bluuebunny/crossref_metadata_2025 huggingface dataset:
Rapid assay of β‐galactosidase and sialyltransferase by lectin affinity high performance liquid chromatography with fluorescence detection
Authors: Harada Hiroshi , Ueno Yasushi , Kamei Masugu , Ohura Reiko , Tanabe Noriko , Uchida Yasuko , Koyama Fumihiro , Yamaguchi Takashi
Published: May 2005

Fluorescein isothiocyanate (FITC)‐labelled asialotransferrin and pyridyl aminated oligosaccharides were prepared from asialotransferrin and human milk using affinity chromatography and high performance liquid chromatography (HPLC), respectively. These substances were incubated with galactosidase or sialyltransferase and then examined by lectin affinity HPLC. The elution patterns changed according to the period of incubation and amount of enzyme. This analytical method using lectin affinity HPLC with fluorescence labelled glycoprotein or oligosaccharides as the substrates has great value for detecting these enzyme under the same chromatographic conditions. In addition, differences were noted in the activity of β‐galactosidase toward oligosaccharides having the Gal β(1 → 3)GlcNAc or Gal β(1 → 4)GlcNAc structure at reducing termini.
Rapid and sensitive determination of β‐phenylethylamine in animal brains by high performance liquid chromatography with fluorometric detection
Authors: Taga Chiaki , Tsuji Motohiro , Nakajima Teruo
Published: May 2005

A reversed phase HPLC method with fluorometric detection for the analysis of β‐phenylethylamine has been developed using p‐methoxyphenylethylamine as an internal standard. Two columns, containing 200 μL of Dowex 50‐X8 and Amberlite CG‐50 respectively, were used to prepare a fraction containing β‐phenylethylamine. The recoveries of β‐phenylethylamine and p‐methoxyphenylethylamine were 53.9 ± 9.4% and 68.1 ± 12.4%, respectively, and elution profile of p‐methoxyphenylethylamine was sufficiently well correlated with that of β‐phenylethylamine. Regional distributions of β‐phenylethylamine in rat and mouse brains were determined. The highest concentrations were found in hypothalamus and hippocampus in both animals.
Lactoferrin from human breast milk and from neutrophil granulocytes. Comparative studies of isolation, quantitation, characterization and iron binding properties
Authors: Bezwoda W. R. , Mansoor N.
Published: May 2005

The isolation and properties of lactoferrin from human breast milk and from neutrophilic granulocytes were investigated. Human breast milk lactoferrin was purified by means of heparin‐sepharose or Cibacron Blue affinity chromatography. Quantitative recovery using these two methods was comparable but Cibacron Blue affinity chromatography allowed for isolation of a more homogenous protein. Lactoferrin could only be isolated from human neutrophilic granulocytes by sequential use of antibody affinity followed by non‐specific affinity chromatography. Both breast milk lactoferrin and granulocyte lactoferrin were separated into apo and iron‐rich species by SDS polyacrylamide gel chromatography. Iron binding is accompanied by a conformational change in tertiary structure associated with more rapid electrophoretic migration. The isoelectric point of both human breast milk lactoferrin and human granulocyte lactoferrin is 5.5 – 6.2. Both types of lactoferrin have similar iron binding properties with release of iron from the one binding site occurring at pH 5.2 – 6.0 while the other binding site holds on to iron down to pH 3.6 – 3.2. Despite the high affinity for iron the percentage saturation of native lactoferrin is low, that for breast milk lactoferrin averaging 12 – 25% and that for granulocyte lactoferrin < 10%.
Purification of rat liver soluble catechol‐O‐methyltransferase by high performance liquid chromatography
Authors: Korkolainen Tapio , Nissinen Erkki
Published: May 2005

The soluble form of catechol‐O‐methyltransferase (EC 2.1.1.6.) from rat liver was purified to homogeneity by high‐performance anion‐exchange chromatography and high‐performance gel‐filtration chromatography. The specific activity of the final pool was 270 U/mg protein. The purification was 1180‐fold and recovery of the enzyme activity was 15%. During this rapid and gentle purification there were no problems with loss of activity, and the estimated half life of the final purified enzyme pool was 5.5 days at +4°C. The only additive used was phenylmethylsulfonylfluoride in the homogenizing buffer.
Evidence for the overestimation of molecular masses of proteins after chemical modification and chemical crosslinks on sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS‐PAGE)
Authors: Richard Claude , Han Kia‐Ki , Yang Hui‐Ling , Zhu De‐Xu , Balduyck Malika , Mizon Jacques
Published: May 2005

Human trypsin inhibitor (home prepared), lactalbumin, trypsinogen, carbonic anhydrase, and bovine serum albumin were submitted to succinylation and their molecular masses were determined by SDS‐PAGE according to the method of Weber and Osborn (1969 J. Biol. Chem. 244, 4406) before and after chemical modification. High estimates of their molecular masses were obtained. The monomer and dimer of arrowhead inhibitor proteinase‐B (Chinese vegetable legume) obtained after chemical crosslink(s) were also submitted to SDS‐PAGE and their apparent molecular masses were also determined and compared to the native arrowhead inhibitor proteinase‐B. Abnormally high estimates of their molecular masses were obtained. Our results agree with those in the literature.
Unconjugated methoxylated catecholamine metabolites in human saliva. Quantitation methodology and comparison with plasma levels
Authors: Drebing Carla J. , Freedman Robert , Waldo Merilyne , Gerhardt Greg A.
Published: May 2005

