bluuebunny/crossref_metadata_2025 huggingface dataset:
‘In situ’ Edman degradation of protein(s) blotted to immobilon membranes suitable for unblocking reversible chemical modification and elimination of coomassie blue in sodium dodecyl sulfate‐polyacrylamide gel electrophoresis
Authors: Chirat Frédéric , Belaiche Denise , Malki Nouzha , Han Kia‐Ki
Published: May 2005

Conditions for unblocking reversible chemical modifications such as maleylation or citraconylation ‘in situ’ at the N‐terminus of proteins after transfer of proteins to immobilon membranes from SDS‐PAGE are described. Demaleylation or decitraconylation occurred at 55°C in 70% formic acid (pH 1.50) during 60 min. During the unblocking reaction, Coomassie blue dye was completely removed, resulting in superior high performance liquid chromatographic separation of phenylthiohydantoin‐amino acid (PTH‐AA) after Edman degradation (automatic gas phase sequencer). The protein fixed on the matrix after demaleylation and removal of Coomassie blue was not degraded. The possible cleavage at the aspartyl‐prolyl peptide bonds was considered, but no side reaction was observed. Furthermore, the incubation time in 70% formic acid at 55°C could be reduced to 10 min in the absence of maleylation of the starting material, and this was suitable for the removal of Coomassie blue and the quantification of phenylthiolhydantoin‐amino acids (PTH‐AAs) by HPLC. The yield from the starting protein through SDS‐PAGE, blotting, and Edman degradation to quantitative analysis of PTH‐aminoacid(s) by HLPC was established.
Capillary gas chromatographic determination of epomediol in human plasma and urine using a rapid solid‐phase extraction
Authors: Girometta Maria Angela , Loschi Loredana , Ventura Paolo , Canali Sergio , Giachetti Claudio , Zanolo Gianni
Published: May 2005

A simple and precise method for the quantitation of epomediol in human plasma and urine is described. Each biological sample is added with the internal standard and applied directly to an Extrelut‐1 solid‐phase column. After absorption the column is eluted with chloroform and the eluate is evaporated to dryness. The residue, reconstituted in ethanol, is analysed by capillary gas chromatography. No interferences from possible metabolites or endogenous constituents can be noted. The method has been applied to human pharmacokinetic studies: the results of a subacute administration to volunteers are presented.
High performance liquid chromatography determination of cyanide in urine by pre‐column fluorescence derivatization
Authors: Sano Akira , Takezawa Masaaki , Takitani Shoji
Published: May 2005

A method for the determination of cyanide in human urine has been developed. The method is based on the reaction of cyanide with 2,3‐naphthalenedialdehyde and taurine to give a fluorescent product for reversed‐phase HPLC separation and fluorometric detection. After centrifugation followed by dilution of urine samples, the specimens could be analysed directly by this method. The recovery of cyanide added to urine at concentration levels of 50 – 1000 pmol/mL was 85 – 96%. The detection limit of cyanide was 30 pmol/mL in urine. The method was successfully applied to the analysis of urine from smokers and nonsmokers. The mean concentrations of cyanide were found to be 215 pmol/mL for the former and 84 pmol/mL for the latter.
Reversed phase high performance liquid chromatography of glycopeptides
Authors: Wang Tsonghui , Chen Tsefang , Barofsky Douglas F.
Published: May 2005

A practical procedure for isolating and purifying glycopeptides is described, viz. enzymatic hydrolysis—gel permeation chromatography—ion exchange chromatography—reversed phase HPLC. Using this procedure 28 glycopeptides from hen ovalbumin have been isolated some of which hitherto have not been identified. Water was a suitable mobile phase for preparing pure glycopeptides, and control of column temperature was important for good separations and reproducible retention times. Structural confirmation was by fast atom bombardment mass spectrometry.
Simultaneous analysis of furosemide and bumetanide in horse plasma using high performance liquid chromatography
Authors: Singh Ashok K. , McArdle Camille , Gordon Brad , Ashraf M. , Granley K.
Published: May 2005

A high performance liquid chromatographic method is described for the simultaneous determination of furosemide and bumetanide in horse plasma. The C8 (3 μm) reversed phase column (4.8 × 150 mm) provided clear separation of furosemide and bumetanide with other components present in the horse plasma. The detection limit for both the drugs was 10 ng/mL. Both drugs were stable in plasma (at natural or acidic pH) for up to 24 h. The method is sufficiently sensitive to detect furosemide levels in plasma obtained from horses receiving a therapeutic dose of furosemide.
Rapid determination of the benzodiazepine antagonist flumazenil, Ro 15‐1788, by high performance liquid chromatography
Authors: Bun H. , Duplan V. , Crevat‐Pisano P. , Llurens M. , Durand A.
Published: May 2005

A sensitive (2 ng/mL) and specific method for the determination of the benzodiazepine antagonist Ro 15‐1788 or flumazenil is described. Following a simple extraction, the compound is analyzed by reversed phase high performance liquid chromatography (HPLC) and detection at 245 nm. The method was applied to plasma specimens collected from patients receiving a single dose of this drug.
Purification of the membrane protein enzyme lipoamidase by affinity chromatography
Authors: Oizumi Jun , Hayakawa Kou
Published: May 2005

Lipoamidase, a membrane glycoprotein enzyme, was purified from brain membrane by means of various affinity columns. A column with immobilized Arg‐Phe‐NH2 was found to be the most effective. After loading the crude material of the membrane, and extensive washing of the column with sodium chloride (0.3 M) solution, the enzyme activity was eluted by a solution containing 1% of nonionic detergent (Nonidet P‐40). The fractions containing the lipoamidase activity were analyzed by SDS‐PAGE, and a single protein band detected in this fraction. On the other hand, lipoyl‐affinity columns with various resins were not effective in enzyme purification. Single step chromatography on the Arg‐Phe‐NH2 column enriched the membrane enzyme lipoamidase by 40‐fold. The mechanism by which this affinity resin effectively enriches the enzyme remains to be elucidated.
High performance ion exchange chromatography of human plasma lecithin: Cholesterol acyltransferase
Authors: Holmquist Leif
Published: May 2005

