bluuebunny/crossref_metadata_2025 huggingface dataset:
Simultaneous determination of salbutamol and terbutaline at overdose levels in human plasma by high performance liquid chromatography with electrochemical detection
Authors: Sagar Kamal A. , Kelly Mary T. , Smyth Malcolm R.
Published: May 2005

A multidimensional column chromatographic method involving electrochemical detection using a carbon fibre microelectrode flow cell was optimized and successfully applied to the simultaneous determination of salbutamol and terbutaline in plasma at overdose levels. This method performs, in a single step, an efficient extraction and clean‐up of salbutamol and terbutaline from human plasma. The calibration graphs over three days were linear over the calibration range 20–100 ng/mL plasma with a limit of detection of 1 ng and 0.8 ng/mL plasma for salbutamol and terbutaline, respectively. The intra‐ and inter‐assay coefficients of variation were less than 8% and the recoveries ranged from 94 to 96%. The accuracy of the assay, which was defined as the percentage difference between the mean concentration found and the theoretical concentration, was 7% or better. The proposed method combines the advantages of being simple, reproducible and selective in the presence of other sympathomimetic and commonly ingested drugs and is suitable for routine analyses to obtain valuable information about the clinical effects and treatment of overdose with these drugs. The whole procedure takes ca. 10 min and compares favorably with detection at a conventional glassy carbon electrode.
Studies on silica‐bonded monoclonal antibody packing material for separation of recombinant interferon by high performance immunoaffinity chromatography
Authors: Feng Wenke , Geng Xindu
Published: May 2005

A method for the preparation of a new packing material for high performance liquid chromatography by bonding an anti‐interferon monoclonal antibody to the surface of silica gel is described. The high coupling efficiency and activity of the interferon‐αA‐monoclonal antibody (IFN‐αA‐McAb) column were obtained by activated diol‐silica gel with an activating agent. After purification with this packing material, the specific activity of recombinant human interferon‐αA rose by up to 1.03 × 107IU/mg protein and the purification efficiency was increased by approximately 100 times.
Use of blue‐sepharose for purification of immunotoxin containing type 1 ribosome‐inactivating protein, gelonin
Authors: Masuda Kazuyoshi , Hirano Koichiro , Takagishi Yasushi
Published: May 2005

This paper describes a method suitable for purifying immunotoxin containing type 1 ribosome‐inactivating protein, gelonin. The separation of free (unreacted) 80G, a monoclonal antibody against α‐fetoprotein (AFP), from semipurified 80G–gelonin conjugate was unsuccessful by conventional CM‐Sepharose ion‐exchange chromatography because the isoelectric point of the conjugate did not increase enough to reach that of gelonin alone. In contrast, Blue Sepharose affinity chromatography could efficiently separate free 80G from the semipurified conjugate because the conjugate was bound to the column by its gelonin moiety while free 80G was not in buffer containing NaCl of a particular concentration range. However, a small amount of conjugate containing gelonin modified with N‐succinimidyl 3‐(2‐pyridyldithio)propionate, but not with 2‐iminothiolane, could not bind to the column. The conjugate purified by the use of Blue Sepharose showed selective cytotoxicity against AFP‐producing human hepatoma cells.
Charge‐transfer chromatographic study on the interaction of amino acids with ethoxylated stearic acid surfactants
Authors: Cserháti Tibor
Published: May 2005

The interaction of amino acids with ethoxylated stearic acid nonionic surfactants was studied by charge‐transfer reversed‐phase thin‐layer chromatography and the relative strength of interaction was calculated. In the majority of cases the surfactant has a negligible effect on the hydrophobicity of amino acids. Only the binding of Asn, Cys, Gln, Leu, Lys, Met, Nle, Phe, Ser, Trp and Tyr to the surfactants was observed, however, the strength of interaction was fairly low. Stepwise regression analysis proved that the electronic parameters of the amino acids have the highest impact on the strength of interaction. This finding supports the hypothesis that the binding of ethoxylated stearic acid nonionic surfactants to proteins involves more than one amino acid residues and that the hydrophilic forces have a considerable impact on the interaction.
Automated HPLC analyses of drugs of abuse via direct injection of biological fluids followed by simultaneous solid‐phase extraction and derivatization with fluorescence detection
Authors: Bourque A. J. , Krull I. S. , Feibush B.
Published: May 2005

An automated system is described for the simultaneous extraction and derivatization of nucleophilic compounds from various biological media. The method includes the use of a solid‐phase reagent containing a 9‐fluorenylacetate activated ester. The reagent is based on a controlled pore, polystyrene divinylbenzene support prepared through a silica template procedure. An X‐Y‐Z robotic arm equipped with a needle is used in conjunction with a syringe pump for aspirating and dispensing samples and standards into the HPLC system. A precolumn cartridge containing the solid‐phase reagent is put on‐line in place of the fixed‐volume injection loop. Injections of biological fluids such as urine or plasma with minimal sample treatment and handling are made directly into this reactor. The analytes are dervatized as they are extracted, allowing virtually unlimited sample volumes to be injected. The polymeric cartridge can be used for up to 100 injections without accruing unacceptable reductions in sensitivity. A detection limit of 500 p.p.t. (parts per trillion) of amphetamine in urine was achieved with this system.
Improved thin layer chromatographic resolution of PTH amino acids with some new solvent systems
Authors: Bhushan R. , Mahesh V. K. , Varma A.
Published: May 2005

