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PubMed1988

Isolation of bile acid glucosides and N-acetylglucosaminides from human urine by ion-exchange chromatography and reversed-phase high-performance liquid chromatography.

A method for the isolation, separation and analysis of glucosides and N-acetylglucosaminides of non-amidated bile acids and of glycine- and taurine-conjugated bile acid glucosides from normal human urine is described. Total bile acids were extracted from 24-h collections of urine by repetitive use of Sep-Pak C18 cartridges. After elution with 80% aqueous methanol, a group separation into non-amidated, glycine- and taurine-conjugated bile acids was performed by ion-exchange chromatography on Lipidex-DEAP. The glycosylated compounds were then separated from the corresponding non-glycosylated ones by high-performance liquid chromatography (HPLC) using a reversed-phase system with a linear methanol gradient. The glycosylated compounds isolated by HPLC were analysed by fast atom bombardment mass spectrometry and, after derivatization, by gas chromatography-mass spectrometry. Information about the sugar moieties of the bile acid glycosides was also obtained by treatment with different glycosidases.

Acetylglucosamine↗
PubMed1984

Combination of liquid chromatography with ultraviolet detection and gas chromatography with electron-capture detection for the determination of delta 9-tetrahydrocannabinol-11-oic acid in urine.

A method is described for the determination of delta 9-tetrahydrocannabinol-11-oic acid (delta 9-THC-11-oic acid) in urine by using a combination of liquid chromatography and glass capillary gas chromatography with electron-capture detection. Prior to extraction, the glucuronide conjugate of delta 9-THC-11-oic acid was enzymatically converted to the free acid and delta 8-THC-11-oic acid added as an internal standard. An aliquot of the extract was separated by liquid chromatography and one eluate fraction containing delta 8-THC-11-oic and delta 9-THC-11-oic acids was collected. Before gas chromatographic analysis the acids were converted to their pentafluoropropyl-pentafluoropropionyl derivatives. Authentic urine samples, positive with the EMIT cannabinoid assay, were analysed and the results compared with those obtained from an earlier described gas chromatographic-mass spectrometric method. The detection limit for the overall method was approximately 20 ng/ml. The precision was 8% for a sample concentration of 90 ng/ml of delta 9-THC-11-oic acid in urine.

Chromatography, Gas↗
PubMed2006

Separation of Delta5- and Delta7-phytosterols by adsorption chromatography and semipreparative reversed phase high-performance liquid chromatography for quantitative analysis of phytosterols in foods.

A method for the separation, isolation, and identification of phytosterols was developed. A commercial phytosterols mixture, Generol 95S, was fractionated first by adsorption silica gel column chromatography and then separated by means of a semipreparative reverse phase high-performance liquid chromatography fitted with a Polaris C8-A column (250 mm x 10 mm i.d., 5 microm) using isocratic acetonitrile:2-propanol:water (2:1:1, v/v/v) as the mobile phase. Milligram scales of six individual phytosterols, including citrostadienol, campesterol, beta-sitosterol, Delta7-avenasterol, Delta7-campesterol, and Delta7-sitosterol, were obtained. Purities of these isolated sterols were 85-98%. Relative response factors (RRF) of these phytosterols were calculated against cholestanol as an authentic commercial standard. These RRF values were used to quantify by gas chromatography-mass spectrometry (GC-MS) the phytosterols content in a reference material, oils, and chocolates.

Adsorption↗
PubMed1993

Micellar electrokinetic capillary chromatography theory based on conventional chromatography.

