bluuebunny/crossref_metadata_2025 huggingface dataset:
Isolation and sequencing of a new biologically active peptide from human lung carcinoma
Authors: Hsi Kuo‐ling , Wu Shih‐xiang , Chen Zhong‐guang , Guo Xiu‐yue , Tsou Kang
Published: May 2005

A biologically active peptide designated hLCP has been isolated and purified to homogeneity from human lung carcinoma by means of acidic extraction and successive chromatography on Sephadex G‐50, Toyopearl HW‐40 F and reverse‐phase high performance liquid chromatography columns. Analysis showed that peptide consists of thirteen amino acids. Primary structure of hLCP has been deduced by double‐coupling Edman degradation combined with enzyme digestion as H‐Ser‐Pro‐Pro‐Asp‐Gly‐Lys‐Lys‐Glx‐Ser‐Ala‐Asp‐Val‐Lys‐OH. hLCP possesed significant excitatory activity on an electrical stimulation induced contraction. No hLCP could be detected in normal lung tissue. The possibility of using hLCP as a biochemical marker in the clinic for the early detection of lung carcinoma is being investigated.
Packing materials suitable for rapid, analytical, low‐pressure chromatography of haemoglobins on midget columns
Authors: Ersser R. S. , Barlow G. B. , Drew R. G. , Hjelm M.
Published: May 2005

Rapid, quantitative, chromatographic separations of mixtures of human haemoglobins have been performed on short (5 – 20 mm) columns of packing material. The desirable characteristics of suitable column packing materials are illustrated and discussed. Simple, inexpensive, manually operated equipment can be used for the analysis, since the specifically designed midget columns generate little back pressure (10 – 30 lb/in2) when eluted at constant flow rates up to 2 mL/min. Cation exchange Chromatography on bonded silicas has been used for the detection of pathological haemoglobinopathies. Separations similar to the HPLC procedures are possible with the correct selection of buffer composition. It also compares favourably with the methods in common clinical use employing electrophoresis on cellulose acetate. Both ion‐exchange and affinity methods for the estimation of glycated haemoglobins have been developed and are compared.
Chromatographic studies of human lung elastin digestion products obtained by leucocyte elastase: Comparison between newborn and adult soluble fragments
Authors: Smyrlaki Marie , Davril Monique , Hayem Annette
Published: May 2005

Human insoluble elastin was prepared from newborn lungs and digested by leucocyte elastase. The soluble fragments were compared to those obtained from adult lung elastin in a previous work (Smyrlaki et al., 1986).

Gel filtration on a Bio‐Gel P‐100 column of newborn elastin allowed the separation of fraction F1N (Mr's 30 000–10 000) which was eluted later than the excluded fraction F1A (Mr's 80 000–30 000) previously isolated from adult elastin. The difference in the sizes of the large peptide fragments originating from both elastins was also shown on SDS‐PAGE.

Reversed phase HPLC was performed on a C18 column using a multi‐step gradient elution procedure. Different patterns were observed for the high (F1N) and the low (F2N) molecular size fragments of newborn elastin. The same peak distribution was obtained with adult elastin. Comparison of the amino acid compositions of the most retained peaks (3, 4 and 5), derived from fractions F1N and F1A, showed analogies for the contents of the major nonpolar amino acids and crosslinks. Thus, this procedure allowed the separation of typical fragments of elastin which might be released in vivo by leucocyte elastase during pulmonary diseases.
Quantitative determination of adriamycin in rat hepatocytes using a volatile extraction buffer, HPLC and fluorescence detection
Authors: Mahdadi R. , Pommery N. , Pommery J. , Lhermitte M.
Published: May 2005

A rapid and sensitive isocratic technique is described for the determination of concentrations of adriamycin and two of its metabolites, adriamycinol and adriamycinone, in freshly isolated rat hepatocytes. The drugs are easily and efficiently extracted from the cells with an organic mixture (chloroform–n‐butanol) after proteolytic digestion with trypsin. Mean recoveries from spiked culture medium cell suspension are greater than 96%. The within run and day‐to‐day coefficients of variation are less than 7.5%.
Thin‐layer chromatography—the forgotten alternative for the quantitative determination of steroids
Authors: Golf S. W. , Graef V. , Schiller J. Th. , Hischer H. , Funk W.
Published: May 2005

We have developed a high performance thin layer chromatography (HPTLC) system for quantitative determination of androgens, corticosteroids, mineralocorticoids and gestagens on silicagel KG‐60 HPTLC‐plates with different solvent systems. A complete separation of androgens, gestagens and metabolites was achieved with dichlormethane/cyclohexane/acetone (70:25:5). Corticosteroids, mineralocorticoids and their derivatives were completely separated with diethylether/isooctane/isopropanol (70:25:5). The quantitative in situ fluorescence determination was carried out after post‐chromatographic derivatization with cinnamic aldehyde, 4‐dimethylaminobenzaldehyde and sulfuric acid. The sensitivity of detection was found between 500 pg and 1 ng per spot.

The steroid metabolism as catalysed by rat liver microsomal oxidoreductases was measured by these procedures, and was compared with determination of steroids by gas chromatography (GC). According to HPTLC, steroids were reduced by NADPH‐5α‐reductase (EC 1.3.1.4) in the order progesterone > testosterone > aldosterone > cortisol > corticosterone. The enzyme activities as measured by HPTLC agree well with those obtained by GC (r = 0.94). When turnover of enzyme assays, speed of determination, detection limit, application to labile steroids and costs of steroid determination are considered, all points speak in favour of HPTLC.
The effect of storage on rat tissues and human plasma amino acid levels determined by HPLC
Authors: Bottiglieri T.
Published: May 2005