A newly developed method for the simultaneous extraction and quantitation of the unconjugated levels of the catecholamine metabolites vanilmandelic acid (VMA), 3‐methoxy‐4‐hydroxyphenylethylene glycol (MHPG) and homovanillic acid (HVA) in plasma by high performance liquid chromatography with electrochemical detection was modified and applied to studies of human saliva. The assay had a mean coefficient of variation under 3% for each of the metabolites. Levels of plasma VMA, MHPG and HVA were measured in 28 normal subjects and compared to their saliva levels, obtained before and after stimulation by mastication. Significant correlations were found between plasma and saliva MHPG and HVA, but there was no correlation between plasma and saliva VMA. Salivary MHPG and HVA can be reproducibly assayed and may be useful tools for indications of changes in central and peripheral catecholamine metabolism.
Isolation of glycoprotein D from herpes simplex virus type 1 by gel filtration high performance liquid chromatography
Authors: McGarry Thomas J. , Al‐Ahdal Mohammed N.
Published: May 2005

Rabbit kidney (RK‐13) and human jejunum and ileum (I‐407) cells infected with herpes simplex virus type 1, strain F, were radiolabelled with [14C]glucosamine or [35S]methionine for 24 h. The cells were extracted with 1% Triton X‐100 and the extracts were separated by gel filtration high performance liquid chromatography. Monoclonal antibody immunoprecipitation of the fractions collected from the column revealed a monomeric glycoprotein D (gD) of 52 – 56 000 molecular weight from RK‐13 cells and two monomeric forms of gD, 54 000 and 58 000 molecular weight, from I‐407 cells. Densitometry scanning of the autoradiograms from SDS‐PAGE showed gD from the RK‐13 host cells to be 98.7% pure with the [35S]methionine label and 97.0% pure with the [14C]glucosamine. On the other hand, gD from the I‐407 host cells was only 78.6% with the [35S]methionine label and 96% pure with the [14C]glucosamine. This method could provide a means for the isolation of native gD for structural and immunological studies.
Multidimensional chromatographic separation of estrogen receptors
Authors: Raubenheimer J. , van der Walt L. A.
Published: May 2005

Analyses of estrogen and progesterone receptors in biopsies of breast carcinoma play a vital role in the selection of patients likely to respond to hormone manipulation. Sucrose density gradient centrifugation has been the reference method in the determination of estrogen receptors in human breast carcinoma cytosols. To reduce assay time and circumvent prolonged manipulation of labile receptor preparations, high performance liquid chromatography techniques in the size‐exclusion and ion‐exchange modes were compared as potential alternate methods for the rapid separation of receptor isoforms. Multidimensional analyses were performed by reapplying estrogen receptor isoforms obtained from high performance size‐exclusion and ion‐exchange chromatography to sucrose density gradients and vice versa. This confirmed that the estrogen‐binding components identified by high performance liquid chromatography appear to correspond to estrogen receptor species from sucrose density gradients.
Capillary gas chromatography/negative ion chemical ionization mass spectrometry for the quantification of bile acids including 1β‐hydroxylated and unsaturated bile acids in serum and urine
Authors: Miyara Takako , Shindo Noriko , Tohma Masahiko , Murayama Kimie
Published: May 2005

12 bile acids, including 1 β‐hydroxylated and unsaturated bile acids, have been quantified by capillary gas chromatography/negative ion chemical ionization mass spectrometry, using the trimethylsilyl(TMS) ether derivatives of bile acid pentafluorobenzyl(PFB) esters. The analysis time is 12 min and the minimum measurable amount is 100 fg for each bile acid. Bile acids in 200 μL of serum and 50 μL of urine from healthy human adults were measured. These small sample sizes enhance the practicality of using this method as a screening test for bile acids in the serum and urine of human infants, where small sample size is a major problem.
Simultaneous detection and quantitation of O6‐monoacetylmorphine, morphine and codeine in urine by gas chromatography with nitrogen specific and/or flame ionization detection
Authors: Vu‐Duc Trinh , Vernay André
Published: May 2005

It is of importance to differentiate heroin intake from the absorption of opiate‐containing pharmaceuticals or opiates from other sources. A method for the routine determination of O6‐monoacetylmorphine (6‐MAM), the specific metabolite of heroin in human urine, by gas chromatography and classical detectors without having recourse to gas chromatography/mass spectrometry‐selected ion mode (GC/MS‐SIM) is described. With dual detection by nitrogen selective and flame ionization detectors, the limits of detection for 6‐MAM were determined to be 2 ng/mL and 4 ng/mL urine for a 10 mL sample. When applied to urines preliminarily screened for opiates, the results appeared consistent in comparison with those obtained by GC/MS‐SIM. The method was also developed for the simultaneous quantitative analysis of morphine and codeine. The linearity was tested up to 600 ng/mL for the three compounds of interest 6‐MAM, morphine and codeine and their absolute recoveries were 76%, 78%, 75% respectively.
Rapid detection of extended ganglio‐series gangliosides with terminal GalNAcβl‐4Gal sequence on high performance thin layer chromatography plates
Authors: Müthing Johannes , Ziehr Holger
Published: May 2005

The mouse monoclonal antibody 2D4, which recognizes the terminal GalNAcβ1‐4Gal‐disaccharide of GgOse3Cer and GgOse5Cer, was used for the detection of ganglio‐series gangliosides. The method involves separation of gangliosides on thin layer chromatography plates, followed by silica gel fixation, Arthrobacter ureafaciens neuraminidase treatment and final immunostaining of desialylated gangliosides with the monoclonal antibody 2D4. Both neuraminidase and the hybridoma 2D4 producing the specific monoclonal antibody are commercially available and therefore accessible to all researchers working in this field.

