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PubMed1997

Measurement of nitrite and nitrate in biological fluids by gas chromatography-mass spectrometry and by the Griess assay: problems with the Griess assay--solutions by gas chromatography-mass spectrometry.

Assay methods based on the Griess reaction are frequently used to measure nitrite and nitrate in urine, plasma, and other biological fluids. With minor exceptions, careful attention has not been paid in extending the Griess assay from aqueous solutions to biological fluids, In the present study, parallel measurements of nitrite and nitrate were performed in urine, plasma, and aqueous solutions with a published batch assay based on the Griess reaction and with gas chromatography-mass spectrometry (GC-MS). We report here further interferences by free reduced thiols, proteins, and other plasma constituents in the Griess assay but not in GC-MS. The best correlation (r2 = 0.985) between the Griess assay and GC-MS was observed for aqueous solutions in the absence of thiols. Unlike GC-MS, the Griess assay was not applicable to whole human plasma and urine samples. For the measurement of nitrate in diluted human urine samples, reduction by cadmium was performed both under acidic (pH 2 or 5) and alkaline (pH 8.8) conditions. The mean recovery rate of nitrate from urine samples was quantitative in the GC-MS but amounted to only 30-80% in the Griess assay. Measurement of nitrate in human urine samples (n = 33) resulted in an excellent correlation between two GC-MS techniques (r2 = 0.979) but only in a poor correlation (r2 < 0.64) between the Griess assay and GC-MS. Unlike GC-MS, the batch Griess assay is associated with many problems in measuring nitrate in biological fluids.

Chromatography, High Pressure Liquid↗
PubMed2002

Comparison of gas chromatography-mass spectrometry and liquid chromatography-tandem mass spectrometry methods to quantify alpha-tocopherol and alpha-tocopherolquinone levels in human plasma.

Two mass spectrometric methods were established for the quantitative analyses of alpha-tocopherol (TH) and its oxidation product alpha-tocopherolquinone (TQ) in human plasma. Both methods make use of isotopically labeled internal standards of different levels of deuteration (d3-TH and d6-TQ). Plasma (100 microl) was saponified in the presence of a mixture of antioxidants, and then TH and TQ were extracted with hexane. With the GC-MS method, the analytes were first converted into O-trimethylsilyl derivatives before analysis in the selective ion monitoring mode. The derivatization procedure led to the quantitative conversion of TQ into the O-trimethylsilyl derivative of tocopherolhydroquinone, giving rise to a more stable molecule with less fragmentation than for TQ. The increased stability of the molecule resulted in an enhanced contribution of the base peak to the total observed ions and therefore an increased sensitivity of the base peak for quantification. With the liquid chromatography-tandem mass spectrometry (LC-MS/MS) method, TH and TQ were detected by multiple reaction monitoring after positive electrospray ionization. The GC-MS and LC-MS/MS methods showed nearly the same accuracy (>95%) and the same within-day precisions, with less than 5 and 10% for TH and TQ, respectively. The between-day precision and the limit of quantification for TQ in plasma were better by LC-MS/MS (4%; 3 nM) than by GC-MS (21%; 10 nM). Analysis and method validation were carried out with plasma samples obtained from a male volunteer pre- and postexercise. Both techniques showed that the ratio of TQ/TH was elevated by 35% immediately after exercise and had returned to basal levels when measured 24 h later.

Chromatography, Liquid↗
PubMed1986

Determination of polycyclic aromatic hydrocarbons (PAH) in edible vegetable oils by liquid chromatography and programmed fluorescence detection. Comparison of caffeine complexation and XAD-2 chromatography sample clean-up.

Two clean-up procedures were compared for the analysis for polycyclic aromatic hydrocarbons (PAHs) in edible vegetable oils. One method comprises a liquid-liquid extraction followed by XAD-2 chromatography and the other a caffeine-formic acid complexation. The clean-up step is followed by gradient reversed-phase HPLC in combination with wavelength-programmed fluorescence detection. Due to better repeatability and simplicity, the XAD-2 method was selected for the determination of PAHs in 14 different vegetable oils. Between the different oil samples large differences were observed in PAH concentrations. PAH concentrations in vegetable oils sampled from the Dutch market appear to be comparable with those found in other countries.