Highly purified monomeric human plasma lecithin:cholesterol acyltransferase (LCAT), completely free of apolipoprotein D, has been chromatographed on a MonoQ HR 5/5 anion exchanger. LCAT eluted as symmetrical peaks after 12.8 min and 14.8 min at pH 5.0 and pH 6.0, respectively, using a linear NaCl gradient. The corresponding concentrations of NaCI effecting desorption of LCAT from the anion exchanger were 125 mM and 175 mM. At both pH values human serum albumin eluted earlier and was well separated from the enzyme. Rechromatography of LCAT in the eluates from these experiments at acid pH, on high performance gel filtration, demonstrated absence of aggregation. The nonspecific adsorption during anion exchange chromatography at pH 5.0 and pH 6.0 was negligible, as demonstrated by a linear relationship between injected amounts of LCAT and recorded peak areas for a 2–20 μg protein range. Zone immunoelectrophoresis assay indicated unaltered immunoreactivity of the eluted LCAT.
Determination of urinary tryptophan and its metabolites along the nicotinic acid pathway by high performance liquid chromatography with ultraviolet detection
Authors: Bizzarri Mariano , Catizone Angela , Pompei Marco , Chiappini Loretta , Curini Letizia , Lagana Aldo
Published: May 2005

A fast and sensitive method is given for analysing urinary tryptophan and six of its metabolites on the nicotinic acid pathway. Kynurenine, tryptophan, 3‐hydroxykynurenine, anthranilic acid, 3‐hydroxyanthranilic acid, kynurenic acid and xanthurenic acid were isocratically eluted and completely resolved with a mobile phase of acetonitrile + sodium acetate buffer, pH 4.76 (4:96, v/v). The flow rate was 0.8 mL/min at the beginning and was then linearly increased to 1.2 after 6 min; after 14 min the flow was augmented from 1.2 to 2 mL/min. The effluent was monitored with a variable UV detector set at 254 nm for the first five peaks and at 280 and 325 nm for the penultimate peak and final peak. Analytical recoveries of the compounds after deproteinization varied between 64% and 98%. The reported method should enable one to examine easily, extensively, quantitatively and routinely urinary tryptophan and the most important metabolites of the nicotinic acid pathway.
Studies on the metabolism of lappaconitine in humans. Identification of the metabolites of lappaconitine in human urine by high performance liquid chromatography
Authors: Xie Fuming , Wang Hong‐Cheng , Li Jian‐Hua , Shu Hen‐Ling , Jiang Ji‐Rong , Chang Jen‐Pin , Hsieh Yu‐Yuan
Published: May 2005

The metabolités of lappaconitine in the urine of humans having been previously administered intramuscularly with lappaconitine hydrobromide were studied using high performance liquid chromatography with electrochemical and ultraviolet detection. The urine was extracted by means of liquid‐ and solid‐phase extractions. Each of the metabolites of lappaconitine was purified by high performance liquid chromatography on a reversed phase column and identified on the basis of the chromatographic behaviour and the detector response. It was proved that lappaconitine, N‐deacetyl‐16‐O‐demethyllappaconitine and N‐deacetyllappaconitine were excreted in urine from humans receiving lappaconitine.
A reversed phase high performance liquid chromatography approach in determining total red blood cell concentrations of 6‐thioguanine, 6‐mercaptopurine, methylthioguanine, and methylmercaptopurine in a patient receiving thiopurine therapy
Authors: Erdmann Gary R. , France Laura A. , Bostrom Bruce C. , Canafax Daniel M.
Published: May 2005

A reversed phase high performance liquid chromatographic procedure was developed to quantify 6‐thioguanine, 6‐mercaptopurine, methylthioguanine, and methylmercaptopurine in red blood cells. The free base of each thiopurine was liberated from the respective nucleoside and nucleotide moiety by acid hydrolysis, which allowed for a determination of the total thiopurine present. 6‐Thioguanine and 6‐mercaptopurine were analyzed on an octadecyl‐ silane column using methanol + 20 mM sodium phosphate (15:85), pH 7.5, containing 0.07% tetrabutylammonium chloride. Detection was by potassium permanganate oxidation and fluorescence detection at 290 nm excitation and 400 nm emission. Methylmercaptopurine and methylthioguanine were analyzed on a cyanopropylsilane column using methanol + 40 mM sodium phosphate (18:82), pH 2.7, and then ultraviolet absorption at 314 nm and 290 nm, respectively. The method was used to quantify the four primary thiopurines present in red blood cells of an acute lymphoblastic leukemia patient. The procedure may be a therapeutic monitoring technique that quantifies the cytotoxic drug burden in patients receiving azathioprine or 6‐mercaptopurine therapy.
A sensitive high performance liquid chromatography microassay for bemoradan, a novel cardiotonic agent, in plasma/serum using a semi‐automatic liquid/solid extraction sample preparation system—AASP®
Authors: Huang Shiew‐Mei , Chien Shu‐Chean , Flor Soledad C.
Published: May 2005

A high performance liquid chromatographic method for the measurement of bemoradan levels in plasma/serum is described. This method uses Varian's AASP® (Varian Associates, Sunnyvale, CA, USA), a semi‐automatic liquid/solid extraction sample preparation system. It requires only small volumes of plasma/serum samples (0.2–1 mL) and needs no organic solvent for sample preparation. The mean recovery of bemoradan at plasma or serum concentrations of 0.5–100 ng/mL is 82%. The assay has a detection limit of 0.5 ng/mL (when 1 mL of plasma/serum is used) and is linear in the concentration range 0.5–500 ng/mL.
Determination of polyamines in urine of normal human and cancer patients by capillary gas chromatography
Authors: Jiang Xiaochun
Published: May 2005

