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PubMed1985

Purification of hemopexin and its domain fragments by affinity chromatography and high-performance liquid chromatography.

A method is described for the preparation of apohemopexin from Cohn Fraction IV-4 of human serum by one-step affinity chromatography on a heme-agarose column and separation of its tryptic domain fragments by high-performance liquid chromatography (HPLC). Limited tryptic digestion cleaved human apohemopexin after Arg-216 into half molecules and the N-terminal half was degraded very rapidly, whereas heme-saturated hemopexin was cleaved after Lys-101. These results suggest that hemopexin is composed of two domains that are connected by an exposed histidine-rich hinge-like region in apohemopexin which becomes inaccessible to trypsin in heme-saturated hemopexin. Also described is the preparation of apohemopexin from whole rabbit serum in two steps, heme-affinity chromatography and ion-exchange HPLC, and separation of its tryptic domain fragments by HPLC. Limited tryptic digestion also cleaves rabbit apohemopexin into half-molecules but the N-terminal half is more stable than the C-terminal half in this case. This lends support to the idea of functional differences between domains.

Amino Acids↗
PubMed2000

A non-destructive method to determine the safranal content of saffron (Crocus sativus L.) by supercritical carbon dioxide extraction combined with high-performance liquid chromatography and gas chromatography.

A supercritical carbon dioxide extraction method to obtain selectively volatile compounds of saffron without sample destruction has been developed. The influence of both pressure and temperature was studied, 20 MPa and 100 degrees C being the best conditions to extract the total safranal content. A decrease in supercritical fluid density was shown to be a critical parameter for enhancing the extraction power of carbon dioxide. For all the assay conditions, the extracts mainly contained safranal and HTCC, as demonstrated by gas chromatography and high-performance liquid chromatography analyses. Both chromatographic methods were suitable for safranal quantification and showed excellent agreement. Supercritical extracts from five different saffron types were studied by high-performance liquid chromatography and their safranal contents were determined.

Chromatography↗
PubMed1989

Human milk proteins: separation of whey proteins and their analysis by polyacrylamide gel electrophoresis, fast protein liquid chromatography (FPLC) gel filtration, and anion-exchange chromatography.

Human milk proteins are of nutritional and physiological significance to the newborn infant. To further study these proteins, a rapid procedure to separate and analyze human milk whey proteins was developed using fast protein liquid chromatography (FPLC). First, to separate whey proteins from casein, different variables such as low- or high-speed centrifugation at different temperatures with or without adjustment of pH to 4.6 or 4.3 and with or without addition of calcium to whole milk or skim milk were tested. Each variable was evaluated by gel filtration, anion-exchange chromatography, sodium dodecyl sulphate (SDS)-polyacrylamide gradient gel electrophoresis, immunoelectrophoresis, and immunodiffusion. The optimum method for a discrete separation of whey and casein is the adjustment of whole milk to pH 4.3 with addition of 60 mmol calcium/L, followed by ultracentrifugation. Rapid and sensitive separation and analysis of whey proteins was achieved by FPLC gel filtration and anion-exchange chromatography.

Chromatography, Gel↗
PubMedDate not supplied

Instrumental planar chromatographic determination of benzodiazepines: comparison with liquid chromatography and gas chromatography.

Diazepam, chlordiazepoxide, and midazolam are determined by high-perfromance, thin-layer chromatography (HPTLC). Results are compared with those obtained by liquid chromatography (LC) and gas chromatography (GC). Analytical conditions for all the methods are also described. Validation parameters for linearity, precision, detection, and quantitative limits are informed. Correlation study of HPTLC with LC and GC gave a correlation factor between 0.98 and 0.99. The obtained results showed that HPTLC, LC, and GC techniques are comparable for determination of such benzodiazepins in the requested working range to be analyzed in raw materials.

Benzodiazepines↗
PubMed1997

[Analysis of triacylglycerol composition of perilla oil by combination of non-aqueous reversed-phase high performance liquid chromatography with gas chromatography].