From the definitions of retention time (tR) and resolution (Rs) in conventional chromatography, two fundamental equations for the retention behaviour and resolution of neutral solutes are derived and proved to be valid in all cases of micellar electrokinetic capillary chromatography (MECC). Two parameters, phase velocity ratio (Pr) and column availability (Aco), are introduced to reveal clearly the relationships and differences between MECC and conventional chromatography. The tR and Rs values may be either positive or negative in MECC. A negative tR indicates that the solute migrates toward the positive electrode and a positive tR toward the negative electrode. Rs > 0 means that the solute with a smaller value of the capacity factor (k') in the pair of solutes reaches the detector first, while Rs < 0 means that the elution order is the opposite. MECC can be classified into eight cases depending on the values of Pr for convenience of discussion. So far, MECC was usually performed in case IV and the resolution was poorer than that in conventional chromatography for given values of theoretical plate number, selectivity and k'. However, a better resolution can be obtained in cases II, VI and VIII when Pr < (1-k')/2. Cases VI, VIII and II are preferable to case IV for high resolution and should be more frequently employed in the future.

Chemical Phenomena↗
PubMed1995

DNA clearance in chromatography of proteins, exemplified by affinity chromatography.

Complications of DNA clearance in protein chromatography using the conventional methodology of spiking experiments are reported. Protein A affinity chromatography demonstrated this complications in a small scale experiment. A concentrated hybridoma culture supernatant was spiked with DNA extracted from hybridoma cells fed with [3H]thymidine. Protein A affinity chromatography was subsequently carried out. The column effluent was collected in fractions, and each fraction was analyzed for radioactivity and IgG levels. A substantial amount of DNA was eluted before the main IgG peak. Frequently a small peak is observed in front of the main peak in protein chromatography. This phenomenon can be explained by either displacement effects, or incomplete washing, or hysteresis during the adsorption and desorption conditions. Fractionation at the beginning of elution is critical to the maintenance of a high standard protein purity.

Chromatography, Affinity↗
PubMed2005

Comprehensive two-dimensional liquid chromatography and hyphenated liquid chromatography to study the degradation of poly(bisphenol A)carbonate.

Size exclusion chromatography (SEC), gradient polymer elution chromatography (GPEC) and liquid chromatography at critical conditions (LC-CC) have been developed and applied to observe chemical changes in poly(bisphenol A)carbonate (PC) due to hydrolytic degradation. Especially LC-CC appeared to be very successful to observe differences in functionality of PC as result of hydrolytic degradation. Observed differences due to degradation could be identified by (semi) on-line coupling to matrix assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The differences in functionality could be attributed to the formation of different end-groups, i.e. OH end-groups. In addition, comprehensive two-dimensional liquid chromatography (2D-LC) has been applied successfully to study the hydrolytic degradation of PC. LC-CC x SEC showed that the formation of PC with different end-groups occurred over the whole molecular mass range. This information could not be obtained with the separate liquid chromatographic techniques, thereby illustrating the added value of 2D-LC.

Benzhydryl Compounds↗
PubMed2003

On-line turbulent-flow chromatography-high-performance liquid chromatography-mass spectrometry for fast sample preparation and quantitation.

A sensitive and selective liquid chromatography-mass spectrometry method has been developed for the simultaneous identification and quantitation of drug substances and metabolites in rat plasma. The method combines on-line turbulent-flow chromatography, high-performance liquid chromatography and mass spectrometry. This combination is considered to be a new approach suitable for fast bio-analysis in drug discovery. Dextromethorphan, and its two metabolites, dextrorphan and 3-methoxymorphinan served as model substances. The analytes present in plasma were collected on a Cyclone column using turbulent-flow chromatography and were subsequently transferred on-line to and focused on an X-Terra MS C8 column. The analytes were eluted by a linear gradient and detected by a fast scanning mass spectrometer. The detector response was quadratic and the dynamic range was estimated to be 0.5-100 ng/ml plasma or 12.5 pg to 2.50 ng injected into the system.

Animals↗
PubMed1999

Affinity chromatography of human estrogen receptor-alpha expressed in Saccharomyces cerevisiae. Combination of heparin- and 17beta-estradiol-affinity chromatography.

