Graphitized Carbon Cartridges Research Articles

Analysis of UDP-Sugars from Cultured Cells and Small Tissue Samples.

UDP-sugars are important substrates for the synthesis of various cellular glycans and glycoconjugates, many of which play essential roles in the pathobiology of diseases associated with deranged glucose metabolism, such as cancer and type 2 diabetes. Hence, their analysis from cultured cells and especially from tissue samples can give valuable information. This chapter describes a method for UDP-sugar isolation from various sources utilizing ion-pair solid-phase extraction with graphitized carbon cartridges, and their analysis using anion-exchange high-performance liquid chromatography.

Determination of Diniconazole in Agricultural Products, Livestock and Marine Products by LC-MS/MS.

An LC-MS/MS method for the determination of diniconazole in agricultural products, livestock and marine products was developed. Diniconazole in agricultural products was extracted with acetone. The extract was concentrated and partitioned with n-hexane and 10% sodium chloride solution. Agricultural products such as grains and beans were defatted using n-hexane-acetonitrile. Livestock and marine products were extracted with a mixture of acetone and n-hexane, and the organic layer was evaporated to dryness. The residue was defatted using n-hexane-acetonitrile. Cleanup was carried out using a Florisil cartridge column and a graphitized carbon cartridge column for these samples. The LC separation was carried out on an Inertsil ODS-3 column with a linear gradient of 0.1% formic acid and acetonitrile containing 0.1% formic acid. MS was carried out in the positive ion electrospray ionization mode. The calibration curve was linear between 0.00125 to 0.00750 mg/L. Average recoveries (n=5) of diniconazole from 16 kinds of agricultural products, livestock and marine products fortified at the MRLs (0.01 ppm) were 88.3-108%, and the relative standard deviations were 0.5-5.1%. The limits of quantitation were 0.01 mg/kg.

CYN determination in tissues from freshwater fish by LC–MS/MS: Validation and application in tissues from subchronically exposed tilapia (Oreochromis niloticus)

Harmful cyanobacterial blooms are occurring in eutrophic freshwater lakes and reservoirs throughout the world and, because of the production of toxins such as cylindrospermopsin (CYN), they can present a public safety hazard through contamination of seafood and fish for human consumption. Therefore it is important to develop methods to determine CYN at trace levels in those organisms. A new method for unconjugated CYN determination in tissues (liver and muscle) of tilapia (Oreochromis niloticus) is herein described and discussed; it is based on solvent extraction and purification with C18 and graphitized carbon cartridges, and quantification by liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS). The method was optimized and suitably validated, with a linear range from 0.125–12.5µg CYN/g dry weight (dw) in the case of the liver, and 0.02–1µg CYN/g dw for the muscle. Limits of detection and quantitation were 0.07 and 0.12µg/g dw for the liver, and 0.002 and 0.007µg/g dw for the muscle, respectively. Mean recoveries ranged 80–110% in liver, and 94–104% in muscle, and intermediate precision values from 6 to 11%. The method is robust against the three factors considered for purification (batch of the graphitized carbon cartridges, time for the sample to pass through the cartridge, and final dissolving water volume). Furthermore, it has been successfully applied to the extraction and quantification of CYN in tissue samples from tilapia subchronically exposed to CYN in the laboratory. This represents a sensitive, reproducible, accurate, and robust method for extraction and determination of unconjugated CYN in tissues of fish exposed to the toxin. This procedure can be used for confirmatory routine monitoring of CYN in fish samples in environmental studies.

O-glycan profiling of serum glycan for potential renal cancer biomarkers

Serum was obtained from 25 male renal cell carcinoma (RCC) patients and 21 healthy males. O-glycans were released by a β-elimination reaction and purified by graphitized carbon cartridge solid phase extraction, then profiled by matrix-assisted laser desorption/ionisation-time of flight mass spectrometry. After noise removal and peak alignment, 1372 peaks were extracted from 200000 data points. Feature peaks were analyzed by calculation of differential sensitivity and specificity. The combination of two feature peaks was chosen as a biomarker and could clearly differentiate RCC and normal samples in our study group.