Perilla oil was verified to be a rich source of polyunsaturated fatty acid (PUFA). Its linolenic content is 64.82%, the highest in the plants. Its triacylglycerol (TAG) components were isolated and identified first time in this paper by a combination of non-aqueous reversed-phase high performance liquid chromatography (RP-HPLC) with gas chromatography (GC). The TAG of perilla oil were isolated by HPLC with a Zorbax ODS column (5 microm, 4.6 mm i.d. x 250 mm; Dupont, Inc) and differential refractometer. The eluent was acetone/acetonitrile (80:20, V/V) with flow rate of 1.0 mL/min. The TAG component acyl groups were converted to fatty acid methyl esters (FAME). The acyl constituents for each TAG were determined by GC analysis. Gas chromatography of FAMEs was performed with a AC20 Carbowax 20M column (30 m x 0.32 mm i.d.). Detector and injection port temperatures were 260 degrees C. Column temperature was programmed from 120 degrees C, 1 min initially hold, then 8 degrees C/min to 220 degrees C and finally hold for 10 min. Nitrogen was the carrier gas. FAMEs peaks were identified by comparison of their retention times with those of standards. The retention times of the palmitic, stearic, oleic, linoleic, n-nonadecanoic acid (internal standard) and linolenic FAMEs were 10.7, 13.0, 13.3, 13.8, 14.2 and 14.7 min respectively. Five main components were determined. They were LnLnLn (34.10%), LnLnL (13.22%), LnLL (6.22%), LnLnO (11.38%) and LnLnP (9.17%). The TAG contents were corrected from their fatty acids content. The combination of non-aqueous RP-HPLC with GC is a simple and rapid method for the analysis of oil TAG structure and is also very helpful for the identification of oil quality.

Chromatography, Gas↗
PubMed1983

Effects of whole blood storage on results for glycosylated hemoglobin as measured by ion-exchange chromatography, affinity chromatography, and colorimetry.

After storage of whole blood at either 4 or 20 degrees C, results for glycosylated hemoglobin by ion-exchange chromatography ("high-performance" liquid and mini-column chromatography), thiobarbituric acid colorimetry, and affinity chromatography were compared. At 4 degrees C, all methods gave acceptable results for samples stored for as long as a week. At 20 degrees C, the colorimetric and affinity methods also showed sample stability for a week or more. The ion-exchange methods were associated with a marked increase in values for glycosylated hemoglobin after a few days of storage. Evidently, care in details of sample collection and handling is especially important for ion-exchange methods, and the colorimetric and affinity methods have advantages over ion exchange in situations where long delays between sample collection and assay are unavoidable.

Blood Preservation↗
PubMed2006

Comparison of supercritical fluid chromatography and liquid chromatography for the separation of urinary metabolites of nobiletin with chiral and non-chiral stationary phases.

Nobiletin (NOB), a polymethoxylated flavone found in sweet orange (Citrus sinensis) peel, is currently recognized as a promising anti-inflammatory and anti-tumor agent. It is believed that, by undergoing metabolic biotransformation in vivo, nobiletin is demethylated by hepatic P450 enzymes, yielding multiple hydroxylated metabolites. However, it has not been possible to date to separate the two demethylated nobiletin metabolites, 3'-demethyl-NOB and 4'-demethyl-NOB (regio-isomers) on reversed-phase liquid chromatography (RPLC). Additionally, both display similar mass spectrometric fragmentation, resulting in difficulties to identify the dominant metabolite. A successful separation method was developed by utilizing supercritical fluid chromatography (SFC) with chiral stationary phase. The separation was also attempted with normal-phase liquid chromatography (NPLC) in both chiral and non-chiral modes. Chromatographic separation for the two nobiletin metabolites was superior by SFC than by LC, especially using chiral stationary phase. By comparing the SFC profile of the synthesized standards, the major nobiletin metabolite in mouse urine was identified as 4'-demethyl-NOB, with the concentration of 28.9 microg/mL.

Animals↗
PubMed1989

Use of thin layer chromatography for detection and high performance liquid chromatography for quantitating gliotoxin from rice cultures of Aspergillus fumigatus fresenius.

Gliotoxin, a mycotoxin with antimicrobial and immunosuppressive capabilities, is produced by several genera of fungi including the pathogenic fungus Aspergillus fumigatus. The ability of selected isolates of A. fumigatus to produce gliotoxin on three different media was tested and a thin layer chromatographic and high performance liquid chromatographic method for quantitation of gliotoxin from rice culture was developed and is described. Rice cultures were extracted with chloroform and the resulting extract was partially purified by precipitation with petroleum ether and cleanup by gel permeation chromatography. Gliotoxin was detected by thin layer chromatography and quantitated by high performance liquid chromatography using a U.V. absorbance detector with a 254 nm filter and a mobile phase of methanol-water 43:57 (V/V) with a flow rate of 2.0 ml/min. The retention time for gliotoxin was approximately 4.8 min. From rice samples spiked with gliotoxin concentrations of 0.67, 1.33, 2.67, 4.00 and 5.33 micrograms/g the average recovery was 83.8%.