Gangliosides from mouse T lymphocytes and the mouse T cell lymphoma YAC‐1 have been used as examples. This technique may be used for fast screening of gangliosides with the GgOse5Cer core structure which have been described as T cell markers, antigens in human neuronal disease and receptors for certain pathogenic bacteria.
A new method for the quantitation of aflatoxin M1 in urine by high performance liquid chromatography and its application to the etiologic study of hepatoma
Authors: Liu Zhong‐he , Tu Wen‐sheng , Li Dan‐rong , Li Yuan‐dong , Xie Chong‐hu , Yang Yin‐zhong , Qin Bing‐bing
Published: May 2005

A high performance liquid chromatographic (HPLC) method for the analysis of aflotoxin M1 (AFM1) in urine is described. Urine samples were treated with saturated lead acetate and AFM1 was extracted with chloroform. After washing with water to remove impurities the compound was derivatized with trifluoracetic acid and the AFM1 derivative was analyzed quantitatively by HPLC. The sample pretreatment is simple and more selective. A good line correlation between AFM1 peak height and its concentration was obtained when AFM1 content was in the range of 50–400 pg. The ratio of recovery was 87.42%. Sensitivity is 0.01 ppb. The method is applicable to trace analysis. Results in urine of residents who live in the high/low liver cancer incidence area in Fushui county were the same as that of previous epidemiological investigation.
Chemiluminescence high performance liquid chromatography of corticosteroids using lucigenin as post‐column reagent
Authors: Takeda Motoko , Maeda Masako , Tsuji Akio
Published: May 2005

A chemiluminescence high performance liquid chromatographic method for the determination of corticosteroids and tetrahydrocorticosteroids has been developed. Corticosteroids and their metabolites extracted from urine samples were separated using an ODS column and a mixture of methanol + water + 0.01 M sodium acetate solution (70:30:5) as eluent. The eluent from the column was mixed with the chemiluminescent solution containing lucigenin and Triton X‐100 and a 0.28 M KOH solution by pumps and monitored by a chemiluminescence detector. No interference was encountered and the method is both precise and reproducible.
Determination of individual bile acids in serum by high performance liquid chromatography
Authors: Wang Ganfeng , Stacey Neill H. , Earl John
Published: May 2005

A high performance liquid chromatographic (HPLC) method for analysis of 4 free and 8 conjugated bile acids in submicromolar quantities in serum is described using precolumn derivatization with 4‐bromomethyl‐7‐methoxy‐coumarin (BMC) and fluorescence detection. Bile acids were extracted from serum with 0.4 M sodium bicarbonate, adsorbed onto a Sep‐Pak C18 cartridge and eluted with methanol. The extract was derivatized with BMC in acetonitrile using 18‐crown‐6 crown ether as catalyst and the BMC labelled glycine conjugates and free bile acids were analysed using acetonitrile + methanol + water gradient elution and detection at 320/385 nm.

Using a novel and simple approach, taurine conjugates were isolated by extracting the dried, derivatized material with water, in contrast to previous methods which required column chromatography cleanup to isolate the taurine conjugates prior to derivatization. The isolated taurine conjugates were then hydrolysed enzymatically, extracted, derivatized and analysed as free‐bile acids.

Recoveries of individual bile acids varied from 83 – 96% for free and glycine conjugates and 72 – 83% for taurine conjugates. Coefficients of variation were in the range of 5.1 – 12.5%. In addition to the simpler and shorter procedure for taurine conjugates, this method has increased sensitivity over most other procedures and improved HPLC separation for the various bile acids and conjugates with equivalent recovery and reproducibility compared with other published methods.
Gel filtration high performance liquid chromatography of envelope polypeptide variants of herpes simplex type 1 strains
Authors: Al‐Ahdal Mohammed N. , Qadri S. M. Hussain , McGarry Thomas J.
Published: May 2005

High performance liquid chromatography (HPLC) with a TSK‐4000SW gel filtration column was used to compare envelope polypeptides from four strains of herpes simplex virus type 1 (HSV‐1). The chromatographic profiles demonstrated polypeptide variability among three clinical strains and the wild‐type F strain. Radioimmunoprecipitation of the HPLC fractions with polyclonal anti‐HSV‐1 followed by SDS‐polyacrylamide gel electrophoresis (PAGE) of the immunoprecipitates revealed molecular weight differences of various polypeptides in fractions from the area containing major peaks. This HPLC method could prove useful for the analysis of polypeptide polymorphism in clinical isolates of HSV‐1, as well as in other viruses.
Studies on the determination and degradation of pyrimethamine in mammals
Authors: Kumar Reena , Kaushik P. , Sharma C. B.
Published: May 2005

Treatment of pyrimethamine with blood plasma in vitro yields a metabolite which is also produced when the drug is administered through intravenous injection in the rat. A thin layer liquid chromatographic method for quantitative and qualitative determination of pyrimethamine and its metabolite in plasma and biological tissues is described.
Determination of amines, amine metabolites and some amine metabolizing enzymes by high performance liquid chromatography
Authors: Bháird Nóirín Nic a' , McCrodden John M. , Wheatley Anthony M. , Harrington Michael C. , Sullivan James P. , Tipton Keith F.
Published: May 2005

Some useful high performance liquid chromatographic methods for the determination of amines, amine metabolites and amine metabolizing enzymes are described. These include the separation of tyramine in wines and beers, determination of tryptamine in urine, assay of monoamine oxidase and catechol‐O‐methyltransferase and analysis of amine‐aldehyde condensation products.
Enzyme degradation, high performance liquid chromatography and liquid secondary ion mass spectrometry in the analysis of glycoproteins
Authors: Smith Kevin D. , Harbin Anne‐Marie , Carruthers Robert A. , Lawson Alexander M. , Hounsell Elizabeth F.
Published: May 2005

