bluuebunny/crossref_metadata_2025 huggingface dataset:
The general theory of assembly and its application to the production of moulds and other tools
Authors: Čatić I. , Zelić B. , Kokolek R.
Published: May 2005

The basis of the General Theory of Assembly (GTA) and its application to the production of moulds and other tools is described. The terms of accuracy and precision are defined. Existing methods of calculating tolerances are explained and their significance summarised. The results of a production run on a small series of homogeneous components are presented. The assembly of two‐ and multi‐component fits is explained by examples. Average improvements in the tolerances of fits from 2 to 8.3 times are presented. Finally, an attempt is made to answer the question why GTA has not been used more in toolmaking in spite of its simplicity and applicability without any additional investment in equipment.
Rethinking the injection molding process with thermosets and rubber
Authors: Buschhaus F. , Benfer W.
Published: May 2005

To optimize the processing, a process model has to be developed. To be able to use a model of this kind, we must also have material data (thermodynamic material data, viscosity, processing time) and must install sensors on the machine and in the mould.

The flow properties of thermosets/elastomers under processing conditions is determined not only by pressure, temperature and throughput, but also to a considerable extent by the boundary conditions.

At a given throughput, it is possible to calculate the pressure loss for passing through a runner system by dividing up the flow into a plug flow and a boundary shear flow. This takes into account the pronounced temperature profile, which brings about a low‐viscosity outer layer and a high‐viscosity core.

With thermosets/elastomers, the compounding process influences the flow properties to a considerable extent. This means that the continuous monitoring of the material state must be included in the process control. This monitoring can be carried out during the mould filling period. Since the mould is filled over a wide area at constant velocity, the gate can be regarded as a “rheometer” and the pressure loss between two pressure sensors installed in the gate used as a measure of the viscosity.

Starting from this measurement of the flow properties, moulding properties such as weight and surface texture are influenced in the compression phase. The form of the pressure build‐up in the compression phase affects the flash formation; this pressure buildup should, in fact, be carried out in such a way that the pressure maximum is not reached until the outer layers have passed through their viscosity minimum.

After the compression, the moulding is crosslinked in the crosslinking phase far enough for it to be demoulded or for the moulding properties, which are connected with a given crosslinking profile over the cross‐section, are reached.
The quinlan decision: Problems and legislative alternatives
Authors: Beresford H. Richard
Published: May 2005

In the Quinlan decision, a state supreme court authorized remval of a respirator from a person in a chronic vegetative state provided that the attending physicians and an “ethics committee” agreed there was no reasonable possibility of her regaining cognition or sapience. While the decision involves an unusual factual situation and poses some troublesome technical and doctrinal problems, it underscores the need for formal legal standards to guide physicians and families of severely brain‐damaged patients.

Legislation seems preferable to a series of court decisions. The legislative process allows the public to express views on controversial ethical and religious questions. It also permits extensive deliberations about medical and scientific data relating to brain damage and its prognosis. Legislative alternatives include expanding the concept of brain death, redefining homicide, allocating to physicians and families legally unreviewable discretion to decide on levels of care, mandating procedures for implementing a desired reduction of care, and approving the use of a “living will” as a means of requiring physicians to withdraw or withhold treatment.
Sarcoidosis, galactorrhea, and amenorrhea: 2 Autopsy cases, 1 with chiari‐frommel syndrome
Authors: Brust John C. M. , Rhee Richard S. , Plank Charles R. , Newmark Michael , Felton Charles P. , Lewis Linda D.
Published: May 2005

Two women with amenorrhea and galactorrhea, 1 with Chiari‐Frommel syndrome, died of cerebral sarcoidosis. At autopsy in both cases, sarcoid granulomas were numerous in the hypothalamus, but the pituitary appeared normal and did not contain increased prolactin as determined by immunoperoxidase technique. These are the first autopsy reports of sarcoidosis with galactorrhea and of Chiari‐Frommel syndrome in the absence of pituitary adenoma.
Recovery of cognition after prolonged vegetative state
Authors: Rosenberg Gary A. , Johnson Stephen F. , Brenner Richard P.
Published: May 2005

A 43‐year‐old man remained in a vegetative state for one and one‐half years after resuscitation from cardiac arrest. During the vegetative period neurological examinations provided little indication of recovery. Initially, his EEG showed activity of delta and theta frequencies. After eighteen months intellectual functions partially recoverd; his verbal WAIS score is now 100, although he remains severely physically disabled. The usual prognostic criteria did not predict the cognitive outcome in this patient with prolonged medical coma.
Subcellular distribution of rat pituitary homogenates by poly(ethylene glycol)‐dextran countercurrent partitioning
Authors: De Marco Luiz , Morris Wesley B. , Mashiter Keith , Peters Timothy J.
Published: May 2005

Rat pituitary homogenates were subjected to two phase countercurrent partition in a poly(ethylene glycol)–dextran mixture using a simple apparatus with enhanced gravity to facilitate the phase separations. Assay of the fractions for organelle marker enzymes and prolactin after 17 transfers showed similar distributions for endoplasmic reticulum, lysosomes, prolactin granules and plasma membrane at the lowest dextran concentrations. Increasing the dextran concentrations had a differential effect on the various organelles. Excellent resolution of endoplasmic reticulum from the other organelles was obtained and marked organelle heterogeneity was demonstrated. Two‐phase countercurrent partition thus offers an alternative approach to the subcellular fractionation of pituitary homogenates and should prove useful in separating endoplasmic reticulum from plasma membrane and other cell components.
A rapid and reliable method for extraction and assay of tricyclic antidepressant drugs in serum or plasma
Authors: Roberts G. M. , Hann C. S.
Published: May 2005