Three new solvent systems, pyridine: benzene (2.5:20, v/v), methanol: carbon tetrachloride (1:20, v/v) and acetone : dichloromethane (0.3:8, v/v), for the resolution of a mixture of 18 PTH amino acids have been reported. Using these solvent systems, various combinations of PTH amino acids which had previously posed resolution problems have been resolved and identified.
UV‐transparent, replaceable agarose gels for molecular‐sieve (capillary) electrophoresis of proteins and nucleic acids
Authors: Hjertén Stellan , Srichaiyo Tasanee , Palm Anders
Published: May 2005

Gels of methoxylated agarose (gelling point 25.6 °C) and other low‐melting agarose derivatives compare favorably with cross‐linked polyacrylamide gels for capillary and slab molecular‐sieve electrophoresis of proteins and DNA. These agarose gels can be pressed out of the capillary following a run and replaced by an agarose solution with a temperature of 35–40 °C. Gelation occurs upon lowering the temperature and the same capillary can thus be reused for another analysis with a fresh gel. The methoxylated, non UV‐absorbing agarose gels are, accordingly, replaceable, which makes them very attractive for series analyses with modern, automated capillary electrophoresis apparatus. The high resolution of these agarose gels is demonstrated with a separation of an albumin sample into monomers, dimers, trimers, tetramers, pentamers, hexamers, heptamers, and of DNA fragments.
A simple method for the determination of YM060 in plasma and urine by high performance liquid chromatography
Authors: Miura Hiroya , Takeshige Tetsuo , Kobayashi Shin‐Ichiro , Higuchi Saburo
Published: May 2005

We developed a simple method for the determination of YM060, a new 5‐HT3 receptor antagonist, in plasma and urine. The method has good accuracy and precision, and sufficient sensitivity to allow use in pharmacokinetic studies of YM060 in humans and laboratory animals.
Theoretical consideration on polarized photometric detection
Authors: Hayakawa Kazuichi , Yamamoto Atsushi , Matsunaga Akinobu , Mizukami Eiichi , Nishimura Masayuki , Miyazaki Motoichi
Published: May 2005

Previously, we developed a novel detector, a polarized photometric detector for optically active compounds, where two polarizers are set on either side of the UV‐visible absorbance detector flow cell. In this paper, we present theoretical considerations on this method with several equations. If the inclined angle of the second polarizer is defined as α (left when α > 0°), the following equation is obtained: ΔAbs = 2 log e · tan α · β, where ΔAbs is the change in absorbance and β (|β| ≪ α) is the angle of rotation of light passing through the cell containing the optically active compound. This equation suggests that ΔAbs is proportional to the analyte concentration and that the dextro‐ and laevorotatory compounds are detected as positive and negative absorbance changes, respectively. The theoretical maximum signal to noise (S/N) ratio is obtained when α = 45° (=0.8 rad) and the smallest detection limit is 5 × 10−5° (S/N = 3) when the path length of the flow cell is 10 mm. This detector was successfully used in flow injection analysis (FIA) as well as HPLC for optically active compounds.
A new matrix for affinity chromatography and its application in the separation of a human monoclonal antibody
Authors: Guo Wei , Shang Zhenhua , Yu Yinian , Zhou Liangmo
Published: May 2005

A method for the preparation of a p‐aminobenzene sulphonyl ethyl containing crosslinked Sepharose 4B (ABSE‐Sepharose 4B‐CL) is described. trypsin, bovine serum albumin (BSA) and concanavalin A (Con A) were immobilized onto this matrix by diazotization. Conditions for the coupling reaction were investigated. The activity of immobilized trypsin reached 1.4 × 104U/g, and 25 mg BSA can be coupled onto 1 g ABSE‐Sepharose 4B‐CL under the optimal conditions. An affinity medium with immobilized Con A as ligand was prepared by this method, and was used in the separation of a human monoclonal antibody.
Separation and identification of proteins obtained from Agkistrodon acutus snake venom by capillary zone electrophoresis and laser desorption/ionization mass monitoring
Authors: Zhang Yukui , Chen Nong , Wang Lei , Yang Boyu , Qian Xiaohong , Ma Liran
Published: May 2005

Fractions of seven protein principles with fibrinolytic or thrombin‐like activities obtained from Agkistrodon acutus snake venom purified by two steps of normal pressure chromatography were separated further by capillary zone electrophoresis (CZE). Mass determination for these fractions were achieved by performing laser desorption/ionization mass monitoring (LDIM). The comparative study between CZE and LDIM on the separation of these fractions was made.
Continuous beds. Their applicability for immobilization of proteins
Authors: Mohammad Jamil , Hjertén Stellan
Published: May 2005

An epoxy‐activated continuous bed can be prepared for immobilization of Proteins in a simple, rapid, and cost‐effective way. The concentration of epoxy groups on the continuous bed was as high as 600 μmol/mL compressed bed (compression of the bed decreases the peak broadening). Human transferrin, human serum albumin and particularly urease were employed as model proteins. The immobilization of urease was virtually completed within 1h in 1 M potassium phosphate, pH 7.4. The binding capacity was 97 mg of urease/mL compressed bed. This bed is of clinical interest, since it is inexpensive to prepare and permits reproducible enzymatic determination of urea in serum and urine (the chromatographic step is finished within 1–2 min).
Use of capillary electrophoresis for thalassaemia screening
Authors: Law Hai‐Yang , Ng Ivy , Ong Joyce , Ong Choon‐Nam
Published: May 2005