Tissue concentrations of the amino acids tryptophan, methionine, valine, phenylalanine, isoleucine and leucine were determined in rat liver, skeletal muscle and brain tissue and human plasma. The effect of storage at −20°C for up to 21 days was examined. Significantly elevated levels after 7 and 21 days of storage were observed in liver and brain tissue but no changes were found in muscle and plasma. Minimal changes occurred in liver tissue stored up to 6 hours at −4°C. This study emphasizes the need for rapid deporteinization of tissues to avoid changes in the free pool of amino acids.
Development of a selective clean‐up method using immobilized antibody and its application to HPLC and GC/MS determination of a carbacyclin derivative, CS‐570, in plasma
Authors: Nakagawa Akihiko , Matsushita Yoko , Muramatsu Shigeki , Tanishima Yukimi , Hirota Takashi , Takasaki Wataru , Kawahara Yukinori , Takahagi Hidekuni
Published: May 2005

Methods for the determination of CS‐570, a chemically stable prostacyclin analogue, in plasma were developed by using an immobilized antibody column followed by fluorescence HPLC and GC/MS. The CS‐570 antibody, obtained from rabbit plasma by giving CS‐570–BSA for a few months, was coupled to Sepharose 4B and used as extraction phase for sample clean‐up and extraction of the drug. A plasma sample was applied to this column, washed with water and the drug was eluted with 90% acetonitrile. 0.02% (w/v) 9‐anthryldiazomethane (ADAM) was added to the extract to form a fluorescent derivative. The CS‐570–ADAM adduct exhibited high sensitivity when applied to HPLC with fluorescence detection and column switching. The detection limit was 1 ng/mL when 1 mL of plasma was available. Additionally, a pentafluorobenzyltrimethylsilyl derivative of CS‐570 showed excellent sensitivity when determined by capillary GC interfaced to negative ion chemical ionization MS using a stable isotope labelled analogue as an internal standard.
Purification of streptococcal protein g expressed by Escherichia coli by high performance liquid affinity chromatography using immobilized immunoglobulin G and albumin
Authors: Falkenberg Cecilia , Björck Lars , Åkerström Bo , Nilsson Staffan
Published: May 2005

A one‐step HPLC method was developed for the purification of protein G, a cell wall molecule from group C and G streptococci with immunoglobulin G‐ and albumin‐binding properties. Lysed Escherichia coli bacteria infected with lambda‐phages containing the protein G gene from group G streptococci were used as a starting material for the preparations. The lysate was applied to a column with immobilized human immunoglobulin G or human serum albumin. Protein G was selectively bound and eluted at pH 2.0. A 750‐fold purification was achieved. Sodium dodecylsulfate + polyacrylamide gel electrophoresis showed that the highly purified protein G consisted of three sets of doublets with the apparent molecular weight of 64 and 67, 56 and 58, and 45 and 47 kilodaltons, respectively. A specific method for quantitation of small amounts of protein G was developed and used for specific tracing of the protein after the affinity chromatography. Goat polyclonal antibodies were bound to an antigen coated to the plastic walls of microtiter plates, causing the Fc‐region of the immunoglobulins to be directed outwards. Unknown samples of protein G were then allowed to compete with radio‐iodinated protein G (solid phase radioassay) or protein G coupled to alkaline phosphatase (enzyme linked sorbent assay) for the Fc‐regions.
Quantitative determination of propranolol in plasma and pharmaceutical preparations by agar‐based cation‐exchange chromatography utilizing the native anion groups in the agaropectin moiety
Authors: Zelikman Ilya , Hjertén Stellan
Published: May 2005

A new cost‐effective method for determining the amount of total propranolol (free and protein‐bound) in plasma samples and the amount of the drug in tablets and injection solutions has been developed. The method is based on cation‐exchange chromatography at pH 8.5 on an inexpensive column of agar, the native sulfate (and carboxylic) groups of which interact with the positively charged propranolol (this interaction is reinforced by the ‘aromatic adsorption’ characteristic of the agar matrix). The method can be used for quantitative analyses at both low (1 × 10−2 mg/L) and high (5 × 103 mg/L) concentrations of the drug. For analyses of plasma and tablets—but not for injection solutions—extraction of the propranolol is required. The extraction time was 6 min. On a 6(ID) mm × 9 mm agar column the run time was less than 10 min.
The determination of selenomethionine in selenium yeast by cyanogen bromide gas chromatography
Authors: Ouyang Zheng , Wu Jiang
Published: May 2005

A new method for the determination of bonded selenomethionine in selenium yeast by gas chromatography is established. Bonded selenomethionine is rapidly and precisely determined by measuring CH3SeCN released during the reaction of selenoprotein with CNBr (CNBr‐GC method). Conditions for the reaction and chromatography are described. The results of CNBr‐GC are compared with those obtained by acid hydrolysis/ion exchange chromatography. This new method has the advantage of being accurate, sensitive and selective.
Analysis of leukotrienes by liquid chromatography/electrochemistry after conversion to dinitrobenzoate derivatives
Authors: Steffenrud Steffen , Salari Hassan
Published: May 2005

A sensitive liquid chromatography method has been developed using electrochemistry for the determination of leukotrienes in biological fluids. Biological specimens are treated with 3,5‐dinitrobenzoyl chloride in acetonitrile which undergoes rapid reaction with hydroxyl groups of non‐peptidic leukotrienes in the presence of pyridine and with amino groups of peptidic leukotrienes in the presence of potassium tetraborate buffer. The resulting dinitroben‐zoate derivatives of leukotrienes are highly electroactive, suitable for reduction or oxidation at moderate potentials by an electrochemical detector. In reductive mode at −0.7 V or oxidative mode at + 1.15 V potentials, the lower limits of detection for leukotriene derivatives were approximately 8±3 pg and 70 ± 16 pg respectively, with a signal‐to‐noise ratio of 5 to 1. This method was applied to the detection of leukotrienes in plasma, nasal and bronchial fluids of patients with asthma.
HPLC separation and characterization of chlorin derivatives with intact ring V from acid degradation products of silkworm excrement crude chlorophyll mixture
Authors: Hu Longqin , Xu Deyu
Published: May 2005

