bluuebunny/crossref_metadata_2025 huggingface dataset:
Multimodal liquid chromatographic separation methods in the study of ethimizol metabolism in man
Authors: Šoltés Ladislav
Published: May 2005

The metabolism of ethimizol in the human body has been investigated. Focus was on the detection and demonstration of the regioselective pathway of metabolic demethylation of ethimizol by determining the presence of the corresponding metabolites in blood, saliva and urine. Isolation, purification and identification of the metabolites present in the biological samples was achieved by applying a combination of the following methods: solid phase extraction, high performance liquid chromatography, high performance thin layer chromatography, nuclear magnetic resonance and mass spectrometry. The suggested chemical structures were definitely established by comparing the physicochemical characteristics of the ethimizol metabolites obtained from the individual biological fluids with the characteristics of synthetized authentic derivatives.
Non‐labelled benzodiazepines partitioned into synaptosomal membranes: their extraction and quantification by high performance liquid chromatography
Authors: Perillo Maria A. , Garcia Daniel A.
Published: May 2005

A method for the extraction and quantification by high performance liquid chromatography (HPLC) of non‐labelled benzodiazepines partitioned into biological membranes has been developed. The benzodiazepine (BZD) was partitioned in a synaptosomal membrane–buffer system. The membranous pellet was separated by centrifugation and from this pellet, previously submitted or not to a proteolytic treatment and resuspended in the same buffer, the BZD was extracted by the addition of the proper volume of ethyl acetate, i.e. that capable of extracting at least 99.5% of the total drug in the aqueous phase. Optimal conditions for maximum proteolysis were: incubation of membranes at a final concentration of 1 mg protein/mL in the presence of 0.08 mg of Protease type I per mL for 90 min. at 45°C. Although the efficiency of the proteolysis was demonstrated not only by kinetic but also by electrophoretic evidence, recovery of BZD in the organic solvent was not increased by the enzymatic treatment at a mass BZD/protein ratio in the range 4–4.6 10−5 μg BZD/mg protein. For the HPLC quantification a reversed phase ODS column was used with methanol: water (1:1) plus 3.5% acetic acid as the mobile phase at a constant pressure and 1 mL/min flow rate. The UV detector was calibrated at 265 nm. Calibration curves were constructed by plotting peak height or peak area vs. nmoles BZD and adjusted to straight lines by a regression analysis by the least squares method. The peak height was selected as the detection method. Intra‐ and inter‐assay precision did not exceed 10% and the total recovery was 100%.
Simultaneous determination of pseudouridine and creatinine in urine of normal children and patients with leukaemia by high performance liquid chromatography
Authors: Li Yongtian , Wang Shaoshen , Zhong Ning
Published: May 2005

A reversed phase high performance liquid chromatographic method for the simultaneous determination of pseudouridine (PU) and creatinine (Cr) in urine is described. The mobile phase was 0.01 mol phosphate buffer (pH 6.1) containing 2.5 mmol octanesulphonic acid as the ion pairing agent. UV detection was set at 250 nm. Variation in pH value affected the retention time of PU and Cr significantly; Their separation from interfering peaks was also affected. The recoveries of PU and Cr were 89.93% and 90.35%, respectively. The standard deviation of the method for PU was 48.69 ± 0.063 (nmol/μmol Cr, mean ± SD, n = 5). The urine samples from 233 normal children of different ages and 119 patients with leukaemia were analysed by this method. The normal reference value was appraised by comparison with the percentage of immature cells in the bone marrow. The results showed that the sensitivity of the method was 94.12%, the specificity was 95.86%, the accuuracy was 95.50%, the positive predictive value was 82.05% and the negative predictive value was 98.78%. The method can be used to evaluate the state of the leukaemia, and to monitor the effect of treatment.
One‐step preparation of Hageman factor fragment concentrate
Authors: Simonianová E.
Published: May 2005

The Hageman factor fragment (HFf) concentrate was prepared from acetone and dextran sulphate activated human plasma by chromatography on a CM‐Sephadex column. The preparation was free of the main kallikrein inhibitors—Cl‐inactivator, α2‐macroglobulin, antithrombin III and α1‐antitrypsin. The HFf concentrate can serve as an activator of prekallikrein in patient plasma.
Quantitative determination of a new anticonvulsant (CGS 18416A) in human plasma using capillary gas chromatography/mass spectrometry
Authors: Hayes M. J. , Khemani L. , Leal M. , Powell M. L.
Published: May 2005

An analytical method has been developed for the determination of a new antiepileptic drug, CGS 18416A, in human plasma. CGS 18416A is a new anticonvulsant representative of a novel class of water‐soluble agents being developed for the treatment of epilepsy. Preclinical trials indicate sustained efficacy at relatively low oral doses, indicating a need for a sensitive assay. The method is based on capillary gas chromatography/mass spectrometry and utilizes stable isotope‐labelled CGS 18416A as the internal standard. Samples (1 mL) are acidified, then washed with pentane/ethyl acetate, followed by liquid/liquid extraction at pH 11 with pentane/ethyl acetate. Extracts are then concentrated and analysed directly by gas chromatography/mass spectrometry. Separation is accomplished on a thick film methylsilicone capillary column. Mass spectrometry was carried out under positive ion ammonia Cl conditions with selected ion monitoring of the protonated molecular ions (m/z = 248 and 252) for CGS 18416A and the 13CD3‐CGS 18416A, respectively. Specificity was demonstrated by the lack of interfering peaks at the retention time of CGS 18416A and the internal standard. Recovery and reproducibility assessments indicate good accuracy and precision over the validated concentration range of 0.2–51 ng/mL. The limit of quantification is 0.2 ng/mL and the method has sufficient sensitivity to support clinical trials. This is illustrated with an example of quantification in a normal volunteer following oral dosing.
Determination of naphazoline in rat plasma using column liquid chromatography with ultraviolet detection
Authors: Chabenat Christiane , Boucly Patrick
Published: May 2005