Estrogen receptor-alpha is a member of the nuclear hormone receptor superfamily and is considered as a very important regulatory protein. Human estrogen receptor-alpha has been cloned into Saccharomyces cerevisiae as a fusion to ubiquitin and expression is controlled by a metallothionin promotor. Pilot scale quantities of receptor have been produced by a yeast strain transformed with expression plasmid YEpE13 [Graumann et al., J. Steroid Biochem. Mol. Biol. 57 (1996) 293] in a 14 l stirred tank reactor. The yeast extract contained 2-4 pmol of receptor protein per mg total protein. A purification scheme has been developed using heparin-affinity chromatography combined with affinity chromatography with immobilized 17beta-estradiol 17-hemisuccinate. Heparin-affinity chromatography was very efficient to remove host cell protein. Accompanying proteins that stabilize unoccupied receptor have not been dissociated during elution. The receptor could be purified 5-10-fold in ligand-free state. In contrast to previous reports, we did not find a difference of the binding affinity of liganded and unliganded receptor for heparin immobilized onto Sepharose. The unoccupied receptor could be further purified 100-fold with ligand-affinity chromatography using 17beta-estradiol 17-hemisuccinate-bovine serum albumin-Sepharose. The receptor could be kept in its native state, although saturated with 17beta-estradiol. The purification sequence allows an efficient production of receptor. Further improvement of productivity can be only accomplished by increasing the expression level.

Base Sequence↗
PubMed1997

Development of a purification procedure for the placental protein 14 involving metal-chelate affinity chromatography and hydrophobic interaction chromatography.

Placental protein 14 was isolated from the biological material of patients undergoing legal abortions. The major part of ballast protein was removed by ion-exchange chromatography on DEAE-Sepharose and CM-Sepharose. Albumin was separated by chromatography on Blue-Sepharose. Complete purification was obtained by metal-chelate affinity chromatography on Nickel-Chelate Sepharose and hydrophobic interaction chromatography on Phenyl-Sepharose and Octyl-Sepharose. The protein was not exposed to denaturing agents or extreme pH.

Abortion, Legal↗
PubMed1997

Simple and rapid determination of enflurane in human tissues using gas chromatography and gas chromatography-mass spectrometry.

A simple, rapid and reliable method was devised to determine the levels of enflurane in human tissues, using gas chromatography and gas chromatography-mass spectrometry. 1,4-Dioxane was used as an internal standard (I.S.). Enflurane and the I.S. were extracted from 0.25 g of body tissues using an automatic headspace sampler and 1 ml of headspace gas was injected into the gas chromatograph. Enflurane was analyzed qualitatively by gas chromatography-mass spectrometry and quantitatively by gas chromatography with a flame-ionization detector. The calibration curves in all tissues examined were linear in the concentration range 1-100 microg/0.25 g. The lower limit of detection was 200-300 ng/0.25 g. The accuracy and precision of this method were evaluated at two different concentrations, 1 and 20 microg/0.25 g. The coefficient of variation ranged from 3.4-13.4%. We used this method to determine the presence of enflurane in tissues from an autopsied individual who died suddenly during extirpation of a malignant tumor.

Anesthetics, Inhalation↗
PubMed1998

Application of gas chromatography-mass spectrometry and gas chromatography-tandem mass spectrometry to assess in vivo synthesis of prostaglandins, thromboxane, leukotrienes, isoprostanes and related compounds in humans.

Prostaglandins, thromboxane, leukotrienes, isoprostanes and other arachidonic acid metabolites are structurally closely related, potent, biologically active compounds. One of the most challenging tasks in eicosanoids research has been to define the role of the various eicosanoids in human health and disease, and to monitor the effects of drugs on the in vivo synthesis of these lipid mediators in man. Great advances in instrumentation and ionization techniques, in particular the development of tandem mass spectrometry and negative-ion chemical ionization (NICI), in gas chromatography and also advances in methodologies for solid-phase extraction and sample purification by thin-layer chromatography and high-performance liquid chromatography have been made. Now gas chromatography-mass spectrometry (GC-MS) and GC-tandem MS in the NICI mode are currently indispensable analytical tools for reliable routine quantitation of eicosanoid formation in vivo in humans. In this article analytical methods for eicosanoids based on GC-MS and GC-tandem MS are reviewed emphasizing the quantitative measurement of specific index metabolites in human urine and its importance in clinical studies in man. Aspects of method validation and quality control are also discussed.