A reversed‐phase HPLC system with 88% methanol in 1 M ammonium acetate buffer pH 5.35 as the mobile phase on an ODS column was used to analyse pheophorbides a, a', b, b' and pyropheophorbides a and b. Pyropheophorbides a and b were found in samples obtained from silkworm excrement but not from spinach leaves though the preparation methods used were the same. This difference suggests that chlorophylls undergo metabolism in the body of silkworm to give pyrochlorophyll derivatives.
Rapid purification of detergent‐solubilized bovine hormone‐sensitive lipase by high performance hydrophobic interaction chromatography
Authors: Nilsson Staffan , Holm Cecilia , Belfrage Per
Published: May 2005

Hormone‐sensitive lipase (HSL), the enzyme controlling the rate of adipose tissue lipolysis and also possibly involved in the regulation of steroidogenesis, has been purified from bovine omental adipose tissue. Partially detergent‐solubilized, delipidated and purified HSL was obtained through step‐elution at conventional DEAE ion‐exchange chromatography, followed by concentration on hydroxylapatite. High performance hydrophobic interaction chromatography (HPHIC) on phenylsilica then resulted in an increase of HSL protein purity from 2% to more than 70%. Final purification of the enzyme to apparent homogeneity (>95% protein purity), concentration and removal of most of the detergent was obtained by high performance cation exchange chromatography on Mono S. At least 0.5 mg of highly stable HSL was obtained from 5 kg of bovine omental fat within four working days. The purified lipase had a lower specific activity than previously reported for the corresponding rat enzyme but the preparations have proved very useful for enzyme structure studies and as an antigen.
Determination of thiols and disulfides in normal rat tissues and hamster pancreas treated with N‐nitrosobis(2‐oxopropyl)amine using 4‐(aminosulfonyl)‐7‐fluoro‐2, 1,3‐benzoxadiazole and ammonium 7‐fluoro‐2, 1,3‐benzoxadiazole‐4‐sulfonate
Authors: Toyo'oka Toshimasa , Furukawa Fumio , Suzuki Takashi , Saito Yukio , Takahashi Michihito , Hayashi Yuzo , Uzu Sonoko , Imai Kazuhiro
Published: May 2005

Biological thiols and disulfides in rat and hamster tissues were simultaneously determined by HPLC‐fluorescence detection using 4‐(aminosulfonyl)‐7‐fluoro‐2, 1,3‐benzoxadiazole (ABD‐F) and ammonium 7‐fluoro‐2,1,3‐benzoxadiazole‐4‐sulfonate (SBD‐F). The coefficients of variation (CV) of the method for reduced glutathione (GSH) and oxidized glutathione (GSSG) in liver and for cysteine (CySH) and cystine (CySSCy) in kidney were less than 3.1%. In 11 tissues of Wistar rats (liver, spleen, heart, lung, stomach, bladder, ovary, uterus, adrenal, kidney and pancreas), only CySH, CySSCy, GSH and/or GSSG were detected. Other thiols and disulfides were at extremely low levels in all samples. Both concentrations of CySH and CySSCy in the livers of old rats (111 weeks old, F344) were significantly higher than those of young rats (8 weeks old) (CySH, 0.246 ± 0.099 vs 0.130 ± 0.020 μmol/g; CySSCy, 0.051 ± 0.027 vs 0.013 ± 0.002 μmol/g). Administration of N‐nitrosobis(2‐oxopropyl)amine (BOP), a selective carcinogen of hamster pancreatic cancer, to Syrian golden hamsters (38 weeks old) resulted in the increase in the pancreas of GSH to a level 19 times as high and of GSSG to a level 14 times as high as those in untreated hamsters (GSH, 1.173 ± 0.272 vs 0.062 ± 0.017 μmol/g; GSSG, 0.155 ± 0.063 vs 0.011 ± 0.001 μmol/g).
High performance liquid chromatographic determination of fluvoxamine in human plasma
Authors: Pommery J. , Lhermitte M.
Published: May 2005

A method has been developed for the separation and measurement of fluvoxamine in human plasma by high performance liquid chromatography. The method uses metapramine as an internal standard and provides a limit of detection of about 1.5 ng/mL for fluvoxamine. At a concentration of 25 ng/mL, fluvoxamine could be measured within a coefficient of variation of ±5.82 of the mean and at 100 ng/mL within a CV of ±2.78 of the mean. The method has been applied to the analysis of plasma from patients undergoing fluvoxamine therapy.
Quantitative determination of itazigrel in rodent diet by reversed‐phase HPLC and evaluation of its stability at 20°C
Authors: Bombardt Paul A. , Bothwell Brian E. , Closson Sharon K. , Adams Wade J.
Published: May 2005

A simple, accurate and precise procedure for the quantitation of itazigrel (a potent lipophilic inhibitor of collagen and arachidonic acid‐induced aggregation being studied for its effects on peripheral vascular disease) from granulated rodent diet is presented. The drug was extracted from rodent diet using methanol + water (80:20) following dissolution of the diet in water. Samples of the supernatant were injected into the HPLC and the eluent was monitored with a fluorescent detector (λex = 320 and λem = 430) to achieve analytical specificity. Interday coefficients of variation of the calibration curve slope were ±6% on standards between 0 and 1000 μg/g. Potency and homogeneity of the drug spiked diet prepared over a 1 year interval at 70,200 and 600 μg/g was 99.3 ± 2.5%, 100 ± 1.8%, and 101 ± 1.9% of label, respectively. Samples prepared for chromatography were stable for 24 h at 20°C, and drug in diet was stable for 102 days when protected from light and stored at 20°C.
High performance liquid chromatography of biological polyamines using immobilized enzyme as post‐column reactor followed by electrochemical detection
Authors: Watanabe Noriyuki , Asano Masao , Yamamoto Katsunobu , Nagatsu Toshiharu , Matsumoto Takatoshi , Fujita Keisuke
Published: May 2005