This paper describes the use of high‐performance capillary electrophoresis (HPCE) for screening of thalassaemias. Preparation of globin was performed by adding 1mL of haemolysate to 20 volumes of cold acidified acetone. Separation of globins was accomplished in a 25 mM disodium phosphate buffer at pH 11.8 using an uncoated capillary column of 50 cm × 75 μm (i.d.). Distinct peaks of alpha, beta and gamma were resolved within 6min. The coefficient of variations for within‐day and between‐day runs were 4.7% and 6.37% respectively. Using this simple and rapid procedure up to 30 specimens could be analysed in a single day. This method appears to be reliable and can be used for mass screening of various haemoglobinopathies.
Separation of reducing monosaccharides by capillary zone electrophoresis
Authors: Qi Li , Zhu Yichuan
Published: May 2005

The separation of reducing monosaccharides derived from glycosidoproteins and glycolipids by capillary zone electrophoresis (CZE) is dependent on the pH and concentration of the borate buffer. Five saccharides were completely separated in a fused silica capillary tube (50 μm i.d., 65 cm) containing 50 mM borate buffer (pH 10.5) as carrier, with high resolution, at an applied potential of 20 kV after the reducing saccharides were derivatized with 1‐naphthylamine. On‐column UV (254 nm) monitoring allowed quantitation of these saccharides at least in the concentration range of 10–100 mM in reaction solution. This method was applied to the determination of the monosaccharides composition of various carbohydrate materials to demonstrate its usefulness.
Improved resolution of tryptic digest fragments from haemoglobin variants using phytic acid in free zone capillary electrophoresis
Authors: Okafo George N. , Perrett David , Camilleri Patrick
Published: May 2005

Capillary electophoresis can be applied to the rapid characterization of tryptic digests of proteins. The addition of phytic acid to the separation buffer was found to improve resolution considerably when the technique was applied to differentiate between tryptic digests derived from variant haemoglobins. Moreover, analysis time was of the order of 15 min, which is considerably shorter than that obtained using gradient reversed‐phase high‐performance liquid chromatography or two‐dimensional paper chromatography–electrophoresis.
Measurement of amiloride in airway surface liquid utilizing HPLC and fluorescence detection
Authors: Selinger Krzysztof
Published: May 2005

A high‐performance liquid chromatographic method for the determination of amiloride in airway surface liquid is described. It involves extraction of the drug with methanol from filter paper on which the sample is absorbed and chromatography on a Zorbax Rx column; the mobile phase is 25% acetonitrile in 0.05 M phosphate buffer; detection by fluorescence at 360/420 nm. Triamterene is used as an internal standard. The range of the assay is 2.0–2033.4 ng/sample, with adequate precision and accuracy. A power curve y = axb best describes the relationship between the peak‐height ratio and concentration. Recovery of amiloride is non‐linear, probably due to an adsorption process. Accuracy of the assay at lower amounts may be affected by the choice of filter paper a well as by presence of endogenous plasma components. The assay was used to measure amiloride amount in airway surface liquid after administration of 2.5 and 4.5 mg of nebulized amiloride.
A quantitative analytical method for the determination of a new thromboxane synthetase inhibitor (CGS 22652) in human plasma using high performance liquid chromatography
Authors: Stelling M. S. , Maniara W. M. , Powell M. L.
Published: May 2005

A quantitative analytical method for the determination of a new thromboxane synthetase inhibitor (CGS 22652) in human plasma has been developed using high performance liquid chromatography (HPLC). The drug and internal standard (CGS 23298) were extracted with methylene chloride at pH 4.8. Separations were achieved by reversed phase chromatography using a mobile phase consisting of acetonitrile: 0.01M citrate/phosphate buffer (pH 3.5): methanol: tetrahydrofuran (45:45:9:1, v/v/v/v/), on a 5 μm C18 column at a flow rate of 1.0 mL/min. Plasma standard curves were linear from 50 to 2000 ng/mL, with recovery of the drug being greater than 94% at all concentrations. The method was validated over a concentration range of 50 to 2000 ng/mL, with a limit of quantification of 50 ng/mL.

The method was successfully applied to the analysis of clinical samples from a single‐dose safety and tolerability study conducted in healthy male volunteers.
A quantitative analytical method for the determination of a new anxiolytic (CGS 19480A) in human plasma using high performance liquid chromatography
Authors: Stelling M. S. , Maniara W. M. , Powell M. L.
Published: May 2005

A sensitive and specific analytical method has been developed for the determination of a new anxiolytic (CGS 19480A) in human plasma using high performance liquid chromatography (HPLC). The drug and internal standard (CGS 18102A), were extracted with hexane at pH 7. Separations were achieved by reversed phase chromatography on a Nucleosil 5 C18 column at a flow rate of 1.0 mL/min. The mobile phase consisted of acetonitrile: 0.01 M phosphate buffer (pH 7): methanol (51:35:14, v:v:v), where the final pH of the mobile phase was adjusted to 4.0 using 85% phosphoric acid. Plasma standard curves were linear from 5.0 to 500 ng/ml, with recovery of the drug being greater than 95% at all concentrations. The method was validated over a concentration range of 5.0 to 500 ng/mL with a limit of quantification of 5.0 ng/ mL.