Caffeine↗
PubMed1993

[Trigonelline in coffee. I. Comparison of thin layer chromatography with high-performance liquid chromatography. Simultaneous determination of caffeine].

Determination of trigonelline in several green and roasted coffees by thin-layer (TLC) and high-performance liquid chromatography (HPLC) were compared. Using TLC, two methods of detection were also compared. In addition, caffeine could be determined using HPLC. The determinations were each made with several variations of extraction and clean-up. Recoveries, variation coefficients and detection limits are listed. The most suitable method was HPLC in connection with hot water extraction. Only water is required as the mobile phase. The values for precision are in most cases better than (trigonelline) or similar to (caffeine) those reported in the literature. The determination of trigonelline by TLC, followed by extraction and photometry, is possible with a similar precision, but the simultaneous determination of caffeine was not tested.

Alkaloids↗
PubMed1980

Semliki Forest virus glycans analyzed by affinity chromatography, hydrazinolysis and paper chromatography.

N-acetyl-lactosamine type glycopeptides of Semliki Forest virus were fractionated on concanavalin A-Sepharose, and their oligosaccharides were liberated by hydrazinolysis and analyzed by paper chromatography. Comparison with labeled glycans from reference glycopeptides suggests that the viral N-acetyl-lactosamine type glycans are bi-, tri- and tetra-antennary.

Chromatography, Affinity↗
PubMed1984

Anion-exchange chromatography of proteins on AG MP-1 using high-performance liquid chromatography equipment.

That the macroporous anion-exchange resin AG MP-1 can be used with HPLC equipment and common aqueous buffers for the chromatography of proteins is shown. The utility of this system is illustrated by the partial purification and complete resolution of the three protein synthesis elongation factors from each other, starting with a crude extract of Escherichia coli. The factors were purified 10- to 30-fold in a yield of 50 to 90% with a single 60-min chromatographic program of increasing NaCl concentration. Other proteins from various biological sources were purified with similar results. Thus, it appears that AG MP-1 is useful, at least in some applications, for the rapid, reproducible, and economical purification of proteins using HPLC equipment.

Animals↗
PubMed1991

An ion-exchange chromatography procedure for the isolation and concentration of basic amino acids and polyamines from complex biological samples prior to high-performance liquid chromatography.

The original objective of this study was to develop a selective and sensitive method for the analysis and quantification of basic amino acids from biological samples via reversed-phase high-performance liquid chromatography. Using various previously described techniques for the separation of amino acids, we were unsuccessful in measuring levels of histidine, arginine, ornithine, and lysine in biological samples due to the presence of interfering compounds. A "cleanup" procedure for the isolation of the basic amino acids using a weakly acidic cation exchange resin, Biorex-70 (Bio-Rad), is described in detail. Upon separation from the bulk of the neutral and acidic amino acids, the basic amino acids were subjected to precolumn fluorescence derivatization using 9-fluorenylmethyl chloroformate (FMOC) and the fluorescent derivatives were separated by RP-HPLC. The advantages of this method over previously described amino acid analysis techniques are (i) isolation and stable recovery (greater than 95%) of the desired basic amino acids, (ii) sensitivity of detection (low pmol range), (iii) complete resolution of derivatized amino acids via HPLC, (iv) limited amount of sample required for analysis, and (v) samples readily concentrated by lyophilization or rotoevaporating. This ion-exchange cleanup procedure was also adapted for the analysis of polyamines in concentrated culture media samples and proved additionally advantageous by eliminating the use of costly C-18 extraction columns required by previously described techniques.

Amino Acids, Diamino↗
PubMed1992

Assessment study on the high-performance liquid chromatography-type hydroxyapatite chromatography in the presence of sodium dodecyl sulfate.

