic resonance. The production mechanisms of cis-S-1-propenylcysteine (cis-S1PC) and S-allylmercaptocysteine (SAMC) were examined by model reactions.Results: Our method has the following advantages: less interference from nonsulfur compounds, high sensitivity, good correlation coefficients (r > 0.98), and high resolution that can separate >20 sulfur compounds, including several isomers, in garlic preparations in a single run. This method was adapted for LC-MS analysis. We identified cis-S1PC and γ-glutamyl-S-allyl-mercaptocysteine in AGE. The results of model reactions suggest that cis-S1PC is produced from trans-S1PC through an isomerization reaction and that SAMC is produced by a reaction involving S-allylcysteine/S1PC and diallyldisulfide during the aging period.Conclusion: We developed a rapid postcolumn HPLC method for both qualitative and quantitative analyses of sulfur compounds, and this method helped elucidate a potential mechanism of cis-S1PC and SAMC action in AGE.

Development of an HPLC post-column antioxidant assay for Solidago canadensis radical scavengers

The aim of this work was to modify and validate the post-column high-performance liquid chromatography (HPLC)-ABTS and DPPH methods for evaluating the antioxidant activity of the methanolic extracts of Solidago canadensis (Canadian goldenrod) leaves and flowers. Separation of the analytes was performed via the HPLC-PDA method on a YMC analytical column using a gradient elution program. Three compounds with antioxidant properties – chlorogenic acid, rutin and isoquercitrin – and two unidentified antioxidants were established. The research showed that the coil temperature regimes and loop length combinations influence the optimised post-column assay method for detecting the antioxidant activity of goldenrod radical scavengers. Investigations established that the temperature in the reaction coil was a substantial factor contributing to the signal strength of the analytes after reacting with the DPPH and ABTS radicals.

Effects of Roasting on Phenolic Composition and In vitro Antioxidant Capacity of Australian Grown Faba Beans (Vicia faba L.)

Faba bean phenolic compounds encompassed phenolic acids, flavonols, proanthocyanidins and anthocyanins. Roasting faba beans for 120 min decreased the total phenolic, flavonoid and proanthocyanidin contents by 42, 42 and 30%, respectively. Roasting beans for 120 min decreased the 2,2-diphenyl-1-picrylhydrazyl radical scavenging activity, total equivalent antioxidant capacity and ferric reducing antioxidant power by 48, 15 and 8%, respectively. High performance liquid chromatography-post column derivatisation revealed the generation of new phenolic compounds as a result of roasting. Antioxidant mechanism of bean less-polar phenolic compounds was largely based on free radical scavenging activity. The bean phenolic compounds with reducing capability were heat stable. Roasted faba bean extracts (70% acetone, v/v) were fractionated into relatively polar and non-polar fractions; the latter contributed the majority of the antioxidant capacity. The extracts from beans with different seed coat colours differed in their phenolic compositions, which suggest different levels of potential benefits to health. Although roasting initially lowers the bean antioxidant capacity, prolonged roasting at 150 °C for 60 min and longer causes generation of new phenolic compounds and an increased antioxidant capacity. The findings encourage a wider ultilisation of faba beans for human foods particularly in baked/roasted products.

Effects of soaking, boiling and autoclaving on the phenolic contents and antioxidant activities of faba beans (Vicia faba L.) differing in seed coat colours

The Australian grown faba beans of different seed coat colours were either soaked, boiled or autoclaved, and analysed for phenolic contents and antioxidant activity using an array of reagent-based assays. Soaking, boiling and autoclaving were shown to lower the level of active compounds in faba beans. A significant amount of active compounds was leached to the soaking and cooking medium. Boiling was a better method in retaining active compounds in beans than autoclaving. The boiled beans had more active compounds than those of resulting cooking broths, which was the opposite observation when autoclaving. The buff-genotypes had a similar level of active compounds to red- and green-genotypes. The high performance liquid chromatography-post column derivatisation (HPLC-PCD) system detected a dense collection of high antioxidant HPLC peaks ('humps') in extracts of raw, soaked and boiled beans. The present findings encouraged consumption of faba beans together with cooking broth for the maximum potential health benefits.