Because of their narrow therapeutic ranges and differing patient tolerances, the monitoring of tricyclic antidepressants (TADs) commonly prescribed for control of endogenous depression is highly desirable for the satisfactory treatment of patients. The majority of methods for analysis of TADs require multiple extractions from plasma with organic solvents, accompanied by obligatory centrifugation steps. We describe a simple procedure for the rapid extraction of serum TADs, with high recovery of the analyte drugs. It involves sample preparation using Analytichem ‘BOND‐ELUT’ C‐2 reverse phase columns, and analysis by gas chromatography using a nitrogen‐phosphorus detector. The method offers a quick, inexpensive and low‐maintenance assay which is applicable to all the common tricyclic class drugs.
Paper chromatography of urinary amino acids. A 30 year survey of dietary influences on the normal pattern, and patients' results
Authors: Mattingley Joan M.
Published: May 2005

In the clinical laboratory, paper chromatography is still the most useful, simple, inexpensive procedure for initial identification of abnormalities of amino acid excretion. The results of its use for more than 8000 paediatric and adult renal patients is surveyed. Nonspecific generalized aminoaciduria was the most frequent abnormality found, comprising some 70% of abnormal results, with cystine‐lysinuria the next most common. The identification of the abnormal excretory pattern of amino acids as distinct from the normal was complicated by the effects of the New Zealand diet. In particular, valine, citrulline, hydroxyproline and glutamic acid are found in considerable amounts as part of the normal pattern. Their dietary origin is discussed. Varying mixtures of monosaccharides and disaccharides occurred in association with a range of amino acid patterns.
Simple determination of forphenicinol in human plasma and erythrocytes by HPLC with native fluorescence detection
Authors: Kai Masaaki , Tamura Kazuhiko , Ohno Masahiro , Ohkura Yosuke
Published: May 2005

A high performance liquid chromatographic method is described for monitoring forphenicinol, a possible therapeutic drug for cancer and muscular dystrophy, in human plasma and erythrocytes. Forphenicinol in the deproteinized samples was separated from interfering biogenic substances on an aminopropyl‐bonded silica (Unicil NH2) column within 10 minutes with isocratic elution, and determined with fluorescence detection. The detection limits for forphenicinol in plasma and erythrocytes are 65 pmol (12.8 ng)/ml and 160 pmol (31.5 ng)/ml, respectively, corresponding to 2 pmol each in a 100 μl injection volume. The method is very simple, and sensitive enough to permit the quantification of forphenicinol in the blood samples from man dosed with forphenicinol.
Opioid peptides in the cerebrospinal fluid of Alzheimer patients
Authors: Muhlbauer Michael , Metcalf James C. , Robertson James T. , Fridland Genevieve , Desiderio Dominic M.
Published: May 2005

Endogenous opioid receptoractive peptides in the cerebrospinal fluid (CSF) of human controls and in those patients diagnosed as having senile dementia of the Alzheimer's type (SDAT) are measured with a radioreceptorassay following HPLC separation. [3H]Etorphine is the ligand used to detect in the HPLC fractions the presence of those endogenous peptides that preferentially interact with several opioid receptors. The RRA uses a receptor‐rich P2 fraction extracted from a canine limbic system. The total opioid peptide content found in the HPLC fractions 6–20 (to avoid salts in fractions 1–5) of SDAT CSF (383 ± 187 pmol ME‐equivalents per ml CSF) is significantly higher than the corresponding total from patients with no known neurological disorders (89.1 ± 46.3 pmol ME‐equivalents per ml).
HPLC of trazodone in serum after microscale protein precipitation
Authors: Lam Stanley , Boselli Lucia
Published: May 2005

Trazodone, an anti‐depressant medication, is found in serum in the 500–1000 ng/mL range in patients taking therapeutic doses. Because of this relatively high concentration, it has been possible to devise an HPLC assay system using the rapid, convenient microscale procedure described previously by Lam et al. (Clin. Chem. 26, 963 1980) to prepare the sample for chromatography. To 0.1 mL serum were added 0.1 mL acetonitrile and 10 μL of 10% zinc sulfate in water. The mixture was centrifuged and 50 μL of the clear supernatant was injected into a reversed‐phase column which was eluted with 65% 0.05 M potassium phosphate−35% acetonitrile, with detection by ultraviolet absorbance at 210 nm. The trazodone elutes in 6 min, clearly resolved from endogenous interferences. The recovery of trazodone added to serum was better than 90%. Peak height was proportional to concentrations in the serum sample from 125 ng/mL to 3000 ng/mL.
Reduction of quinones with zinc metal in the presence of zinc ions: Application of post‐column reactor for the fluorometric detection of vitamin K compounds
Authors: Haroon Y. , Bacon D. S. , Sadowski J. A.
Published: May 2005

A simple and effective chemical method has been developed for quantitatively reducing quinones, which is based on their reaction with zinc metal and zinc ions. Comparison of this method with conventional electrochemical reduction revealed the chemical method to be considerably superior. The reduction of quinones to their corresponding hydroquinones was verified by ultraviolet spectrophotometry. The reduction methodology has been applied to derivatize phylloquinone and its metabolite, namely phylloquinone 2,3‐epoxide, ‘on‐line’, with subsequent fluorometric detection of the generated hydroquinones.
Detection and identification of three thioether conjugates of 2‐hydroxyacetanilide by liquid chromatography/electrochemistry
Authors: Rottero Anthony E. , Kissinger Peter
Published: May 2005

The three 2‐mercaptoethanol (ME) thioether conjugates of 2‐hydroxyacetanilide (2HAA) are detected and identified using liquid chromatography/electrochemistry (LC/EC). The conjugates are produced either by incubations of 2HAA with liver microsomes in the presence of NADPH and ME or by chemically oxidizing a solution of 2HAA and allowing it to react with ME. Identification of the three isomers is based on retention time and comparison of the electrochemical behavior of model compounds.