Analysis of small amounts of glycoproteins by high performance liquid chromatography (HPLC) and liquid secondary ion mass spectrometry (LSIMS) together with enzyme digestion has been investigated using fetuin as a model. Preliminary data indicates that 71% of the expected peptides were detected by LSIMS analysis of 200 pmol total digest. HPLC profiles of peptides and glycopeptides were obtained from 2 nmol of digest using a reversed phase (C18) column eluted in a solvent system containing TFA, water and acetonitrile. This has provided glycopeptides for subsequent oligosaccharide analysis. Strategies are reviewed for the chromatographic characterization of oligosaccharides following their release from glycopeptides by chemical and enzymatic procedures.
A reversed phase liquid chromatographic method for the simultaneous determination of several common penicillins in human serum
Authors: Mendez‐Alvarez E. , Soto‐Otero R. , Sierra‐Paredes G. , Aguilar‐Veiga E. , Galan‐Valiente J. , Sierra‐Marcuño G.
Published: May 2005

A rapid and sensitive high performance liquid chromatographic method is described for the simultaneous determination of benzylpenicillin, ampicillin, phenoxymethylpenicillin, cloxacillin, dicloxacillin and nafcillin in small samples of human serum. The chromatographic system involves the use of a Spherisorb ODS reversed phase column and a gradient elution with 1 mM ammonium acetate buffer/acetonitrile (from 90:10 to 75:25 in 15 min). Detection and quantification are monitored by UV absorption at 208 nm. The compounds are extracted with dichloromethane, using tetrabutylammonium hydrogen sulfate neutralized with sodium hydroxide and buffered with borate as an ion pairing reagent; β‐hydroxyethyltheophylline is added as an internal standard. Our results show that the method is accurate and reproducible, allowing quantification of serum levels of assayed penicillins (0.5–50 μg/mL) without interference from other drugs commonly used in therapy. Recoveries were generally greater than 79.4%.
Chromatographic methods for the analysis of basic neurotransmitters and their acidic metabolites
Authors: Yi Zhen , Brown Phyllis R.
Published: May 2005

Chromatographic techniques for the determination of trace amounts of neurotransmitters were reviewed. The two techniques found to be most useful were GC‐MS and the reversed‐phase mode of HPLC with an electrochemical or fluorescent detector. For structure determination or unequivocal peak identification, GC‐MS is the method of choice. In addition the limits of detection of GC‐MS were better than those obtained by HPLC. However for routine analyses, HPLC is now being used in studies of mental illness and other diseases. Good resolution, reproducibility and sensitivity can be obtained without the derivatisation steps required for GC‐MS, and catecholamines, serotonin, and their acidic metabolites can be concomitantly determined in one analysis.
High performance liquid chromatography as a tool in the definition of abnormalities in monoamine and tryptophan metabolites in cerebrospinal fluid from patients with neurological disorders
Authors: Baig S. , Halawa I. , Qureshi G. A.
Published: May 2005

In this study we report the levels of 3‐methoxy‐4‐hydroxyphenylglycol, 3,4‐dihydroxyphenylacetic acid, homovallinic acid, tryptophan, 5‐hydroxyindole‐3‐acetic acid and serotonin in lumbar cerebrospinal fluid (CSF) from patients with multiple sclerosis, cerebrovascular disease and muscular tension headache the latter, as healthy controls. The separation of these substances was performed on a reversed phase column by ion pair high performance liquid chromatography and detection was made by a glassy carbon electrode set at +900 mV vs Ag+/AgCI. The whole separation was achieved within 25 min. Concentrations of all substances (10‐1000 pmole/L) were linearly proportional to areas obtained. The system is sensitive, stable and reproducible. The significance of CSF levels of these metabolites from patient groups compared with healthy controls are discussed.
Biomedical applications of chiral liquid chromatography
Authors: Camilleri Patrick
Published: May 2005

Many drugs are recemic and therefore much effort has to be devoted towards the stereoselective synthesis of the most effective or less harmful component of a racemic mixture. High performance liquid chromatography will play an important role in the clinical analysis of racemic drugs in anticipation of regulations that are currently being discussed and are expected to be enforced by the end of this decade. In this review a number of methods for chiral resolution are outlined. These include the formation of diastereoisomers and the use of chiral stationary phases or chiral mobile phase additives.
Identification and quantification of lipids from rabbit Harderian glands by gas chromatography/mass spectrometry
Authors: Harvey D. J.
Published: May 2005

Lipids from hydrolysed extracts of Harderian glands from the rabbit were examined as trimethylsilyl, acetonide, nicotinate and picolinyl esters and shown to consist mainly of acylated glycerol ethers and acylated hydroxyglycerol ethers. Constitutents amounting to 98.8% of the recovered secretion were identified. Fatty acids were mainly normal, saturated compounds with chain legths from 12 to 24 carbon atoms; n‐16:0 and n‐18:0 accounted for about 40% of the identified acids. Small concentrations of iso‐17:0 and unsaturated acids with 18‐carbon chains were also identified. Fatty alcohols were again mainly normal‐unsaturated compounds; the chains varied from C18 to C25 with 20:0‐OH and 22:0‐OH being the most abundant. Three types of 1‐alkyl glycerols were found. The major constituents had normal, saturated chains with from 14 to 23 carbon atoms with the even carbon chains predominating. These were accompanied by hydroxylated derivatives of the 16‐ and 18‐carbon glycerol ethers with hydroxy groups in the 10, 11 and 12 positions. Branched‐chain glycerol ethers were of low concentration and contained predominently iso chains. Many of these compounds have not been reported before in this secretion. Low concentrations of the previously reported hydroxy acids with n‐C14, ‐C15 and ‐C16 chains were also found. Nicotinate derivatives were applied to the structural determination of glycerol ethers for the first time and shown to reveal the position of methyl branch points in an analogous manner to that previously shown for mono‐ and di‐hydric alcohols. Acids were identified as picolinyl esters.
Microdetermination of hyaluronic acid in human urine by high performance liquid chromatography
Authors: Akiyama Hiroshi , Toyoda Hidenao , Yamanashi Shigeyuki , Sagehashi Yoshinori , Toida Toshihiko , Imanari Toshio
Published: May 2005