Aspergillus fumigatus↗
PubMed1998

Distributions of conjugated linoleic acid (CLA) isomers in tissue lipid classes of pigs fed a commercial CLA mixture determined by gas chromatography and silver ion-high-performance liquid chromatography.

Pigs were fed a commercial conjugated linoleic acid (CLA) mixture, prepared by alkali isomerization of sunflower oil, at 2% of the basal diet, from 61.5 to 106 kg live weight, and were compared to pigs fed the same basal diet with 2% added sunflower oil. The total lipids from liver, heart, inner back fat, and omental fat of pigs fed the CLA diet were analyzed for the incorporation of CLA isomers into all the tissue lipid classes. A total of 10 lipid classes were isolated by three-directional thin-layer chromatography and analyzed by gas chromatography (GC) on long capillary columns and by silver-ion high-performance liquid chromatography (Ag+-HPLC); cholesterol was determined spectrophotometrically. Only trace amounts (<0.1%; by GC) of the 9,11-18:2 cis/trans and trans,trans isomers were observed in pigs fed the control diet. Ten and twelve CLA isomers in the diet and in pig tissue lipids were separated by GC and Ag+- HPLC, respectively. The relative concentration of all the CLA isomers in the different lipid classes ranged from 1 to 6% of the total fatty acids. The four major cis/trans isomers (18.9% 11 cis,13 trans-18:2; 26.3% 10 trans,12 cis-18:2; 20.4% 9 cis,11 trans-18:2; and 16.1% 8 trans, 10 cis-18:2) constituted 82% of the total CLA isomers in the dietary CLA mixture, and smaller amounts of the corresponding cis,cis (7.4%) and trans,trans (10.1%) isomers were present. The distribution of CLA isomers in inner back fat and in omental fat of the pigs was similar to that found in the diet. The liver triacylglycerols (TAG), free fatty acids (FFA), and cholesteryl esters showed a similar pattern to that found in the diet. The major liver phospholipids showed a marked increase of 9 cis,11 trans-18:2, ranging from 36 to 54%, compared to that present in the diet. However, liver diphosphatidylglycerol (DPG) showed a high incorporation of the 11 cis,13 trans-18:2 isomer (43%). All heart lipid classes, except TAG, showed a high content of 11 cis,13 trans-18:2, which was in marked contrast to results in the liver. The relative proportion of 11 cis,13 trans-18:2 ranged from 30% in the FFA to 77% in DPG. The second major isomer in all heart lipids was 9 cis,11 trans-18:2. In both liver and heart lipids the relative proportions of both 10 trans,12 cis-18:2 and 8 trans, 10 cis-18:2 were significantly lower compared to that found in the diet. The FFA in liver and heart showed the highest content of trans,trans isomers (31 to 36%) among all the lipid classes. The preferential accumulation of the 11 cis,13 trans-18:2 into cardiac lipids, and in particular the major phospholipid in the inner mitochondrial membrane, DPG, in both heart and liver, appears unique and may be of concern. The levels of 11 cis,13 trans-18:2 naturally found in foods have not been established.

Adipose Tissue↗
PubMed1992

Hydrophobic interaction chromatography fractionates lipoteichoic acid according to the size of the hydrophilic chain: a comparative study with anion-exchange and affinity chromatography for suitability in species analysis.