A sensitive (1 ng/mL) and rapid method for the determination of naphazoline in rat plasma is described. Following extraction, the compound is analysed by reversed phase high performance liquid chromatography and ultraviolet detection at 214 nm.
A new method for the purification of cytochrome‐P450 from human liver microsomes
Authors: Isa Majed , Cumps Jean , Fossoul Claudine , Atassi Ghanem
Published: May 2005

A procedure for the solubilization and purification of cytochrome‐P450 (cyt‐P450) from human liver microsomes is described. Successive treatment of microsomes with protease XXVII and 3‐(3‐cholamidopropyl)dimethylammoniopropanesulphonic acid gave a solubilized cyt‐P450 in more than 80% yield and with a three‐fold increae in specific activity. With this treatment it was possible to eliminate 80% of cytochrome‐b5 and 75% of NADPH cyt‐P450 reductase. The solubilized cyt‐P450 was filtered on a Sephacryl‐200 column and then subjected to high performance liquid chromatography with a Mono‐P column (chromatofocussing). The recovery of separated cyt‐P450 was about 50% with a specific activity of 11.5 nmol cyt‐P450/mg protein. Also with this technique it was possible to determinate the isoelectric points of cyt‐P450. These results allowed us to confirm the usefulness of our method, for the study the cyt‐P450 from surgical biopsies.
Determination of low molecular weight aliphatic primary amines in urine as their benzenesulphonyl derivatives by gas chromatography with flame photometric detection
Authors: Kataoka Hiroyuki , Ohrui Satoshi , Miyamoto Yoshiaki , Makita Masami
Published: May 2005

A selective and sensitive method for the determination of low molecular weight aliphatic primary amines in urine is described. These amines were converted into their benzenesulphonyl derivatives by a modified Hinsberg procedure, and measured by gas chromatography with flame photometric detection (FPD–GC) using a DB‐1 capillary column. The derivatives were very stable and provided excellent FPD responses. By FPD‐GC, linear calibration curves were obtained in the range 10–200 ng of methylamine, ethylamine, n‐propylamine, isobutylamine and n‐butylamine using tert‐butylamine as an internal standard, and the detection limits of these amines were ca. 6–25 pg as the injection amount. Benzenesulphonamide derived from ammonia was converted into its N‐dimethylaminomethylene derivative which has a longer retention time, and separated from benzenesulphonyl derivatives of low molecular weight primary amines on the chromatogram. The recoveries of aliphatic primary amines added to urine samples were 91–107% and the relative standard deviations were 0.2–4.5%. Analytical results of aliphatic primary amine contents in urine samples of normal subjects are presented.
High performance liquid chromatographic analysis of six conjugated and unconjugated estrogens in serum
Authors: Su Syang Y. , Shiu Gerald K. , Simmons John , Viswanathan C. T. , Skelly Jerome P.
Published: May 2005

Six estrogens—estrone, equilin, estradiol, sodium estrone sulphate, sodium equilin sulphate and sodium 17‐α‐dihydroequilin sulphate in dog serum were successfully separated and quantified by a high performance liquid chromatographic method developed in our laboratories. The mobile phase was optimized by studying the effects of an organic modifier (acetonitrile) and an ion pairing reagent (tetrabutylammonium hydroxide). The serum sample work‐up procedure was designed to recover both conjugated and unconjugated estrogens. The optimum method involved acetonitrile to precipitate serum proteins/peptides and extract estrogens. Residues were reconstituted in 50% acetonitrile in water for injection. The detection limits for these six estrogens via UV detection ranged from 0.5 to 5 ng on‐column with a signal‐to‐noise ratio (S/N) of 10 for a 20 μL injection and via fluorescence 0.1 ng on‐column for 17‐β‐estradiol. Validation data are included for all six estrogens.
Mixed‐bed polymeric o‐nitrobenzophenone reagents for the on‐line derivatization of amines in high performance liquid chromatography
Authors: Szulc Michael E. , Krull Ira S.
Published: May 2005

Several polymer‐bound o‐nitrobenzophenone reagents containing different detector‐sensitive tags have been combined in the same reactor for the on‐line derivatization of amine samples. The formation of multiple derivatives allows numerous opportunities for quantitation from the same injection, and also allows improved identification from the retention times of the multiple derivatives. Changing the reaction conditions changes the ratio of the products formed, so that changes in the ratio of peak heights and areas can also be used for analyte identification. In this work, propylamine was derivatized in acetonitrile on‐line, precolumn. Changing the reaction conditions, of reaction time, temperature, solvent, presence of catalyst and the components of the reactor, changed the ratio of the derivatives formed. These changes in product formation with changing reaction conditions were applied to the identification and quantitation of amphetamine and methamphetamine in urine. The drugs were identified by the retention times of their derivatives, the ratio of te peak areas of the derivatives and the change in the ratios after changing reaction conditions. Each derivative was also used for quantitation of levels of spiked concentrations of amphetamine and methamphetamine, with relative errors less than 8%.
An automated method for the determination of a new potential antiepileptic agent (CGP 33101) in human plasma using high performance liquid chromatography
Authors: Brunner Linda A. , Powell Mark L.
Published: May 2005

