SAX Column Research Articles

Cloning and Characterization of Two Human VIP1-like Inositol Hexakisphosphate and Diphosphoinositol Pentakisphosphate Kinases

Eukaryotes possess numerous inositol phosphate (IP) and diphosphoinositol phosphate (PP-IPs or inositol pyrophosphates) species that act as chemical codes important for intracellular signaling pathways. Production of IP and PP-IP molecules occurs through several classes of evolutionarily conserved inositol phosphate kinases. Here we report the characterization of a human inositol hexakisphosphate (IP6) and diphosphoinositol pentakisphosphate (PP-IP5 or IP7) kinase with similarity to the yeast enzyme Vip1, a recently identified IP6/IP7 kinase (Mulugu, S., Bai, W., Fridy, P. C., Bastidas, R. J., Otto, J. C., Dollins, D. E., Haystead, T. A., Ribeiro, A. A., and York, J. D. (2007) Science 316, 106-109). Recombinant human VIP1 exhibits in vitro IP6 and IP7 kinase activities and restores IP7 synthesis when expressed in mutant yeast. Expression of human VIP1 in HEK293T cells engineered to produce high levels of IP7 results in dramatic increases in bisdiphosphoinositol tetrakisphosphate (PP2-IP4 or IP8). Northern blot analysis indicates that human VIP1 is expressed in a variety of tissues and is enriched in skeletal muscle, heart, and brain. The subcellular distribution of tagged human VIP1 is indicative of a cytoplasmic non-membrane localization pattern. We also characterized human and mouse VIP2, an additional gene product with nearly 90% similarity to VIP1 in the kinase domain, and observed both IP6 and IP7 kinase activities. Our data demonstrate that human VIP1 and VIP2 function as IP6 and IP7 kinases that act along with the IP6K/Kcs1-class of kinases to convert IP6 to IP8 in mammalian cells, a process that has been found to occur in response to various stimuli and signaling events.

Renaturation and Purification of rhGM-CSF with Ion-Exchange Chromatography

The renaturation and purification of recombinant human granulocyte macrophage colony stimulation factor (rhGM-CSF) expressed in Escherichia coli with strong anion-exchange chromatography (SAX) were studied. The effects of pH values, ratios of concentrations of GSH/GSSG, and urea concentrations in the mobile phase on the renaturation and purification of rhGM-CSF with SAX were investigated, respectively. The results show that the above three factors have remarkable influences on the efficiency of renaturation and mass recovery of rhGM-CSF. The addition of GSH/GSSG in the mobile phase can improve the formation of correct disulfide bonds in rhGM-CSF so that its renaturation yield increases. In addition, to enhance the mass recovery of rhGM-CSF with SAX, the low concentration of urea was added in the mobile phase to prevent denatured protein aggregation. Under the optimal conditions, rhGM-CSF was renatured with simultaneous purification on SAX column within 30 min only by one step. After that its specific bioactivity, mass recovery, and purity reached 1.66 x 10(7) IU x mg, 58.8%, and 96.2%, respectively.

Could linear solvation energy relationships give insights into chiral recognition mechanisms?: 1. π–π and charge interaction in the reversed versus the normal phase mode