A capillary gas chromatographic method is described for the determination of polyamines (putrescine, spermidine and spermine) in the urine of normal human and cancer patients. Morning urine after acid hydrolysis is cleaned up on a silica gel column and derivatized with trifluoroacetic‐anhydride. Creatinine in human urine is used as internal standard. Recoveries of polyamines are 96.7% putrescine, 102.6% spermidine (Spd), and 98.7% spermine. SD of the method for Spd is 1.949 ± 0.041 (μg/mg creatinine, mean ±SD, n = 5). The results show that the mean level of polyamines in cancer patients urine is much higher than that in normal human urine. The mean of total polyamines in the normal human and the cancer patients is 2.01 and 44.74, respectively (g/mg creatinine).
A simple, sensitive liquid chromatographic method for quantitation of D‐galactose and D‐glucose in blood plasma
Authors: Griffin Brenda W. , Williams Gary W.
Published: May 2005

The concentrations of D‐glucose and D‐galactose in plasma of galactosemic rats were quantitatively measured by a liquid chromatographic method, based on retention of the weakly ionized monosaccharides by an anion exchange column under alkaline conditions, elution with 9 mmol/L NaOH, and electrochemical detection. This method is both simple and sensitive, since very dilute plasma samples can be directly analysed.
Determination of lipid hydroperoxides in low density lipoprotein from human plasma using high performance liquid chromatography with chemiluminescence detection
Authors: Miyazawa Teruo , Fujimoto Kenshiro , Oikawa Shin‐Ichi
Published: May 2005

A high performance liquid chromatographic system with chemiluminescence detection (HPLC‐CL) was used for determining phospholipid hydroperoxides in human plasma low density lipoprotein (LDL). This system involved separation of phospholipids from LDL‐total lipids with normal phase silica gel HPLC and post‐column detection of hydroperoxide‐dependent chemiluminescence produced by luminol oxidation during the reaction of hydroperoxide with cytochrome c‐haeme. By using HPLC‐CL, we could detect phosphatidylcholine hydroperoxide (PCOOH) in human plasma LDL, and the LDL‐PCOOH concentration was significantly higher in patients with atherosclerosis and hyperlipidemia than that of healthy volunteers. The LDL‐PCOOH level was proportional to the plasma total cholesterol concentration.
Separation of the Hageman factor fragment from human serum albumin by chromatography on Blue Sepharose CL‐6B
Authors: Simonianová E. , Hrkal Z.
Published: May 2005

The separation of the Hageman factor fragment (HFf) activity from human serum albumin by chromatography on Blue Sepharose CL‐6B is described. The complete separation cannot be achieved in a single chromatography step due to complex formation between HFf and albumin.
Analysis of lorazepam in rat brain using liquid/liquid and solid‐phase extraction in combination with high performance liquid chromatography
Authors: Gunawan S. , Walton N. Y. , Treiman D. M.
Published: May 2005

A method for the determination of lorazepam in rat brain is described using liquid/liquid and solid‐phase extraction, followed by high performance liquid chromatography. After addition of chlordiazepoxide as the internal standard, 100 mg brain tissue was homogenized and incubated with alkaline protease. Lorazepam and chlordiazepoxide were extracted three times with toluene. After treatment through a C 18‐Bond Elut column, lorazepam and chlordiazepoxide were analyzed isocratically on a reversed‐phase column with a mobile phase consisting of methanol + 0.025 M sodium phosphate buffer (66:34, v/v). The eluted drugs were monitored by their absorption at 240 nm. The sensitivity limit of this method was 10 ng of lorazepam per 100 mg of brain tissue sample. The standard curve was linear over the range of 20 to 200 ng lorazepam. The coefficient of variation for day‐to‐day precision established by 21 replicate analyses was 4.5 to 13.6%.
Determination of lipoperoxide in animal tissue by ion‐pair reversed phase high performance liquid chromatography
Authors: Li Ke
Published: May 2005

An ion‐pair reversed phase high performance liquid chromatographic method was developed for the quantitative determination of lipoperoxide (as malondialdehyde, MDA) in animal tissue using a ODS column and a mobile phase of methanol + phosphate buffer (53:47 v/v, pH 6.5). Tetrabutylammonium bromide was used as an ion‐pair reagent and the detection wavelength was set at 532 nm.

The method was sensitive, accurate and simple. A linear relationship was obtained between the peak height and the amount of MDA from 50 nmol/L to 1200 nmol/L and the lower limit of detection of MDA was 15 nmol/L (signal‐to‐noise ratio > 2), and the coefficient of variation at the 200 nmol/L level was 3.7% (n = 7). The results correlated well with those from the 2‐thiobarbituric acid colorimetric method. The method has been used in the pathogenic studies of cerebral ischemia and acute renal failure for the determination of trace lipoperoxide.
A simple and rapid method for the purification of cytochrome P‐450 (form LM4)
Authors: Alterman Michael A. , Dowgii Andrew I.
Published: May 2005

A one step chromatographic method for the purification of membrane protein cytochrome P‐450 LM4 has been developed and it indicates that CM Sepharose Fast‐Flow is an excellent material for cytochrome P‐450 LM4 chromatography.
Use of enzymatic reactors in the high performance liquid chromatographic determination of ethanol and methanol with electrochemical detection
Authors: Tagliaro F. , De Leo D. , Marigo M. , Dorizzi R. , Schiavon G.
Published: May 2005