Development and optimization of a method for the determination of Cylindrospermopsin from strains of Aphanizomenon cultures: Intra-laboratory assessment of its accuracy by using validation standards

The occurrence of cyanobacterial blooms in aquatic environments is increasing in many regions of the world due to progressive eutrophication of water bodies. Because of the production of toxins such as Cylindrospermopsin (CYN), contamination of water with cyanobacteria is a serious health problem around the world. Therefore it is necessary to develop and validate analytical methods that allow us to quantify CYN in real samples in order to alert the public of this toxin. In this work, an analytical method has been developed an optimized for the determination of CYN from Aphanizomenon ovalisporum cultures. The analytical procedure is based on solvent extraction followed by a purification step with graphitized cartridges and CYN quantification by LC–MS/MS. The extraction and purification steps were optimized using a two-level full factorial design with replications. A suitable and practical procedure for assessing the trueness and precision of the proposed method has been applied by using validation standards. The method has been suitably validated: the regression equation was calculated from standards prepared in extracts from lyophilized M. aeruginosa PCC7820 (r2≥0.9999) and the linear range covered is from 5 to 500μg CYN/L, equivalent to 0.18–18.00μg CYN/g dry weight lyophilized cells. Limits of detection and quantification were 0.04 and 0.15μg CYN/g, respectively, the recovery range (%) oscillated between 83 and 94% and intermediate precision (RSD %) values from 5.6 to 11.0%. Moreover, the present method showed to be robust for the three factors considered: the batch of the graphitized carbon cartridges, the flow rate of the sample through the cartridge, and the final re-dissolved water volume after SPE treatment, which permits its validation. The validated method has been applied to different lyophilized cultures of A. ovalisporum (LEGE X-001) to evaluate CYN content. This procedure can be used for determining CYN in lyophilized natural blooms samples in environmental studies.

Cylindrospermopsin determination in water by LC‐MS/MS: Optimization and validation of the method and application to real samples

A new method for determining dissolved cylindrospermopsin (CYN) in waters using solid-phase extraction (SPE) with graphitized carbon cartridges and quantification by liquid chromatography coupled with tandem mass spectrometry is described and discussed. The method has been suitably validated: the linear range covered is from 0.900 to 125 µg CYN/L. Limits of detection and quantification were 0.5 and 0.9 µg CYN/L, respectively, and allow CYN determination at concentrations below the guideline proposed of 1 µg CYN/L in natural waters. The method exhibits mean recoveries from 83 to 95%, and intermediate precision (relative standard deviation (%)) values from 5 to 12%, ensuring adequacy against the Association of Official Analytical Chemists guidelines. The method is robust against the following three influential factors considered in the cleanup stage: the batch of the graphitized carbon cartridges, the flow rate of the water sample through the cartridge, and the final redissolved water volume after SPE treatment. The method has been successfully applied to detection and quantification of CYN in water samples from aquaria of a toxicological in vivo laboratory experiment.

An Efficient Approach for Large-Scale Production of Sialyglycopeptides from Egg Yolks

Glycopeptides are very useful substrates for the synthesis of various complex glycoproteins and glycopeptides that are indispensable materials for the structural and functional investigation of glycoproteins. This report provides an efficient and time- and cost-saving strategy for the large-scale production of a sialylglycopeptide (SGP) that carries an intact N-linked disialyl-biantennary complex-type sialyloligosaccharide. Consequently, 680 mg of SGP was obtained from 100 egg yolks after phenol treatment, Sephadex G-50 gel filtration, and filtration through graphitized carbon cartridges. The chemical structure and the glycosylation site of the isolated SGP were identified by NMR and ESI-MS/MS, and the purity measured with HPLC was over 95%.