Production of the three isomers by microsomal protein occurs only in the presence of NADPH and shows that 2HAA proceeds through a cytochrome P‐450 mediated pathway to a reactive intermediate similar to acetaminophen, possibly N‐acetyl‐o‐quinoneimine.

The relative percentages of each isomer produced were calculated as the percentage of the total peak areas of the isomers. Compound A (2HAA‐5‐ME) is produced in 53.7% and 24.6%, compound B (2HAA‐4‐ME) is produced in 16.2% and 70.3% and compound C (2HAA‐3‐ME) is produced in 30.1% and 5.1% by microsomal incubations and chemical oxidation respectively.
Determination of 3, 4‐dihydroxyphenylglycol (DHPG) by HPLC with coulometric detection, and correlation with 3‐methoxy‐4‐hydroxyphenylglycol (MHPG) in human plasma
Authors: Karege F. , Gaillard J.‐M. , Tissot R. , Azorin J.‐M. , Valli M.
Published: May 2005

A method for determining plasma 3,4‐dihydroxyphenylethyleneglycol (DHPG), a central noradrenaline (NA) metabolite, is described. The method used HPLC with dual coulometric detection set in screen mode of operation. The isolation of DHPG and related catecholamines (noradrenaline, dopamine and dihydroxybenzylamine) was performed on acid washed alumina extracted with 0.2 M HClO4 containing EDTA (0.2%), and reduced glutathione as stabilizer. A reversed phase column with an eluting system containing 0.025 M citric acid‐sodium hydrogen phosphate buffer in the ratio 3:2 (v:v) and 5% methanol was used. The experimental results of plasma DHPG levels compared favourably with the results from the literature, and a positively significant correlation with plasma MHPG was found. This method could be used as an alternative in central NA assessment.
Rapid sample preparation methods for analysis of residues of sulfamethazine and its N4‐acetyl and desamino metabolites in swine tissue by HPLC
Authors: Haagsma N. , Pluijmakers H. G. J. M. , Aerts M. M. L. , Beek W. M. J.
Published: May 2005

Rapid sample preparation methods for the determination of sulfamethazine (SMZ) and its N4‐acetyl and desamino metabolites in swine tissues at the 0.1 mg kg−1 level are presented. The methods use sonication‐aided extraction with dichloromethane. For SMZ and N4‐acetyl SMZ analysis extracts are cleaned up and concentrated on a silica disposable column followed by HPLC on a CP™ Spher C8 column using acetonitrile‐sodium acetate buffer as the mobile phase. For desamino‐SMZ analysis the extract was cleaned up and concentrated on a Florisil disposable column, followed by HPLC on a Nucleosil 5‐CN column after formation of an ion‐pair complex with 1‐heptanesulfonic acid. For desamino SMZ peak identification by diode‐array UV/VIS detection is also described. Mean recoveries from spiked tissue samples were about 87% (muscle) and 76% (kidney) for SMZ and N4‐acetyl SMZ and about 70% for desamino SMZ.
HPLC receptorassay of opioid peptides in the cerebrospinal fluid of lower back pain patients
Authors: Desiderio Dominic M. , Onishi Hiroshi , Fridland Genevieve , Wood George , Pagidipati Devaiah
Published: May 2005

Total opioid peptide receptoractivity in human cerebrospinal fluid is measured in patients who are experiencing lower back pain. Desalted CSF is eluted from a C18 Sep‐Pak and is subjected to a radioreceptorassay (RRA) that employs tritiated etorphin, which is aligand that is effectively displaced by opioids from several different types of opioid receptors. Three clinical groups have significantly different endogenous levels of 2.4, 4.5, and 6.4 pmol of methionine enkephalin‐equivalents per mL CSF. Those three levels indicate that more opioid activity is correlated with the amount of drug to relieve the patient's perception of pain. When the total opioid content exceeds an empirical threshold, the sample is further fractionated with gradient reversed phase HPLC, and the opioid receptoractivity in each HPLC fraction is measured to determine the characteristics pattern of those receptoractive opioid peptides present in that patient's CSF. Different HPLC RRA patterns are found for different clinical categories. A possible interpretation of these two different sets of data‐is that a lesion exists in one or several of the opioid peptidergic systems (metabolism, receptors) in this particular patient population.
HPLC determination of ferrochelatase activity in human liver
Authors: Roberts R. G.
Published: May 2005

A method utilizing HPLC to estimate ferrochelatase activity in human liver cells is presented. A partially purified homogenate of liver cells is incubated with mesoporphyrin IX and cobalt(II) ion. The ferrochelatase in the homogenate incorporates the cobalt(II) ion into the mesoporphyrin. After a fixed time (90 min) the porphyrins are extracted into an ethyl acetate‐acetic acid mixture. The porphyrins are then separated using reversed phase HPLC with a mobile phase of methanol‐acetonitrile‐phosphate buffer pH 3.0 (200:60:30 v/v). The enzyme activity is estimated by measuring the rate of utilization of mesoporphyrin. The optimum pH and substrate concentration for the reaction have been determined.
Separation and quantitation of plasma free fatty acids as phenacyl esters by HPLC
Authors: Uji Yoshinori , Noma Akio , Shiraki Masataka , Maeda Masako , Tsuji Akio , Okabc Hiroaki
Published: May 2005