The HPLC-type hydroxyapatite chromatography in the presence of sodium dodecyl sulfate (SDS) was assessed with special attention to the behavior of the surfactant. A significant amount of SDS was found to be adsorbed to the hydroxyapatite packed in the column from the starting buffer, 50 mM sodium phosphate buffer, pH 7.0, only when the buffer contained SDS in a concentration at or above its critical micelle concentration. When the phosphate buffer concentration was increased while the SDS concentration was kept at 1 mg/ml, the adsorbed surfactant was desorbed in advance of the release of proteins. Polypeptides derived from proteins could be successfully separated only when the column had been thoroughly equilibrated with the above-mentioned starting buffer solution. When a protein polypeptide complexed with SDS, which had been similarly equilibrated, was applied to the column, an amount of SDS corresponding to 75-90% (w/w) of the surfactant originally bound to the polypeptide was released upon its binding to the hydroxyapatite. On the other hand, porin, an Escherichia coli outer membrane protein, retaining its trimeric native structure in the presence of SDS, released a significantly smaller amount of SDS. When the membrane protein was denatured to give a single polypeptide, it behaved in a manner similar to that of the other protein polypeptides. The mechanism of binding of the protein polypeptides was discussed on the basis of these results. The native and denatured entities of porin could be efficiently separated as the result of the difference in their mode of interaction with the hydroxyapatite.

Chromatography↗
PubMed1985

Calcium-dependent changes in properties of human prothrombin: a study using high-performance size-exclusion chromatography and gel-permeation chromatography.

High-performance size-exclusion chromatography using a TSK 3000 SW column and aqueous gel filtration with Sephacryl S-200 SF have been used to characterize the effects of calcium ions on the hydrodynamic properties of human prothrombin and prethrombin 1. The results suggest that the effective hydrodynamic radius of prothrombin is less in the presence than in the absence of calcium ions. In addition, when using the TSK-3000 SW column, Ca2+-dependent formation of a hydrophobic site in the fragment 1 region of prothrombin results in an apparent further decrease in hydrodynamic radius.

Calcium↗
PubMed1990

Rapid determination of clenbuterol residues in urine by high-performance liquid chromatography with on-line automated sample processing using immunoaffinity chromatography.

A liquid chromatographic column-switching system for automated sample pretreatment and determination of clenbuterol in calf urine, using an immunoaffinity precolumn with Sepharose-immobilized polyclonal antibodies against clenbuterol, is described. A second precolumn packed with C18-bonded silica was used for the reconcentration of desorbed clenbuterol prior to the analytical separation. Urine, after 2-fold dilution with buffer (pH 7.4), was loaded directly onto the immuno precolumn, where clenbuterol was trapped by the immobilized antibodies. This immuno precolumn has been used for more than 200 runs with standard solutions and samples. Bound analyte was desorbed with 0.01 M acetic acid and transferred, via the second precolumn, to the analytical column. The total runtime per sample was 35 min. Using a sample load of 27 ml of dilute urine and UV detection at 244 nm, the detection limit was 0.5 ng/ml. The mean recovery of clenbuterol added to a blank urine sample at the 5 ng/ml level was 82 +/- 2% (n = 5) as determined with standard solutions loaded onto the same system. Urine samples from treated animals were analysed and the clenbuterol concentrations were comparable to those obtained by high-performance liquid chromatography using solid-phase extraction for sample clean-up.

Animals↗
PubMed1993

Multimycotoxin detection and clean-up method for aflatoxins, ochratoxin and zearalenone in animal feed ingredients using high-performance liquid chromatography and gel permeation chromatography.

A sensitive and reliable method is described for the determination of aflatoxins B1, B2, G1 and G2, ochratoxin A and zearalenone in animal feed ingredients. A multi-toxin extraction and clean-up procedure is used, with dichloromethane-1 M hydrochloric acid (10:1) being used for the extraction and gel permeation chromatography being used for the clean-up. The liquid chromatographic method developed for the separation of the six mycotoxins involves gradient elution with a reversed-phase C18 column and fluorescence detection. Recoveries, repeatability and reproducibility have been determined on maize, palm and wheat. The detection limits varied depending on the type of feed.