Carbohydrate separation by hydrophilic interaction liquid chromatography on a ‘click’ maltose column

A general and efficient hydrophobic interaction liquid chromatography (HILIC) method has been developed for the separation of various kinds of carbohydrates, ranging from galactooligosaccharides, carrageenan oligosaccharides, sodium alginate, chitooligosaccharides to higher molecular weight fructooligosaccharides. Three types of oligosaccharides (acidic, basic, and neutral) were purified by semi-preparative HILIC. As a result, 18 oligosaccharides in high purity were obtained and their degrees of polymerization ranged from DP 2 to 7. Furthermore, oligosaccharides of plant origin were enriched and fractionated by solid-phase extraction (SPE) on a graphitized carbon cartridge. The oligosaccharide fractions that eluted from the cartridge were purified and then characterized by ESIMS and NMR spectroscopy. This strategy has led to the separation of a homogeneous raffinose family of oligosaccharides. Since the present method is universal, efficient and stable, separation and purification of various types of carbohydrates by HILIC seem to be possible.

Liquid Chromatography Electrospray Ionization Fourier Transform Ion Cyclotron Resonance Mass Spectrometric Characterization of N-Linked Glycans and Glycopeptides

We combine liquid chromatography, electrospray ionization, and Fourier transform ion cyclotron resonance mass spectrometry (LC ESI FT-ICR MS) to determine the sugar composition, linkage pattern, and attachment sites of N-linked glycans. N-linked glycans were enzymatically released from glycoproteins with peptide N-glycosidase F, followed by purification with graphitized carbon cartridge solid-phase extraction and separation over a TSK-Gel Amide80 column under hydrophilic interaction chromatography (HILIC) conditions. Unique glycopeptide compositions were determined from experimentally measured masses for different combinations of glycans and glycopeptides. The method was validated by identifying four peptides glycosylated so as to yield 12 glycopeptides unique in glycan composition for the standard glycoprotein, bovine alpha-2-HS-glycoprotein. We then assigned a total of 137 unique glycopeptide compositions from 18 glycoproteins from fetal bovine serum, and the glycan structures for most of the assigned glycopeptides were heterogeneous. Highly accurate FT-ICR mass measurement is essential for reliable identification.

Mass spectrometry analysis of melanoma related O-glycans in sera

Glycomics comparison was carried out by screening melanoma serum biomarkers between C57 mice with and without B16 implanted. O-glycans were released from 10 μL sera by β-elimination, purified by Graphitized Carbon Cartridge Solid Phase Extraction (GCC-SPE) and analyzed by MALDI- QIT-TOF-MS. MS raw data were acquired and exported by Launchpad software. MATLAB was then applied for further data analysis. 10 Glycans were considered to have stable changes after B16 implantation and 5 of them were under structural analysis via MS/MS.

Advances in solid phase extraction of the cyanobacterial toxin cylindrospermopsin

Unlike other cyanotoxins, cylindrospermopsin (CYN) is often found in high concentrations in the extracellular fraction, possibly due to a higher release from the producing cells and a limited degradation once liberated. This fact demands effective recovery methods from environmental samples to guarantee a proper quantification. A reliable and simple solid phase extraction (SPE) method was obtained by the sole use of graphitized carbon cartridges and by paying special attention to both the selection of the most suitable solvent and the need of sample preparation prior to SPE. An acidified combination of dichloromethane and methanol not only showed best recoveries, but also allowed drastic reduction to the elution volumes needed with complete recovery being achieved in only 8 mL. Acidification of the sample and addition of sodium chloride are suggested as valuable improvements to the SPE method and turn out to be essential for correct and robust recovery of CYN in environmental samples of great diversity in terms of pH, DOC, and conductivity. Excellent behavior of the proposed method over a wide range of CYN loadings, applied sample volumes, and DOC loadings was also confirmed.

Analytical Method of Dodine in Agricultural Products by LC/MS

A method for the determination of dodine in agricultural products was developed by using liquid chromatography-electrospray ionization mass spectrometry (LC/ESI-MS). Dodine was extracted with acetonitrile and then acetonitrile-water (7 : 3) from a sample, and re-extracted with ethyl acetate. The extract was cleaned up on a PSA cartridge column (500 mg), and dodine was analyzed by LC/MS. In the case of oil seeds and nuts, hexane/acetonitrile-hydrochloric acid partition was performed to remove lipids before re-extraction with ethyl acetate. In the case of samples that contained a lot of chlorophyll, the eluate of the PSA cartridge column was further cleaned up on a graphitized carbon cartridge column (500 mg). The calibration curve was linear from 0.0001-0.02 microg/mL of dodine. The recoveries of dodine from sixteen kinds of agricultural products fortified at 0.1 mg/kg were 80.3-100.0%, and their relative standard deviations were 0.3-6.4%. The limits of detection (S/N=3) were 0.0006 mg/kg.