We have developed a rapid method for the separation of plasma free fatty acids as their phenacyl esters by high‐performance liquid chromatography (HPLC) using a reversed‐phase (C18) column. The derivatives of series of both saturated and unsaturated fatty acids (C12:0C22:6) are simultaneously separated within 45 min and detected with ultraviolet at 241 nm. The limit of detection of fatty acids was approximately 0.5 nmol in 20 μL injected volume of extracts, and the coefficient of variation of the present method did not exceed 3.0%. Comparison of the results of the present HPLC method with those of gas chromatography, gave very good correlations for all fatty acids in human plasma.
Measurement of ascorbic acid and dehydroascorbic acid in gastric juice by HPLC
Authors: Sanderson M. J. , Schorah C. J.
Published: May 2005

New methods are presented for measuring total vitamin C and the ascorbic acid/dehydroascorbic acid ratio in gastric juice. Extracts are prepared from a gastric juice which are suitable for direct injection onto a Waters Nova‐pak C18 Radial‐pak cartridge for high performance liquid chromatography (HPLC) using ultraviolet absorbance at 270 nm for detection. Both enable removal of interfering mucus and mucopolysaccharide breakdown products in a novel way. The first uses mini‐columns of Sephadex G‐50, run in acidic conditions to remove large molecular weight material while maintaining the ascorbic acid/dehydroascorbic acid ratio as it was in the fresh sample. Addition of dithiothreitol converts the dehydroascorbic acid quantitatively to ascorbic acid, thus enabling measurement of both components. The second method converts all the dehydroascorbic acid to ascorbic acid at the outset. A perchloric acid extract is neutralized and passed through a Sep‐Pak C18. A new internal standard, reductic acid, is introduced for ascorbic acid analysis which behaves identically on Sep‐Pak C18. Samples are analysed by ion‐pair chromatography using 0.02 M NH4H2PO4 buffer (pH 7.1): methanol (80:20 v/v) containing 0.62 g/L tetrapentylammonium bromide. The detection limit was 1 ng ascorbic acid, and chromatography was completed in 5 min. The values obtained by the two independent HPLC methods were in good agreement with each other and with those obtained by the 2,4‐dinitrophenylhydrazine colorimetric method.
Direct concurrent measurement of urinary vanillylmandelic acid, 5‐hydroxyindoleacetic acid and homovanillic acid by HPLC. Three methodologies compared
Authors: van Haard P. M. M. , Wielders J. P. M. , Wikkerink J. B. W.
Published: May 2005

Three different direct HPLC methods for the determination of 3‐methoxy‐4‐hydroxymandelic acid (VMA, vanillylmandelic acid), 5‐hydroxyindoleacetic acid (5‐HIAA) and 3‐methoxy‐4‐hydroxyphenylacetic acid (HVA, homovanillic acid) in urine were compared: two spectrofluorometric methods, applying discontinuous gradients, and one serial coulometric linear gradient method. The imprecision study (n = 6) revealed comparable coefficients of variation (CV), intra‐assay ranging 1.4–11.1%, and inter‐assay ranging 5.9–11.8% for physiological and moderately elevated levels of VMA, 5‐HIAA and HVA. All methods showed good linearities up to 100 μmol/L for each of the three compounds studied. Analytical recoveries were 97–114% for VMA, 87–103% for 5‐HIAA, and 80–95% for HVA. Recoveries were not dependent on urinary relative densities in the range 1.010–1.030 kg/L or on protein content (prior to acidification) in the range 0.1–3 g/L, or on the pH of conservation in the range 2–5 or on storage temperature in the range −20–+22°C for three weeks.

The distributed‐sample comparison revealed acceptable correlations and clinically unimportant accuracy differences between the methods. It is concluded that direct fluoro netric and electrochemical HPLC methods can be used in the determination of major catecholamine and serotonin metabolites in human urine for clinical diagnosis and follow‐up of neural crest and carcinoid tumours.
Selective purification of reduced oligosaccharides using a phenylboronic acid bond elut column: Potential application in HPLC, mass spectrometry, reductive amination procedures and antigenic/serum analysis
Authors: Stoll Mark S. , Hounsell Elizabeth F.
Published: May 2005

A phenylboronic acid (PBA) derivatized Bond Elut column has been investigated for the selective purification of oligosaccharide alditols and of oligosaccharide derivatives formed by reductive amination reactions. These compounds are specifically retained by the column via interaction of sterically unhindered vicinal hydroxyl groups, hence achieving their separation from glycopeptides, unreduced oligosaccharides and non‐carbohydrate reactants. This purification procedure has widespread application for the clean‐up of derivatives prior to HPLC, mass spectrometry and antigenic/serum analysis.
HPLC analysis of polyamines and their acetylated derivatives in the picomole range using benzoyl chloride and 3,5‐dinitrobenzoyl chloride as derivatizing agent
Authors: Wongyai Surapote , Oefner Peter , Bonn Günther
Published: May 2005