Dinoprost↗
PubMedDate not supplied

Comparison of gel permeation chromatography, sweep codistillation, and Florisil column adsorption chromatography as sample cleanup techniques for the determination of organochlorine pesticide residues in animal fats.

Methods using a commercial sweep codistillation apparatus, gel permeation chromatography (GPC), and Florisil column adsorption chromatography were compared as cleanup techniques for the determination of organochlorine pesticide residues in animal fats by Megabore capillary gas chromatography with electron capture detection. Animal fat that had been previously found to contain no detectable organochlorine residues was spiked with 17 organochlorine pesticides at levels ranging from 0.1 to 0.4 mg/kg and cleaned up by each of the 3 techniques. Recoveries obtained for all 3 methods were in the range 73-113%, with coefficients of variation between 1.1 and 11.2%. Equivalence of method performance was further demonstrated by performing replicate analyses of beef and sheep fat containing naturally incurred residues of heptachlor epoxide, dieldrin, p,p'-DDE, p,p'-DDD and p,p'-DDT. All 3 methods offer effective cleanup and acceptable recovery of organochlorine pesticides in animal fat. The sweep codistillation method has the advantages of low solvent and reagent use, simultaneous cleanup of 10 samples, and rapid turnaround, although thermal degradation of p,p'-DDT requires monitoring and control. GPC offers a high degree of automation but is a relatively slow sequential sample cleanup with high solvent use. Florisil column adsorption chromatography is a simple, proven technique but requires considerable solvent and reagent and has a low potential for automation.

Adipose Tissue↗
PubMed2005

Analysis of selected ionic liquid cations by ion exchange chromatography and reversed-phase high performance liquid chromatography.

The chromatographic behavior of 8 ionic liquids - 7 homologues of 1-alkyl-3-methylimidazolium and 4-methyl-N-butylpyridinium - has been investigated with a strong cation exchange adsorbent. In particular, the dependence of the retention properties of these solutes on mobile phase composition, pH, and buffer concentration was evaluated with the aim of optimizing and improving the selectivity and retention of solute separation. While using the SCX stationary phase, several interactions occurred with varying strengths, depending on the mobile phase composition. Cation exchange, nonspecific hydrophobic interactions, and adsorption chromatography behavior were observed. Reversed phase chromatography occurred at low concentrations of acetonitrile, electrostatic and adsorption interactions at higher organic modifier concentrations. Elevated buffer concentrations lowered the retention factors without affecting the selectivity of ionic liquids. Obtained results were further compared to the chromatographic behaviour of ionic liquids in the reversed phase system. All analyzed ionic liquids follow reversed-phase behavior while being separated. Much lower selectivity in the range of highly hydrophilic compounds is obtained. This suggests preferred use of ion chromatography for separation and analysis of compounds below 4 carbon atoms in the alkyl side chain.

Buffers↗
PubMed1990

Isolation and partial characterization of angiotensinase A and aminopeptidase M from urine and human kidney by lectin affinity chromatography and high-performance liquid chromatography.

Angiotensinase A (ATA) and aminopeptidase M (APM) were partially purified from human urine specimens and human kidney particles using wheat germ lectin affinity chromatography, anion-exchange Fast Protein Liquid Chromatography (FPLC) (Mono Q), chromatofocusing (Mono P, FPLC) and Superose 12 gel filtration. APM, a globular 5-nm glycoprotein, is localized in the brush border membrane of the proximal tubule; angiotensin II-degrading ATA is present on glomerular endothelia and podocytes and, to lesser extent, in the brush border. For the first time, both peaks of ATA and APM activity from urine samples were separated by the above-mentioned techniques with only slight overlap; ATP (146,000 dalton: pI4.8) was enriched more than 20-fold and APM (153,000 dalton, pI4.7) more than 50-fold compared with the activity of the starting material. Using similar separation steps, ATA and APM solubilized from kidney particles could not be resolved into two distinct peak fractions, however, except after hydrophobic interaction chromatography. Thus urine is a major source for the preparation of individual ATA and APM fractions, necessary to generate specific anti-enzyme antibodies for diagnostic purposes.