Linear solvation energy relationships (LSER) have been used for years in liquid chromatography to access the factors that lead to retention and, more recently, to selectivity. In chiral separations, two enantiomers will receive exactly the same descriptors correctly predicting that they will not be separated by any non-chiral stationary phase. However, LSER studies could be used considering that each enantiomer sees a chiral stationary phase (CSP) differently. Working with the enantioselectivity factor, k′2/k′1 could give interesting information on the chiral recognition mechanisms. The b and v system parameters always predominantly contribute to a solute's retention in reversed phase liquid chromatography (RPLC) compared to the a, e and s parameters. However, these minor parameters for retention may become the essential ones for enantiomeric separations where a minimum of three simultaneous interactions is needed for an enantioseparation to occur. In this non-chiral study, six different stationary phases, a classical C18, a diphenyl bonded stationary phase (DP), a polystyrene-divinylbenzene (DVB), a polar embedded new stationary phase, an anion exchanger (SAX) and a teicoplanin aglycone (TAG) CSP were studied with achiral test solutes. Significantly higher e terms were obtained for the SAX and TAG columns. It seems that the ability of stationary surface charges to induce dipoles in polarizable molecules is encoded mainly in the e term. Since the DP and DVB columns did not produce e parameters significantly higher than the C18 column, it seems that π–π interactions are (a) extremely weak in RPLC or (b) they are not simply encoded in this single e system parameter but spread in at least three parameters. The TAG CSP produced logically very different parameters when used in the reversed phase mode compared to the normal phase mode showing the critical role of the mobile phase.

Development and application of analytical methods for the determination of mycotoxins in organic and conventional wheat

The aim of this study was to develop a multicomponent analytical method for the determination of deoxynivalenol (DON), ochratoxin A (OTA) and zearalenone (ZEN), nivalenol (NIV), 3-acetyl-DON (3-acDON), 15-acetyl-DON (15-acDON), zearalenol (ZOL) and citrinin (CIT) in wheat. It also aimed to survey the presence and amounts of DON, OTA and ZEN in Belgian conventionally and organically produced wheat grain and in wholemeal wheat flours. After solvent extraction, an anion-exchange column (SAX) was used to fix the acidic mycotoxins (OTA, CIT), whilst the neutral mycotoxins flowing through the SAX column were further purified by filtration on a MycoSep cartridge. OTA and CIT were then analysed by high-performance liquid chromatography (HPLC) using an isocratic flow and fluorescence detection, while the neutral mycotoxins were separated by a linear gradient and detected by double-mode (ultraviolet light fluorescence) detection. The average DON, ZEN and OTA recovery rates from spiked blank wheat flour were 92, 83 and 73% (RSDR = 12, 10 and 9%), respectively. Moreover, this method offered the respective detection limits of 50, 1.5 and 0.05 µg kg−1 and good agreement with reference methods and inter-laboratory comparison exercises. Organic and conventional wheat samples harvested in 2002 and 2003 in Belgium were analysed for DON, OTA and ZEN, while wholemeal wheat flour samples were taken from Belgian retail shops and analysed for OTA and DON. Conventional wheat tended to be more frequently contaminated with DON and ZEN than organic samples, the difference being more significant for ZEN in samples harvested in 2002. The mean OTA, DON and ZEA concentrations were 0.067, 675 and 75 µg kg−1 in conventional samples against 0.063, 285 and 19 µg kg−1 in organically produced wheat in 2002, respectively. Wheat samples collected in 2003 were less affected by DON and ZEN than the 2002 harvest. Organic wholemeal wheat flours were more frequently contaminated by OTA than conventional samples (p < 0.10). The opposite pattern was shown for DON, organic samples being more frequently contaminated than conventional flours (p < 0.10).

Enzymatically Active N-Deacetylase/N-Sulfotransferase-2 Is Present in Liver but Does Not Contribute to Heparan Sulfate N-Sulfation

Heparan sulfate (HS) proteoglycans influence embryonic development through interactions with growth factors and morphogens. The interactions depend on HS structure, which is largely determined during biosynthesis by Golgi enzymes. NDST (glucosaminyl N-deacetylase/N-sulfotransferase), responsible for HS N-sulfation, is a key enzyme directing further modifications including O-sulfation. To elucidate the roles of the different NDST isoforms in HS biosynthesis, we took advantage of mice with targeted mutations in NDST1 and NDST2 and used liver as our model organ. Of the four NDST isoforms, only NDST1 and NDST2 transcripts were shown to be expressed in control liver. The absence of NDST1 or NDST2 in the knock-out mice did not affect transcript levels of other NDST isoforms or other HS modification enzymes. Although the sulfation level of HS synthesized in NDST1-/- mice was drastically lowered, liver HS from wild-type mice, from NDST1+/-, NDST2-/-, and NDST1+/- / NDST2-/- mice all had the same structure despite greatly reduced NDST enzyme activity (30% of control levels in NDST1+/- / NDST2-/- embryonic day 18.5 embryos). Enzymatically active NDST2 was shown to be present in similar amounts in wild-type, NDST1-/-, and NDST1+/- embryonic day 18.5 liver. Despite the substantial contribution of NDST2 to total NDST enzyme activity in embryonic day 18.5 liver (approximately 40%), its presence did not appear to affect HS structure as long as NDST1 was also present. In NDST1-/- embryonic day 18.5 liver, in contrast, NDST2 was responsible for N-sulfation of the low sulfated HS. A tentative model to explain these results is presented.