The method was successfully applied to the analysis of clinical samples from a single‐dose safety and tolerability study conducted in six healthy male volunteers.
Simultaneous determination of p‐aminobenzoic acid and its metabolites in urine by high performance liquid chromatography
Authors: Kastel Rudolf , Rosival Ivan , Blahovec Jan
Published: May 2005

We developed a new HPLC method for the determination of p‐aminobenzoic acid (PABA) and its metabolites (p‐aminohippuric acid, N‐acetyl‐p‐aminohippuric acid, N‐acetyl‐p‐aminobenzoic acid) in urine. As the internal standard m‐hydroxybenzoic acid was used. In the isocratic elution the mobile phase consisted of methanol and 0.02 M. ammonium acetate (20:80 v/v, pH 4.0). The separation was carried out on the C18, reversed‐phase column, particle size 5 μm. The separated components were detected at 280 nm. The method can be used in the assessment of the response of pancrease (secretion of digestive enzymes) to soya feeding as well as in the diagnosis of the exocrine pancreatic diseases of animals.
A sensitive detection method for peptide using 4‐fluoro‐7‐nitrobenozo‐2‐oxa‐1,3‐diazole and its application to measure prolyl endopeptidase activity
Authors: Yoshinaga Koji , Kobayashi Naomi , Nagatani Yasunori , Tanaka Yoshiaki , Ikeda Yugo
Published: May 2005

A Measuring method sensitive to prolyl endopeptidase (EC 3.4.21.26, PEP) activity using native peptides (Arg‐Vasopressin or substance P) as substrates was established. The investigation of three different derivatization reagents, Which had been developed for an amino acid analysis, demonstrated that 4‐fluoro‐7‐nitrobenzo‐2‐oxa‐1,3‐diazole (NBDF) was the most suitable for the detection of Arg‐Gly‐NH2, which was released from Arg‐vasopressin by PEP. Arg‐Gly‐NH2 was reacted with NBDF at 65°C for 5 min at pH 7.6 and the reaction mixture was analysed by HPLC on a reverse‐phase column by monitoring the gluorescence intensity. The detection limit was 1 picomol per injection and the linear standard calibration curve could be constructed in the range of 1 to 100 picomol per injection with a 3.0% relative standard deviation. This sensitive detection method for peptide was applied to the measurement of PEP activity Using Arg‐vasopressin as a substrate and 1 × 10−3 unit of PEP activity was detectable. This method was also applicable to the measurement of PEP activity Using subsytance P as a substrate by detecting the derivative of its fragment peptide (Arg‐Pro‐Lys‐Pro).
Degradation of organophosphorus and carbamate pesticides in soils—HPLC determination
Authors: Thapar Sangita , Bhushan Ravi , Mathur R. P.
Published: May 2005

Best conditions suited for the extraction and trace analysis of dimethoate, malathion, methyl‐parathion, carbaryl, carbendazim and carbofuran in soils, using HPLC, were established, Theis was followed by studies related to persistence of residues in different soils, Half‐life and take for 95% dissipation were determined in each case, Higher Ph, moderate mosture and hihg calcium carbonate content aided degradation while organic matter increased persistence.
Measurement of clozapine and norclozapine in plasma/serum by high performance liquid chromatography with ultraviolet detection
Authors: McCarthy P. T. , Hughes S. , Paton C.
Published: May 2005

A simple method for the measurement of clozapine and its N‐desmethyl metabolite in human pläsma or serum by high performance liquid chromatography is described. An internal standard (aqueous nortriptyline, 4 mg/L) (50 μL) and Tris buffer (2 mol/L, pH 10.6) (100 μL) are added to plasma/serum (200 μL) and the analytes and internal standard extracted into methyl tert‐butyl ether (200 μL). The extracts are analysed on a 150 mm column containing Spherisorb S5SCX using methanol containing ammonium perchlorate (35 mmol/L, pH 6.7) as eluent at a flow rate of 1.5 mL/min. Detection is by ultraviolet absorption (215 nm). The limit of detection is better than 0.05 mg/L for both analytes and the intra‐assay precision (CV) for clozapine and norclozapine was 5.3 and 7.3% at 0.5 mg/L and 2.6 and 2.8% at 1.5 mg/L, respectively. The method can be applied to the measurement of these compounds in plasma after acute overdosage, for the assessment of compliance in patients apparently refractory to therapy and to identify interactions between clozapine and other neuroleptic and antidepressant drugs which may effect toxicity.
Investigation of enantioselective ofloxacin—albumin binding and displacement interactions using capillary affinity zone electrophoresis
Authors: Arai Takashi , Nimura Noriyuki , Kinoshita Toshio
Published: May 2005

The direct chiral separation of ofloxacin by capillary affinity zone electrophoresis using serum albumins from different animal sources as chiral selector in the supporting electrolyte is described. In addition, the effects of displacers on the mobility and enantioselectivity of ofloxacin were studied. Firstly, the separation behaviour of the enantiomers of the ofloxacin (OFLX) and tryptophan (Trp) was compared. The influence of albumin types, including chemically modified bovine serum albumins (BSAs), and buffer types on the migration behaviour of enantiomers was investigated. The results showed that stereoselectivity of Trp is independent of the type of albumin used. However, chiral separation of OFLX depends on the biological species of albumin. Use of chemically modified BSA led to poorer resolution of enantiomers. Only with acetylated BSA could chiral separation of Trp be achieved. Using Good's buffer solutions (DIPSO and HEPES) as a supporting electrolyte affected the migration times of OFLX enantiomers. Finally, a variety of displacers were added to the buffer along with the protein, and the effects on separation behaviour were observed. The displacers included warfarin, ketoproten, diazepam, propranolol, benzoinphenylbutazone, digitoxin and octanoic acid. From the results obtained, it is concluded that capillary affinity zone electrophoresis using albumin as a chiral selector may allow screening of OFLX‐displacer interactions.
Dye—ligand affinity chromatography on continuous beds
Authors: Mohammad Jamil , Zeerak Asadullah , Hjertén Stellan
Published: May 2005