Aminopeptidases↗
PubMed1979

Determination of bromocriptine in plasma: comparison of gas chromatography, mass fragmentography and liquid chromatography.

Gas chromatographic, mass fragmentographic and liquid chromatographic techniques for the determinations of bromocriptine (2-bromo-alpha-ergocriptine; Parlodel) in human plasma are described. These methods were found to be suitable for determining concentrations of bromocriptine down to 0.5, 1.0 and 10.0 microgram/l, respectively. Accuracy, specificity and analytical capacity were satisfactory for all three methods. Gas chromatography was compared with liquid chromatography, and the two methods were demonstrated to give identical results in patients treated with bromocriptine for Parkinson's disease. Gas chromatography was also compared with mass fragmentography, and the results from these two assays were also in agreement.

Bromocriptine↗
PubMed1985

Determination of sterol and triterpene alcohol acetates in natural products by reversed-phase liquid chromatography and gas chromatography-mass spectrometry.

A partial separation of nine sterol acetates and seven triterpene alcohol acetates by reversed-phase liquid chromatography is described. Good results are obtained using acetonitrile-water (90:10, v/v) as mobile phase with an UV detector at 205 nm. The variation in sterol sensitivity shows that this technique is not suitable for quantitative analyses. A combination of this technique for the fractionation of the natural sterol mixture, gas-liquid chromatography for quantitation and gas chromatography-mass spectrometry for identification is necessary for the determination of sterol compounds contained in natural products. An example of the separation, identification and quantitation of sterol acetates from sunflower seed oil is given.

Chromatography, High Pressure Liquid↗
PubMed1986

Combined use of trypsin-agarose affinity chromatography and reversed-phase high-performance liquid chromatography for the purification of single-chain protease inhibitor from corn seeds.

We have developed a large-scale method for recovering the corn inhibitor of trypsin and activated Hageman factor from a trypsin-agarose column predominantly in the single-chain form. To do so, inhibitor retained by the column was eluted with 1.0 M glycine buffer, pH 2.1. We have used reversed-phase high-performance liquid chromatography to further purify the inhibitor eluted from the trypsin-agarose column by separating the single-chain inhibitor from two-chain inhibitor (a small amount of which is present in the preparation after trypsin-agarose chromatography) and from still smaller amounts of another protein (apparently trypsin) that appears as a contaminant during trypsin-agarose chromatography.

Amino Acids↗
PubMed1988

Assay for trenbolone and its metabolite 17 alpha-trenbolone in bovine urine based on immunoaffinity chromatographic clean-up and off-line high-performance liquid chromatography-thin-layer chromatography.

An high-performance liquid chromatography (HPLC)-thin-layer chromatography (TLC) method was developed to detect the illegal use of the xenobiotic growth promotor Trenbolone acetate (TBA). Very effective clean-up of bovine urine was achieved by immunoaffinity chromatography (IAC). The active form of TBA, the steroid 17 beta-Trenbolone (17 beta-TB), as well as its major metabolite 17 alpha-Trenbolone (17 alpha-TB), were assayed simultaneously with HPLC and on-line UV detection. The fraction containing 17 alpha-TB and 17 beta-TB (TB-fraction) was collected, and for confirmation 17 beta- and 17 alpha-TB were subsequently separated and identified by TLC. The limit of detection by on-line HPLC-UV (350 nm) was 1-2 micrograms TB/l. Off-line TLC detection was even more sensitive, 0.5 microgram 17 beta- or 17 alpha-TB/1. The assay was validated by investigating urine samples from veal calves implanted with TBA. The presence of 17 beta- and 17 alpha-TB was clearly demonstrated. A survey of the illegal use of TBA in cattle was performed by applying the assay to urine obtained at slaughter. No residues of TBA or its metabolites were found in any of the 144 random samples from the Dutch public health surveillance programme.

Animals↗