A high performance liquid chromatographic (HPLC) system is described for determination of the unsaturated disaccharide (ΔDi‐HA) derived from hyaluronic acid (HA) in human urine by digestion with hyaluronidase SD. The effects of eluents on the separation of ΔDi‐HA and ΔDi‐OS, which is derived from the reaction of chondroitin with the enzyme, have been studied. The established chromatographic conditions were as follows–column: a stainless steel tube (4 mm i.d. × 250 mm) packed with TSKgel NH2‐60; eluent: a mixture of acetonitrile and 0.1 M Tris‐HCI buffer containing 0.1 M boric acid and 10 mM sodium sulphate, pH 7.0 (64:36, v/v). The strong fluorescence of unsaturated disaccharide after the reaction with 2‐cyanoacetamide in alkaline medium was used for post‐column detection. The calibration curve for ΔDi‐HA was linear in the range 5 pmol‐5 nmol with a practical detection limit of 2 pmol. The assay coefficients of variation (n = 5) at 200 pmol for ΔDi‐HA and ΔDi‐OS were 1.7 and 1.5%, respectively. This HPLC system has been applied to the determination of HA in human urine.
Plasma B‐6 vitamer and plasma and urinary 4‐pyridoxic acid concentrations in young women as determined using high performance liquid chromatography
Authors: Driskell Judy A. , Chrisley Barbara Mc.
Published: May 2005

Plasma B‐6 vitamer and plasma and urinary 4‐pyridoxic acid concentrations of 21 young white women, 21–27 years, having radiomonitored pyridoxal 5′‐phosphate and coenzyme stimulation of erythrocyte alanine amino‐transferase activities indicative of adequate vitamin B‐6 status were determined in an effort to establish normal ranges for plasma B‐6 vitamers. B‐6 vitamers and 4‐pyridoxic acid were quantitated using reversed phase high performance liquid chromatography with fluorometric and ultraviolet detection. Pyridoxal phosphate values obtained by radioenzymatic and chromatographic, fluorometric and ultraviolet, assays were highly correlated as were pyridoxine phosphate values determined using both detectors. The B‐6 vitamer and 4‐pyridoxic acid values of these subjects should be of use in the establishment of normal ranges of these congeners in women.
Comparative performance of ion exchange and ion‐paired reversed phase high performance liquid chromatography for the determination of nucleotides in biological samples
Authors: Perrett David , Bhusate Lekha , Patel Jayshree , Herbert Karl
Published: May 2005

We have compared anion exchange chromatography on APS‐Hypersil (4.6 × 100 mm) eluted with a phosphate gradient with reversed phase chromatography on ODS‐Hypersil (4.6 × 100 mm) in the presence of either tetrabutylammonium (TBA) or triethylammonium (TEA) ions with a methanol gradient. The systems have been compared both for ease of operation and for their resolving power with standard mixtures and acid extracts of both normal red cells (RBC) and ischaemic tissues. The two chromatographic modes exhibited similar separating efficiencies for standard mixtures of nucleotides but retention times were most stable using reversed phase liquid chromatography (RPLC) with TEA. Anion exchange columns slowly lost ion exchange capacity but selectivity was unchanged. RPLC in the presence of TBA gave reproducibile capacity factors only when operated isocratically due to irreversible changes to the silica surface. For RBCs the RPLC with TEA and anion exchange sysems resolved 17 and 15 peaks, respectively, and for the ischaemic samples 22 and 14 peaks, respectively. However, nucleosides and bases were also resolved by the ODS column causing chromatographic crowding and uncertain peak identification.
Rapid high voltage isoelectric focusing of proteins in rod gels
Authors: Das Joachim
Published: May 2005

A rapid procedure of isoelectric focusing (IEF) of proteins in polyacrylamide rod gels (i.d., 1.1 mm; lenght, 7.5 cm) is described. The time required for IEF can be reduced to 0.5 h by using high voltages up to 3000 V in the presence or absence of urea in the gels. When used as the first dimension of a two‐dimensional technique for IEF sodium dodecyl sulphate electrophoresis, high voltage IEF gives smaller protein spots on the second dimension gel, associated with an increase in resolution. The method has been tested by a two‐dimensional separation of an eye sample of the goodeid fish Xenotoca eiseni.
Determination of 2‐chloro‐2′‐deoxyadenosine in human plasma
Authors: Liliemark Jan , Pettersson Birgitta , Juliusson Gunnar
Published: May 2005