Continuous beds derivatized with three triazine dyes (Cibacron Blue 3G‐A, Pricon Red HE‐3B and Pricon Red H‐3B) were used for chromatographic purification of dehydrogenases from yeast enzyme concentrate. All three columns, which were prepared by a very simple and cost‐effective method, provided strong binding of glucose‐6‐phosphate dehydrogenase and lactate dehydrogenase. The Cibacron Blue 3G‐A column showed high affinity for alcohol dehydrogenase. Under the same conditions, the Pricon Red HE‐3B column showed a lower affinity and the Pricon Red H‐3B column showed none. The adsorbed dehydrogenases were eluted specifically from the columns in high yields (71–113% by desorption with the coenzymes NADP, NADH and NAD respectively). Non‐specific binding of human serum albumin and transferrin to these columns was also investigated. Enzyme assays and analyses by capillary electrophoresis showed that the continuous beds derivatized with triazine dyes gave a high degree of purification.
Use of liquid chromatography for the determination of interactions between bioactive compounds
Authors: Cserháti Tibor , Forgács Esther
Published: May 2005

Molecular interactions can be easily determined both by thin‐layer and high performance liquid chromatography. The theory and practice of the determination of molecular interactions and the various methods for the calculation of complex stability are presented. Examples of the applications of liquid chromatographic methods for the measurements of molecular interactions are also discussed.
Determination of free fatty acids in blood, tagged with 4‐(2‐carbazoylpyrrolidin‐1‐yl)‐7‐(N, N‐dimethylaminosulfonyl)‐2, 1, 3‐benzoxadiazole, by high‐performance liquid chromatography with fluorescence detection
Authors: Toyo'oka Toshimasa , Takahashi Maki , Suzuki Asako , Ishii Yasuko
Published: May 2005

The free fatty acids in blood were determined by high‐performance liquid chromatography (HPLC) after pre‐column tagging with 4‐(2‐carbazoylpyrrolidin‐1‐yl)‐7‐(N,N‐dimethylaminosulphonyl)‐2, 1, 3‐benzoxadiazole (DBD‐ProCZ). The tagging conditions were optimized with palmitic acid (C16:0) and linoleic acid (C18:2) as representative free fatty acids, saturated and unsaturated, respectively. Under the mild reaction conditions of room temperature for 90 min in dimethylformamide (DMF) containing 1‐ethyl‐3‐(3‐dimethylaminopropyl)‐carbodiimide hydrochloride (EDC, 0.2 M)/pyridine (2%), all the fatty acids tested were tagged with the DBD‐ProCZ to produce highly fluorescent derivatives which emit light at 550 nm (excitation at 450 nm). The fluorescence wavelenghts were essentially the same for all fatty acids, whereas the intensities were different for individual fatty acids. The derivatives obtained from ten free fatty acids were completely separated by reversedphase chromatography with two isocratic elution conditions. The on‐column detection limit (signal‐to‐noise ratio of 3) with proposed HPLC separation and fluorescence detection is in the range of 19 (palmitic acid) −176 fmol (palmitoleic acid). The free fatty acids in rat serum and human plasma were successfully determined using the present method.
Identification of 9, 10‐epoxyoctadecanoic acid in human urine using gas chromatography‐mass spectrometry
Authors: Ulsaker Gunnar A. , Teien Gerd
Published: May 2005

9, 10‐epoxyoctadecanoic acid has been detected in human urine. Two simple purification procedures were used; the one based upon liquid‐liquid extraction and the other based upon sorbent extraction technology isolating the free fatty acid fraction. Prior to trimethylsilylation and gas chromatographic‐mass spectrometric analysis, both the epoxy and carboxy functions were reduced to hydroxy groups. The shift in fragmentation of a deuterated sample verified the presence of intact epoxide prior to chemical reduction. Special attention was paid to the risk of false identification of the epoxide. The content of 9, 10‐epoxyoctadecanoic acid in human urine was estimated to be 2.1 nM/L.
Determination of D‐amino acids, derivatized with 4‐fluoro‐7‐nitro‐2, 1, 3‐benzoxadiazole (NBD‐F), in wine samples by high‐performance liquid chromatography
Authors: Kato Masaru , Fukushima Takeshi , Santa Tomofumi , Homma Hiroshi , Imai Kazuhiro
Published: May 2005

The concentrations of D‐amino acids and their enantiomeric ratios in wine samples were determined by high‐performance liquid chromatography with fluorometric detection. We used Pirkle type chiral stationary phases and fluorogenic reagent, 4‐fluoro‐7‐nitro‐2,1,3‐benzoxadiazole (NBD‐F) for simplicity and high sensitivity. The amino acids determined were D‐enantiomers of alanine (Ala), aspartic acid, glutamic acid, isoleucine (Ile) and leucine (Leu). D‐Asparagine, ‐glutamine and ‐lysine were not detected. D‐Leu was detected in red and rosé wine samples, and D‐Ile was determined only in rosé wine. In contrast, neither D‐Leu nor Ile were detected in white wine samples. The concentration of D‐Ala was the most prominent among these amino acids with the highest content of approximately 180 μM (D/D+L ratio; 25%) in rosé wine.
Chromatographic investigation of the glycosylation pattern of alpha‐1‐acid glycoprotein secreted by the HEPG2 cell line; a putative model for inflammation?
Authors: Elliott Heather G. , Elliott Moira A. , Watson John , Steele Les , Smith Kevin D.
Published: May 2005