A novel analytical method for biological polyamines (putrescine, spermidine and spermine) was developed. Polyamines were separated by ion‐pair reversed phase chromatography using a polymer‐based octadecyl bonded column. A polyamine oxidase immobilized column worked effectively as a post‐column reactor to convert polyamines to hydrogen peroxide which was eventually detected by electrochemical oxidation on platinum electrode. This method required neither tedious derivatization nor gradient elution, permitting us to perform simple and rapid analysis of polyamines. The detection limits were 0.3, 0.6, and 4 pmol injected for putrescine, spermidine, and spermine, respectively with a linear range of two to three orders of magnitude. Chromatograms obtained with samples from human urine and rat brain homogenates demonstrated the high sensitivity and selectivity of the method.
Analysis of drug in tissue homogenates by high performance liquid chromatography with direct injection and column switching
Authors: Imai Hideo , Tamai Gen
Published: May 2005

An HPLC method with direct sample injections and column switching was investigated for the analysis of drugs in tissue homogenates. The appropriate precolumn packings and analytical column packings were surveyed in order to obtain quantitative recovery. The use of a large bore end‐fitting filter for the precolumn avoided the interference due to minute tissue particles. A minicolumn was used to trap the analyte or clean up the sample. The method was applicable to hydrophilic as well as hydrophobic drugs in liver, kidney or heart tissues, and has been extensively used in the determination of drugs in centrifugal cell fractions such as the nucleus, mitochondria and microsome.
A rapid method for isolation of human hemoglobin benzo[a]pyrene diol epoxide derived adducts using high performance liquid chromatography
Authors: Naylor S. , Gan L.‐S. , Day B. W. , Skipper P. L. , Tannenbaum S. R.
Published: May 2005

A method is described to isolate rapidly human hemoglobin‐benzo[a]pyrene diol epoxide adducts. A combination of 300 Å pore size C4 reversed phase HPLC to effect separation of adducted protein from native protein, and μ‐bore C18 reversed phase HPLC to isolate and partially characterize proteolytic peptide adducts (by UV), was used.
Simultaneous determination of some organophosphorus pesticides by high performance liquid chromatography
Authors: Kumar Reena
Published: May 2005

An HPLC method for the simultaneous detection of six organophosphorus pesticides (Dimethoate, Ethion, Malathion, Phorate, Phosalone and Parathion) on a Zorbex ODS column using methanol + water (80: 20) as solvent is described.
Optimization of an Isocratic Reversed Phase Liquid Chromatographic System for the Separation of Fourteen Steroids Using Factorial Design and Computer Simulation
Authors: Wei Ji‐Qing , Wei Ji‐Lu , Zhou Xian‐Teng
Published: May 2005

An optimization strategy for an isocratic reversed phase high performance liquid chromatographic system (RP‐HPLC) is described. Factorial design and a computer program are used to predict the retention time and resolution of fourteen steroids. An optimized rapid (<25 min) isocratic RP‐HPLC system for the satisfactory separation of cortisone, cortisol, corticosterone, 11‐deoxycortisol, 11‐deoxycorticosterone, 17α‐hydroxyprogesterone, progesterone, androstenedione, testosterone, estrone, estradiol, estriol, prednisone acetate and dexamethasone acetate has been developed using this strategy through eight experiments.
High performance liquid chromatographic separation of cisplatin and its hydrolysis products on alumina and application to studies of their interaction with cysteine
Authors: Shearan Paula , Alvarez José M. Fernández , Zayed Nihal , Smyth Malcolm R.
Published: May 2005

An alumina stationary phase has been assessed in the study of the retention behaviour of the anticancer drug cisplatin and its major hydrolysis products. Parameters such as buffer concentration in the mobile phase, pH, organic modifier and competing ion have been investigated in order to optimize chromatographic separation with ultraviolet detection. The separation scheme developed has been used to monitor the hydrolysis of cisplatin in aqueous and saline media, and to monitor the interaction of hydrolysed solutions of cisplatin with the amino acid cysteine. A new peak was observed in the chromatograms of such mixtures when they had been allowed to stand for periods of greater than 16 h and, from analysis of the data obtained, it was concluded that this new peak was due to a complex formed between the mono‐aquo hydrolysis product of cisplatin and the amino acid.
Studies on the effects of imidazole on the peroxyoxalate chemiluminescence detection system for high performance liquid chromatography
Authors: Imai Kazuhiro , Nishitani Atsuhiko , Tsukamoto Yukie , Wang Wei‐Hong , Kanda Susumu , Hayakawa Kazuichi , Miyazaki Motoichi
Published: May 2005

The catalytic effect of bases (imidazole, pyridine, Tris and triethylamine) on the peroxyoxalate chemiluminescence (PO‐CL) reaction for high performance liquid chromatography (HPLC) was investigated. Imidazole increased PO‐CL intensity extraordinarily, whereas the other bases (pyridine, Tris and triethylamine) did not. The peak heights of dipyridamole (coronary vasodilator) obtained using the eluents containing buffers were largest at pH 7.0, a few times less at pH 6.0 and pH 5.0, 100 times less at pH 4.0 and a few hundred times less at pH 3.0. The eluents containing buffers at pH 3, 4, 5, 6 or 7 each with imidazole increased the peak heights by a few to ten times as compared with those without imidazole, and those peak heights were within one order of magnitude. On the other hand, the eluent containing buffer at pH 2 did not affect the peak heights with or without imidazole. Bis(4‐nitro‐2‐(3,6,9‐trioxadecyloxycarbonyl)phenyl) oxalate (TDPO) alone and bis(2,4‐dinitrophenyl)oxalate (DNPO) plus TDPO were recommended to be used against eluents containing buffers of pH 5 – 7 and pH 3 – 4, respectively. Dipyridamole and benzydamine hydrochloride (anti‐inflamatory drug) were separated on the ODS column and detected by the present system. The detection limits of dipyridamole and benzydamine hydrochloride were 40 amol and 270 fmol, respectively.
Continuous flow determination of nitrite with membrane separation/chemiluminescence detection
Authors: Aoki Toyoaki
Published: May 2005