Despite numerous applications both in industry and in analytical chemistry, immobilized enzyme technology has encountered only limited success in modern liquid chromatography. The strict requirements of enzymatic reactors for high performance liquid chromatography (HPLC) (i.e., mechanical resistance, limited void volumes, high contact surface, high binding capacity, stable enzyme‐to‐matrix bond without loss of enzyme activity) are not yet fully met by commercially available products. In our laboratory, the development of post‐column reactors with immobilized alcohol oxidase on‐line with an HPLC/EC system has been undertaken. Three home‐made reactors were produced: in the first alcohol oxidase from Candida boidinii was quasi‐immobilized onto an HPLC‐sized anion exchange support, in the second the enzyme was bound to a silica‐based support via Schiff's base formation, and in the third a covalent linkage to an epoxy‐activated hydroxyalkyl methacrylate material was chosen. Chemical immobilization proved superior to the ionic binding. The importance of limiting the post‐column band spreading due to the reactor void volume, by using HPLC‐sized supports, was confirmed.
L‐dopa as Substrate for human duodenal catechol‐O‐methyltransferase and aromatic L‐amino acid decarboxylase
Authors: Schultz Eija
Published: May 2005

Catechol‐O‐methyltransferase (COMT) and aromatic L‐amino acid decarboxylase (AADC) activities were determined in human gastrointestinal samples. L‐dopa was used as the substrate and the reaction products 3‐O‐methyldopa (3OMD) and dopamine were separated by reversed phase HPLC and detected by electrochemical or UV detection. COMT activities varied between 40–350 pmol/mg/min and AADC activities between 100 – 3300 pmol/mg/min in different parts of the gastrointestinal tract. COMT inhibitors nitecapone (OR‐462) and OR‐611 effectively inhibited human gastrointestinal COMT activity in vitro, the IC50 values ranging from 10–20 nM and 5–75 nM, respectively. In vitro carbidopa inhibited AADC slightly more effectively than benserazide.
Coproporphyrinogen oxidase, protoporphyrinogen oxidase and ferrochelatase activities in iron‐overloaded and ethanol‐treated rats
Authors: Li Famei , Lim C. K. , Peters T. J.
Published: May 2005

The activities of coproporphyrinogen oxidase, protoporphyrinogen oxidase and ferrochelatase in liver homogenates of iron‐overloaded and acutely or chronically ethanol‐treated rats were assayed by high performance liquid chromatographic methods. Decreased coproporphyrinogen oxidase activity was found in chronically carbonyliron‐fed rats while the activities of protoporphyrinogen oxidase and ferrochelatase remained unchanged. Coproporphyrinogen oxidase and protoporphyrinogen oxidase activities in both chronically and actuely ethanol‐treated animals were not significantly different from controls. In chronically ethanol‐fed rats normal ferrochelatase activity was found.
The high performance liquid chromatography of enzyme systems relating to purine and pyrimidine metabolism: an overview
Authors: Perrett David , Simmonds H. Anne
Published: May 2005

Purines and pyrimidines are now routinely separated by HPLC. By careful selection of chromatographic conditions which match the expected changes in hydrophobicity and/or ionic nature of the substrate and products most enzymes of the purine and pyrimidine salvage pathways can be routinely and accurately determined. Ion‐paired reversed‐phase systems are often the most advantageous. The relevance of such assays to biomedical analysis including their role in the diagnosis of inborn errors of metabolism is stressed.
A new method for the determination of adenine phosphoribosyltransferase activity in human erythrocytes by reversed phase high performance liquid chromatography
Authors: Kojima Tsukasa , Nishina Toshihiro , Kitamura Motoshi , Yamanaka Hisashi , Nishioka Kusuki
Published: May 2005

A new method for the determination of adenine phosphoribosyltransferase (APRT) activity in human erythrocytes is described. APRT activity was assayed by a non‐radiochemical method in which adenosine monophosphate (AMP) and AMP metabolites produced from a substrate adenine were converted to inosine by alkaline phosphatase and adenosine deaminase. The inosine thus produced was quantitated by reversed phase HPLC. This method was simple, precise, sensitive and free from interference with other co‐existing erythrocyte enzymes. Four patients with 2,8‐dihydroxyadenine urolithiasis and others with several disorders in purine metabolism have been studied, showing that the present method is clinically useful for the diagnosis and the evaluation of the severity of some human diseases.
Characterization of human progesterone receptor by high performance hydrophobic interaction chromatography
Authors: Levy A. , Boyle D. M. , van der Walt L. A.
Published: May 2005

High performance hydrophobic interaction chromatography has been used to separate progestin receptors (PRs) from human uterus and from the T47D human breast cancer cell line. Reproducible separations of high resolution were achieved using a TSK Phenyl‐5PW column and a reverse salt gradient of 400 mM to 0 mM sodium sulfate in phosphate buffer, pH 7.4. Peaks of radioactivity exhibiting hydrophobic behaviour were isolated, as well as a smaller proportion of specific bound receptors located in the void volume fraction. No differences in retention times were observed between uterine and breast cell line samples. When the technique was used in conjunction with rapid vertical tube sucrose density gradient centrifugation, the 8S sedimenting PR from fresh, low‐salt cytosol always eluted with a retention time of 24 min. The natural 4S receptor chromatographed as a single peak at 29 min while the 4S receptor species from high‐salt cytosol appeared as two distinct peaks of radioactivity with retention times of 29 and 33 min. While specific binding was shown to occur in the void volume of the column, the origin of these receptors were indeterminate. These results would suggest that under these conditions the 8S receptor occurs as a single hydrophobic class of protein, whereas the data provides evidence that transformed 4S receptor may be proportioned into two unequal entities as a function of exposure to salt.
Direct enantiomeric high performance liquid chromatographic separation of aminoglutethimide and its major metabolite on a series of Chiralcel OD and Chiralcel OJ columns and its application to biological fluids
Authors: Aboul‐Enein Hassan Y. , Islam M. Rafiqul
Published: May 2005