Automated high performance liquid chromatography with on-line reduction of disulfides and chemiluminescence detection for determination of thiols and disulfides in biological fluids

In general, the reduction of disulfide bonds with tris(2-carboxyethyl)phosphine (TCEP) is performed using off-line operation, which is not only time-consuming but also vulnerable to the spontaneous re-oxidation of thiols during sample preparation and subsequent analysis procedures. To the best of our knowledge, there has been not any case on the on-line reduction for biological disulfides coupled with high performance liquid chromatography (HPLC). In this study, these obstacles are overcome by packing Zn(II)-TCEP complexes into a home-made column. The as-synthesized Zn(II)-TCEP complexes enable efficient reduction of disulfide bonds at pH 3.0. This acidic pH value was compatible with that of the mobile phase for HPLC separation of thiols and disulfides. Therefore, using fluorosurfactant-prepared triangular gold nanoparticles as HPLC postcolumn specific chemiluminescence (CL) reagents for thiols, the feasibility of the established on-line reduction column has been confirmed for the direct identification of both thiols and disulfides by incorporating this reduction column into a single chromatographic separation. Detection limits for these analytes range from 8.3 to 25.4nM and the linear range in a log–log plot can comprise three orders of magnitude. Finally, the utility of this automated on-line reduction of disulfides-HPLC-CL system has been demonstrated for the reliable determination of thiols and disulfides in human urine and plasma samples.

Determination of sphingoid bases from hydrolyzed glucosylceramide in rice and wheat by online post-column high-performance liquid chromatography with O-phthalaldehyde derivatization

We developed a determination method for sphingoid bases using online post-column high-performance liquid chromatography (HPLC) with O-phthalaldehyde (OPA) derivatization. Good separation was achieved using a reversed-phase column and eluting with 50% acetonitrile containing formic acid and heptafluorobutyric acid. Using these conditions, an excellent linearity (R² > 0.999) was achieved using standard solutions of sphinganine (d18:0), sphingosine (d18:1(4t)), 4-hydroxy-sphinganine (t18:0), glucosylsphingosine (glc-d18:1(4t)), and galactosylsphingosine (gal-d18:1(4t)). Plant glucosylceramides were hydrolyzed with 1 M aqueous HCl in methanol for 18 h at 90°C, followed by extraction of sphingoid bases with diethyl ether in preparation for analysis using the proposed HPLC conditions. The glc-d18:1(4t) standard was also hydrolyzed and analyzed by HPLC using the same procedure, and the d18:1(4t) peak obtained from the hydrolyzed glc-d18:1(4t) standard was used as a reference for calculation. We also confirmed the applicability of this method to the analysis of sphingoid bases in rice and wheat, obtaining relative standard deviations of 8.0% for rice and 4.6% for wheat. The recoveries of spiked rice and wheat samples were 104% and 106%, respectively. Our proposed method enables the straightforward determination of sphingoid bases without expensive facilities, employing fluorescence detection of OPA derivatives.

Investigation of contribution of individual constituents to antioxidant activity in herbal drugs using postcolumn HPLC method

The most important attention is paid to the search of natural antioxidants and their evaluation in medicinal and food raw materials of plant origin. A number of plants, their extracts, food products, and medicinal preparations appear to be the objects of scientific research. Effectiveness and informative character of research, undoubtedly, depend on relevance, sensitivity, and efficiency of the methods chosen. The aim of this work was to develop and validate the postcolumn high-performance liquid chromatography (HPLC)-DPPH method as well as its application in the evaluation of antioxidant activity of known and unknown compounds scavenging free radicals and existing in medicinal plant raw materials. HPLC-separated compounds were identified at the wavelength of 275 nm, and then the mobile phase with analytes flowed through a mixing tee to the reaction coil, where DPPH reagent solution was supplied. The solution flow rate was 0.4 mL/min. The reaction coil was connected with UV/VIS type detector, which measured absorption of flowing solution at the wavelength of 520 nm. It was determined that vitexin rhamnoside, the dominant compound in the leaves of Crataegus monogyna, was not a significant radical scavenger. The most active antioxidant in the leaves and flowers of Crataegus monogyna was chlorogenic acid. The most active antioxidant in Origanum vulgare raw material was rosmarinic acid. Identified analytes in the extracts of Achillea millefolium that possessed radical-scavenging properties were chlorogenic acid, luteolin-7-O-glucoside, rutin, and luteolin.