An automated analytical method utilizing laboratory robotics has been developed and validated for quantifying concentrations of a new antiepileptic drug candidate (CGP 33101) in human plasma. The robotic system aliquots the biological sample, adds the internal standard (CGP 23901) and pH 12 buffer, extracts the compounds from the basified matrix into an organic phase (methyl‐t‐butyl ether:dichloromethane, 2:1) and concentrates the extracts for reversed‐phase, high performance liquid chromatographic (HPLC) analysis. The robotic system is directly interfaced with the HPLC system. Separation is achieved on a Hypersil 3 μm C18 column (4.6 × 50 mm) with ultraviolet detection of the analytes at 230 nm. Specificity was demonstrated by the lack of interfering peaks at the retention times for both the drug and internal standard. Recovery and reproducibility assessments indicated good accuracy (overall mean relative recovery of 102.7%) and precision (coefficient of variation of 4.4 to 7.7%) for CGP 33101 over the concentration range of 50–4000 ng/mL. The limit of quantification (LOQ) is 50 ng/mL. The method has been successfully applied to a clinical study in which normal volunteers received single oral doses of 400‐‐1200 mg of this new drug candidate.
Solid phase extraction and simultaneous high performance liquid chromatographic determination of antipyrine and its major metabolites in urine
Authors: Sarkar Mohamadi A. , March Clark , Karnes H. Thomas
Published: May 2005

A reversed phase gradient high performance liquid chromatographic method utilizing solid phase extraction has been described for the simultaneous determination of antipyrine (AP), 4‐hydroxyantipyrine (4‐OHAP), norantipyrine (NorAP) and 3‐hydroxymethylantipyrine (3‐OHMAP) in human urine after hydrolysis with β‐glucuronidase. The C‐18 sorbent cartridges were conditioned and urine samples were applied, washed with 1 × 4 mL of phosphate buffer and eluted with 3 × 100 μL of 20% v/v of acetonitrile in methylene chloride. The eluent was evaporated to dryness, reconstituted in 100 μL phosphate buffer and injected. The calibration ranges were 2.0–250 μg/mL (AP), 2.5–250 μg/mL (NorAP), 2.0–250 μg/mL (3‐OHMAP) and 5.0–500 μg/mL (4‐OHAP) with regression coefficients of 0.998 or greater. Specificity was indicated by the absence of interferences in chromatogram of blank urine from normal as well as cirrhotic patients. The average recovery was 86.7% for AP, 90.5% for NorAP, 85.2% for 4‐OHAP and 74.2% for 3‐OHMAP. The within‐assay precision as indicated by the reproducibility of the assayed spiked urine was less than 9% in all cases and the between‐assay precision was less than 12%. The method was applied to studies on antipyrine metabolism in stable cirrhotic patients. Following administration of a single oral dose of about 1000 mg to nine stable cirrhotic patients and eigh age‐matched healthy volunteers, the cumulative account excreted in the urine up to 48 h for AP and the three metabolites was comparable to other literature reports.
Robotic solid phase extraction and high performance liquid chromatographic analysis of ranitidine in serum or plasma
Authors: Lloyd Thomas L. , Perschy Teresa Benedetti , Gooding Ann E. , Tomlinson Julie J.
Published: May 2005

A fully automated assay for the analysis of ranitidine in serum and plasma, with and without an internal standard, was validated. It utilizes robotic solid phase extraction with on‐line high performance liquid chromatographic (HPLC) analysis. The ruggedness of the assay was demonstrated over a three‐year period. A Zymark Py Technology II® robotic system was used for serial processing from initial aspiration of samples from original collection containers, to final direct injection onto the on‐line HPLC system. Automated serial processing with on‐line analysis provided uniform sample history and increased productivity by freeing the chemist to analyse data and perform other tasks. The solid phase extraction efficiency was 94% throughout the assay range of 10–250 ng/mL. The coefficients of variation for within‐ and between‐day quality control samples ranged from 1 to 6% and 1 to 5%, respectively. Mean accuracy for between‐day standards and quality control results ranged from 97 to 102% of the respective theoretical concentrations.
Determination of 1,5‐Anhydroglucitol in urine by high performance liquid chromatography and an enzyme sensor
Authors: Tajima Shigeru , Hashiba Masashi , Suzuki Takayuki , Akanuma Hiroshi , Yabuuchi Masahiko
Published: May 2005

A simple high performance liquid chromatographic method combined with an enzyme sensor has been developed to measure 1,5‐anhydroglucitol in urine. The enzyme sensor consists of a hydrogen peroxide electrode and a chitosan membrane of an immobilized pyranose oxidase. As the system does not resist interfering substances, urine samples are first purified by passing them through a two‐layer column packed with (1) strongly basic anion (OH− form, the upper layer) and (2) strongly acidic (H+ form, the lower layer) exchange resins. 1,5‐Anhydroglucitol is efficiently recovered in the flow‐through fraction of the column. In this system, the minimum detectable concentration of 1,5‐anhydroglucitol is 0.1 mg/L, and the measurable range extends from 0.1 to 60 mg/L. The coefficient of variation values of the within‐day and day‐to‐day precisions are 3.0‐6.5% and and 4.4‐6.7% respectively, and there is good agreement between the results measured by our method and those obtained by the gas‐liquid chromatographic/mass spectrometric method (r=0.994). The method we have described here has been successfully used to elucidate a mechanism for the reducing 1,5‐anhydroglucitol level in the serum and plasma of patients.
High performance gel filtration using monodisperse highly cross‐linked agarose as a one‐step system for mucin purification
Authors: Parker N. , Finnie I. A. , Raouf A. H. , Ryder S. D. , Campbell B. J. , Tsai H. H. , Iddon D. , Milton J. D. , Rhodes J. M.
Published: May 2005

High performance gel filtration on monodisperse cross‐linked agarose (Superose 6) has been assessed as a system for purification of mucus glycoproteins. Comparison with the conventional two‐step purification of mucus glycoprotein by Sepharose CL4B gel filtration followed by caesium chloride density gradient centrifugation shows that purification by high performance gel filtration is at least as thorough, yielding mucin that is free from non‐mucin glycoproteins as defined by buoyant density, mobility on sodium dodecyl sulphate‐polyacrylamide gel electrophoresis and absence of Concanavalin A binding (mannose‐containing) material. This technique allows mucus glycoprotein to be purified from lyophilized crude mucin in 120 min compared with approximately 72 h using the conventional techniques. This makes the comparative study of mucus glycoprotein changes in disease states much more feasible.
Free‐solution capillary electrophoretic separation of small hydrophilic peptides
Authors: Zhang Y. K. , Chen N. , Wang L.
Published: May 2005