A direct, isocratic, sensitive and precise liquid chromatographic method is presented for the enantiomeric separation of aminoglutethimide (AG) and its acetylated metabolite (AcAG) using cellulose tris‐3,5‐dimethyl phenyl carbamate (Chiralcel OD) and cellulose tris(4‐methylphenyl benzoate) ester (Chiralcel OJ) columns in series. The enantiomeric elution order is determined by separate chromatography of the racemate AG and racemate AcAG and of their separate enantiomers under similar conditions. This method has been used to determine and identify the enantiomers of AG and AcAG in the urine sample collected from a metastatic breast cancer patient after administration of AG for 24 h. Large amounts of (+)‐R‐AG are excreted unchanged in the urine together with smaller quantities of (+)‐R‐AcAG, while most of the (−)‐S‐AG is metabolically converted into (−)‐S‐AcAG.
Separation of β‐ and γ‐tocopherols in serum by high performance liquid chromatography
Authors: Al‐Meshari Abdul Aziz , Aleem Mohammed A.
Published: May 2005

A simple and sensitive high performance liquid chromatographic method for the analysis of tocopherols (α, β, γ and δ) in serum with UV detection is described. In normal phase mode excellent separation of positional isomers (β and γ) was achieved by adjusting the proportion of isopropanol in the mobile phase. Detection limits for both β and γ were 0.62 mg/L and 0.42 and 1.2 mg/L for α and δ isomers, respectively. Measurement of all the tocopoherol structural isomers (α, β, γ and δ) in a sample required only 400 μL of serum and each run was completed in less than 22 min.
Inorganic supports coated with N‐substituted polyacrylamides: Application to biospecific chromatography of proteins
Authors: Ivanov Alexander E. , Kozlov Leonid V. , Shojbonov Batozhab B. , Zubov Vitaly P. , Antonov Vladimir K.
Published: May 2005

Wide porous glass (WPG) chemically coated with a poly‐N‐(2‐hydroxyethyl)acrylamide layer is proposed as a carrier of biospecific ligands in affinity chromatography. The method of WPG chemical modification includes synthesis of the γ‐aminopropyl derivative followed by chemical adsorption of poly(p‐nitrophenyl acrylate). Ester groups of the polyacrylate‐coated WPG can be used for coupling the ligands bearing primary amino groups. Condensation of esters with ethanolamine yields a poly‐N‐(2‐hydroxyethyl)acrylamide‐coated support with non‐specific adsorption properties resembling those of Sepharose 4B. Human IgG immobilized on the polyacrylate support was used for isolation of the first complement component from human serum and for its separation into subcomponents C1r, C1s and C1q by a one‐step method. An unbound part of serum may be used as the R1 reagent for determining haemolytic C1 activity. The stepwise elution of C1r, C1s and C1q from the column reflects the course of C1 breakdown after its activation on immune complex formation.
Rapid determination by high performance liquid chromatography of di‐2‐ethylhexyl phthalate in plasma stored in plastic bags
Authors: Dine T. , Luyckx M. , Cazin M. , Brunet Cl. , Cazin J. C. , Goudaliez F.
Published: May 2005

A rapid sensitive technique was developed for the analysis of di‐2‐ethylhexyl phthalate (DEHP) in plasma stored in plastic bags by using high performance liquid chromatography (HPLC) with UV detection and a Hypersil ODS column. The compound was easily and efficiently extracted with a mixture of sodium hydroxide and acetonitrile, which allowed the deproteinization of plasma samples. The recovery was greater than 95% and the intra‐ and inter‐assay coefficients of variation were better than 6.5%. The results obtained showed that the amount of DEHP accumulated in plasma varied according to different parameters and depended on the storage conditions (time, temperature and shaking) and also on the lipid content of the stored plasma and the sterilization process of the PVC bags.
Effect of a dual‐head short‐stroke pump on post‐column peroxyoxalate chemiluminescence detection
Authors: Hayakawa Kazuichi , Imaizumi Noriko , Miyazaki Motoichi
Published: May 2005

A dual‐head short‐stroke pump has two advantages in the post‐column peroxyoxalate chemiluminescence (PO‐CL) detection system. The first is to increase the mixing efficiency of solutions. The second is to increase the stability of the PO‐CL reaction by keeping the aryl oxalate and hydrogen peroxide solutions separate. The detection sensitivities for six polycyclic aromatic hydrocarbons (PAHs) increased in the present system by using bis(2,4‐dinitrophenyl) oxalate (DNPO) or bis (2,3,4,5,6‐pentafluorophenyl) oxalate (PFPO) instead of such popular aryl oxalates as bis(2,4,6‐trichlorophenyl) oxalate (TCPO) and bis[2‐(3,6,9‐trioxadecyloxycarbonyl)‐4‐nitrophenyl] oxalate (TDPO). Both DNPO and PFPO increased the sensitivities by factors of 4.1–10.2 and 3.5–8.1, respectively. In addition, DNPO was more stable than PFPO in acetonitrile. These results suggest that DNPO is the most useful aryl oxalate for the sensitive PO‐CL detection of PAHs.
A high performance liquid chromatographic assay for AMP‐deaminase activity in the erythrocytes of healthy subjects and patients with inherited purine disorders
Authors: Smolenski Ryszard T. , Montero Celia , Rodgers A. Victoria , Simmonds H. Anne
Published: May 2005