Hydrophobic interaction chromatography fractionated the lipoteichoic acid of Enterococcus faecalis into species of decreasing poly(glycerophosphate) chain length and decreasing extent of substitution with alpha-kojibiosyl residues (Glcp alpha 1----2Glcp alpha 1----). The chain length varied between 14 and 33 glycerophosphate residues per lipid anchor, the extent of glycosylation between 0.18 and 0.44 mol of alpha-kojibiosyl residues per mole of phosphorus, and, accordingly, the number of alpha-kojibiosyl substituents per chain between 3 and 15. Almost identical values were obtained when the same lipoteichoic acid was chromatographed on DEAE-Sephadex and concanavalin A, which separate molecular species according to increasing number of phosphate groups and alpha-kojibiosyl residues, respectively. Species from all three columns, which were identical in chain length and glycosylation, also had similar fatty acid patterns. These results prove the suitability of all three procedures for species analysis. One advantage of hydrophobic interaction chromatography over the other two procedures lies in its broader applicability since it is not dependent on negative charges or specifically binding oligosaccharide structures. Another advantage is the capacity of hydrophobic interaction chromatography to separate molecular species differing in the number of fatty acids [W. Fischer, H.U. Koch, and R. Haas (1983) Eur. J. Biochem. 133, 523-530] and render them accessible to molecular analyses.

Chromatography, Affinity↗
PubMed1986

Diagnosis of inherited adenylosuccinase deficiency by thin-layer chromatography of urinary imidazoles and by automated cation exchange column chromatography of purines.

Patients with inherited adenylosuccinase deficiency excrete large quantities of succinyloaminoimidazolecarboxamide riboside (SAICAR) and succinyloadenosine (SAdo). A two-dimensional thin-layer chromatography method for the detection of SAICAR is described. The method consists of isolation of imidazoles with a cation exchange resin; TLC on cellulose plates, solvent I, isopropanol-ammonia 10% (4:1) and II, butanol-acetic acid-water (4:1:1); detection with Pauly reagent. SAICAR gives rise to an isolated spot with a characteristic bluish color. Also a simple one-dimensional thin-layer chromatography method using urine without any pretreatment for screening of high risk populations is given. Four new cases could be diagnosed. Clinical and chemical data, including concentrations of SAICAR and SAdo in urine, plasma and cerebrospinal fluid, determined by cation exchange column chromatography, are presented.

Adenosine↗
PubMed1979

Analysis of the anti-coccidial drug, halofunginone, in chicken feed using gas-liquid chromatography and high-performance liquid chromatography.

Methods are described for the analysis of the anti-coccidial drug, halofuginone, at concentrations of 3 ppm in chicken feed, using gas-liquid chromatography and high-performance liquid chromatography. Both methods are based on ethyl acetate extraction, partition into hydrochloric acid and purification and concentration using XAD-2 column chromatography. The precision and accuracy of both methods is given.

Animal Feed↗
PubMed1992

Modified on-column interface for coupled high-performance liquid chromatography-gas chromatography and its application to the determination of levamisole in milk.

A modified on-column interface is reported for the coupling of high-performance liquid chromatography with gas chromatography, incorporating an adapted, commercially available multidimensional gas chromatography switching system. Novel features include cryogenic analyte focusing, total solvent exclusion from the analytical column and independent carrier gas supplies to the analytical GC column and uncoated pre-column. The instrumentation was used for the determination of the veterinary anthelmintic drug levamisole in milk with analyte detection by both flame ionisation and nitrogen-phosphorus detectors. Detection limits for the assay were 2.2 micrograms l-1 and 0.4 micrograms l-1 by flame ionisation and nitrogen-phosphorus detectors, respectively. The assay was applied to a survey of fourteen milk samples from different dairy outlets.

Animals↗
PubMed1994

Comparison of micellar electrokinetic chromatography (MEKC) with capillary gas chromatography in the separation of phenols, anilines and polynuclear aromatics potential field-screening applications of MEKC.

Capillary electrophoresis (CE) is known to be complementary to liquid chromatography, but comparison of CE with capillary gas chromatography (GC) for applicable analytes has not been extensive. Capillary GC has been the preeminent separation technique for environmental analysis, but CE has yet to be applied systematically to the determination of environmental analytes. We present data on separations of three classes of semivolatile analytes of interest to environmental analysis: phenols, anilines and polynuclear aromatic hydrocarbons (PNAs). Standard GC conditions were used to illustrate typical separations observed on 30-m and 40-m columns. Rapid analyses were addressed using a high-temperature 15-m column of thinner film. CE separations employed borate buffer with sodium cholate as the micellar agent in micellar electrokinetic chromatography (MEKC). The effects of organic additives were studied using methanol, acetone and tetrahydrofuran. gamma-Cyclodextrin was also used in MEKC to enhance the separation of polynuclear aromatic hydrocarbons and to examine its effects on separations of phenols and anilines. Short capillaries effected very rapid (< 3 min) compound-class characterization, an approach which has potential use in site characterization/remediation (field-screening) studies.