Versican/PG-M Assembles Hyaluronan into Extracellular Matrix and Inhibits CD44-mediated Signaling toward Premature Senescence in Embryonic Fibroblasts

Versican/PG-M is a large chondroitin sulfate proteoglycan of the extracellular matrix which interacts with hyaluronan at the N-terminal G1 domain, composed of A, B, and B' subdomains. Recently, we generated knock-in mice Cspg2(Delta3/Delta3), whose versican, without the A subdomain, has decreased hyaluronan (HA) binding affinity, thereby exhibiting reduced deposition of versican in the extracellular matrix. Here, we show that the Cspg2(Delta3/Delta3) fibroblasts within 20 passages proliferate more slowly and acquire senescence. Whereas the extracellular matrix of the wild type fibroblasts exhibited a network structure of hyaluronan and versican, that of the Cspg2(Delta3/Delta3) fibroblasts exhibited approximately 35 and approximately 85% deposition of versican and HA, without such a structure. The Cspg2(Delta3/Delta3) fibroblasts showed a substantial increase of ERK1/2 phosphorylation and expression of senescence markers p53, p21, and p16. Treatment of wild type fibroblasts with hyaluronidase and exogenous hyaluronan enhanced ERK1/2 phosphorylation, and treatment with an anti-CD44 antibody that blocks HA-CD44 interaction inhibited the phosphorylation. These results demonstrate that versican is essential for matrix assembly involving hyaluronan and that diminished versican deposition increases free hyaluronan fragments that interact with CD44 and increase phosphorylation of ERK1/2, leading to cellular senescence.

Determination of paralytic shellfish poisoning toxins in Norwegian shellfish by liquid chromatography with fluorescence and tandem mass spectrometry detection

A novel extraction and clean-up method has been developed for the determination of paralytic shellfish poisoning (PSP) toxins in shellfish samples. Raw shellfish material was extracted with an acidic acetonitrile/water (80:20, v/v) solution, whilst being homogenised. During the homogenisation the sample extraction solution was cooled with ice water. Subsequently, the extract was frozen at −20 °C for at least 4 h. During freezing, two layers were formed, only the lower predominantly aqueous layer was used for the determination. The final extract solution was cleaned-up using a combination of Oasis HLB and Carbograph activated carbon SPE columns. The developed extraction and clean-up methods combined with gradient elution liquid chromatography (LC)-mass spectrometry/mass spectrometry (MS/MS) has resulted in a method which can determine the analogs GTX 1-5, C1-2, DcGTX 2-3, DcSTX, Neo, STX in a single analysis with an overall detection limit of 313 μg STXdiHCL-eq./kg shellfish meat. The use of the developed extraction method with post-column high performance liquid chromatography (HPLC) with fluorescence detection (FLD) method provided an overall limit of detection of 89 μg STXdiHCL-eq./kg shellfish meat for the same toxins. Both post-column HPLC-FLD and LC–MS/MS was used to investigate the Norwegian PSP toxin profile. It was found that the PSP toxins could be detected in shellfish samples from the Norwegian coastline for 10 months of the year, from March till December. The toxin profile consisted mainly of the carbamate toxins, GTX 1-4, Neo and STX, in terms of both concentrations and contribution to the overall toxicity. In addition, several of the n-sulfo-carbamoyl toxins were either detected in the samples at relatively low concentrations or their presence in the samples were indicated but could not be confirmed by the post-column HPLC-FLD and LC–MS/MS analyses.

Heparan Sulfate Regulates Self-renewal and Pluripotency of Embryonic Stem Cells

Embryonic stem (ES) cell self-renewal and pluripotency are maintained by several signaling cascades and by expression of intrinsic factors, such as Oct3/4 and Nanog. The signaling cascades are activated by extrinsic factors, such as leukemia inhibitory factor, bone morphogenic protein, and Wnt. However, the mechanism that regulates extrinsic signaling in ES cells is unknown. Heparan sulfate (HS) chains are ubiquitously present as the cell surface proteoglycans and are known to play crucial roles in regulating several signaling pathways. Here we investigated whether HS chains on ES cells are involved in regulating signaling pathways that are important for the maintenance of ES cells. RNA interference-mediated knockdown of HS chain elongation inhibited mouse ES cell self-renewal and induced spontaneous differentiation of the cells into extraembryonic endoderm. Furthermore, autocrine/paracrine Wnt/beta-catenin signaling through HS chains was found to be required for the regulation of Nanog expression. We propose that HS chains are important for the extrinsic signaling required for mouse ES cell self-renewal and pluripotency.