A novel method for measuring AMP‐deaminase activity in human erythrocytes is presented, based on the determination of the reaction product, IMP, using high performance liquid chromatography. IMP formation was found to be proportional both to the incubation time and the amount of haemolysate over a wide range. The minimal detectable AMP‐deaminase activity was more than 1000 times lower than the mean activity found in healthy controls (1083 nmol/h/mg Hb). No marked difference of activity was found in the patients with the following inherited purine disorders: familial juvenile gouty nephropathy and deficiencies of adenosine deaminase, hypoxanthine‐guanine phosphoribosyltransferase or adenine phosphoribosyltransferase. The activity in the erythrocytes of patients with chronic renal failure was also similar to controls. The existence of subjects with low erythrocyte AMP‐deaminase activity in the population has been confirmed.
pH‐dependent colum fractionation for characterization of endogenous digoxin‐like immunoractive factors in pregnant urine
Authors: Homma Masato , Hirano Toshihiko , Oka Kitaro
Published: May 2005

Samples of pregnant urine were treated with three diatomaceous earth columns to fractionate organic solvent‐soluble components. The first column was pre‐injected with the urine sample. The second and the third columns were pre‐injected with sodium bicarbonate and sodium hydroxide solutions, respectively; these played an important role in capturing acidic components in the urine extract from the first column. Neutral components could be eluted out from the columns with the organic solvent, while the basic components were retained in the first column. Once strongly and weakly acidic components were captured on the alkaline columns, they were separately treated with an acidic mobile phase and recovered. The digoxin‐like activities of each fraction were examined for cross‐reactivity to antidigoxin antibody and inhibitory effects on dog kidney Na+, K+‐ATPase. Both types of reactivity were found in all three fractions and decreased in the following order: strongly acidic, neutral and weakly acidic. The neutral fraction of pregnant urine showed significantly greater cross‐reactivity than that of the non‐pregnant urine. Following hydrolysis of the pregnant urine with β‐D‐glucuronidase, the cross‐reactivity of the neutral fraction increased significantly. High performance liquid chromatographic analysis showed that several components in the neutral fraction had apparently increased after the hydrolysis, showing the possible presence of unknown digoxin‐like components in the pregnant urine.
Determination and purification of metallothioneins by high performance liquid chromatography
Authors: Pan Aihua , Wang Zhenxin , Ru Binggen
Published: May 2005

A rapid, reproducible and sensitive high performance liquid chromatography (HPLC) method for the determination and purification of metallothionein‐I (MT‐I) and metallothionein‐II (MT‐II) in mouse and rabbit livers has been developed. Methallothioneins (MTs) were separated by an HPLC anion exchange column, eluted through a linear gradient of Tris buffer and the peak containing MTs was determined by atomic absorption spectrophotometry. Furthermore, the content of MT‐I or MT‐II was calculated by protein peak area in a short time (about 20 min). The sample to be tested was homogenized, centrifuged and saturated by cadmium. MT‐I and MT‐II were eluted at 15.9 and 19.3 min, respectively. The following mouse liver cytosols were tested: controls, Cd‐injected samples and 60Co‐irradiated samples. A detection limit of 5 μg/g liver was established for this method. We have analysed more than 100 biological samples and obtained satisfactory results.
A high performance liquid chromatographic method for the quantitation of flecainide in plasma
Authors: Broly F. , Marecaux P. , Houdret N. , Lhermitte M. , Houssin R.
Published: May 2005

A routine high performance liquid chromatographic method for the rapid determination of flecaïnide (Flecaine®), using a novel internal standard, N‐methylflecaïnide, has been developed. After deproteinization of spiked samples, flecaïnide was totally recovered at neutral pH. Flecaïnide and the internal standard were separated on a reversed phase XL 3 μm ODS column using 10 mM phosphate buffer, pH 3.0: acetonitrile (70:30) as mobile phase, in less than 10 min. With spectrofluorometric detection, the limit of quantitation for flecaïnide was 10 n/mL. Intra‐ and inter‐assay precision variations were 0.24% and 1.4%.
Specific high performance liquid chromatographic determination of the molecular weight and concentration of hyaluronic acid in complex mixtures by labelled hyaluronate binding proteins
Authors: Orviský E. , Kéry V. , Stančiková M.
Published: May 2005

A simple High perfomance liquid chromatographic (HPLC) method for the specific determination of the molecular weight and concentration of hyaluronic acid (HA) in complex mixtures has been developed. Hyaluronate‐binding proteins isolated from bovine cartilage labelled by 125I or fluoresceinisothiocyanate were used as specific markers. The specific binding affinities of the markers were compared and were found to have association constants of 1.6 × 107 M−1 and 1.2 × 107 M−1 respectively. The HA levels and molecular weight distributions can be easily determined in the range 10‐500 ng/mL in complex mixtures by the use of markers, molecular sieving HPLC columns and appropriate detectors. It has been demonstrated clearly that the method is useful for the highly specific determination of the parameters in complex biological samples such as serum and synovial fluids and is recommended for clinical applications.
High performance liquid chromatographic analysis of tetroxoprim and sulphadiazine in serum and urine
Authors: Alkaysi Hanan N. , Salem Mutaz Sheikh , Badwan Adnan A.
Published: May 2005

Quantitative determination of tetroxoprim and sulphadiazine in serum and urine was performed using reversed phase high performance liquid chromatography. Protein precipitation using 10% perchloric acid was utilized for purification of serum samples while urine samples were diluted prior to analysis. The mobile phase consisted of triethylammonium acetate buffer (85%), acetonitrile (12%) and methanol (3%), with a final pH of 4.2. The eluent was monitored at 280 nm. Benzoic acid was used as an internal standard. Standardization, validation and application of the method is described.
Preparation of glycine‐conjugated bile acids and their gas/liquid chromatographic analysis on an aluminum‐clad flexible fused silica capillary column
Authors: Iida Takashi , Tamaru Tamaaki , Chang Frederic C. , Goto Junichi , Nambara Toshio
Published: May 2005