A method for the determination of nitrite in water utilizing a membrane separation process and a chemiluminescence detector, with the addition of air‐stripping and air‐carrier, is proposed. The microporous poly(tetrafluoroethylene) tube was used as separator to transfer nitric oxide (reduced by iodine in acidic media) into a gas phase. Air‐stripping was used to enhance the separation. Chemiluminescence signals produced from the reaction of nitric oxide with ozone were linearly proportional to the concentration of nitrite from 10 ppb (μg as N/L) to 5 ppm (mg N/L). The relative standard deviation (n = 5) was 0.7% at 0.1 ppm. The time elapse from starting the sample flow until the signal reached a stable level was 1.5 min.
Isolation and characterization of β‐hydroxypropionic acid‐ and hydroxyacetic acid‐uroporphyrin I in the urine of a patient with congenital erythropoietic porphyria by high performance liquid chromatography and liquid secondary ion mass spectrometry
Authors: Guo Rong , Chaim W. , Rideout J. M. , Lawson A. M. , Lim C. K.
Published: May 2005

β‐Hydroxypropionic acid‐ and hydroxyacetic acid‐uroporphyrin I have been isolated from the urine of a patient with congenital erythropoietic porphyria by reversed phase high performance liquid chromatography. The compounds were characterized by their chemical behaviour and confirmed by liquid secondary ion mass spectrometry.
Determination of 6‐mercaptopurine and its metabolites in plasma or serum by high performance liquid chromatography
Authors: Sahnoun Z. , Serre‐Debeauvais F. , Lang J. , Faucon G. , Gavend M.
Published: May 2005

A sensitive and accurate reversed phase liquid chromatographic assay was developed for the determination of 6‐mercaptopurine (6MP) (the active metabolite of azathioprine) in human plasma. The assay involved extraction into acetonitrile and dichloromethane from plasma pretreated with 0.038 M of dithiothreitol solution. The residue was analyzed by isocratic chromatography on a C18 analytical column with UV detection at 326 nm. The average extraction recovery of 6MP was 85%.

The method has been applied successfully to the determination of 6MP and its metabolites in pharmacokinetic studies.
Chiral separation and determination of propranolol enantiomers in rat or mouse blood and tissue by column switching high performance liquid chromatography with ovomucoid bonded stationary phase
Authors: Tamai Gen , Edani Masami , Imai Hideo
Published: May 2005

Resolution of propranolol (PL) enantiomers in biological samples was accomplished by column switching high performance liquid chromatography using a short precolumn and an analytical column of ovomucoid chiral phase. Plasma, whole blood or tissue homogenate sample was directly injected into the precolumn, and PL was adsorbed on Butyl Toyopearl 650‐M. After column switching, the PL was backflushed and transferred to the analytical column (Ultron ES‐OVM) by the eluant. Fluorometric detection was carried out at λex = 297 nm and λem = 340 nm with a detection limit of 0.5 pmol (signal to noise ratio = 2). The recovery 98.8–103%), reproducibility (coefficient of variance less than 3%) and enantiomer resolution (separation factor 1.15) were satisfactory using as eluant 50 mM sodium dihydrogenphosphate (pH 4.6) containing 12% ethanol. The time course of elimination of PL enantiomers in rat or mouse blood and tissues was also studied.
Isocratic reversed phase high performance liquid chromatography determination of twelve natural corticosteroids in serum with on‐line ultraviolet and fluorescence detection
Authors: Wei Ji‐qing , Wei Ji‐lu , Zhou Xian‐teng , Cheng Ji‐ping
Published: May 2005

An isocratic reversed phase high performance liquid chromatography procedure utilizing ultraviolet and fluorescence detectors linked in series is described for the analysis of cortisone (E), cortisol (F), corticosterone (B), 11‐deoxycortisol (S), 11‐deoxycorticosterone (DOC), androstenedione (A), testosterone (T), 17‐hydroxyprogesterone (17‐OHP), progesterone (P), estriol, estradiol, estrone, prednisone acetate and dexamethasone acetate in serum. Serum specimens were extracted with ethyl ether. The optimized mobile phase was methanol + tetrahydrofuran + water 26:18:56, v/v/v). A Shim‐pack ODS column was used. The recoveries were 80 to 103%. lntra‐ and inter‐day coefficient of variance were less than 8%. The detection limit is 0.5 pmol per injection volume for estriol, estradiol, E, F and B; 1 pmol for S, A, DOC and estrone; 2 pmol for T and 17‐OHP; and 4 pmol for P. Serum from normal subjects and patients with congenital adrenal hyperplasia due to 21‐ or l7‐hydroxylase deficiency were measured, as well as samples of maternal and umbilical cord serum.
High performance liquid chromatographic determination of methylxanthines in canine serum, gastric and pancreatic juices
Authors: Casoli Philippe , Vérine Henri
Published: May 2005