ADP-ribosyltransferase-specific Modification of Human Neutrophil Peptide-1

Epithelial cells lining human airways and cells recruited to airways participate in the innate immune response in part by releasing human neutrophil peptides (HNP). Arginine-specific ADP-ribosyltransferases (ART) on the surface of these cells can catalyze the transfer of ADP-ribose from NAD to proteins. We reported that ART1, a mammalian ADP-ribosyltransferase, present in epithelial cells lining the human airway, modified HNP-1, altering its function. ADP-ribosylated HNP-1 was identified in bronchoalveolar lavage fluid (BALF) from patients with asthma, idiopathic pulmonary fibrosis, or a history of smoking (and having two common polymorphic forms of ART1 that differ in activity), but not in normal volunteers or patients with lymphangioleiomyomatosis. Modified HNP-1 was not found in the sputum of cystic fibrosis patients or in leukocyte granules of normal volunteers. The finding of ADP-ribosyl-HNP-1 in BALF but not in leukocyte granules suggests that the modification occurred in the airway. Most of the HNP-1 in the BALF from individuals with a history of smoking was, in fact, mono- or di-ADP-ribosylated. ART1 synthesized in Escherichia coli, glycosylphosphatidylinositol-anchored ART1 released with phosphatidylinositol-specific phospholipase C from transfected NMU cells, or ART1 expressed endogenously on C2C12 myotubes modified arginine 14 on HNP-1 with a secondary site on arginine 24. ADP-ribosylation of HNP-1 by ART1 was substantially greater than that by ART3, ART4, ART5, Pseudomonas aeruginosa exoenzyme S, or cholera toxin A subunit. Mouse ART2, which is an NAD:arginine ADP-ribosyltransferase, was able to modify HNP-1, but to a lesser extent than ART1. Although HNP-1 was not modified to a significant degree by ART5, it inhibited ART5 as well as ART1 activities. Human beta-defensin-1 (HBD1) was a poor transferase substrate. Reduction of the cysteine-rich defensins enhanced their ability to serve as ADP-ribose acceptors. We conclude that ADP-ribosylation of HNP-1 appears to be primarily an activity of ART1 and occurs in inflammatory conditions and disease.

Aprataxin Forms a Discrete Branch in the HIT (Histidine Triad) Superfamily of Proteins with Both DNA/RNA Binding and Nucleotide Hydrolase Activities

Ataxia with oculomotor apraxia type 1 (AOA1) is an early onset autosomal recessive spinocerebellar ataxia with a defect in the protein Aprataxin, implicated in the response of cells to DNA damage. We describe here the expression of a recombinant form of Aprataxin and show that it has dual DNA binding and nucleotide hydrolase activities. This protein binds to double-stranded DNA with high affinity but is also capable of binding double-stranded RNA and single-strand DNA, with increased affinity for hairpin structures. No increased binding was observed with a variety of DNA structures mimicking intermediates in DNA repair. The DNA binding observed here was not dependent on zinc, and the addition of exogenous zinc abolished DNA binding. We also demonstrate that Aprataxin hydrolyzes with similar efficiency the model histidine triad nucleotide-binding protein substrate, AMPNH2, and the Fragile histidine triad protein substrate, Ap4A. These activities were significantly reduced in the presence of duplex DNA and to a lesser extent in the presence of single-strand DNA, and removal of the N-terminal Forkhead associated domain did not alter activity. Finally, comparison of sequence relationships between the histidine triad superfamily members shows that Aprataxin forms a distinct branch in this superfamily. In addition to its capacity for nucleotide binding and hydrolysis, the observation that it also binds DNA and RNA adds a new dimension to this superfamily of proteins and provides further support for a role for Aprataxin in the cellular response to DNA damage.