Small hydrophilic peptides differing in only one glycine amino acid group have been successfully separated by free‐solution capillary electrophoresis (FSCE) using phosphate buffer at acid pH. The effects of column temperature and operating current on the migration times of these small peptides have been investigated. The results from FSCE have been compared with those from reversed phase high performance liquid chromatography.
Thermal desorption‐gas chromatographic determination of ethane and pentane in breath as potential markers of lipid peroxidation
Authors: Massias L. , Postaire E. , Regnault C. , Hazebroucq G.
Published: May 2005

Formation of free radicals and lipoperoxidation occur at the onset of cellular damage. These effects are produced during normal metabolism and in pathological states. The peroxidation of polyunsaturated fatty acids, i.e. linoleic acid and linolenic acid, which are both cellular membrane compounds, induces ethane and pentane formation in pulmonary air exhalation. These two volatile hydrocarbons can be considered as potential lipoperoxidation markers. Methodological difficulties limit the use of these gases for assessment of free oxygen radical activity but we have developed and validated a non‐invasive technique. A study was performed with ten healthy volunteers.
Determination of free and total proline and hydroxyproline in plasma and tissue samples by gas chromatography with flame photometric detection
Authors: Kataoka Hiroyuki , Nagao Katsuyuki , Makita Masami
Published: May 2005

A selective and sensitive method for the determination of free and total proline (Pro) and 4‐hydroxyproline (Hyp) by gas chromatography (GC) was developed. For free Pro and Hyp analysis, plasma and tissue homogenate were extracted with methanol. For total Pro and Hyp analysis, these samples were hydrolysed in 6 M HCI. After removal of primary amino compounds by the reaction with o‐phthaldialdehyde, Pro and Hyp in methanol extract and acid hydrolysate were converted into their N‐dimethylthiophosphoryl methyl ester derivatives and then determined by GC with flame photometric detection using a DB‐5 capillary column. This method was successfully applied to small samples without prior clean‐up, and Pro and Hyp in these samples could be analysed without any influence from coexisting substances. Overall recoveries of Pro and Hyp added to plasma and tissue samples were 92–106%. The analytical results of free and total Pro and Hyp in human plasma and mouse tissue samples are presented.
Metabolic fate of fenitrothion in liver, kidney and brain of rat
Authors: Kumar Reena , Roy Shalini , Rishi Ritu , Sharma C. B.
Published: May 2005

The metabolic fate of fenitrothion, O,O‐dimethyl‐O‐(3‐methyl‐4‐nitro phenyl) phosphorothioate, was investigated in rat tissues during the first 24 hours following the intramusculer administration of the pesticide in the animal. High performance liquid chromatography (HPLC) of the pesticide and its metabolites formed in liver, kidney and brain showed the time‐dependent sequential conversion of the pesticide into three major metabolites. These metabolites were separated and purified to homogeneity by HPLC and characterized by IR spectroscopy as O,O‐dimethyl‐O‐)3‐methyl‐4‐aminophenyl) phosphorothioate (metabolite I), O,O‐dimethyl phosphorothioate (metabolite II), and O,O‐dimethylphosphate (metabolite III). These results indicated reduction of the nitro group in fenitrothion aas the first step, followed by the hydrolytic cleavage of the P‐O‐aryl bond in metabolite I and the oxidative desulphurylation of metabolite II. Fenitrothion was found to induce ultrastructural changes in liver cells especially after 12 h exposure nuclear membrane was completely distorted, nuclear intactness was totally lost and smooth endoplasmic reticulum and Golgi apparatus were abnormally enlarged. In 24 h, however, the regeneration of the nuclear material was observed.
Determination of nitrite by high‐performance liquid chromatography system with electrochemical detector: Measurement of nitric oxide synthase activity in rat cerebellum cytosol
Authors: Kaku Shinsuke , Tanaka Makoto , Muramatsu Makoto , Otomo Susumu
Published: May 2005

A simple and sensitive assay method for NO synthase activity is described. Using glassy carbon as electrode and 30% methanol solution with 10 mM NH4Cl as mobile phase, NO can be measured without disturbing ECD‐detectable substance in NO synthase assay mixture. The NO production in the assay mixture of rat cerebellum NO synthase increased with protein and in a time‐dependent manner. The Km value for the substrate, L‐arginine, was 1.25 μM. The enzyme activity was inhibited in a concentration‐dependent manner by a NO synthase inhibitor, NNA. The Ki value for NNA was 0.166 ± 0.060 μM. This ECD–HPLC method for determining NO synthase activity has advantages compared with the diazo‐coupling method of the Greiss reagent and the isotopic method in which the conversion of the substrate, [14C]L‐arginine, to the product, [14C]L‐citrulline is measured; it is simple, sensitive and is convenient for studying the NO synthase activity with various compounds as the substrate.
Sensitive determination of (−)‐isoproterenol and (−)‐(R)‐1‐(3,4‐dihydroxyphenyl)‐2‐[(3,4‐dimethoxyphenethyl)amino] ethanol (T‐0509), a cardiotonic agent, in rat plasma utilizing a fully automated catecholamine analyser
Authors: Prados Pablo , Higashidate Sakae , Imai Kazuhiro , Sato Yoji , Nagao Taku
Published: May 2005