Identification of Novel Metabolites of Colchicine in Rat Bile Facilitated by Enhanced Online Radiometric Detection

Three novel conjugation metabolites of colchicine were identified in rat bile facilitated by enhanced on-line liquid chromatography-accurate radioisotope counting. The known 2- and 3-demethylcolchicines (DMCs) underwent O-sulfate conjugation in addition to the previously described O-glucuronidation. 2-DMC was preferably O-glucuronidated, whereas 3-DMC predominantly yielded O-sulfation conjugates, indicating phase II conjugation regiopreferences. Moreover, M1 was identified as a novel glutathione conjugate and a possible biotransformation pathway for its formation was proposed. The known 2-DMC (M6), 3-DMC (M7), 2-DMC glucuronide (M4), and novel 3-DMC sulfate (M3) were confirmed as the major metabolites. Radiometric data were acquired by the XFlow liquid chromatography-accurate radioisotope counting (XFlow LC-ARC) system, a novel technology for dynamic control of both on-column and postcolumn high-performance liquid chromatography flow rates to maximize sensitivity and resolution of radiochromatograms. A comparative evaluation was also performed between the XFlow LC-ARC system and a conventional flow radiometric detection system using bile samples from an in vivo disposition study of colchicine in male Sprague-Dawley rats. Results demonstrated a 20-fold sensitivity improvement of the XFlow LC-ARC system in comparison with radioactivity detection by conventional flow scintillation analyzers. The dynamic flow mode also provided the best chromatographic resolution. Unambiguous metabolite identification was performed by high-resolution mass spectrometry and nuclear magnetic resonance analysis.

Hepatic SR-BI-mediated cholesteryl ester selective uptake occurs with unaltered efficiency in the absence of cellular energy

Scavenger receptor class B type I (SR-BI) plays a critical role in the delivery of HDL cholesterol and cholesteryl esters (CEs) to liver and steroidogenic tissues by a selective process that does not result in significant degradation of HDL protein. Recently, SR-BI-mediated endocytosis and recycling of HDL have been demonstrated. However, it remains unclear whether efficient SR-BI-mediated selective uptake occurs strictly at the plasma membrane or at additional sites along its endocytic itinerary. To examine the requirement for SR-BI endocytosis in HDL selective uptake, we determined the effects of energy depletion on the levels of cell-associated HDL protein and CE in primary mouse hepatocytes. Compared with CHO cells, we observed a much larger energy-dependent effect on CE uptake in primary mouse hepatocytes. Although varying the levels of caveolin-1 and carboxyl ester lipase altered the efficiency of selective uptake, neither was able to account for the energy-dependent component of HDL-CE uptake. Finally, we demonstrate that the hepatocyte-specific, energy-dependent effects on HDL-apolipoprotein A-I and -CE uptake are independent of SR-BI and are not required to achieve efficient SR-BI-mediated selective uptake of CE. Together, these data support the conclusion that neither the intracellular trafficking of HDL nor any energy-dependent cellular process affects the ability of the cell to maximally acquire CE through SR-BI-mediated selective uptake from HDL.

Psicose Contents in Various Food Products and its Origin

To elucidate the history of human exposure of psicose, a determination method of psicose (D/L-psicose) in various food products has been developed : post column high-performance liquid chromatography (HPLC) using gel permeation column in ligand exchange mode coupled with pulsed amperometric detection (PAD). The calibration curve of D-psicose was linear in a concentration range of 5 to 150μg/ml with a correlation coefficient of 0.9999. With ultrasonic extraction and C18 Sep-pack filtration methods, the samples for HPLC analysis could be prepared from various food products within 0.5-1.5h without any interference materials. The psicose recovery (n=5) from coffee and corn-snack was found to be 96.6% with relative standard deviation (R.S.D.) of 1.9% and 96.7% with R.S.D. of 3.0%, respectively. The resultant psicose content varied from 0.5mg/100g (coffee) to 130.6mg/100g (Worcester sauce). In particular, confectionery products and seasoning sauces exhibited higher psicose content than other studied products. In high sugar food products, heat processing had a marked effect on the production of psicose. The psicose formation due to heating was suggested to be a non-enzymatic reaction.