Analytical methods are described for the quantitative determination of putrescine, cadaverine, spermidine, spermine and the acetylated derivatives of spermidine and spermine in biological fluids using pre‐column derivatization with either benzoyl chloride or 3,5‐dinitrobenzoyl chloride, which were added to each sample as solutions in diethyl ether. Putrescine, spermidine and spermine can be analysed in seminal plasma at nanogram levels when benzoyl chloride is used as derivatizing agent. In the analysis of putrescine, cadaverine, spermidine and acetyl derivatives of spermidine and spermine, higher sensitivity is obtained with 3,5‐dinitrobenzoyl‐chloride. This method can readily be used in the determination of acetylated polyamines in urine samples.
MicroHLPC determination of amygdalin in Semen pruni armeniacae and Semen pruni persicae
Authors: He Li‐Yi , Li Bao‐Ming
Published: May 2005

The application of micro HPLC to the determination of amygdalin in Semen pruni armeniacae and Semen pruni persicae is described. Amygdalin is separated at ambient temperature on a reversed phase column of U‐Finepak SIL C18(150×0.5 mm) with methanol + water (25: 75 v/v) as the mobile phase at a flow rate of 10 μL/min. The results are calculated by the internal standard method. The linear range is 1–7 μg. The CV and recovery of pure amygdalin are 1.47% (n = 10) and 98.13%, respectively. The results of analysis are lower than those obtained by TLC, but microHPLC is much simpler, faster, and more sensitive and reproducible than TLC.
Compositional analysis of the three main gangliosides from adult human myometrium by a rapid capillary gas chromatographic method
Authors: Zhu Zhengmei , Li Ruixiang , Cui Zhaochun , Zhou Liangmo , Huang Mingxian
Published: May 2005

A new capillary GC method is described for the compositional analysis of the three main gangliosides isolated from adult human myometrium. The sample was subjected to methanolysis, acetylation and trimethylsilylation which allows all the constituents to be analyzed simultaneously. The predominant ganglioside was found to be GD3, with GM3 and GT1b the next most abundant.
Determination of catechol‐O‐methyltransferase activity in erythrocytes by high performance liquid chromatography with electrochemical detection
Authors: Schultz Eija , Nissinen Erkki , Kaakkola Seppo
Published: May 2005

A rapid assay employing HPLC with electrochemical detection for catechol‐O‐methyltransferase (COMT) activity in red blood cells is described. Enzyme activity is determined from erythrocyte lysates using S‐adenosyl‐L‐methionine as methyl donor and 3,4‐dihydroxybenzoic acid as substrate. The 3‐O‐ and 4‐O‐methylated reaction products are measured by high‐performance liquid chromatography with electrochemical detection. Human erythrocyte soluble form of COMT had Km values of 6.1 μM and 26.0 μM for S‐adenosyl‐L‐methionine and dihydroxybenzoic acid, respectively. The mean O‐methylation ratio for the soluble form of COMT was 5.3. An O‐methylation ratio of 15.5 was estimated in the membrane fraction of an erythrocyte pool from three samples. The activities of soluble COMT in erythrocytes of some animal species are also reported. The procedure is easily automated, and a large number of samples can be processed during one working day.
Determination of polyol profiles in human urine by capillary gas chromatography
Authors: Haga Hidehiko , Nakajima Terumi
Published: May 2005

A new rapid capillary gas chromatographic method is described for the profile analysis of urinary polyols as their trifluoroacetyl derivatives. Ten urinary polyols: erythritol, threitol, fucitol, ribitol, arabinitol, xylitol, mannitol, glucitol, galactitol and myo‐inositol were completely determined for the first time. Iditol was newly found in normal urine. Urinary polyols were verified by gas chromatography/mass spectrometry.
High performance liquid chromatographic measurement of lignocaine in tissue samples following transabdominal placental biopsy
Authors: Monkman S. C. , Armstrong R. , Flanagan R. J. , Holt D. W. , Rosevear S.
Published: May 2005

A method is described for the measurement of lignocaine in small samples of fetal and placental tissue. Tissue samples (ca 100 mg) are digested using a preteolytic enzyme. Lignocaine and an internal standard are extracted into methyl tert‐butyl ether and analysed by high‐performance liquid chromatography with electrochemical detection (+1.0 V vs Ag/AgCl). The limit of accurate measurement is better than 0.1 mg/kg wet weight for a 100 mg sample. This method has been used to assess fetal exposure to the drug when used as a local anaesthetic during transabdominal placental biopsy (chorionic villus sampling). The range of lignocaine concentrations found in the tissue samples was large (from <0.04 mg/kg wet weight to 15.4 mg/kg wet weight) although most samples contained less than 1.0 mg lignocaine/kg wet weight.
A high performance liquid chromatography/electrochemical assay for glutamatergic neurotransmitters in the rat brain
Authors: Harsing L. G. , Lajtha A. , Vizi E. S.
Published: May 2005

The release and content of the excitatory amino acid neurotransmitters glutamate and aspartate in rat striatum were determined by liquid chromatography/electrochemistry. This determination was based on precolumn off‐line derivatization of the amino acids with o‐phthaldialdehyde and 2‐mercaptoethanol (OPT/2‐MCE), and the adducts formed were separated under isocratic conditions and oxidized on a glassy carbon electrode at moderate potential (+0.6 V). The standard and the extracted glutamate when derivatized with OPT/2‐MCE produced similar electrochemical and chromatographic characteristics. The detection limit of glutamate was 0.5 pmol. Depolarization induced by the high potassium medium (40 mmol/L) enhanced the release of glutamate and aspartate from superfused rat striatum, whereas the efflux of glutamine remained unchanged. Perfusion (for 60 – 70 min) removed 50 – 80% of the free amino acid content of striatal tissue. The method described here is useful in neurochemical investigations of the brain amino acid neurotransmitters.
Application of high performance liquid chromatography combined with diode‐array detection for analysis of proteins and peptides in human cerebrospinal fluid
Authors: Silberring Jerzy , Nyberg Fred , Lyrenäs Sven
Published: May 2005