Determination of uric acid in urine, saliva and calcium oxalate renal calculi by high-performance liquid chromatography/mass spectrometry

A very simple and direct method for determination of uric acid, in various biological matrices, based on high-performance liquid chromatography and mass spectrometry is described. Chromatographic separations were performed with a stationary phase Zorbax Sax Column, an anion exchange resin, with 50% sodium citrate 1 mM at pH 6.5 and 50% acetonitrile as mobile phase delivered at a flow rate of 1 ml/min. The detector counted negative ions by monitoring m/z 167.1, which corresponds to the urate anion. The method does not use an internal standard but quality control samples were used. Intra-day precision ranged between 1.1 and 1.5%, whereas inter-day precision was between 1.3 and 2.8% (n=5) working with some selected standards. Recovery tests of added standard have been successfully performed in urine and saliva samples, thus showing an appropriate accuracy of the method. The limit of quantitation found was 70 microg/l. Different urine and saliva samples were analyzed using an alternative analytical methodology based on an enzymatic reaction and photometric detection at 520 nm, resulting both methods comparable at a 95% confidence level. The method has been also applied to the determination of trace amounts of uric acid in the core of some selected calcium oxalate renal calculi.

Efficiency of crude corn extract clean-up on different columns in fumonisins determination

The efficiencies of different clean-up procedures for crude corn extract from corn samples naturally contaminated by fumonisins B1 and B2 were compared. These procedures precede liquid chromatography determination with fluorescence detection. The efficiencies of immunoaffinity columns (IMA) strong anion exchange columns (SAX), as well as columns with reversed-phase C18 (RP C18) were investigated. No significant differences in the obtained results were found, regardless of the crude extract clea-nup procedure. However, the use of IMA columns for clean-up provided better chromatographic resolution, with the clean-up procedure being the simplest and the fastest. Also, because of the possibility of IMA column regeneration, it is possible to prepare ten samples on one column, so all in all, the lower price of SAX and RP C18 columns is of no great significance.

Quantitative determination of salicylic acid and metabolites in animal tissues by liquid chromatography–tandem mass spectrometry

In order to study the excretion pattern of salicylic acid and its metabolites in animals, a liquid chromatographic–tandem mass spectrometric (LC–MS/MS) method was developed. The method was used to study the biotransformation of salicylic acid in poultry (chicken and pigeon) after therapeutic use, i.e. in matrices such as plasma, excreta (=combined urine and faeces), kidney and liver. Thereafter, the method was also adopted for the analysis of residues of salicylic acid in porcine tissues, such as muscle, kidney, liver and skin + fat. For the biotransformation study, salicylic acid, the oxidation and amino acid conjugation metabolites such as gentisic acid, salicyluric acid, and a double conjugated ornithine metabolite, together with the internal standard phenoxyacetic acid were extracted from 1.0 g of tissue or 100 μl of plasma into ethyl acetate, after acidifying using 1 M HCl. After centrifugation and for kidney and liver tissue, the analytes were back-extracted and subjected to a solid-phase clean-up on SAX columns. For the analysis of excreta, the sample preparation included only a dilution step, performed before LC–MS/MS analysis. For the residue study, salicylic acid and the internal standard phenoxyacetic acid were extracted from 1.0 g of porcine muscle, liver, kidney and skin + fat with 6 ml of diethyl ether, after acidifying using 3 ml of 1 M HCl. After extraction and centrifugation, the ether phase was evaporated under N 2 at 40 °C. The residue was redissolved with 500 μl of 0.1% acetic acid in water. For skin + fat and when needed for the other tissues, a supplemental extraction of the redissolved residue with 500 μl of hexane was performed. The analysis of the extracts was done on a Nucleosil 100-5 C18 column, using a gradient elution with 0.1% acetic acid in water and methanol. A Quattro Ultima ® triple quadrupole instrument was used, equipped with an ESI z-spray source, which was operated in the negative ion MS/MS mode. The methods were validated for the linearity (100–1000 ng ml −1 and 10–50 μg ml −1 for plasma; 5–250 μg g −1 for excreta; 100–1000 ng g −1 and 5–25 μg g −1 for the biotransformation study in poultry kidney and liver; 25–1000 ng g −1 for the residue study in porcine tissues); trueness and precision; specificity; limit of detection and limit of quantification. The limit of quantification for the residue analysis of salicylic acid in porcine tissues was set at 50 ng g −1.