A convenient high performance liquid chromatographic method for the determination of methylxanthines in biological samples is described. Separation was achieved by reversed phase chromatography using a mobile phase consisting of tetrahydrofuran + methanol + 0.01M potassium dihydrogen phosphate, pH 3.5 (1:20:79, v/v/v), on a 7 μm C18 column and a C18 Lichrosorb precolumn at a flow rate of 0.8 mL/min. Levels varying from 0.25 – 16 mg/L could be detected by UV at 280 nm. In this range, standard curves were established for 4 methylxanthines: theobromine, paraxanthine, theophylline and caffeine in 4 media: mobile phase, serum, gastric and pancreatic juices, and were found to be linear (r ≥ 0.9975). Overall characteristics of the method were determined as: percent recovery (89.54%), accuracy (≥99.4%) and reproducibility (≥95%). Retention times ranged from 4.21 ± 0.01 (1‐methyluric acid) to 10.8 ± 0.03 min (caffeine). Animal experiments (5 and 10 mg/kg boluses) were used to determine caffeine half life in dog's blood (310 ± 46 and 453 ± 59 min, respectively) and its secretion into pentagastrin stimulated gastric juice (mean concentrations 2.51 and 6.04 mg/L; mean outputs 351 and 1206 μg/2.25 h; both statistically different at p < 0.001 level).
Separation and quantification of histamine and Nτ‐methylhistamine in brain extracts
Authors: Pastó Manel , Sabria Josefa
Published: May 2005

Histamine and its Nτ‐methyl derivative can be separated from perchloric acid extracts of rat brain by high performance liquid chromatography on a C18 column under isocratic conditions eluting with 0.1 M sodium phosphate buffer containing 0.19 mM sodium dodecyl sulphate and 25% methanol. Using electrochemical detection, histamine and Nτ‐methylhistamine can be detected at levels of less than 40 pg/μL tissue extract (<1 pmol). The retention times for histamine and Nτ‐methylhistamine were 15 min and 23 min, respectively, at a flow rate of 1.2 mL/min, and both compounds eluted as acceptably sharp peaks. The concentrations of histamine and Nτ‐methylhistamine in brain from seven‐day‐old rats were found to be very similar to those obtained by other analytical procedures.
Separation of proteins by consecutive sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis and reversed phase high performance liquid chromatography
Authors: Kaplan Batia , Pras Mordechai
Published: May 2005

A procedure for the preparative separation of proteins was developed by using consecutively sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis (SDS‐PAGE) and reversed phase high performance liquid chromatography (HPLC). The proteins were separated by SDS‐PAGE and afterwards extracted from the gel. The extracted proteins were separated from SDS and other small molecular weight contaminants on a Fractogel TSK HW‐40 (F) column in acidic aqueous acetonitrile. The proteins eluted from the Fractogel column were fractionated by HPLC. The identity and purity of the recovered proteins was confirmed by SDS‐PAGE analysis.
Application of ion pair high performance liquid chromatography to the analysis of porphyrins in clinical samples
Authors: Jacob K. , Luppa P.
Published: May 2005

Reversed phase ion pair chromatography is a highly selective separation technique for the determination of free porphyrin carboxylic acids from human materials. Isocratic and gradient elution methods can be used to analyse porphyrin isomers and to establish porphyrin profiles for the biochemical diagnosis of porphyrias. Ion pair high performance liquid chromatography led to the discovery of the atypical isomers II and IV of uroporphyrin and coproporphyrin in human urine. Advantages and limitations of the ion pair technique are discussed.
Field flow fractionation in biomedical analysis
Authors: Levin Shulamit
Published: May 2005

Samples of biomedical interest which have been analysed by field‐flow fractionation techniques are surveyed. The list begins with whole cells and microorganisms, going through viruses, nucleic acids, cell fragments and organelles, down to proteins and their aggregates. The principles of separation in the normal and steric mode of retention are illustrated, and instrumentation and techniques are described. The review concentrates mainly on the two systems of choice for biomedical applications: sedimentation and flow field‐flow fractionation.
Analysis of vitamin D and its metabolites using thermospray liquid chromatography/mass spectrometry
Authors: Watson David , Setchell Kenneth D. R. , Ross Richardus
Published: May 2005

A new method is described for the analysis of vitamin D and its metabolites utilizing thermospray (TSP) mass spectrometry as an on‐line detector for high performance liquid chromatogrpahy. Ionization conditions were optimized for use with isocratic reversed phase chromatography. TSP mass spectrometry was employed in series with a UV absorbance detector to facilitate comparisons between the two methods of detection. Positive ion TSP mass spectra were recorded for vitamin D2, vitamin D3, 25‐hydroxyvitamin D3 (25(OH)D3), 1,25‐dihydroxyvitamin D3 (1,25(OH)2D3) and 24,25‐dihydroxyvitamin D3 (24,25(OH)2D3). The spectra contained protonated molecular ions, ammonium adduct ions and fragment ions due to the loss of one or more molecules of water. A comparison of quantitative precision was made by determining UV absorbance and TSP standard curves for vitamin D3 using two different methods: (1) External standard method with post‐column (post UV detector) addition of ammonium acetate. (2) As (1) but using the method of internal standards with a closely eluting internal standard (vitamin D2). In each case the quantitative precision (correlation coefficient) for UV absorbance detection was superior owing to intrinsic instability of the TSP ion beam. A stable isotopically labelled internal standard was employed in the development of an assay for 1,25(OH)2D3. The assay was used to quantify in vitro enzymic conversion of 25(OH)D3 to 1,25(OH)2D3 in guinea pig and sheep renal mitochondrial incubations. TSP LC/MS was also applied to analysis of an extract of human blood plasma in which D3 and each of its principal metabolites were identified in a single analysis.
Medium‐ and long‐chain 3‐hydroxymonocarboxylic acids: Analysis by gas chromatography combined with mass spectrometry
Authors: Dorland L. , Ketting D. , Bruinvis L. , Duran M.
Published: May 2005