(−)‐Isoproterenol (ISO), used as bronchodilator, and (−)‐(R)‐1‐(3,4‐dihydroxyphenyl)‐2‐ [(3,4‐dimethoxyphenethyl)amino]ethanol (T‐0509), a new cardiotonic agent, were determined in rat plasma (only 25 μL) using a fully automated catecholamine (CA) analyser that includes in‐line pre‐extraction of CAs, coupled with an HPLC–ethylenediamine condensation reaction and a peroxyoxalate chemiluminescence detection system (HPLC–ED–PO‐CL). The chromatographic conditions were: precolumn, SERUMOUT‐CEX; buffer for delivering CAs in the precolumn, 10 mM potassium phosphate buffer (pH 7.5)/ethanol (92 + 8, v/v) (1 mL/min); precolumn clean‐up solution, 4% phosphoric acid/acetonitrile (50 + 50, v/v) (1 mL/min); analytical column, TSKgel ODS‐80Ts, 250 × 4.6 mm i.d.; eluent, 75 mM potassium acetate buffer (pH 3.2)/50 mM potassium phosphate buffer (pH 3.2)/acetonitrile (83.6 + 4.4 + 12, v/v/v for ISO and 76 + 4 + 20 v/v/v for T‐0509) (1 mL/min); fluorogenic reagent solution, 105 mM ED and 175 mM imidazole in acetonitrile/ethanol (90 + 10, v/v) (0.25 mL/min); chemiluminogenic reaction solution, 0.25 mM TDPO, 150 mM hydrogen peroxide and 110 mM TFA in dioxane/ethyl acetate (50 + 50, v/v) (1.4 mL/min). Colterol, (±)‐1‐(3,4‐dihydroxyphenyl)‐2‐[(1,1‐dimethyl)ethylamino]ethanol, (±)‐N‐t‐butylnorepinephrine was used as an internal standard (IS) for ISO, and (±)‐1‐(3,4‐dihydroxyphenyl)‐2‐ [(3,4,5‐trimethoxyphenethyl)amino]ethanol, (±)‐(3,4,5‐trimethoxyphenethylaminomethyl)‐3,4‐dihydroxybenzylalcohol (T‐1583) for T‐0509. The detection limits for ISO and T‐0509 were 1.3 and 0.9 fmol on injection, respectively.
Isolation and purification of three glucuronides of antipyrine. Proposal for an original analytical method for quantitation of sulpho‐ and glucuroconjugated metabolites
Authors: Palette C. , Cordonnier P. , Tillequin F. , Koch M. , Advenier C. , Pays M.
Published: May 2005

The determination of the concentrations of antipyrine metabolites in biological fluids is hampered by the difficulty in obtaining pure conjugated compounds to be used as standards. Most authors have proposed determination of total forms by high performance liquid chromatography (HPLC) after deconjugation of these metabolites using chemical or enzymatic hydrolysis. Up to now there is no satisfactory hydrolysis method for the study of all antipyrine metabolites. The situation is further complicated by the fact that the deconjugated metabolites are highly unstable whichever technique is being used. Because of the lack of stability of all these molecules it has been necessary to isolate the glucuroconjugated compounds from urine. We describe a method which allows us to obtain highly purified glucuroconjugated metabolites of antipyrine. Sulphoconjugated compounds have been synthesized previously. We are thus able to propose a chromatographic procedure which allows us to determine simultaneously all stable phase I and phase II metabolites of antipyrine in biological fluids without any step of extraction. This analytical technique allows us to study the activity of the different isoenzymes implicated in the metabolism of antipyrine.
Human hemofiltrate as a source of circulating bioactive peptides: Determination of amino acids, peptides and proteins
Authors: Schepky Andreas G. , Bensch Klaus W. , Schulz‐Knappe Peter , Forssmann Wolf‐Georg
Published: May 2005

Human hemofiltrate (HF) was evaluated regarding its content of free amino acids, proteins, and regulatory peptides. Human HF was obtained from patients with end stage renal disease (ESRD). In contrast to plasma it mainly contains low and middle weight molecules ⩽45 kDa. The content of free amino acids, peptides, and proteins in pooled filtrate was determined by amino acid analysis using ortho‐phtaldialdehyde/fluorenyl methyl chloroformate (OPA/FMOC) precolumn derivatization. The total amount of peptides and proteins in human HF is 49.4 mg/L (n = 8). The levels of all free amino acids (230 mg/L) and the concentration of some regulatory peptides like insulin, endothelin, gastrin, vasopressin and angiotensin II were similar compared with blood plasma. The amount of peptides and proteins detected in the filtrate was around 0.07% of total plasma proteins, and consisted mainly of smaller proteins and peptides as shown by size exclusion chromatography (SEC). The presence of large proteins in plasma is reduced by a factor of 1500 after filtration. We conclude that human hemofiltrate is a valuable source for the large‐scale extraction of regulatory peptides.
Determination of sulphur amino acids by gas chromatography with flame photometric detection
Authors: Kataoka Hiroyuki , Tanaka Hirofumi , Fujimoto Ayumi , Noguchi Ikuko , Makita Masami
Published: May 2005

A selective and sensitive method has been developed for the determination of sulphur amino acids by gas chromatography (GC). Sulphur amino acids were converted into their N(S)‐isopropoxycarbonyl methyl ester derivatives and measured by GC with flame photometric detection using a DB‐17 capillary column. The derivatives were sufficiently volatile and stable to give single symmetrical peaks. The detection limits of sulphur amino acids were ca. 0.5–1 pmol per injection, and the calibration curves were linear in the range 0.5–1 nmol for each sulphur amino acid. This method was successfully applied to small urine samples without prior cleanup, and sulphur amino acids in these samples could be analysed without any influence from coexisting substances. Overall recoveries of sulphur amino acids added to urine samples were 85–113%. The analytical results of free sulphur amino acid contents in urine samples of normal subjects are presented.
Carbonic anhydrase‐immobilized precolumn for selective on‐line sample pretreatment in high‐performance liquid chromatographic determination of certain sulphonamide drugs
Authors: Ohta Takafumi , Takamiya Ittetsu , Takitani Shoji
Published: May 2005