The first high performance liquid chromatographic method for determination of the plasma concentration of 2‐chloro‐2′‐deoxyadenosine (CdA) in patients, which is significantly more sensitive than the previously used RIA method, is presented. CdA is a purine analogue with useful clinical activity against lymphoproliferative disorders and it has recently been found to be the single most active agent in the treatment of hairy cell leukaemia. Guaneran (6‐nitroimidazol‐6‐thioguanine) was added to 1 mL plasma as the internal standard and CdA was extracted using ethyl acetate. A Perkin‐Elmer C18, 3 μ, 8 cm column was used for the separation of CdA and the internal standard from endogenous compounds in the sample with a mixture of sodium phosphate buffer 10 mM, methanol and acetonitrile (85:10:5, pH = 3.0) as the mobile phase. The sensitivity of the method (1 nM) allows the determination of CdA in plasma 24 h after the administration of 0.14 m/kg as a 2 h infusion.
Reversed phase high performance liquid chromatographic separation of hydroxy steroidal unsaturated esters and their hemisuccinates
Authors: Drašar Pavel , Pouzar Vladimir , Černỳ Ivan , Procházka Miroskav
Published: May 2005

The high performance liquid chromatographic (HPLC) separation and identification of 12 isomeric and/or highly chemically related steroids with an unsaturated ester moiety at position 17β has been achieved. The main stereochemical features of the steroid skelton cover 3α/β or Δ, and 20 E/Z, bearing the alcohol or hemisuccinate group at the 3 position. The isocratic reversed phase C18 HPLC separation employed ethanol, methanol and its mixtures with water or 0.01 M phosphoric acid as the mobile phase. The best separaton of the respective alcohols from their hemiscuccinates has been achieved with 20% of aqueous phase content. The best separation among isomeric or related steroids has been achieved with methanol: water 8:2 and 85:15 and similar systems containing phosphoric acid.
Determination of the level of the core of 2′,5′‐oligoadenylates by high performance liquid chromatography
Authors: Suzuki H. , Buonamassa D. Tornese
Published: May 2005

A simple and rapid method for analysis of the core of 2′,5′‐oligoadenylates, mainly based on the use of high performance liquid chromatography (HPLC), is described. Perchloric acid extracts of tissues or cells were first treated with nuclease P1. Portions of the extracts were then digested with alkaline phosphatase. HPLC analysis of the extracts was performed on a column system composed of an Ultrasphere ODS precolumn (4.6 × 45 mm) and an Ultrasphere Octyl column (4.6 × 250 mm) by stepwise elution using a 50 mM ammonium phosphate buffer, pH7, containing 3.5 and 7% methanol. Three species of the core of 2′,5′‐oligoadenylates (dimer, trimer and tetramer) from a number of samples were eluted separately with 7% methanol, and the concentration of each core was directly estimated using constant values calculated with the standard core. The level of the core of 2′,5′‐oligoadenylates in tissues and cells determined by our method is similar to that reported by other authors who used biological, radiobinding or radioimmunological assays.
Favourable biospecific reactivity of blood group B antigenic trisaccharide chemically attached to poly‐N‐(2‐hydroxyethyl)acrylamide‐coated porous glass
Authors: Ivanov Alexander E. , Bovin Nickolay V. , Korchagina Elena Yu. , Zubov Vitaly P.
Published: May 2005

Blood group B antigenic trisaccharide‐β‐aminopropyl glycoside (B‐TSAP) covalently attached to poly‐N‐(2‐hydroxyethyl)acrylamide‐coated porous glass interacts with anti‐B monoclonal antibodies faster than the ligand coupled to CNBr‐activated Sepharose 4B and Affi‐Gel 10. Rates of hydrophobic adsorption of antibodies on the butyl derivatives of the same supports were measured to evaluate the diffusion input to overall kinetics. The lowest average affinity adsorption time [t1(aff) = 250 s] observed for polymer‐coated glass probably arises because of the flexibility of the extended segments of chemisorbed N‐substituted polyacrylamide acting as effective spacer arms.
Peroxyoxalate chemiluminescence detection of condensates of malondialdehyde with thiobarbituric acids using a flow system
Authors: Nakashima Kenichiro , Nagata Masahiko , Takahashi Masakatsu , Akiyama Shuzo
Published: May 2005

The peroxyoxalate chemiluminescence(CL) detection method for the evaluation of the CL intensity of malondialdehyde(MDA) condensates with seven 2‐thiobarbituric acid derivatives is described. The method consists of a flow injection technique together with a CL detection system using bis(2,4,6‐trichlorophenyl) oxalate(TCPO) and hydrogen peroxide as chemiluminogenic reagents. Linear correlations between CL intensity and concentration are obtained for pmol levels of condensates. Among the condensates, 1,3‐diethyl‐2‐thiobarbituric acid(DETBA)–MDA shows the largest CL intensity. High performance liquid chromatography (HPLC)/CL detection of DETBA–MDA and 1,3‐diphenyl‐2‐thiobarbituric acid(DPTBA)–MDA using a mixture of TCPO and hydrogen peroxide in acetonitrile as a postcolumn reagent solution is also described. The detection limits for DETBA–MDA and DPTBA–MDA are 20 and 200 fmol, respectively, per 20 μL injection at a signal‐to‐noise ratio of 2. This HPLC/CL detection system was applied to the defermination of MDA in rat brains by using DETBA as a fluorescent derivatizing reagent.
Combined application of analytical high performance thin layer chromatography and electroblotting for the detection of anti‐ganglioside antibodies in human sera
Authors: Ritter K. , Schaade L. , Thomssen R. , Grunow E.
Published: May 2005

Antibodies against gangliosides isolated from small tumour and nervous tissue specimens can be reliably detected in serum samples by the ganglioside electrotransfer technique without the need for previous purification steps. After separation by high performance thin layer chromatography gangliosides are transferred from silica gel plates to hydrophobic polyvinylidene difluoride membranes. These membranes are highly suitable for immuno‐staining. The use of a 15‐slit device allows simulataneous testing of up to 15 serum samples. Samples of serum from 39 patients with clear‐cell carcinoma of the kidney, mammary carcinoma, ovarian carcinoma and neurological disorder together with samples from healthy controls were tested for anti‐ganglioside antibodies from various tissues.
Rapid chiral separation of metoprolol in plasma—application to the pharmacokinetics/pharmacodynamics of metoprolol enantiomers in the conscious goat
Authors: Leloux M. S.
Published: May 2005