Medium‐ and long‐chain 3‐hydroxymonocarboxylic acids represent intermediates in the β‐oxidation of fatty acids: they accumulate in the plasma of patients with an inherited deficiency of long‐chain 3‐hydroxyacylcoenzyme A dehydrogenase. 3‐Hydroxy acids with chain lengths varying from 6 to 16 were synthesized by a Reformatzky reaction. Capillary gas chromatography of the pertrimethylsilyl derivatives was performed on a CP‐Sil 19 CB column, coupled to a quadrupole mass spectrometer in the electron impact mode. Calculation of the retention indices showed that the separation of the 3‐hydroxy acids from the homologous straight‐chain fatty acids may be troublesome, stressing the need for mass spectrometric identification.
High performance liquid chromatography of glucose using a post‐column reactor of immobilized enzyme followed by electrochemical detection (glucose‐LCEC)
Authors: Watanabe Noriyuki
Published: May 2005

A sensitive and selective, reasonably fast method for the determination of glucose content has been developed. A glucose oxidase immobilized column was coupled to a small‐size anion exchange column/borate buffer chromatograph. The hydrogen peroxide produced in the enzyme reaction was detected directly by an amperometric detector using a platinum working electrode. The detection limit was 0.03 ppm (1.5 × 10−7 M, 3 pmol/injection). The linear dynamic range was three orders of magnitude at least. The system was stable and reproducible both in short‐and long‐term operation. The proposed method is suitable for analysis of complicated matrices of biological samples because of its good selectivity and sensitivity.
4‐(N,N‐Dimethylaminosulphonyl)‐7‐Fluoro‐2,1,3‐Benzoxadiazole as chemilumigenic reagent for high performance liquid chromatographic peroxyoxalate chemiluminescence detection
Authors: Uzu Sonoko , Imai Kazuhiro , Nakashima Kenichiro , Akiyama Shuzo
Published: May 2005

4‐(N,N‐Dimethylaminosulphonyl)‐7‐fluoro‐2,1,3‐benzoxadiazole (DBD‐F), presented as a fluorogenic labelling reagent for amines and amino acids, is preferred for peroyoxalate chemiluminescence (PO‐CL) detection in high performance liquid chromatography. Amino acids and epinephrine derivatized with DBD‐F were separated on a reversed phase column and detected at the femtomole level by the PO‐CL detection system.
Solid phase extraction of oxcarbazepine and its metabolites from plasma for analysis by high performance liquid chromatography
Authors: Hartley R. , Green M. , Lucock M. D. , Ryan S. , Forsythe W. I.
Published: May 2005

A rapid, sensitive and simple‐to‐operate high performance liquid chromatographic method for the simultaneous determination of oxcarbazepine, 10‐hydroxycarbazepine and 10,11‐dihydro‐10,11‐trans‐dihydroxy‐carbamazepine in plasma is described. The drug and its metabolites were extracted from plasma using commercially available reversed phase octadecylsilane bonded‐silica columns (Bond Elut C18, 1 mL capacity). Chromatographic separation of oxcarbazepine and its metabolites was achieved using a mobile phase consisting of acetonitrile/methanol/water (13:25:62 by volume) at a flow rate of 1.2 mL/min in conjunction with a Waters Associates Nova‐Pak C18 column. The analytical column, in Radial‐Pak cartridge form, was used in combination with a LiChrospher 5 μm C18 guard column. By measuring the UV absorbance at 214 nm, plasma levels in the region of 50–100 ng/mL for the drug and its metabolites can be detected with only 100 μL of plasma. The method has been applied to pharmacokinetic studies of oxcarbazepine and its metabolites in children with epilepsy; preliminary pharmacokinetic findings in two patients at steady‐state are presented.
Use of high performance liquid chromatography in defining the abnormalities in the free amino acid patterns in the cerebrospinal fluid of patients with aseptic meningitis
Authors: Halawa Imad , Baig Shahid , Qureshi G. Ali
Published: May 2005

Free amino acids were quantitatively determined in crebrospinal fluid (CSF) and plasma samples from patients with aseptic meningitis by a newly developed high performance liquid chromatographic (HPLC) method. The method of analysis was based on precolumn derivatization of orthophthalaldehyde in the presence of 2‐mercaptoethanol and detection was made at Eex = 340 nm and Eem = 450 nm. The method was sensitive and the limit for detection was < 1 pmol for most of the amino acids. It took 45 min to separate 26 amino acids with highly reproducible results, giving a coefficient of variance for retention times and integrated areas < 0.4% and 2%, respectively, after five replicate runs. The results accumulated in 10 patients were compared statistically with 11 age‐matched healthy controls. Among the amino acids almost all the neurotransmitter candidates, such as aspartic acid, glutamic acid, glutamine, glycine, tyrosine, phenylalanine and γ‐aminobutyric acid (GABA), were significantly increased in the patients' CSF, whereas arginine and threonine were low. No change was observed in plasma amino acids in patients as compared to healthy controls. The higher levels of most of the neurotransmitters, especially GABA, aspartic acid and glutamic acid, could be used diagnostically in assessing the progression and remission in aseptic meningitis.
Automated quantitative microcolumn chromatography of haemoglobin A2
Authors: Ersser R. S. , Blight Leonie , Hjelm N. M. , Chambers K. , James D.
Published: May 2005

Automated cation exchange microcolumn chromatography of haemoglobins has been modified for the analysis of haemoglobin A2. It provides the quantitative data of sufficient precision and specificity for the investigation of potential heterozygotes for β‐thalassaemia. Results have been compared with an established method of electrophoresis followed by densitometry of the eluted bands.
Liver high performance liquid chromatographic porphyrin profiles in experimental porphyria induced by peroxidizing herbicides
Authors: Krijt J. , Sanitrák J. , Vokurka M. , Janoušek V.
Published: May 2005

The effect of peroxidizing herbicides on the liver porphyrin content of experimental animals was examined. Mice treated with the herbicide oxadiazon accumulated uroporphyrin and protoporphyrin in the liver. Fomesafen‐treated mice accumulated uroporphyrin and heptacarboxylic porphyrin.
Study of the solid phase extraction of pentoxifylline and its major metabolite as a basis of their rapid low concentration gas chromatographic determination in serum
Authors: Marko Vladimír , Bauerová Katarína
Published: May 2005