Carbonic anhydrase (CA)‐immobilized aminopropyl silica precolumn was developed for direct injection determination of certain sulphonamide drugs in biological fluids by column‐switching (CS) high‐performance liquid chromatography. Under the optimized conditions, only the sulphonamide drugs with an unsubstituted sulphonamide group were retained on the CA precolumn and separated on a reversed‐phase analytical column. Of these, the retention of hydrochlorothiazide (HCT), chlorothiazide, acetazolamide, furosemide (FS) and chlorthalidone was almost quantitative. The peak area of HCT was proportional to the concentration in the range of 1–100 nmol/mL with relative standard deviations of 3.7% (5 nmol/mL) and 0.7% (100 nmol/mL).

This CS system was applied to urine and plasma samples spiked with HCT and FS. Endogenous components of these were effectively removed, and HCT and FS were selectively retained on the CA precolumn. Almost quantitative recoveries and reproducibility were obtained.
Quantitative determination of 21‐hydroxy‐deflazacort in human plasma using gradient semi‐microbore liquid chromatography
Authors: Reynolds Donald L. , Burmaster Steve D. , Eichmeier Larry S.
Published: May 2005

A sensitive and selective liquid chromatographic procedure to quantitate the deflazacort metabolite 21‐hydroxy‐deflazacort (DF‐21OH) in human plasma was developed and validated. DF‐21OH and fludrocortisone acetate (internal standard, IS) were isolated from human plasma (2 mL) by solid‐phase extraction onto C‐18 cartridges. Potential interferences were selectively removed and analytes were eluted with ethyl acetate. Following evaporation, the residue was reconstituted for HPLC analysis. Separation was achieved by gradient elution using a 5 μm YMC Basic column (2.0 × 100 mm) with mobile phases consisting of 20% methanol and 50% acetonitrile in 50 mM phosphate buffer (pH 3) at a temperature of 50°C. Flow rate was maintained at 0.3 mL/min., and analytes were quantified spectrophotometrically at 246 nm.

The assay was validated over the range 1.0 to 500 ng/mL DF‐21OH. Calibration curves were prepared using a weighted (1/concentration) nonlinear quadratic regression algorithm. Peak‐height ratios were proportional to the amount of DF‐21OH added to plasma. Assay precision (%RSD) ranged from 4.2 to 11%, with a corresponding assay accuracy (% relative error) of ±2.8%. Absolute recovery of DF‐21OH from plasma was 78–86% over the concentration range. The minimum quantitation limit was 1.0 ng/mL.
Solid phase extraction and high performance liquid chromatographic determination of dobutamine in plasma of dialysed patients
Authors: Leflour C. , Dine T. , Luyckx M. , Brunet C. , Gressier B. , Cazin M. , Robert H. , Durocher A. , Cazin J. C.
Published: May 2005

An isocratic reversed‐phase high performance liquid chromatographic method has been developed for the determination of dobutamine in the plasma of dialysed patients. A solid phase extraction method with a Sep‐Pak C18 cartridge was used to isolate the drug and isoxsuprine (internal standard) from plasma. The separation was carried out on an ODS‐Hypersil column with 0.1 M phosphate buffer: acetonitrile: methanol (72 : 20 : 8 v/v/v) as the mobile phase. The recovery of dobutamine added to plasma by the extraction procedure was 87 ± 2.3% (mean ± SD). The accuracy and reproducibility of the method were within acceptable limits over the concentration range 0–1000 ng/ mL. Quantification was by fluorescence detection at 275 nm excitation and 310 nm emission wavelengths with a detection limit of 5 ng/ mL for dobutamine. This procedure was applied to ascertain the pharamacokinetics of dobutamine infusion in nine patients with cardiogenic shock and end‐stage renal disease undergoing haemodialysis.
Optical purity determination of thyroxine enantiomers in bulk materials by chiral thin layer chromatography
Authors: Aboul‐Enein Hassan Y. , Serignese Vince
Published: May 2005

L‐thyroxine and D‐thyroxine were separated on ligand exchange chiral thin layer chromatographic plates, using a solvent system consisting of acetonitrile:methanol:water 60:15:15 v/v, at a wavelength of 254 nm. The methodology, chiral recognition mechanism (s) involved and its application are discussed.
High‐performance liquid chromatographic assay for the quantitation of nadolol in human plasma using fluorescence detection
Authors: Srinivas Nuggehally R. , Shyu Wen Chyi , Shah Vinod R. , Campbell Dale A. , Barbhaiya Rashmi H.
Published: May 2005

A rapid and sensitive HPLC‐fluorescence assay was developed and validated for the determination of nadolol, a β‐blocker, in human plasma. Nadolol and the internal standard (desmethyl nadolol) were extracted from alkalinized plasma into methyl‐tert.‐butyl ether. The organic solvent was evaporated under nitrogen at 40°C. The residue was reconstituted in the mobile phase and injected on to a C18 silica column (25 cm × 4.6 mm i.d.) at a flow rate of 1.4 mL/min. The mobile phase was 0.05 M monobasic ammonium phosphate (pH 4.2) and acetonitrile (84:16, v/v). Fluorimetric detection was performed at excitation 230 nm and emission 330 nm. The nominal retention times were 3.3 and 4.3 min for the internal standard and nadolol, respectively. The lower limit of quantitation was 5 ng/mL and linearity (R2 ≥ 0.994) of the standard curve was demonstrated between 5 and 500 ng/mL. The analysis of quality control (QC) samples at 60, 200 and 400 ng/mL resulted in precision estimates ⩽7.0% relative standard deviation (RSD) for the inter‐assay and ⩽6.3% RSD for intra‐assay. The predicted concentrations of the QC samples deviated <10% from the nominal values. The extraction recovery of nadolol from human plasma was 64%. Nadolol was stable in human plasma at −20°C for at least 5 months and for at least three freeze‐thaw cycles. Nadolol and the internal standard were stable in the autosampler at 5°C for at least 40 h. Overall, the assay was accurate, precise, sensitive, specific and reproducible for the analysis of nadolol in plasma. The validated assay procedure was applied to study the pharmacokinetics of nadolol after administration of single and multiple doses to human subjects.
A sensitive high‐performance liquid chromatographic assay for the cholecystokinin‐b antagonist, CI‐988, in human and monkey plasma
Authors: Hinton Joanna P. , Jennings Kathleen , Johnson Elvin L. , Wright D. Scott
Published: May 2005