Determination of N-acetyl-S-(N-methylcarbamoyl)cysteine (AMCC) in urine by high-performance liquid chromatography combined with mass spectrometry.

A simple, sensitive and specific method for the quantification of N-acetyl-S-(N-methylcarbamoyl)cysteine (AMCC) in urines of the general population and of exposed workers has been developed. Samples were first diluted with phosphate buffer followed by purification by solid-phase extraction (SPE) with SAX columns prior to analysis by liquid chromatography/mass spectrometry (LC/MS). An external standard was used for the quantification together with selected ion monitoring of m/z 219, [M-H]-. The linear calibration curve showed a good correlation (r2 = 0.98, p < 0.001) between 0.1-110 mg/L of AMCC, and the detection limit was calculated to be 2 microg/L. The within-day and between-day precision, calculated for exposed workers and general population AMCC levels in urine samples, were determined as 1.2 and 3.6%, respectively (as relative standard deviation (RSD)).

Targeted Disruption of a Murine Glucuronyl C5-epimerase Gene Results in Heparan Sulfate Lacking l-Iduronic Acid and in Neonatal Lethality

The glycosaminoglycan, heparan sulfate (HS), binds proteins to modulate signaling events in embryogenesis. All identified protein-binding HS epitopes contain l-iduronic acid (IdoA). We report that targeted disruption of the murine d-glucuronyl C5-epimerase gene results in a structurally altered HS lacking IdoA. The corresponding phenotype is lethal, with renal agenesis, lung defects, and skeletal malformations. Unexpectedly, major organ systems, including the brain, liver, gastrointestinal tract, skin, and heart, appeared normal. We find that IdoA units are essential for normal kidney, lung, and skeletal development, albeit with different requirement for 2-O-sulfation. By contrast, major early developmental events known to critically depend on heparan sulfate apparently proceed normally even in the absence of IdoA.

Sugars and amino acids as factors affecting the synthesis of fumonisins in liquid cultures by isolates of the Gibberella fujikuroi complex

The capacity of four isolates belonging to the Gibberella fujikuroi complex to produce fumonisin B 1 and fumonisin B 2 when grown in liquid medium supplemented with one sugar and one amino acid at various concentration levels has been investigated. The sugars used for supplementing the medium were glucose, fructose, rhamnose, sucrose, maltose, and trehalose at 5, 10 or 20 g/l. The amino acids used were serine, threonine, glutamic acid, aspartic acid, valine, isoleucine, methionine, glycine, alanine, and cystine at 1 or 10 g/l. Fumonisins were extracted from culture filtrates, purified by SAX column and determined by reversed-phase C18 HPLC with fluorescence detection of the o-phthaldialdehyde derivatives. Two isolates produced very low concentrations of fumonisins with all sugars. The remaining isolates provided increased contents of fumonisins when sugar level increased. Concerning the amino acids, production of fumonisins was also dependent on the isolate, although at 1 g/l, the production of fumonisins was greater than at 10 g/l. The results indicate that the sugar–amino acid-isolate combination is basic in fumonisin biosynthesis and that the particular behaviour of each isolate in the different nutritional conditions may constitute a piece of interesting information in the fields of the Taxonomy, Physiology and Toxicology of these fungi. This is the first report on the influence of the carbon and nitrogen sources on fumonisin production by isolates of the G. fujikuroi complex.