EXT gene family member rib-2 is essential for embryonic development and heparan sulfate biosynthesis in Caenorhabditis elegans

EXT gene family members including EXT1, EXT2, and EXTL2 are glycosyltransferases required for heparan sulfate biosynthesis. To examine the biological functions of rib-2, a member of the Caenorhabditis elegans EXT gene family, we generated a mutant worm lacking the rib-2 gene using the UV-TMP method followed by sib-selection. Inactivation of rib-2 alleles induced developmental abnormalities in F2 and F3 homozygous worms, while F1 heterozygotes showed a normal morphology. The F2 homozygous progeny generated from the F1 heterozygous hermaphrodites somehow developed to adult stage but exhibited abnormal characteristics such as developmental delay and egg-laying defects. The F3 homozygous progeny from the F2 homozygous hermaphrodites showed early developmental defects and most of the F3 worms stopped developing during the gastrulation stage. Whole-mount staining analysis for heparan sulfate using Toluidine blue (pH 2.5) revealed a defect of heparan sulfate biosynthesis in the F2 homozygotes. The analysis using fluorometric post-column high-performance liquid chromatography also uncovered reduced production of heparan sulfate in the rib-2 mutant. These results indicate that rib-2 is essential for embryonic development and heparan sulfate biosynthesis in C. elegans.

A new analytical method for gonyautoxins based on postcolumn HPLC.

A new ion-pairing high-performance liquid chromatography (HPLC) method on a C30 column with a volatile mobile phase was developed to separate the gonyautoxin group (GTXs) from contaminants, allowing the utilization of liquid chromatography/mass spectrometry (LC/MS) with higher performance. A mobile phase consisting of 5 mmol/L heptafluorobutyric acid and 2% acetonitrile in 10 mmol/L ammonium acetate was adopted for separation of GTXs because the C30 column strongly retains GTXs under acidic conditions. The newly adopted method could efficiently separate GTXs from contaminants, especially in the toxic short-necked clam, whereas the routine HPLC so far used has poor resolution to separate GTXs from unknown interfering substances. In our method, GTXs were eluted in the order of GTX5, GTX3, GTX4, GTX2 and GTX1 from the C30 column, and were successfully determined by sonic spray ionization mass spectrometry (SSI-MS) with high sensitivity. This method is characterized by the combination of HPLC using a fluorescence detection system for PSP, and SSI-MS for measurement of the mass number.

Degradation of triphenyltin by a fluorescent pseudomonad.

Triphenyltin (TPT)-degrading bacteria were screened by a simple technique using a post-column high-performance liquid chromatography using 3,3',4',7-tetrahydroxyflavone as a post-column reagent for determination of TPT and its metabolite, diphenyltin (DPT). An isolated strain, strain CNR15, was identified as Pseudomonas chlororaphis on the basis of its morphological and biochemical features. The incubation of strain CNR15 in a medium containing glycerol, succinate, and 130 microM TPT resulted in the rapid degradation of TPT and the accumulation of approximately 40 microM DPT as the only metabolite after 48 h. The culture supernatants of strain CNR15, grown with or without TPT, exhibited a TPT degradation activity, whereas the resting cells were not capable of degrading TPT. TPT was stoichiometrically degraded to DPT by the solid-phase extract of the culture supernatant, and benzene was detected as another degradation product. We found that the TPT degradation was catalyzed by low-molecular-mass substances (approximately 1,000 Da) in the extract, termed the TPT-degrading factor. The other fluorescent pseudomonads, P. chlororaphis ATCC 9446, Pseudomonas fluorescens ATCC 13525, and Pseudomonas aeruginosa ATCC 15692, also showed TPT degradation activity similar to strain CNR15 in the solid-phase extracts of their culture supernatants. These results suggest that the extracellular low-molecular-mass substance that is universally produced by the fluorescent pseudomonad could function as a potent catalyst to cometabolite TPT in the environment.