A sensitive and specific high‐performance liquid chromatographic assay for the non‐peptide cholecystokinin subtype B receptor antagonist, CI‐988, in human and cynomolgus monkey plasma has been developed and validated. The method involves isolation of CI‐988 and internal standard by batch robotic solid phase extraction with a C18 cartridge, liquid chromatographic separation on a C18 column and quantitation by fluorescence detection. The human plasma assay is linear from 0.25 to 500 ng/mL for a 1.00‐mL plasma aliquot. Assay precision for CI‐988 based on human plasma quality control samples was within ±7.2% relative standard deviation with an accuracy of ±5.6% relative error. The monkey plasma assay is linear from 1.00 to 250 ng/mL for a 0.500‐mL plasma aliquot. Assay precision based on monkey plasma quality control samples was within ±11.0% relative standard deviation with an accuracy of ±2.6% relative error.
Isolation and characterization of new porphyrin metabolites in human porphyria cutanea tarda and in rats treated with hexachlorobenzene by HPTLC, HPLC and liquid secondary ion mass spectrometry
Authors: Luo Jinli , Lim Chang Kee
Published: May 2005

Porphyrin metabolisms in human porphyria cutanea tarda (PCT) and in rats treated with hexachlorobenzene (HCB) have been studied in detail by high performance thin layer chromatography (HPTLC), high performance liquid chromatography (HPLC) and liquid secondary ion mass spectrometry (LSIMS). The analyses of porphyrin metabolites in the urine, faeces and liver biopsies of patients with PCT have shown that apart from uroporphyrin I and III and their expected decarboxylation intermediates and products, a complex mixture of many other porphyrins are present. The new porphyrins identified are: meso‐hydroxyuroporphyrin III, β‐hydroxypropionic acid uroporphyrin III, hydroxyacetic acid uroporphyrin III, peroxyacetic acid uroporphyrin III, β‐hydroxypropionic acid heptacarboxylic acid porphyrin III, hydroxyacetic acid heptacarboxylic porphyrin III and peroxyacetic acid pentacarboxylic porphyrin III.
Determination of guanase activity in normal and pathological sera by high‐performance liquid chromatography
Authors: Canepari S. , Castellano P. , Cernia E. , Girelli A. M. , Bozza A.
Published: May 2005

A simple HPLC assay for serum guanase based on the direct determination of enzymatically formed xanthine was applied to normal and pathological sera. The procedure is sensitive, precise (CV below 5%) and suitable for routine purposes, and the method requires only 50 μL of sample. Using this method the reference range as determined from the sera of 40 healthy adult controls is 0–1.1 U/L. In patients with various liver diseases serum guanase activities were found to be increased 5‐ to 50‐fold compared with the normal mean value.
Determination of salicylic acid in plasma by gas chromatogrphy using steam as carrier gas
Authors: Li Yun‐Hua , Ren Zi‐Hong , Li Xiu‐Lu , Cai Yi‐Feng , Zhai Zheng‐Xing
Published: May 2005

A gas Chromatogrphy method using steam as the carrier gas was established for the determination of salicylic acid in plasma. A 0.5 mL plasma sample was mixed with 0.5 mL of 6N HCl and then extracted with 2 mL of dichloromethane. The dichloromethane was evaporated to dryness and the residue was redissolved in 0.5 mL of water and then analysed by gas Chromatogrphy using steam as the carrying gas. The result was calculated by external standardization.
TLC characterization of liposomes containing D‐myo‐inositol derivatives
Authors: Brailoiu Eugen , Huhurez Gabi , Slatineanu Sebastian , Filipeanu Catalin M. , Costuleanu Marcel , Branisteanu Dimitrie D.
Published: May 2005

The thin‐layer chromatographic (TLC) behaviour of liposomes containig inositol phosphates (IPs) was studied. The liposomes contained different concentrations of D‐myo‐inositol 1, 4, 5‐trisphosphate (IP3), D‐myo‐inositol 1, 2, 6‐trisphosphate (α‐trinositol, PP 56, a novel Perstorp Pharma derivative), D‐myo‐inositol 1, 3, 4, 5‐tetrakisphosphate (IP4), D‐myo‐inositol 1, 3, 4, 5, 6‐pentakisphosphate (IP5) and D‐myo‐inositol 1, 2, 3, 4, 5, 6‐hexakisphosphate (IP6). Migration of all liposome batches was compared to that of control liposomes (containing only triple‐distilled water), and to that of free phosphatidylcholine (PC); the same amount of lipid was used in all situations.