This paper describes a method for the direct gas/liquid chromatographic (GC) analysis of 46 glycine‐conjugated bile acids, which differ from one another in the number, position and configuration of the hydroxyl groups at positions C‐2, C‐3, C‐4, C‐6, C‐7 and/or C‐12. Free bile acids were converted quantitatively on a micro scale to ethyl ester‐trimethylsilyl (Et‐TMS) and methyl ester‐dimethylethylsilyl (Me‐DMES) ether derivatives of the corresponding glycine conjugates. The Et‐TMS and Me‐DMES ethers of the glycine conjugates were chromatographed on an aluminum‐clad flexible fused‐silica capillary column coated with a thin film (0.1 μm) of chemically bonded and cross‐linked methylpolysiloxane. Relative retention time (RRT) and methylene unit (MU) values were determined for the 46 compounds and their GC behaviour was discussed. The derivatization procedure and the retention data would be useful for the direct GC identification of unknown glycine‐conjugated bile acid mixtures extracted from biological samples.
A sensitive and specific high performance liquid chromatographic assay for imidazole dipeptides and 3‐methylhistidine in human muscle biopsies, serum and urine
Authors: Teahon K. , Rideout J. M.
Published: May 2005

A sensitive and specific high performance liquid chromatographic method is described for measuring imidazole dipeptides and 3‐methylhistidine in human muscle biopsies, serum and urine. Muscle extract, serum or urine was reacted with o‐phthaldialdehyde and the derivatives were separated by reversed phase chromatography with column swithing and fluorescence detection.
Quantitative determination of valproic acid and 14 metabolities in serum and urine by gas chromatography/mass spectrometry
Authors: Fisher E. , Wittfoht W. , Nau H.
Published: May 2005

A method for the determination of the antiepileptic drug valproic acid and 14 of its metabolites in serum and urine by gas chromatography/mass spectrometry with selected ion monitoring of the trimethylsilylated derivatives has been developed. Sample preparation, including hydrolysis of VPA‐conjugates and removal of urea in urine is carried out at pH 5.0 and is rapid and simple. The samples are extracted with ethyl acetate and the concentrated extracts are trimethylsilylated. Analysis with adequate separation of metabolites is achieved with a DB 1701 fused silica (Megabore) capillary column. The method exhibits high recovery and reproducibility and is sufficiently sensitive and selective for analysis of small sample volumes. Application of the method for screening patient serum and urine samples for unusual metabolite patterns, with possible predictive value for early detection of liver injury, is presented.
Enhanced selectivity of the isolation of basic drugs from body fluids by means of extraction minicolumns and sophisticated elution schemes
Authors: Šcoltés Ladislav
Published: May 2005

The paper presents results and experience obtained in the isolation of a number of basic polar/non‐polar solutes (drugs, their metabolites and structural derivatives) from biological samples by using the separation minicolumns SeparcolR SI and SeparcolR SI C18 L/T. The aim was to provide insight into some simple procedures yielding optimal selectivity/specificity of the isolation of the studied solutes either by proper selection of the sorbent or by applying sophisticated elution schemes which use to advantage different properties of the eluents/solvents.
Isolation of clobazam–orosomucoid complexes from Patients' sera
Authors: Treuheit Michael , Halsall H. Brian
Published: May 2005

Orosomucoid, a member of the lipocalin family, may function in the in vivo transport of lipophilic compounds such as basic and neutral drugs. We describe the identification of 7‐chloro‐1‐methyl‐1,5‐benzodiazepine‐2,4‐dione (clobazam) bound to the serum orosomucoid from individuals actively taking this tranquillizer. This suggests not only that other endogenous factors limit access to the benzodiazepine binding site on human serum albumin, but also that the differential binding of benzodiazepines and their metabolites by orosomucoid should be considered in determining therapeutic doses, particularly in the acute phase response.
Determination of urinary free cortisol by high performance liquid chromatography with sulphuric acid–ethanol derivatization and column switching
Authors: Nozaki Osamu , Ohata Tsuneko , Ohba Yasuhiro , Moriyama Hiroyuki , Kato Yoshio
Published: May 2005

A rapid and highly sensitive determination method for urinary free cortisol has been developed using reversed phase high performance liquid chromatography (HPLC) with a precolumn for sulphuric acid‐ethanol fluorescence derivatization and column switching. Urinary cortisol, eluted from the octadecylsilane‐bonded silica (ODS) minicolumn with 90% aqueous ethanol, was derivatized with the addition of sulphuric acid only at ambient temperature. Cortisol derivatives injected directly onto the ODS precolumn were purified on‐line. After switching the columns, the cortisol derivative was separated on an ODS analytical column with a retention time of 15.3 min and monitored at an emission wavelength of 520 nm (exitation wavelength of 365 nm) to decrease the detection limit to 0.26 μg/dL (signal‐to‐noise ratio = 3). The automated HPLC operation resulted in good reproducibility and recovery of the stable cortisol derivative at 5°C.
Simple and highly sensitive determination of free fatty acids in human serum by high performance liquid chromatography with fluorescence detection
Authors: Iwata Tetsuharu , Inoue Kouji , Nakamura Masaru , Yamaguchi Masatoshi
Published: May 2005