Glycomic Approach for Potential Biomarkers on Prostate Cancer: Profiling of N‐Linked Glycans in Human Sera and pRNS Cell Lines

Prostate cancer is a leading cause of cancer death among men. Currently available screening test measures prostate-specific antigen (PSA) to detect prostate cancer. However, this test produces false positive values that often lead to negative biopsies. Therefore, a more reliable diagnostic tool is needed. Glycans in serum are of particular interest as around half of all proteins are glycosylated. In this study, N-linked glycans were enzymatically released by PNGase F from prostate epithelial cell lines (pRNS) expressing wild type or mutant androgen receptors and a small set of human serum samples. Released glycans were purified and partitioned into neutral and acidic components by solid phase extraction (SPE) using graphitized carbon cartridges. The SPE fractions were analyzed by matrix-assisted laser desorption/ionization Fourier transform ion cyclotron resonance mass spectrometry (MALDI FT-ICR MS). Significant changes in some high-mannose and fucosylated biantennary complex N-linked glycans were observed in the serum of prostate cancer patients.

Occurrence of polychlorinated naphthalenes, polychlorinated biphenyls and short-chain chlorinated paraffins in marine sediments from Barcelona (Spain)

Polychlorinated naphthalenes (PCNs), short-chain chlorinated paraffins (SCCPs) and polychlorinated biphenyls (PCBs) were analysed in marine sediment samples collected from the coastal area of Barcelona (Spain) and near of a submarine emissary coming from a waste water treatment plant located at the mouth of the Besòs River (Barcelona). An integrated sample treatment based on Soxhlet extraction followed by a simple clean-up with Florisil and graphitized carbon cartridge was employed. Gas chromatography coupled to ion-trap tandem mass spectrometry (GC–MS/MS) and gas chromatography–mass spectrometry in electron capture negative ionization mode, were used for PCN and SCCP determinations, respectively, while for PCB analysis gas chromatography with electron capture detection (GC-ECD) was used. The method developed provided low limits of detection (0.001–0.003 ng g −1 dry weight (dw) for PCNs, 1.8 ng g −1 for SCCPs and 0.006–0.014 ng g −1 dw for PCBs) and good run-to-run precisions (lower than RSD 8%) for the analysis of sediment samples. Concentration levels ranging from 0.17 to 3.27 ng g −1 dw for PCNs, between 0.21 and 1.17 μg g −1 dw for SCCPs, and from 2.33 to 44.00 ng g −1 (dw) for PCBs, were found in the coastal sediments, while for samples collected near to the submarine emissary higher levels (from 2.02 to 6.56 ng g −1 dw for PCNs, between 1.25 and 2.09 μg g −1 dw for SCCPs and from 22.34 to 37.74 ng g −1 dw for PCBs) were obtained. The results obtained provide new data about the occurrence of PCN and SCCP in the coastal area of Barcelona.

Sample clean-up method for analysis of complex-type N-glycans released from glycopeptides

N-Glycans in glycoprotein can be liberated either from glycoproteins or from their glycopeptides with glycoamidases. The latter approach is preferable, because it requires a smaller amount of the enzyme, and yields N-glycans in excellent yields. Moreover it alleviates the necessity of removing from the reaction mixture the detergents needed to denature the glycoproteins. On the other hand, this approach necessitates removal of interfering peptidic materials, because some of the peptide peaks often overlap with the peaks of carbohydrate chains in high-performance anion-exchange chromatography (HPAEC). These peptidic materials also hinder labeling of N-glycans by reductive amination. We have tried to remove the interfering peptidic materials by several different methods—octadecyl (C 18) silica cartridge, cation-exchange resin column, and graphitized carbon cartridge. Unfortunately, none of these could completely remove the interfering peptidic materials. Therefore, we resorted to modify the amino groups of the peptidic materials with sodium 2,4,6-trinitro-benzene-1-sulfonate (TNBS) to render them more hydrophobic, so that they can be retained more strongly on the C 18 or graphitized carbon cartridges. In the model study presented here, we were able to obtain N-glycans for HPAEC analyses without any interfering materials by a combination of TNBS reaction and graphitized carbon treatment.