In certain pathophysiological conditions, such as rheumatoid arthritis, there are alterations in the glycosylation pattern of the acute phase protein, alpha‐1‐acid glycoprotein (AGP). These changes are likely to be functionally significant, however, verification of the latter role requires a system which reflectsin vivo glycosylation changes in AGP and also produces sufficient quantities of the protein for further study. The human hepatoma cell line HepG2 is documented as displaying a shift in the glycosylation pattern of glycoproteins from normal state to acute phase after stimulation with inflammatory mediators. We have isolated AGP from the culture medium of HepG2 cells both before and after stimulation with a cytokine preparation and analysed the glycosylation pattern of each preparation, after enzymatic release, by high pH anion‐exchange chromatography. Before stimulation, the glycosylated population was similar to a profile which was of AGP isolated from normal plasma; however, cytokine stimulation resulted in a shift to a profile which was consistent with that of AGP from a rheumatoid arthritis sufferer. Thus a HepG2 cell culture system is capable of being a crude model of the changes in glycosylation of acute phase proteins although it has a tendency to produce oligosaccharide chains which are not fully sialylated.
Binding site for basic drugs on α1‐acid glycoprotein as revealed by chemometric analysis of biochromatographic data
Authors: Kaliszan Roman , Nasal Antoni , Turowski Maciej
Published: May 2005

Interactions between α1‐acid glycoprotein (AGP) and 52 basic drugs were quantified by means of highperformance liquid chromatography (HPLC). The HPLC retention parameters were related quantitatively to the hydrophobicity and molecular modelling parameters, giving rise to the prediction of relative drug‐AGP binding from the chemical structure of a drug. A structural model of one binding site on AGP, common for various classes of drugs, was defined which accounted for the observed and reported differences in binding to AGP. A combination of biochromatography and chemometrics has been presented as a promising new approach in biochemical/pharmacological studies.
High‐performance liquid chromatography with fluorescence detection of ebiratide using 4‐(N, N‐dimethylamino‐sulphonyl)‐7‐fluoro‐2, 1, 3‐benzoxadiazole as a fluorogenic reagent
Authors: Hamachi Yozo , Tsujiyama Tsuyosi , Nakashima Kenichiro , Akiyama Shuzo
Published: May 2005

A sensitive high‐performance liquid chromatography (HPLC) method with fluorescence detection has been developed and applied to the determination of ACTH4–9 analogue (ebiratide), 4‐(N,N‐Dimethylamino‐sulphonyl)‐7‐fluoro‐2,1,3‐benzoxadiazole (DBD‐F) was used as a fluorogenic labelling reagent.

Ebiratide in 0.1 m borate buffer (pH 9.0) was reacted with DBD‐F in acetonitrile solution at 50°C for 30 min. After cooling, the mixture was seperated by a reversed‐phase HPLC. Then DBD‐ebiratide was monitored at 580 nm with the excitation at 440 nm. The calibration curve was liner up to 10 pmol on column (r=0.999) with the detection limit of 0.25 pmol (signal‐to‐noise=3).
Determination of nitroimidazole residues in poultry tissues, serum and eggs by high‐performance liquid chromatography
Authors: Semeniuk Stanislaw , Posyniak Andrzej , Niedzielska Jolanta , Żmudzki Jan
Published: May 2005

A simple high‐performance liquid chromatographic (HPLC) method was developed for determination of dimetridazole and metronidazole residues in poultry muscle, liver, serum and eggs. The drug residues were extracted from tissues and egg samples with acetonitrile followed by solid‐phase extraction clean‐up and HPLC analysis with photodiode array detector. Serum samples were treated with trichloroacetic acid, centrifuged and analysed without further clean‐up. The detection limits were 2 ng/g and 5 ng/mL of analysed tissue (egg) and serum samples, respectively. Average recoveries for spike levels of 10 and 20 ng/g ranged from 82 to 94%, and coefficients of variation were below 10%.
Determination of unbound L‐tryptophan in human plasma using high‐performance liquid chromatography with fluorescence detection
Authors: Jagannathan Vanitha , March Clark , Venitz Jürgen
Published: May 2005

An assay for the measurement of unbound L‐tryptophan concentrations in plasma was developed using reverse‐phase HPLC with fluorescence detection. Unbound L‐tryptophan from plasma was obtained using the Amicon MPS‐1 ultrafiltration device. L‐tryptophan binding to the membrane in the ultrafiltration device was not significant. A linear relationship between the peak‐area ratios (peak‐area of L‐tryptophan to internal standard) and concentrations of L‐tryptophan was obtained in the range of 0.1 μg/mL to 2.5 μg/L. The intra‐assay precision was less than 10% for each control (0.18 μg/mL, 0.75 μg/mL and 2.0 μg/mL). The inter‐assay precision was less than 15% for each control; the accuracy for each control, expressed as percent difference from nominal (%DFN), was less than 12%. This method was used to determine unbound L‐tryptophan concentrations in plasma from nine male subjects who participated in a clinical study.
Two‐Dimensional correlation method applied to Yb3+ vibronic sideband spectroscopy: Discrimination of fluorescence spectral features arising from different yb3+ binding sites
Authors: Roselli Cécile , Burie Jean‐René , Mattioli Tony , Boussac Alain
Published: May 2005

Ytterbium (Yb3+) vibronic sideband (VSB) spectroscopy allows the identification of the lanthanide ligands both in model compounds and in Yb3+ ‐reconstituted metalloproteins. In some cases there exist multiple binding sites which complicate the Yb3+ fluorescence spectrum. The presence of different sites can be deduced from the variation of the intensity of the VBSs, when the wavelength of excitation is varied. A general two‐dimensional (2D) correlation method developed by Noda can be potentially used to discriminate the different Yb3+ binding sites. A very simple procedure has been further developed so that, by using a few different spectra, usable information can be extracted from the 2D spectrum; the spectra were obtained with different wavelengths of excitation. This method is applied successfully in the present work on Yb3+‐reconstituted transferrin, which contains two spectroscopically different Yb3+ sites, in order to determine any structural difference between the two sites. © 1995 John Wiley & Sons, Inc.
Characterization of exfoliated cells and tissues from human endocervix and ectocervix by FTIR and ATR/FTIR Spectroscopy
Authors: Wong Patrick T. T. , Lacelle Suzanne , Fung Michael Fung Kee , Senterman Mary , Mikhael Nadia Z.
Published: May 2005