3′-Azidothymidine Potently Inhibits the Biosynthesis of Highly Branched N-Linked Oligosaccharides and Poly-N-acetyllactosamine Chains in Cells

3′-Azidothymidine Potently Inhibits the Biosynthesis of Highly Branched N-Linked Oligosaccharides and Poly-N-acetyllactosamine Chains in Cells

HPLC DETERMINATION OF SULFADIMETHOXINE AND ITS HYDROXY METABOLITES FOLLOWING SPE OF EDIBLE CHICKEN TISSUES

New cleanup techniques by an anion-exchange SPE column and a centrifugal ultra-filtration unit and a high performance liquid chromatographic (HPLC) determination of residual sulfadimethoxine (SDM) and its hydroxy metabolites (OH-SDMs), 4-sulfanilamido-6-methyl-2-hydroxy-4-pyrimidine (2-OH-SDM), 4-sulfanilamido-2-methyl-6-hydroxy-4-pyrimidine (6-OH-SDM), and 4-sulfanilamido-2,6-dihydroxy-4-pyrimidine (2,6-diOH-SDM), in edible chicken tissues (liver and muscle) were presented. The sample preparation was performed by homogenizing with a mixture of 90% (v/v) acetonitrile solution and n-hexane (5:4, v/v) to minimize the fat amount followed by ISOLUTE® SAX column and Ultrafree™ C3 as a centrifugal ultra-filtration unit. A Mightysil® RP-4 GP column and an isocratic mobile phase of 4% (v/v) acetic acid solution – acetonitrile – N,N-dimethylformamide (83:12:5, v/v/v) with a photo-diode array detector was used. Average recoveries of spiked SDM and OH-SDMs (0.1 and 0.5 ppm), respectively, were > 80.1% with coefficients of variation between 0.5 and 4.8%. The practical limit of detection was 0.05 ppm for all compounds.

Effect of extraction and extract purification on the measurable fumonisin content of maize and maize products. Tests on the efficiency of acid extraction and use of immunoaffinity columns

The extraction of naturally contaminated maize-based products using methanol / water (3+1) or methanol / 0.1 M hydrochloric acid (3+1) produced varying results. Compared with methanol / water extraction tested in IUPAC/AOAC intercomparison studies, as a rule acid extraction achieves a higher fumonisin content. Strong anion exchange columns are very effective for the analysis of untreated maize, such as maize grits or maize meal. This method guarantees satisfactory extract purification. In products with a high fat content (such as maize based snack products) or cornflakes, SAX columns were not particularly effective. For there immunoaffinity columns provide a highly selective extract purification and achieve a very pure chromatogram. Immunoaffinity columns were also used on cornflakes as they achieved better results than the SAX columns.

Quantification of carbamylated dehydroascorbate derivative produced from cyanate and dehydroascorbate

We established a high-performance liquid chromatographic method for separating and quantifying carbamylated dehydroascorbate derivative (CDA), a reaction product of cyanate with dehydroascorbate. The separation of CDA from interfering substances was achieved by anion-exchange HPLC using a TSK gel SAX (250×4.6 mm I.D.) column and 0.12 M NaCl eluent. The detection of CDA was achieved through two steps: (1) degradation of CDA to cyanate and amino compounds in alkaline solution, and (2) detection of these products by an indophenol reaction. For the processing of plasma and urine samples, anion-exchange solid-phase extraction was used. The detection limit for quantitative determination was 0.1 μ M CDA ( S/ N=3). The linear range found applying the optimized conditions was 0.2 to 200 μ M. The intra- and inter-day assay precision (R.S.D.) of CDA (10 μ M) were 4.8 and 7.2% for rat plasma, and 4.0 and 4.9% for rat urine, respectively. The usefulness of the present method was proved by the application to plasma and urine samples. The study of the biokinetics of CDA in rats revealed that the elimination of CDA is due to urinary excretion.