Different strategies for HPLC separation, including molecular sieving, ion‐exchange, and hydrophobic interaction as well as reversed phase chromatography, were used to study molecular components in human cerebrospinal fluid (CSF). The separations were followed by photodiode‐array UV detection, which is a recently developed technique allowing a direct and rapid discrimination between peptides and proteins differing in their content of aromatic amino acids. By the various HPLC techniques in conjunction with diode‐array detection it was possible to identify and characterize several protein and peptide components present in CSF. The procedure also allowed quantitative analysis of CSF proteins using minute amounts of the fluid.
High performance liquid chromatography stability study of malonyl‐coenzyme A, using statistical experimental designs
Authors: De Spiegeleer Bart M. J. , Sintobin Karel , Desmet Jan
Published: May 2005

Malonyl‐CoA is a biochemically important compound, formed by an acetyl‐coenzyme A carboxylase catalysed reaction. The stability of this short‐chain coenzyme A derivative under various experimental conditions is discussed in this article. High‐performance liquid chromatography was used for the analysis of the reaction mixture because of its excellent selectivity and sufficient sensitivity. Several variables were investigated as possible stability‐influencing factors: pH, magnesium and buffer concentration, reaction temperature and time. The Plackett‐Burman screening design was first used for selecting the most important variables, with which a central composite design was constructed. In this way, a response surface was obtained with the percentage remaining malonyl‐CoA as a function of magnesium concentration, reaction temperature and time. The usefulness of this approach is demonstrated by obtaining kinetic data from the mathematical function and by the evaluation of the stopping of reaction procedure in the activity assay of acetyl‐coenzyme A carboxylase.
Thin layer chromatography of dansyl and dinitrophenyl derivatives of amino acids. A review
Authors: Bhushan R. , Reddy G. P.
Published: May 2005

The dinitrophenyl (DNP) derivatives of amino acids have found continual application in protein sequencing since Sanger used them for the first time for the sequencing of insulin. Dansyl derivatives of amino acids have been widely used in protein sequencing because of their fluorescent nature. The success of protein sequencing largely depends upon correct identification of such derivatives. The choice for the method of identification is related to cost, the availability of instrumentation and to the sensitivity needed for the analysis. Thin layer chromatography (TLC) is simple and has several advantages over other chromatographic methods. Therefore the literature after 1972 is reviewed for TLC analysis of dansyl‐ and DNP‐amino acids, the two important amino acid derivatives required for identifying protein sequences. Additionally, the literature on the TLC resolution of enantiomeric mixtures of dansyl amino acids is reviewed. Application of various adsorbents, composition of solvent systems and other experimental conditions together with successful resolution data have been discussed. TLC provides a direct and inexpensive method for the resolution of enantiomers, and is fast becoming a sensitive instrumentalized quantitative analytical technique.
High resolution chromatography of ribonucleosides and its application to RNA analysis
Authors: Tanaka Kouji , Yazawa Kouhei , Nakajima Terumi
Published: May 2005

A simple and precise method was developed for the separation of nucleosides including modified nucleosides and oligonucleotides. Nineteen kinds of nucleosides were completely separated by HPLC using an ODS column (TSK‐gel ODS 80TM) and aqueous mobile phases. The RNA molecule was digested by base restrictive RNase (RNase A, RNase T1) and the digests were separated chromatographically into each oligonucleotide. The nucleoside composition of an oligonucleotide was then determined by this analytical system. It is thus possible to fit the oligonucleotide in the original RNA molecule by using modified bases as markers. The reaction site of quinacrine mustard for tRNAPhe (from yeast) could be determined by this analytical system.
Rapid quantitative determination of epirubicin and its metabolites in plasma using high performance liquid chromatography and fluorescence detection
Authors: Dine T. , Brunet C. , Luyckx M. , Cazin M. , Gosselin P. , Cazin J. L.
Published: May 2005

A rapid sensitive and selective isocratic technique was developed for the analysis of plasma epirubicin and three of its known fluorescent metabolites epirubicinol, 4′‐O‐β‐D‐glucuronyl‐4′‐epidoxorubicin and 4′‐O‐β‐D‐glucuronyl 1,3‐dihydro‐4′‐epidoxorubicin, with daunorubicin as an internal standard, by using high performance liquid chromatography (HPLC) with fluorescence detection and a ‘Hypersil ODS’ column. The drugs were easily and efficiently extracted with a Sep‐Pak C18 cartridge and the mean recoveries were greater than 85%. Intraassay and Interassay coefficients of variation (plasma samples) were better than 8.25%. An example of pharmacokinetic study obtained in a cancer patient after intravenous injection of epirubicine is described.
Application of reversed phase high performance liquid chromatography to the analysis of sulphidopeptide leukotrienes in pig bile
Authors: Tagari Philip , Ethier Diane , Ford‐Hutchinson Anthony , Cirino Maria
Published: May 2005