The plasma concentrations of metoprolol enantiomers have been determined by means of a direct phenyl carbamate‐cellulose‐based chiral high performance liquid chromatography assay using fluorimetric detection. This assay has been used to investigate the pharmacokinetics and pharmacodynamics of metoprolol enantiomers in the conscious goat. There is evidence that the pharmacokinetics of metoprolol in the goat occurs stereoselectively and that enantiomer–enantiomer pharmacokinetic interactions occur. R‐Metoprolol is less effective in reducing the mean arterial blood pressure than S‐ and R/S‐metoprolol.
Dissolution assay of theophylline, diprophylline and proxyphylline from a sustained release dosage form by high performance thin layer chromatography
Authors: Agbaba D. , Živanov‐Stakić D. , Vukićević N.
Published: May 2005

The content and dissolution rate of theophylline, diprophylline and proxyphylline from a sustained release formulation were determined by UV in situ densitometry. After separation the chromatographic zones corresponding to the spots of theophylline, diprophylline and proxyphylline on the high performance thin layer chromatographic plates were scanned in reflectance/absorbance mode at 275 nm. Quantification was performed with a second degree polynomial function over the range 40–200 ng for theophylline and 60–300 ng for diprophylline and proxyphylline. Percentages of dissolved theophylline, diprophylline and proxyphylline were monitored over 1, 3 and 6 h. The method was found to be simple, accurate, reliable, time‐saving (up to 18 samples can be determined simultaneously) and low‐cost.
Chiral separation by high performance liquid chromatography. I. Review on indirect separation of enantiomers as diastereomeric derivatives using ultraviolet, fluorescence and electrochemical detection
Authors: Srinivas Nuggehally R. , Igwemezie Linus N.
Published: May 2005

The increased attention on the therapeutic implications of stereoisomerism has provided an impetus for the development of analytical methods for enantiomeric separation. The indirect method of separation of enantiomers as diastereomers using high performance liquid chromatography (HPLC) has emerged as an efficient and versatile approach. This is due mainly to the availability of numerous chiral derivatization reagents (CDRs). This article reviews CDRs useful for the development of an indirect HPLC method using ultraviolet, fluoresence and electrochemical detection. In addition, factors crucial for the development of the indirect method are discussed.
Use of immobilized metal ions as a negative adsorbent for purification of enzymes: Application to phosphoglycerate mutase from chicken muscle extract and horseradish peroxidase
Authors: Chaga Grigory , Andersson Lennart , Ersson Bo , Berg Mikael
Published: May 2005

Two enzymes, phosphoglycerate mutase and peroxidase, were purified by using an immobilized metal ion adsorbent for the removal of unwanted proteins. The mutase was obtained pure from a single column, whereas the purification of peroxidase required the use of a thiophilic adsorbent in a tandem. The capacity was 2.5 mg pure peroxidase per mL gel.
Liquid chromatographic determination of hyoscine (scopolamine) in urine using solid phase extraction
Authors: Whelpton Robin , Hurst Peter R. , Metcalfe Roger F. , Saunders Sally A.
Published: May 2005

A sensitive method for the determination of hyoscine (scopolamine) in urine is described. After concentration and “clean‐up” on C18 and CN solid phase extraction columns, hyoscine was quantified by high performance liquid chromatography with coulometric detection (oxidation at +0.9 V). The limit of detection was 5 ng per sample and the precision for 5 mL samples containing 2 ng/mL was 12.3%. The method was applied to urine samples collected from 12 volunteers wearing Scopoderm TTS patches. The mean excretion rate of unmetabolized hyoscine was 0.45 μg/h and 87% of the total hyoscine was present as conjugates. Apohyoscine (aposcopolamine) was identified as a urinary metabolite. The significance of this with regard to hyoscine assays is discussed.
High performance liquid chromatographic determination of 3α, 5β‐tetrahydroaldosterone and cortisol in human urine with fluorescence detection
Authors: Yoshitake Takashi , Ishida Junichi , Sonezaki Shinji , Yamaguchi Masatoshi
Published: May 2005

A simple and sensitive method for the simultaneous determination of 3α, 5β‐tetrahydroaldosterone (THALD) and cortisol in human urine is described. The method uses high performance liquid chromatography with fluorescence detection. THALD and cortisol, released by enzyme hydrolysis, and fludrocortisone (internal standard) are isolated by a Sephadex G‐25M column and a Bond‐Elut C18 cartridge, and then oxidized by cupric acetate to form the corresponding glyoxal derivatives. The glyoxal derivatives are converted into the fluorescent quinoxalines by reaction with 1,2‐diamino‐4,5‐methylenedioxybenzene. The quinoxalines are successfully separated on a reversed phase column (L‐column ODS) with isocratic elution and monitored fluorimetrically. The detection limits for THALD and cortisol are 0.45 and 1.18 ng/mL urine (0.65 and 2.65 pmol/100 μL injection volume), respectively, at a signal‐to‐noise ratio of 3 in a 100 μL injection volume. This method permits the precise and sensitive determination of THALD and cortisol in human urine (2 mL).
Detection of abnormal haemoglobin by capillary electrophoresis and structural identification
Authors: Ishioka Noriaki , Iyori Naoko , Noji Jun , Kurioka Susumu
Published: May 2005