The transmission infrared spectra of exfoliated endocervical mucin‐producing columnar epithelial cells and the attenuated total reflectance (ATR) infrared spectra of the single‐columnar cell layer on the endocervical tissues have been measured and compared with the corresponding infrared spectra of the ectocervical squamous cells and squamous epithelium. The infrared spectra of the exfoliated cervical cells obtained from the present work are comparable with those directly measured from the epithelia on the cervical tissues by ATR technique. The transmission infrared spectra of endocervical columnar epithelial tissue containing some components of the underlying connective tissue have also been measured and compared with the ATR/FTIR (Fourier‐transform infrared) spectra of the endocervical columnar epithelial tissue. The effects of the contaminated connective tissue on the infrared spectra of the endocervical columnar epithelial tissue have demonstrated that ATR/FTIR is a more desirable method than the transmission method to obtain meaningful and good‐quality infrared spectra of tissue samples, especially samples consisting of thin layers of different types of tissues. Substantial differences in the infrared spectra between the columnar cells and squamous cells on the endocervical and ectocervical tissues, respectively, were evident. The strong glycogen bands in the infrared spectrum of the ectocervical squamous cells are absent in the spectrum of the endocervical columnar cells. This spectral change is similar to that observed in malignant squamous cells. Therefore, if the decrease in the intensity of the glycogen bands is used as the only criterion for the determination of cellular abnormalities in the cervix, the presence of a large number of normal endocervical columnar cells in the cervical specimen would lead to a false result. Consequently, in addition to the glycogen bands, other features in the infrared spectra should be considered for the evaluation of abnormalities in exfoliated cervical epithelial cells. In order to identify the spectral features that are unique to endocervical columnar cells, the infrared spectra of an aqueous solution of glycogen, the mucus from the endocervix, and the connective tissues from both the endocervix and the ectocervix have also been measured and analyzed. © 1995 John Wiley & Sons, Inc.
Context and its significance in identifying ‘what works’ in child protection
Authors: Barton Adrian , Welbourne Penelope
Published: May 2005

The repetitive nature of the underlying problems with child protection systems identified by many inquiries into child deaths suggests that the ability to transpose successful strategies from one area to another may be contingent on more than a ‘technical’ approach to best practice. Current policy responses to failing child protection systems are arguably based on an assumption that practices that work in one area may be applied in other areas without reference to the existing base for practice. Drawing on our own experiences in the field, we attempt to explore some methodological issues relevant to the evaluation of service provision and the dissemination of effective practice in interagency working. Copyright © 2005 John Wiley & Sons, Ltd.
Cellular utilization of cytosolic NADPH in kidney and liver cells from rats fed a normal or a vitamin D‐deficient diet
Authors: Bachelet Marie , Bader Cyrille , Merlot Anne‐Marie , Laborde Kathleen , Snarska Joanna , Ulmann Andréa
Published: May 2005

The amount of reducing equivalents from NADPH generated by glucose 6‐phosphate dehydrogenase activity (G6PD) used in mixed function oxidation (pathway I) or in reductive biosynthesis (pathway II) has been determined by cytochemical methods and microdensitometry in cells from the pars recta (PR) and distal convoluted tubule (DCT) of the kidney and from centrilobular (CL) and periportal (PP) hepatocytes from rats fed a normal or a vitamin D‐deficient diet. In the kidney, pathway I activity was similar to that of pathway II in PR, whereas in DCT pathway II was markedly predominant. Feeding a vitamin D‐deficient diet resulted in an increase in the total amount of reducing equivalents in PR and DCT. This increase was due to a rise in pathway I activity in the PR, whereas in the DCT the increase resulted from a stimulation of pathway II activity. Pathway I activity in PR was inversely correlated with plasma calcium, and was significantly decreased when calcium (1 mM) was added in vitro. In the liver the total amount of reducing equivalents generated by G6PD and both hydrogen pathways, was higher in CL than in PP hepatocytes. In CL cells, a vitamin D‐deficient diet induced a significant increase in both NADPH pathways. Furthermore, in these cells pathway I activity was inversely related to plasma calcium and was significantly lowered when 1 mM calcium was added in vitro. It is concluded that vitamin D status and calcium influence the production and utilization of cytosolic reducing equivalents both in kidney and liver.
Activation of carboxylesterases in root cortical parenchymal cells of Pisum sativum during xylem induction in vitro
Authors: Gahan P. B. , Rana M. A. , Phillips R.
Published: May 2005

A quantitative cytochemical study of naphthol AS‐D esterase activity in explants from roots of Pisum sativum grown on basal medium for 3 or 6 days showed similar levels of activity to those seen in sections of cortex and stele from intact roots of similar ages. Explants grown in the presence of auxins or cytokinin alone showed a threefold or twofold increase in cortical parenchymal activity, respectively. On adding both hormones to initiate xylem element formation, there was also a threefold increase in activity in the cortex. In all three cases, the stimulated activity was totally inhibited by either 10−4 M diisopropylfluorophosphate (DFP) or 10−4 M diethyl p‐nitrophenyl‐phosphate (E600), indicating carboxylesterase activity. The low level of activity normally present in cortical cells was inhibitor resistant, so indicating acetylesterase activity. Thus, auxins and cytokinins appear to activate mainly similar carboxylesterases during the initiation of xylem elements from cortical parenchyma cells.
Comparison of androgen biosynthesis in isolated Leydig cells from rat and mouse testis: Incubation and superfusion studies
Authors: Kühn‐Velten N. , Schumacher H. , Wolff J. , Staib W.
Published: May 2005