Liquid Chromatographic Determination of Glyphosate in Water-Soluble Granular Formulations: Collaborative Study

Abstract A liquid chromatographic (LC) method for the determination of glyphosate in various water-soluble granular formulations was tested by 28 laboratories. Samples were dissolved in mobile phase and injected directly into an LC system with a 25 cm SAX column and a mobile phase of 96% aqueous buffer solution, 0.0062M KH2PO4, and 4% methanol. Detection was by UV absorption at 195 nm. Manual or automated injections were made via fixed-volume loops. Calculations were based on peak area comparisons with external standards. The collaborative study analyzed 5 matched pairs of 4 water-soluble granular formulations, with one determination per sample. The study generated 138 matched pairs, which were analyzed by using the AOAC spreadsheet and Youden’s matched-pair calculations. Coefficients of variation for the 4 formulations ranged from 0.65 to 1.37%. The LC method for the determination of glyphosate in water-soluble granular formulations has been adopted first action by AOAC INTERNATIONAL.

Post-column enzyme reactors for chemiluminometric detection of glucose, 1,5-anhydroglucitol and 3-hydroxybutyrate in an anion-exchange chromatographic system.

A liquid chromatographic system consisting of a co-immobilized 3-hydroxybutyrate dehydrogenase-NADH oxidase reactor and an immobilized pyranose oxidase reactor in series and a chemiluminometer was developed for the simultaneous determination of glucose, 1,5-anhydroglucitol and 3-hydroxybutyrate in plasma. The enzymes were immobilized on toresylated poly(vinyl alcohol) beads. Separation was achieved on a TSK gel SAX column (40 x 4 mm I.D.) with an eluent of 50 mM NaOH containing 30 mM sodium butyrate. The hydrogen peroxide produced was detected by measuring the chemiluminescence emitted on admixing with luminol and potassium hexacyanoferrate(III). The calibration curves were linear from 0.8 to 500 microM (7 ng-4 micrograms) for glucose, from 0.8 to 400 microM (7 ng-3 micrograms) for 1,5-anhydroglucitol and from 1 to 700 microM (5 ng-4 micrograms in a 50-microliter injection) for 3-hydroxybutyrate. The sample throughput was four per hour. The reactors were stable for at least ten days.

Simultaneous determination of rufloxacin, fenbufen and felbinac in human plasma using high-performance liquid chromatography

A simple, specific and sensitive high-performance liquid chromatographic method has been developed for the simultaneous determination of rufloxacin, fenbufen and felbinac in human plasma. Plasma, spiked with internal standard, was vortex-mixed for 1 min with a mixture of dichloromethane-diethyl ether (80:20, v/v). The evaporated extract was dissolved in 0.02 M NaOH. Drugs were resolved at room temperature on a 5 μm Zorbax SAX column (250×4.6 min I.D.) equipped with a 20×4.6 mm anion-exchange Vydac AXGU ( 10 μm particle size) precolumn. The mobile phase consisted of acetonitrile and phosphate buffer (pH 7.0), delivered at a flow-rate of 1.2 ml/min. Detection was made at 280 nm, 2-[4-(2′-Furoyl)phenyl]propionic acid was used as internal standard. The calibration curve was linear from 0.2 to 10μg/ml for rufloxacin, from 0.5 to 30 μg/ml for fenbufen and from 0.2 to 10 μg/ml for felbinac, respectively. The detection limit was 0.1 μg/ml for rufloxacin. 0.3 μg/ml for fenbufen and 0.1 μg/ml for felbinac, respectively.