Extraction and purification of the zwitterions cylindrospermopsin and deoxycylindrospermopsin from Cylindrospermopsis raciborskii.

The hepatotoxin cylindrospermopsin (CYN) has been isolated from the cyanobacterium Cylindrospermopsis raciborskii (C. raci.). Efforts to study this toxin have been hampered by the time-consuming requirement to extract it from cultures of the organism. It is usually extracted from lyophilized cells collected from a laboratory culture. Our preliminary work suggested far more of the toxin is available in solution in the culture media than in the cells collected. We have therefore investigated the use of commercially available solid phase extraction sorbents to extract CYN from culture media in which C. raci. has been grown. A range of reverse phase and ion-exchange sorbents were tested across a range of pHs for their ability to retain CYN without success. Subsequently, graphitized carbon cartridges were found to retain CYN strongly. Elution with 5% formic acid in methanol allowed the CYN to be regained for final purification by HPLC. Deoxy-CYN, an analog of CYN can also be extracted using this procedure.

Determination of daminozide in apples and apple leaves by liquid chromatography–mass spectrometry

A straightforward and efficient method was developed for the determination of intact daminozide in apples and apple leaves. After extraction with methanol and a clean-up step using a graphitized carbon cartridge, the extract was analysed by ion-trap liquid chromatography–tandem mass spectrometry (LC–MS–MS) using atmospheric pressure chemical ionisation in the positive ion mode. Recoveries for apple were 98–102% with a R.S.D.≤11% ( n=6) and for leaves were 112–116% with a R.S.D.≤18% ( n=6). The limits of detection were 0.008 and 0.02 mg/kg for apples and leaves, respectively.

Quantification of two chromatographic unresolved dichlorophenols using gas chromatography-direct deposition-fourier transform infrared spectrometry and multivariate calibration

A method for the quantification of two chromatographically unresolved dichlorophenol isomers in water is described. Acetylation and concentration on graphitized carbon cartridges are carried out as a preliminary step. Detection is made by gas chromatography (GC) coupled to Fourier transform infrared spectroscopy (FTIR), using a direct deposition interface (DD). Infrared spectra in the maximum of the unresolved peak of 2,5- and 2,4-dichlorophenol for a series of standards with different amounts of these two compounds are used, to elaborate a multivariate calibration model (PLS-1 algorithm). By the method described, concentrations of dichlorophenol isomers in water at ng/ml level can be determined.

Determination of chlorophenols in drinking water with high resolution gas chromatography-tandem mass spectrometry

A new method for the determination of sub-ppb levels of chlorophenols in drinking water by use of GC-MS-MS is proposed. Monitoring of these analytes for assurance of compliance with legally allowed limits can readily be accomplished by extraction as acetylated chlorophenols from low sample volumes (10 ml). Much lower detection limits can be achieved by preconcentrating 1 1 of sample using graphitized carbon cartridges for solid extraction. Appropriate selection of parent ions and fragmentation conditions ensures not only a high sensitivity, but also clean product ion spectra that allow positive identification of every species considered (polychlorophenol isomers included).

Analysis of structural isomers of polychlorinated phenols in water by liquid-nitrogen-trapping gas chromatography-Fourier transform infrared spectroscopy

A procedure is described for the identification and determination of structural isomers of polychlorinated phenols in drinking water. First, their acetylation and concentration on graphitized carbon cartridges are carried out. Detection is accomplished by gas chromatography (GC) coupled with Fourier transform infrared spectroscopy (FTIR) using a direct deposition (DD) interface. In this way it is possible to accurately identify and differentiate variably substituted isomers of chlorophenols, which is very difficult or even impossible to do by means of the widespread gas chromatography-mass spectrometry (GC-MS) instrumentation. Chlorophenols occurring in water samples have been identified according to their IR spectra and additionally the identification was confirmed by means of the MS-MS spectra obtained with a GC-MS-MS tandem instrument.