Reversed phase HPLC methodology has been developed for separation of peptide leukotrienes and indomethacin in porcine bile. Reproducible recoveries were obtained using radioactive leukotrienes ([3H]LTC4, 57.1 ± 2.5%; [3H]LTE4, 62.7 ± 1.9%; [3H]LTD4, 54.3 ± 2.7%). Radioimmunoassay of column eluant demonstrated that as little as 300 pg of exogenous leukotrienes could be measured in bile fluids, with similar recoveries. Analysis of bile sampled 60–90 min after initiation of experimental endotoxic shock in indomethacin treated pigs revealed a leukotriene concentration of 5.24 ± 1.16 ng/mL(LTD4). This was significantly greater (p < 0.05, n = 3) than that observed in samples collected prior to endotoxin (0.42 ± 0.23 ng/mL), or from untreated animals (0.85 ± 0.51 ng/mL). This method is thus applicable to investigation of the role of 5‐lipoxygenase products in porcine models of human disease, including shock conditions such as endotoxaemia, during cyclooxygenase inhibition by indomethacin.
Removal of sodium dodecyl sulphate from proteins isolated by sodium dodecyl sulphate polyacrylamide gel electrophoresis
Authors: Kaplan Batia , Pras Mordechai
Published: May 2005

A procedure is described for the removal of sodium dodecyl sulphate (SDS) from proteins isolated by SDS‐polyacrylamide gel electrophoresis (SDS‐PAGE). The proteins separated by SDS‐PAGE were stained with Coomassie Blue and extracted with Tris‐HCI buffer containing SDS. The obtained extracts were subjected to gel permeation chromatography in an acidic aqueous acetonitrile solution. The procedure allows purification of the isolated proteins not only from SDS, but also from Coomassie Blue, buffer salts and other small molecular weight contaminants.
Low‐level interferences in peroxyoxalate chemiluminescence
Authors: Gooijer C. , Velthorst N. H.
Published: May 2005

The role of interferences at concentrations lower than 10−3 M in peroxyoxalate chemiluminescence is examined based on experimental results available in the literature. Implications for fluorophore and for hydrogen peroxide determinations are discussed. An interpretation in terms of the reaction mechanism is proposed.
A chemiluminometric method for NADPH and NADH using a two‐enzyme bioreactor and its application to the determination of magnesium in serum
Authors: Tabata Masayoshi , Totani Masayuki , Murachi Takashi
Published: May 2005

Chemiluminometric methods are described for the automated flow injection analysis of NADPH and NADH using an immobilized enzyme column reactor and serum magnesium. This application is for the clinical analysis of NADPH and NADH. The reactor for NADPH and NADH contains immobilized L‐glutamate dehydrogenase and L‐glutamate oxidase, and that for serum magnesium immobilized hexokinase, glucose‐6‐phosphate dehydrogenase, L‐glutamate dehydrogenase and L‐glutamate oxidase. When the sample is introduced into the four‐enzyme bioreactor, hydrogen peroxide is produced in proportion to the concentration of serum magnesium by the successive reactions. A co‐immobilized hexokinase/glucose‐6‐phosphate dehydrogenase/glutamate dehydrogenase column reactor gave better efficiency compared with an enzyme column which was prepared by packing co‐immobilized hexokinase/glucose‐6‐phosphate dehydrogenase and immobilized glutamate dehydrogenase to make two layers. Magnesium in serum was determined with 1μ L of the sample without carry‐over and for an assay time of approximately 15 s. The present method is sensitive (detection limit 0.1 nmol) because Mg2+ is recycled in a column, and gives perfect linearity of the data up to 3.0 mmol/L with satisfactory precision, reproducibility, and accurate reaction recoveries.
High performance liquid chromatography determination of doxorubicin and daunorubicin in plasma using UV detection and column switching
Authors: Mikan A. , Lanao J. Martinez , Lopez F. Gonzalez , Hurle A. Dominguez‐Gil
Published: May 2005

A method for the determination of doxorubicin and daunorubicin in plasma is described. The plasma is injected directly into a loop column and then washed with water. After switching the injection valve, the sample is separated on a phenyl column using detection at 254 nm. The detection limit is 10 ng/mL, the coefficient of variation is 7% for 100 ng/mL of doxorubicin and 4% for 200 ng/mL of daunorubicin.
A new method for the high performance liquid chromatographic determination of TA‐870, a dopamine prodrug (catechol ester compound)
Authors: Yoshikawa Masayoshi , Endo Hiroshi , Hoshino Kumiko , Sugawara Yoichi , Takaiti Osasi , Kanda Susumu , Imai Kazuhiro
Published: May 2005

A new method for the high performance liquid chromatographic (HPLC) determination of N(N‐acetyl‐L‐methionyl)‐O,O‐bis(ethoxycarbonyl)dopamine (TA‐870), a dopamine prodrug, in biological fluid has been developed. In order to measure with an electrochemical detector (ECD), TA‐870 was passed first through an immobilized carboxylesterase column to be converted to the electrochemically active deethoxycarbonylated TA‐870 (DEC‐TA‐870). The properties of this carboxylesterase immobilized on Sepharose 4B were examined by this flow injection system. Hydrolysis of TA‐870 with this immobilized carboxylesterase was a maximum at pH 7 – 8 and 50 °C, and the activity decreased in the presence of organic solvent such as acetonitrile. For the determination of TA‐870 in biological fluids, an HPLC‐immobilized enzyme‐ECD system using a column‐switching technique was developed. The blood was deproteinized with ethanol, and TA‐870 in the ethanol extracts was adsorbed in Bond Elut C18. The dichloromethane eluate from Bond Elut C18 was injected into the HPLC system. The HPLC apparatus was composed of three pumps, two separation columns (LiChrosorb Si 60 and μ Bondasphere), a trap column (Bond Elut), an enzyme column, ECD and the column‐switching system. The calibration curve for TA‐870 in blood was linear in the range from 2 to 200 ng/mL. This new assay method might be useful also for the determination of other catechol ester compounds.
High performance liquid chromatographic studies of erythrocyte haem biosynthetic enzyme activities in alcoholic macrocytosis
Authors: Wright D. J. , Lim C. K. , Peters T. J.
Published: May 2005