Aflatoxins↗
PubMed1994

Separation of selenium analogues of sulphur-containing amino acids by high-performance liquid chromatography and high-resolution gas chromatography.

The historically conditioned adaptation of living organisms to chemically corresponding elements is influenced in nature by anthropogenic activities in many regions, the selenium-sulphur pair being one example of such a case. The separation of selenomethionine, selenoethionine and selenocystine was studied by HPLC and high-resolution GC. Ion-exchange chromatography followed by temperature-programmed GC gives the possibility of the analytical separation of trace amounts of selenomethionine in a complex mixture of common amino acids. Diastereoisomers of selenocystine were identified by HPLC in the AccQ-Tag mode.

Amino Acids, Sulfur↗
PubMed1994

Determination of neutral lipids from subcutaneous fat of cured ham by capillary gas chromatography and liquid chromatography.

The determination of neutral lipids in fat of cured ham is reported. Fat samples were extracted with chloroform-methanol (2:1) and neutral lipids and free fatty acids were separated on an aminopropyl minicolumn, the first fraction with chloroform-2-propanol (neutral lipids) and the second fraction with 2% acetic acid in diethyl ether (free fatty acids). Neutral lipids were fractionated with minicolumns, with aminopropyl and silica stationary phases. Two fractions were obtained with the first column: (A) triglyceride and cholesteryl esters and (B) cholesterol and mono- and diglycerides. Fraction A was applied to the silica column to obtain two new fractions: (C) cholesteryl esters and (D) triglycerides. Fractions B and C were analysed by capillary gas chromatography (cGC) and fraction D by cGC and HPLC. The R.S.D.s obtained were below 5% except for the monoglycerides (8%). Cholesteryl esters were determined by cGC in 5 min with R.S.D. 5%. The main triglycerides identified were PPO, POS, POO, POL and OOO (P = palmitic acid, O = oleic acid, L = linoleic acid; S = stearic). Monoglyceride and diglycerides having 18, 34 and 36 carbon atoms were the most abundant. The determination of triglycerides by HPLC was more difficult than by cGC because the linearity with HPLC was concentration dependent. The procedure allowed the determination of neutral lipid classes without derivatization of mono- and diglycerides.

Adipose Tissue↗
PubMed1996

Structure-retention relationships of steroid hormones in reversed-phase liquid chromatography and micellar electrokinetic capillary chromatography.

Quantitative structure-retention relationship (QSRR) studies are useful in retention prediction, finding the relevant structural descriptors for analytes and estimating the relative biological activities of a series of analytes. Most of the studies have been conducted by RP-HPLC and a few by micellar electrokinetic capillary chromatography (MECC). The aim of this work was to find structural parameters and characteristics related to the RP retention and electrophoretic migration of steroids in order to predict the retention/migration of steroid hormones on the basis of their molecular structure. Retention data were obtained with an ODS column using a mobile phases aqueous methanol-acetonitrile (mobile phase A) and methanol-tetrahydrofuran (mobile phase B). MECC was conducted with a sodium dodecyl sulfate (SDS)-borate system and with a mixed micellar solution of SDS and sodium cholate. Several topological indices, such as the connectivity indices, chi, were used as structural parameters. Steric factors seem to have a great effect on the retention of steroid hormones, especially with the MeCN-containing mobile phase. Retention in mobile phase B could be predicted more accurately. Topological indices can be used in the modelling and prediction of the retention/migration of steroid hormones when the solutes form a congeneric series and stereochemical properties do not govern the separation process.

Chromatography↗
PubMed1996

Laboratory-scale purification of microcystins using flash chromatography and reversed-phase high-performance liquid chromatography.