A gas—liquid chromatographic method for the determination of pentoxifylline and its secondary alcohol metabolite in serum has been developed. The method is based on the combination of solid phase extraction, capillary column separation and nitrogen—phosphorus detection of the analytes. Optimization of the solid phase extraction conditions permitted a low concentration determination, with limits of determination of 2 n/mL and 10 n/mL for pentoxifylline and its metabolite, respectively. The simplicity and rapidity of the extraction step was preserved.
Determination of 10α‐methoxy‐9,10‐dihydrolysergol, a nicergoline metabolite, in human urine by high performance liquid chromatography
Authors: Sioufi A. , Sandrenan N. , Godbillon J.
Published: May 2005

A specific method for the determination of 10α‐methoxy‐9,10‐dihydrolysergol, a nicergoline metabolite (metabolite 2), in urine is described. Metabolite 2 was well separated from the urine components on a reversed phase column, Hypersil ODS 5 μm, using an acetonitrile: pH 3.5 phosphate buffer (40:60, v/v) as the mobile phase at a flow rate of 1 mL/min. UV detection was set up at 220 nm. After addition of a known amount of lysergamide as the internal standard, the compounds were extracted from alkalysed urine on a pre‐packed glass column (Extrelut 1) with dichloromethane. With 0.5 mL urine, concentrations down to 0.56 μmol/L could be determined.
Phosphotungstate as a useful eluent for hepatitis‐B virus surface antigen purification by heparin‐sepharose affinity chromatography
Authors: Tajima Yutaka , Nagumo Fumio , Nishimura Tadataka , Tadano Jutaro
Published: May 2005

On the basis of the heparin‐like effect of phosphotungstate (PTA), we have shown that it is useful for the purification of hepatitis‐B virus surface antigen (HBsAg) using heparin‐Sepharose affinity chromatography. HBsAg was eluted with 0.2–0.6 M NaCl. HBsAg was also eluted with PTA at approximately 1 mM, and the HBsAg fraction thus obtained contained fewer impurities than the corresponding fraction eluted with NaCl. Moreover, PTA yielded HBsAg and hepatitis‐B virus e‐antigen simultaneously. PTA may specifically reduce the affinity of HBsAg for heparin as well as simply competing with heparin for an anion‐binding site of HBsAg. Residual PTA in the eluate was easily decomposed by alkalization, which was useful for subsequent studies.
The combination of normal phase with reversed phase high performance liquid chromatography for the analysis of asparagine‐linked neutral oligosaccharides labelled with p‐aminobenzoic ethyl ester
Authors: Matsuura Fumito , Ohta Masaya , Murakami Koichi , Hirano Kaoru , Sweeley Charles C.
Published: May 2005

We have developed a novel approach for the analysis of asparagine‐linked neutral oligosaccharides derived from glycoproteins. The oligosaccharides are labelled with p‐aminobenzoic ethyl ester and the derivatives are separated on two high performance liquid chromatographic columns, one containing amide‐silica and the other containing octadecyl‐silica. The elution positions of 39 different ABEE‐oligosaccharides on the two columns were plotted on a two‐dimensional map. Unique non‐overlapping positions of these oligosaccharides demonstrate that this technology would be useful for the identification of Asn‐linked oligosaccharides at high sensitivity.
Membrane affinity chromatography used for the separation of trypsin inhibitor
Authors: Guo Wei , Shang Zhenhua , Yu Yinian , Guan Yafeng , Zhou Liangmo
Published: May 2005

Polysulphone (PS) was chemically modified by acrylation–amination and by chloromethylation‐amination, respectively. An ultrafiltration membrane of chemically modified polysulphone (CMPS) was prepared by the phase inversion method. Trypsin was then covalently bonded onto the CMPS membrane by diazotization. The activity of immobilized trypsin reaches up to 10200 U/g; 15 mg trypsin was immobilized on 1 g CMPS membrane. Separation of soybean trypsin inhibitor was carried out on the affinity membrane, yielding 6.5 mg pure trypsin inhibitor in one run. The enzyme membrane has good activity and stability.
TLC separation of certain tetracycline and amino glycopeptide antibiotics
Authors: Bhushan R. , Ali Imran
Published: May 2005

The separation of tetracycline and amino glycopeptide antibiotics was achieved on silica gel thin layers. Tetracycline antibiotics were resolved on a Co+2 (1.0%) impregnated silica gel layer using ethanol:acetic acid: water (10:6:6, v/v/v) as the mobile phase. Amino glycopeptide antibiotics were separated on an untreated silica gel layer using the mobile phase n‐butanol: formic acid: water (6:5:7, v/v/v). The spots of these antibiotics were located by exposing the chromatoplate to iodine vapours.
An improved method for isolation and identification of Zn‐metallothionein from cadmium‐induced rat liver
Authors: Pan Aihua , Tie Feng , Ru Binggen , Li Lingyuan , Shen Tong
Published: May 2005

Zn‐metallothioneins (MT‐1 and MT‐2) were isolated and purified from Wistar rat liver induced by subcutaneous injection with cadmium chloride over a short time. Instead of Sephadex G‐50 and DEAE Sephadex A‐50, new chromatographic media produced by Pharmacia, Sephacryl S‐200, S‐100 and DEAE Sepharose Fast Flow were used in the purification of metallothioneins. The time required for purification with the new method was only 1/3 that required with the usual method and had the same purification effect and rate of recovery. The number of mercapto groups measured with modified Ellman's reagent and cysteine as standard is 20 in MT molecules. Zn and Cd concentrations in each fraction were measured by single sweep polarography rather than atomic absorption spectrophotometry. MT‐1 and MT‐2 contained 6 gram atoms of zinc, but no cadmium. Purified MT‐1 and MT‐2 were shown by high performance liquid chromatographic analysis to be highly homogeneous and had an amino acid composition similar to that of Cd‐MT.