Aniline Compounds↗
PubMed1994

Enantioselective assay for the determination of nisoldipine in dog, rat and mouse plasma by chiral microbore high-performance liquid chromatography combined with gas chromatography-mass spectrometry.

A sensitive, selective and validated method for the enantioselective determination of (+)- and (-)-nisoldipine in rat, mouse and dog plasma following administration of nisoldipine racemate is described. The alkalized plasma samples containing [13C4]nisoldipine racemate as internal standard (ISTD) were extracted once with toluene. The enantiomers of nisoldipine were quantitatively separated by high-performance liquid chromatography on a 250 x 2 mm I.D. column containing tris(4-methylbenzoate)-modified cellulose on silica. The fractions containing either the (+) or (-)-enantiomer of the analyte and [13C4]ISTD were analysed by gas chromatography with mass-selective detection in the single-ion monitoring mode. The limits of determination and detection were 0.5 and 0.2 ng/ml, respectively, the total precision was better than 7% (R.S.D. at 5 and 50 ng/ml, n = 35) and the accuracy was better than 10% (0.5-100 ng/ml, n = 23). The sum of the concentrations of the enantiomers determined with this assay corresponds to the concentration of the racemate determined independently by capillary gas chromatography with electron-capture detection (accuracy better than 15%, 1-80 ng/ml). The method was used for the analysis of more than 500 plasma samples obtained from toxicokinetic studies.

Animals↗
PubMed1989

Analysis of the major urinary thromboxane metabolites, 2,3-dinorthromboxane B2 and 11-dehydrothromboxane B2, by gas chromatography-mass spectrometry and gas chromatography-tandem mass spectrometry.

2,3-Dinorthromboxane B2 and 11-dehydrothromboxane B2, the two major metabolites of thromboxane B2, are considered to be indices of thromboxane A2 activity in humans. The determination of these metabolites in urine was comparatively performed by gas chromatography-mass spectrometry and gas chromatography-mass spectrometry-mass spectrometry using the corresponding chemically synthesized tetradeuterated analogues as internal standards. The urine samples of five females and two males, all healthy, were prepurified by solid-phase extraction. The corresponding pentafluorobenzyl ester derivatives were repurified by high-performance liquid chromatography. The concentrations of 2,3-dinorthromboxane B2 and 11-dehydrothromboxane B2 ranged from 21 to 266 pg/ml and 47 to 942 pg/ml, respectively. The ratio of urinary 2,3-dinorthromboxane B2 to 11-dehydrothromboxane B2 varied from 1:3 to 1:5, except for one sample with nearly equal concentrations of 2,3-dinorthromboxane B2 and 11-dehydrothromboxane B2.

Chemical Phenomena↗
PubMed1992

Determination of manidipine enantiomers in human serum using chiral chromatography and column-switching liquid chromatography.

A stereoselective and highly sensitive method using chiral chromatography and successive column-switching liquid chromatography is described for the determination of manidipine enantiomers in human serum. A human serum sample obtained after ingestion of manidipine was extracted twice with a mixture of n-hexane-diethyl ether under alkaline conditions. The enantiomers in the extract were separated on a chiral stationary phase column (Chiralcel OJ), and the effluents containing the respective enantiomers were collected. Each fraction was then analysed by column-switching liquid chromatography. The proposed stereoselective method offered high sensitivity: detection limits for both isomers were 0.2 ng/ml in human serum, both at a signal-to-noise ratio of 3. The method is suitable for the pharmacokinetic studies of manidipine enantiomers.

Administration, Oral↗
PubMed1995

Complementary use of counter-current chromatography and hydroxyapatite chromatography for the separation of three main classes of lipoproteins from human serum.

High-density, low-density and very-low-density lipoproteins (HDLs, LDLs and VLDLs) were purified from human serum by the combined use of counter-current chromatography (CCC) and hydroxyapatite chromatography. Polymer-phase CCC of human serum using the cross-axis coil planet centrifuge yielded two lipoprotein fractions, one containing HDLs and LDLs and the other VLDLs and serum proteins. Each fraction was concentrated and subjected to hydroxyapatite chromatography to obtain three lipoprotein fractions, all free from serum proteins. Each lipoprotein was confirmed by agarose gel electrophoresis.

Chromatography↗