Microcystins were extracted from 7 l (equivalent to 313 g dry weight) of cyanobacterial scum collected from Rutland Water in Leicestershire, UK in 1989. The resulting aqueous extract was rapidly concentrated on a C18 flash chromatography cartridge and microcystins were eluted using a step gradient. Fractions were collected manually and monitored by UV spectrophotometer and analytical HPLC. Fractions containing microcystins of similar polarity were pooled to give three fractions. Simple isocratic methods for separating each fraction were developed on an analytical column and scaled up to a 15 x 7.5 cm I.D. column. Closed-loop recycling was used to maximise yield and purity of two hydrophobic microcystins.

Amino Acid Sequence↗
PubMed1990

Purification of immunotoxins containing the ribosome-inactivating proteins gelonin and momordin using high performance liquid immunoaffinity chromatography compared with blue sepharose CL-6B affinity chromatography.

Comparable amounts of the ribosome-inactivating proteins (RIPs) ricin A chain, gelonin and momordin were allowed to bind to Blue Sepharose CL-6B (immobilised Cibacron Blue F3GA) in phosphate buffer, pH 7.5, and were then eluted quantitatively with buffer containing 0.5 M NaCl. Differences in the elution profiles indicated that the RIPs possessed different affinities for the Cibacron Blue F3GA dye. Conjugation of the RIPs to the monoclonal antibody Fib75 resulted in decreased affinity for Blue Sepharose. Under conditions allowing the complete separation of the Fib75-ricin A chain immunotoxin from unconjugated Fib75, the Fib75 immunotoxins made with gelonin and momordin failed to bind completely to the Blue Sepharose column. The Fib75-gelonin and Fib75-momordin fractions that eluted from the column with 0.5 M NaCl were free of unconjugated Fib75 but were enriched in multiply substituted conjugate molecules. A high performance liquid immunoaffinity chromatography procedure based on the selective binding of conjugated RIP to immobilised affinity-purified anti-RIP antibody permitted the complete separation of the gelonin and momordin immunotoxins from unconjugated Fib75 without altering the composition, molecular integrity or cytotoxic activity of the immunotoxins.

Animals↗
PubMed1984

Analysis of metabolic profiles of steroids in faeces of healthy subjects undergoing chenodeoxycholic acid treatment by liquid-gel chromatography and gas-liquid chromatography-mass spectrometry.

The multicomponent analysis of faecal steroids is described. Steroids were removed from faeces by solvent stripping in a Soxhlet apparatus and the resulting extracts were fractionated by diethylaminohydroxypropyl Sephadex column chromatography into neutral sterols, free bile acids, glycine conjugated bile acids, taurine conjugated bile acids and sulphated steroids. In this study the method has been applied for faecal steroid analyses of healthy subjects undergoing chenodeoxycholic acid therapy. Chenodeoxycholic acid administration causes a considerable increase in the concentration of faecal lithocholic acid which is a known comutagenic bile acid. Furthermore it has been shown that conjugated bile acids can account for between 10 and 20% of the faecal bile acid pool. The method described is convenient and may be useful for epidemiological studies which require a large number of faecal samples to be analysed.

Bile Acids and Salts↗
PubMedDate not supplied

High-performance liquid chromatography of proteins on deformed nonporous agarose beads: immuno-affinity chromatography, exemplified with human growth hormone as ligand and a combination of ethylene glycol and salt for desorption of the antibodies.

An affinity chromatography column packed with nonporous agarose beads derivatized with human growth hormone via carbonyldiimidazol was used for the purification of antibodies against human growth hormone from antiserum. Desorption with 1 M sodium chloride in 60% ethylene glycol at pH 9.8 gave 100% total recovery of the antibodies, as measured by radioimmunoassay. The adsorption/desorption process is discussed in terms of hydrophobic and electrostatic interaction (these interactions may be involved in the bond between antibody and antigen in a cooperative fashion). The binding capacity of the column was estimated at about 50 micrograms of antibodies per gram sedimented agarose beads.

Antibodies↗