The activities of 5‐aminolaevulinate dehydrase (ALA‐D), hydroxymethylbilane synthase (HMB‐S), uroporphyrinogen III synthase (URO‐S) and uroporphyrinogen decarboxylase (URO‐D) in the erythrocytes of alcoholics were assayed and compared with those in controls and abstaining alcoholics. The HMB‐S and URO‐S activities in alcoholics were significantly higher than the controls and there were significant correlations between enzyme activities and mean corpuscular volume (MCV). Alcoholics abstaining from alcohol over a nine‐month period showed a return to normal enzyme activities in parallel with the normalization of their MCV. Abstaining alcoholics thus all had normal HMB‐S and URO‐S activities. The ALA‐D and URO‐D activities of alcoholics were not significantly different from controls or from abstaining alcoholics. Alcohol appears to have a selective effect on the haem biosynthetic enzymes in the erythron.
Chromatographic techniques in inborn errors of metabolism
Authors: Lehotay Denis C.
Published: May 2005

Chromatography has played a pivotal role in the advances made during the last 30 years in our knowledge of inborn errors of metabolism. This review discusses the application of some of these techniques to the analysis of organic acids and acylcarnitines. The separation of organic acids needed a comprehensive approach that would permit all of the many organic acids present in urine or other complex mixtures to be extracted, analysed and identified in a single run. This required analytical methods of great resolving power, wide linear range and universal detectors such as gas chromatography (GC), or GC coupled with mass spectrometry. Sample preparation was another problem that has been tackled by a variety of approaches. Organic solvents have been employed widely for the extraction of organic acids from physiological fluids. Unfortunately, recoveries of the different organic acids by this method are sometimes less than quantitative and variable depending on the compound. Other methods, such as the use of DEAE‐Sephadex columns, have the advantage of resulting in close to 100% recoveries, but are more tedious. Liquid partition chromatography on short silicic acid columns has also been recommended as a useful clean‐up step prior to GC, permitting both the identification and quantitation of organic acids in urine, plasma or amniotic fluid. Although many derivatization procedures have been used to prepare organic acids for gas chromatography, the most common is trimethylsilylation. Oxo acids are usually reacted with one of several commonly used reagents to form oximes. GC analysis of organic acids was initially done using packed columns with methylsilicone‐based, non‐polar stationary phases. In recent years most laboratories have switched to the commercially available, bonded‐phase capillary columns. Some authors have recommended the use of two columns of slightly differing polarities to improve the reliability of identification without mass spectrometry. Carnitine and acylcarnitines are substances whose measurement may yield useful diagnostic information about inborn errors and about the basic biochemical mechanisms of disease. While free carnitine is usually measured by a radioenzymatic assay, the separation of acylcarnitines requires paper chromatography or high performance liquid chromatography. The identification of various acylcarnitines following separation has been accomplished by mass spectrometric methods. Another technique relies on gas chromatographic identification of the acyl groups liberated following alkaline hydrolysis. Recently, liquid chromatography/mass spectrometry has also been utilized to separate and identify the various acylcarnitines present in urine and other biological samples.
Determination of short‐chain fatty acids in equine caecal liquor by ion exchange high performance liquid chromatography after solid phase extraction
Authors: Horspool L. J. I. , McKellar Q. A.
Published: May 2005

A high performance liquid chromatography (HPLC) method was developed for the determination of seven short‐chain fatty acids in equine caecal liquor. Samples were cleaned up on a Sep‐pak (C18) cartridge, and the analyte was eluted from the extraction cartridge and filtered through a 0.45 μm cellulose nitrate filter. The analyte was chromatographed by ion exchange HPLC. Detection was by UV at 210 nm. Recovery from phosphate buffer (0.05 M, pH 7.0) and equine caecal liquor was 76.95% (lactic), 76.76% (acetic), 84.40% (propionic), 89.35% (isobutyric), 88.73% (butyric), 80.33% (isovaleric) and 72.61% (valeric). The limit of detection of the short‐chain fatty acids in phosphate buffer was 0.00006 M (lactic), 0.0001 M (acetic), 0.0002 M (propionic), 0.0001 M (isobutyric), 0.0002 M (butyric), 0.0002 M (isovaleric) and 0.0003 M (valeric). The specificity and sensitivity of this method was sufficiently high to allow the characterization of the pattern of these short‐chain fatty acids in equine caecal liquor following intravenous administration of oxytetracycline at the recommended dose rate in a pony.
Application of photodiode array detection and fast atom bombardment mass spectrometry for the identification of the arginine residue in neuropeptides
Authors: Silberring Jerzy , Nyberg Fred
Published: May 2005

Chemical derivatization by phenylgyoxal (PGX) was applied to the identification of arginine in the neuropeptides dynorphin A (1–6) and substance P. The obtained products were separated on a short reversed phase C18 column and analysed on‐line with the photodiode array UV technique. The selective attachment of a chromogenic molecule into the arginine residue resulted in significant change in the absorbance spectra around 250 nm, depending on the number of PGX molecules attracted. Further analysis employed fast atom bombardment mass spectrometry (FAB MS) and C‐terminal sequencing for detailed verification of the derivatives formed during modification with PGX. The results clearly demonstrated that the photodiode array technique, when combined with chemical modification of certain amino acids, provides new possibilities for the analysis of peptide structures.