A highly sensitive and simple reversed phase high performance liquid chromatographic (HPLC) method for the quantitative determination of free fatty acids in human serum is presented. The method is based on the direct derivatization of serum fatty acids with 6,7‐dimethoxy‐1‐methyl‐2(1H)‐quinoxalinone‐3‐propionylcarboxylic acid hydrazide. The derivatization reaction proceeds in aqueous solution in the presence of pyridine and 1‐ethyl‐3‐(3‐dimethylaminopropyl)carbodiimide at 37°C. The resulting derivatives are separated within 75 min on a reversed phase column (YMC Pack C8) with a gradient elution of aqueous acetonitrile and detected fluorimetrically. The detection limits are 2.5–5 fmol in a 10 μL injection volume. The sensitivity permits precise determination of free fatty acids in 5 μL serum. The method is simple and is without the conventional liquid–liquid extraction steps of serum fatty acids.
A sensitive determination method for mexiletine derivatized with dansyl chloride in rat plasma utilizing a HPLC peroxyoxalate chemiluminescence detection system
Authors: Nishitani Atsuhiko , Kanda Susumu , Imai Kazuhiro
Published: May 2005

A sensitive determination method for a non‐fluorescent anti‐arrhythmic drug, mexiletine, in rat plasma is presented utilizing a HPLC peroxyoxalate chemiluminescence (PO‐CL) detection system. After an internal standard (4‐methylmexiletine, 4.35 pmol) and 0.1 N sodium hydroxide solution were added to 5 μL rat plasma, the solution was poured onto an Extrelut 1 column. Both mexiletine and the internal standard were eluted with diethy ether and then the eluate was evaporated to dryness. The residue was dissolved in 0.2 M borate buffer (pH 8.5) and mixed with dansyl chloride (75 nmol) in acetronitrile. After standing of 90 min at room temperature, 0.5 N HCI was added to the reaction mixture to stop the reaction and a 2/45 aliquot of the mixture was subjected to a HPLC PO‐CL detection system using bis(4‐nitro‐2(3,6,9‐trioxadecyloxycarbonyl)phenyl)oxalate (TDPO) and hydrogen peroxide. The calibration curve for mexiletine in rat plasma was linear over the range 20–100 ng/mL plasma (20.6–103 fmol/injection). The detection limit (S/N = 2) was 1.0 fmol over the whole procedure. The method was applied to the measurement of the time courses of plasma mexiletine concentration after oral administration of the drug [25 mg (115.9 μmol)/kg] to rats.
The influence of synthetic conditions on the stability of methotrexate‐monoclonal antibody conjugates determined by reversed phase high performance liquid chromatography
Authors: Hudecz F. , Garnett M. C. , Khan T. , Baldwin R. W.
Published: May 2005

Methotrexate (MTX) has been convalently attached to an IgG‐type monoclonal antibody (791T/36) directed to tumour‐associated antigen gp72. Conjugates were synthesized by the active ester method using MTX N‐succinimidyl ester at various pH values (7.5–10.5). Following purification by gel filtration, high performance liquid chromatography was used to assess the free drug or its derivatives in samples of MTX–791T/36 conjugates previously treated (or not) with hydroxylamine. Quantitative analysis, performed on a reversed phase column (pore size 300 Å) with isocratic acetonitrile–sodium acetate buffer (pH 4.8) as mobile phase, indicated no detectable amount of free methotrexate in hydroxylamine‐treated conjugates even six months after their preparation. Similar observations were made with conjugates, whose synthesis were performed at pH ⩾ 10. In contrast, the presence of increasing amounts of drug/metabolite could be demonstrated in samples produced at lower pH values. Based on these findings, the pH‐dependent kinetics of MTX release has been determined and used to design conditions under which stable MTX–791T/36 conjugates could be prepared without hydroxylamine reaction.
Measurement of N‐acetylneuraminic acid in human serum and urine by high performance liquid chromatography with chemiluminescence detection
Authors: Ishida Junichi , Nakahara Toshihiro , Yamaguchi Masatoshi
Published: May 2005

A highly sensitive method for the determination of N‐acetylneuraminic acid in human serum and urine is investigated. This method employs high performance liquid chromatography with chemiluminescence detection. N‐Acetylneuraminic acid, released by hydrochloric acid hydrolysis of serum and urine, and N‐glycolylneuraminic acid (internal standard) are converted into chemiluminescent derivatives with 4,5‐diaminophthalhydrazide dihydrochloride, a chemiluminescence derivatization reagent for α‐keto acids. The derivatives are separated within 35 min on a reversed phase column, TSKgel ODS‐120T, with isocratic elution, followed by chemiluminescence detection; the chemiluminescence is produced by the reaction of the derivatives with hydrogen peroxide in the presence of potassium hexacyanoferrate(III) in alkaline solution. The detection limit for N‐acetylneuraminic acid is 9 fmol (signal‐to‐noise ratio = 3). This sensitivity permits precise determination of N‐acetylneuraminic acid in 10 nL of serum or 50 nL of urine. The method is applied to the determination of the N‐acetylneuraminic acid in human sera from normal subjects and cancer patients and in normal urine.
An improved method for preparation of perbenzoylated ganglioside‐derived sialic acids and nanogram detection of N‐acetyl‐ and N‐glycolylneuraminic acid by high performance liquid chromatography
Authors: Unland Frank , Müthing Johannes
Published: May 2005

An improved method for the preparation of perbenzoylated ganglioside‐derived sialic acids is described. After mild acid hydrolysis, isolation of sialic acids can be achieved by Folch partition (Method A) or by anion exchange chromatography (Method B). Perbenzoylated sialic acids were freed from benzoylation reagents by a second Folch partition. Total recoveries of both methods were found to be ⩾90%, calculated from metabolically labelled gangliosides. Derivatized N‐acetylneuraminic and N‐glycolylneuraminic acids were separated and quantified by isocratic high performance liquid chromatography using a RP18 column as the stationary phase and methanol: water (8:2) as the mobile phase. Both sialic acids were completely separated and eluted as single peaks within 15 min, monitored by UV detection. As little as 20 ng of neuraminic acid could be detected, the detector being linear up to 5 μg tested.