Thin‐layer chromatography was performed on silica gel as adsorbent. As solvent we used an n‐buthanol: ethanol: water mixture in a 4:3:3 volume ratio. Significant differences were found between PC and all liposome batches, as well as between control liposomes and the ones containing IP3, α‐trinositol, IP4, or IP5, in various concentrations. Liposomes containing IP6 migrate completely differently compared not only to phosphatidylcholine and control liposomes, but also to the ones containing other IPs (<10−3 M). Unlike the other IPs studied, liposome‐entrapped IP6 elicits dose‐independent contractions of the isolated rat aorta. This suggests that liposomes loaded with IP6 undergo, during or after their preparation, physico‐chemical alterations that eventually change their drug‐delivery capacity.
Availabilty of phenylisothiocyanate for the amino acid sequence/configuration determination of peptides containing D/L‐amino acids
Authors: Imai Kazuhiro , Matsunaga Hirokazu , Santa Tomofumi , Homma Hiroshi
Published: May 2005

A potential use for phenylisothiocyanate (PITC), the most popular Edman reagent, is presented for analysis of the amino acid sequence and configuration in peptides containing D/L‐amino acids. After derivatization with PITC of the N‐terminal amino acid (L‐Tyr) of an enkephalin analogue, [D‐Ala2, D‐Leu5]‐enkephalin, followed by cleavage/cyclization with trifluoroacetic acid at 50°C for 5 min, the liberated 2‐anilino‐5‐thiazolinone‐L‐Tyr (ATZ‐L‐Tyr) was further treated with 20% aqueous trifluoroacetic acid at 50°C for 10 min. The resultant phenylthiohydantoin (PTH)‐L‐Tyr was then separated on a chiral stationary phase (a penylcarbamoylated cyclodextrin column), retaining its configuration. The residual sequence and configurations of the peptide (D‐Ala‐Gly‐L‐Phe‐D‐Leu) were also determined by separating the corresponding PTH‐D‐ or L‐amino acids on chiral columns. This method may be applicable to an automatic Edman sequence analyzer for the configuration determination of peptides containing D/L‐amino acids.
Red‐band resonance raman spectroscopy of chlorophyll cofactors in photosynthetic proteins
Authors: Lutz Marc
Published: May 2005

Only recently have various methodological approaches been successful in producing Raman spectra of chlorophylls resonantly with their lowest singlet electronic transitions (Qy bands). The information content of these new approaches and their impact on current research on photosynthetic proteins are discussed. It is shown that resonance methods can contribute very valuable data on the dynamics of the primary photophysical events, while preresonance methods are yielding many new data on the structural bases of these events at atomic level. © 1995 John Wiley & Sons, Inc.
Serious case reviews: learning to use expertise
Authors: Brandon Marian , Dodsworth Jane , Rumball Daphne
Published: May 2005

While undertaking an analysis of 20 serious case reviews for the Welsh Assembly Government, the authors became aware of important, largely unexplored, practice issues with implications for training, service delivery and the role of the lead professional. The reviews were studied using a ‘layered reading’ methodology to ensure that emerging themes were grounded entirely in the reports. In looking beyond the familiar recurring themes and practice failings, findings emerged about the use of expertise in its widest sense. The need for more skilled use of expertise was identified, from external specialists and child protection specialists, and from children, siblings and parents. Professionals from agencies who contribute less routinely to the process of safeguarding children should be consulted as experts and be empowered to play a fuller part. Consultation could often be used prior to, or in place of referral. The barrier to the collation and analysis of relevant information often appeared to be a failure to recognize and understand expertise rather than a lack of communication as often postulated in review reports. Skilled use of expertise and consultation in a coordinated manner could result in more rigorous assessments and promote greater professional trust, confidence and challenge. Copyright © 2005 John Wiley & Sons, Ltd.
Origins of the differences in function of rat adrenal zona glomerulosa cells incubated as intact tissue and as collagenase‐prepared cell suspensions
Authors: Raven P. W. , McCredie E. , McAuley M. , Vinson G. P.
Published: May 2005

While in vitro incubation of dispersed cell preparations of adrenal cell types has been widely used as an experimental model, few studies have addressed the possibility that the enzymic and mechanical treatments involved may affect tissue functions. Using rat adrenal whole capsule tissue, consisting of glomerulosa cells still attached to the connective tissue capsule together with some fasciculata cells, and dispersed glomerulosa cell preparations formed by a variety of enzymic and incubation treatments, striking differences have been demonstrated between the functions of the various preparations in vitro. Under ACTH stimulation, whole capsules produced (ng per pair ± s.e.) 405 ± 35 ng aldosterone, 650 ± 60 ng 18‐hydroxycorticosterone (18‐OH‐B) and 850 ± 90 ng corticosterone. In cells dispersed by collagenase incubation followed by repeated pipetting and filtration, aldosterone and 18‐OH‐B yields under ACTH stimulation fell to values less than 10% of those produced by whole tissue, whereas corticosterone values were unchanged. Omitting the filtration step gave a less well marked decline in aldosterone and 18‐OH‐B to 50% of intact tissue values. When the tissue was not dispersed after collagenase incubation, aldosterone and 18‐OH‐B outputs were similar in the two preparations. The decline in aldosterone and 18‐OH‐B is not attributable to loss in cell–cell contact alone, since short term culture of collagenase dispersed cells on contracting collagen discs did not restore the capacity to produce these steroids, and a decline in their output also occurred in similar culture of intact capsule tissue. In acute incubations, hyaluronidase had similar effects to collagenase, whereas trypsin, papain and a bacterial protease evoked aldosterone release during the preincubation period, but did not affect subsequent yields of aldosterone and 18‐OH‐B in incubations of dispersed (but not filtered tissue) in the presence of ACTH. Chymo‐trypsin had no effect on preincubation but eliminated subsequent response to ACTH in all incubation conditions. Together with previously published data on the effects of trypsin, the results support the view that in intact rat adrenal glomerulosa tissue, aldosterone and 18‐OH‐B are sequestered into intracellular stores in the form of novel steroid‐protein complexes. These are hydrolysed by trypsin and other preoteases with consequent release of steroid, but are virtually eliminated by conventional methods of cell suspension preparations, using collagenase preincubation with subsequent mechanical dispersal and filtration.