Haemoglobin obtained from a male adult Ghanian with retinopathy, which was probably caused by haemoglobinopathy was analysed by capillary electrophoresis (CE) for clinical diagnosis. Two major peaks, which were in the ratio of nearly one, were detected. The elution times of these peaks (HbXI and HbXII) were faster than that of normal haemoglobin (HbA). The existence of two different abnormal types of haemoglobin was clear in the patient blood. The following sequence analysis revealed that the first peak (HbXI) was HbC and the second (HbXII) was HbS on the electropherogram, and that the patient was a heterozygote of HbS and HbC (HbSC disease). One of the diagnostic processes in a haemoglobin disease was shown by the combined use of CE, HPLC and a protein sequencer.
Detection of gangliosides with the fluorochrome NBD dihexadecylamine and its application for preparative high performance thin layer chromatography
Authors: Müthing Johannes , Unland Frank
Published: May 2005

A new method for the detection of gangliosides based on the lipophilic fluorescence agent 4‐(N,N‐dihexadecyl)amino‐7‐nitrobenz‐2‐oxa‐1,3‐diazole (NBD dihexadecylamine) and its application for preparative high performance thin layer chromatography is described. Brain gangliosides were chromatographed on silica gel coated thin layer plates and located with non‐destructive fluorochrome under longwave ultraviolet light. The fluorescent zones were scraped off and the gangliosides were extracted with a mixture of chloroform/methanol/water (30/60/8; v/v/v). The gangliosides were separated from uncharged NBD dihexadecylamine by anion exchange chromatography and impurities were removed by Iatrobeads chromatography. The method described offers a simple and succesful preparative thin layer chromatographic strategy to obtain pure gangliosides in microgram and milligram quantities.
The retention behaviour of conjugated bile acids in reversed phase high performance liquid chromatography
Authors: Zhang Y. K. , Chen N. , Zao R. H. , Li T. , Wang Z. Y. , Lu P. C.
Published: May 2005

The retention behaviour of conjugated bile acids has been studied in a reversed phase high performance liquid chromatographic (RP‐HPLC) system by using the mixture of methanol and aqueous phosphate buffer as the mobile phase. The retentions of the conjugates in RP‐HPLC have been found to be mainly controlled by the glycine and taurine groups. The selectivity between five different glycine and taurine conjugated bile acids is a constant in RP‐HPLC. This selectivity has been used for peak identification in the practical separation of conjugated bile acids.
Determination of MDL 201,012 at femtomole/millilitre levels in human plasma by liquid chromatography with electrochemical detection
Authors: Reynolds Donald L. , Eichmeier Larry S. , Giesing Dennis H.
Published: May 2005

A sensitive and selective liquid chromatographic method to quantitate MDL 201,012 in human plasma was developed and validated. MDL 201,012 (I), diethyl‐MDL 201,012 (internal standard, II) and desmethyldiol‐MDL 201,012 (masking agent, III) were isolated from basified plasma (2 mL) by solid phase extraction using Bond‐Elut® C‐18 cartridges. Endogenous components were selectively removed prior to eluting the analytes from the sorbent. Components were separated using on‐line LC column switching with a cyanopropyl precolumn and a phenyl analytical column. The analytical column effluent was monitored electrochemically at a glassy carbon electrode at a potential of + 1025 mV vs. Ag/AgCI. Peak‐height ratios were proportional to the amount of MDL 201,012 added to plasma over the range 125–7500 pg/mL MDL 201,012. Absolute recovery of MDL 201,012 from human plasma was >94% across the calibration range. The minimum quantitation limit was 125 pg/mL. Assay precision (%RSD) ranged from 5.2 to 13% based on the analysis of quality control standards containing 125, 250, 500, 1000, 2500, 5000 and 7500 pg/mL MDL 201,012. Corresponding assay accuracy (% relative error) was ±8.5%. The method has been successfully used to quantitate MDL 201,012 in samples from acute dose tolerance studies in human volunteers.
Lead and the terminal mitochondrial enzymes of haem biosynthesis
Authors: Rossi E. , Taketani S. , Garcia‐Webb P.
Published: May 2005

Lead exposure causes increases in urinary coproporphyrin excretion and the accumulation of zinc protoporphyrin in red cells. In the conventional view of the effect of lead on haem biosynthesis, the accumulation of these metabolites results from lead inhibition of two of the mitochondrial enzymes of haem biosynthesis, corproporphyrinogen oxidase (EC 1.3.3.3.) and ferrochelatase (EC 4.99.1.1). This review critically assesses the evidence for the inhibition of these enzymes. We consider this evidence to be inconclusive and alternative explanations for the increased concentrations of coproporphyrin and zinc protoporphyrin are proposed.
Biodegradation of a carbamate pesticide, propoxur, in rat tissues
Authors: Kumar Reena , Madhavi N. B. Bindu , Sharma C. B.
Published: May 2005

Propoxur (Baygon, 2‐isopropoxyphenyl N‐methylcarbamate) is a carbamate pesticide commonly used against house insects. When the insecticide was administered intramuscularly in rats it was converted to a new metabolite which was found to be present in the serum, liver, kidney and brain 6 h after the administration of the pesticide. The metabolite was purified by high performance liquid to chromatography and comparison of the infrared spectra of Propoxur and the metabolite showed that a deamination reaction was responsible for the formation of the metabolite from the parent pesticide. The pesticide also induced haematological changes such as an increased level of total bilubrin, amylase and glutamic–oxalacetic transaminase and decrease of cholinesterase activity, indicating damage of the liver and nervous system in rats.