Production of testosterone by highly purified Leydig cells prepared from rat and mouse testes is compared. Testosterone formation is improved to a higher degree in rat (2.7‐fold) than in mouse (1.7‐fold) cells by callagenase treatment of the testis compared with mechanical isolation. Mouse Leydig cells respond to exogenous stimuli (chorigonadotropin, dibutyryl cyclic AMP) with 2.4‐fold higher testosterone secretion than rat cells. A 1.7‐fold increased conversion of androgen precursors to testosterone by mouse compared with rat Leydig cells is demonstrated in static incubations as well as in steady‐state superfusion experiments and can be derived from enhanced androstenedione reduction and a less inhibitory effect of progesterone on this process is mouse Leydig cells.
Use of microscopic interferometry for measuring changes in water content of small samples of tissue
Authors: Darracott‐C̄anković S. , Braimbridge M. V. , Kyösola K. , Bitensky L. , Chayen J.
Published: May 2005

Oedema following periods of ischaemic arrest and subsequent reperfusion has been shown experimentally and clinically to affect the functional state of the heart. Tissue water content has been measured in myocardial sections by microscopic interferometry and densitometry, and the results correlated with those obtained by wet and dry weight analysis (r = 0.87; p < 0.001). Microscopic interferometry also revealed the distribution of the water in the tissue. Experimentally induced ischaemic arrest in isolated rat hearts resulted in predominantly intra‐fibrillar oedema, whilst subsequent reperfusion resulted in interfibrillar oedema. Microscopic interferometry facilitates accurate measurement of water content in tissue samples as small as 2 mg wet weight and shows (as conventional wet/dry weight analysis cannot) the distribution of the water in the tissue.
Integrity of glycoprotein complex sugars is required for homing but not for several other membrane‐mediated functions
Authors: Hooghe Robert J. , Plaetse Frank Vander , de Saint‐Georges Louis , Janowski Michel , Schaaf‐Lafontaine Nicole , Hooghe‐Peters Elisabeth L. , Dorling Peter
Published: May 2005

In order to correlate the biochemistry of cell surface carbohydrates with cell function, we have treated cells with swainsonine and followed the biochemical and functional modifications induced by this compound. After treatment with swainsonine, surface glycoproteins had a lower apparent molecular weight and a higher isoelectric point. This is compatible with the replacement of complex carbohydrates by hybrid or high‐mannose carbohydrates. Several functional tests were unaffected. However, swainsonine‐treated cells displayed an altered pattern of in vivo homing, suggesting that carbohydrates play a role in this process.
Effect of methylglyoxal on tumour microtubular protein
Authors: Miglietta A. , Bonelli G. , Vironda N. , Gabriel L.
Published: May 2005

Methylglyoxal inhibits cell division, exerting an antiproliferative action on tumour cells. Supernatants from ascites hepatoma cell homogenate, previously incubated with the aldehyde, showed a decrease in colchicine binding activity dependent on methylglyoxal concentration. In contrast, in vivo treatment of tumour‐bearing rats apparently did not cause a significant impairment of microtubular protein, suggesting that the aldehyde interaction with microtubules cannot be considered responsible for its carcinostatic action.
Fibronectin assays and their clinical application: A review
Authors: Boughton B. J.
Published: May 2005

Cold insoluble globulin (fibronectin) was discovered 30 years ago but recently there has been a remarkable growth of knowledge concerning its interaction with the cell cytoskeleton and its role in cell‐cell and cell‐matrix adhesion. The protein is also a major plasma opsonin with a role in regulating fixed macrophage activity and it is this area in which clinical applications are now beginning to develop. Methods are discussed for measuring the concentration of the protein and its opsonic function in vitro, and for the evaluation of fixed macrophage function in vivo. Also discussed are the metabolism of the protein, the implications of opsonin depletion in patients with serious injury or infection and the attempts to reverse this with plasma protein replacement therapy.
Increased myosin orientation during muscle contraction: A measure of cardiac contractility
Authors: Chayen J. , Bitensky Lucille , Braimbridge M. V. , Darracott‐Cankovic Sally
Published: May 2005

Myosin form birefringence has been studied in cryostat sections of left ventricular myocardium from the dog and human. The muscle in such sections has been shown to demonstrate the sliding filament phenomenon. The sarcomere length of canine myocardium agreed with that found in comparable electron micrographs. Unexpectedly, it was found that glycerol, normally used as an inert and optically ideal mountant, caused profound change in myosin birefringence. This apparently invalidates results obtained with this mountant.

The absolute birefringence found in these sections, whether mounted in glycerol or in an ATP‐calcium buffer, corresponded to values found by other workers with skeletal muscle and isolated myosin. However, the birefringent properties (optical path difference: o.p.d.) of well functioning muscle was found to be low, the o.p.d. increasing when exposed to ATP and calcium. Poorly functioning muscle could be distinguished from well functioning muscle on the basis of its higher ‘in air’ o.p.d. This difference correlated well with physiological assessments of myocardial function or with clinical assessments of cardiac failure. Evidence is presented indicating that changes in apparent birefringence, caused by ATP‐calcium or by anoxia, are due to altered orientation of the myosin micelles and can be inhibited by agents that inhibit myosin ATPase activity.