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PubMed1989

Determination of benzylpenicillin in plasma and lymph at the ng ml-1 level by reversed-phase liquid chromatography in combination with digital subtraction chromatography technique.

A method for the determination of benzylpenicillin (Pc-G) at very low levels in plasma and lymph is described. Detection at 325 nm of the mercuric mercaptide of benzylpenicillinic acid was made by liquid chromatography via a pre-column derivatization method. By using a digital subtraction chromatography technique, the bioanalytical method could be applied to different kinds of samples whether interfering peaks were present or not. Two different clean-up steps were used for two concentration ranges 0.1-100 micrograms and 1-1000 ng Pc-G ml-1; namely, precipitation of a 100-microliters sample with acetonitrile, or precipitation of a 1- or 2-ml sample followed by concentration via liquid-liquid extraction. The pre-column derivative of Pc-G was achieved using mercury(II)chloride in the presence of imidazole. The blank samples required for the digital subtraction chromatography technique were obtained by penicillinase treatment. Standard curves were made in the two concentration ranges. The relative standard deviation (RSD) at 5 ng Pc-G ml-1 plasma was 4.9% and at 5 micrograms Pc-G ml-1 lymph it was 2.8%. The stability of Pc-G, including the problems with non-sterile samples, was studied.

Animals↗
PubMed1996

Comparison of liquid chromatography, capillary electrophoresis and super-critical fluid chromatography in the determination of Losartan Potassium drug substance in Cozaar tablets.

Reversed phase liquid chromatography, micellar electrokinetic chromatography, and packed column supercritical fluid chromatography were used to measure the amount of Losartan Potassium drug substance in pharmaceutical tablets. Tablet extract samples were analyzed and results were used to compare accuracy, precision, and linearity of calibration of the instrumental techniques. All three techniques have satisfactory precision (less than 1% RSD) and accuracy to be used for the analysis of the drug substance in tablets and the advantages of each technique are discussed.

Antihypertensive Agents↗
PubMed2006

Application of high-speed counter-current chromatography coupled with high-performance liquid chromatography-diode array detection for the preparative isolation and purification of hyperoside from Hypericum perforatum with online purity monitoring.

Following preparative isolation and purification by high-speed counter-current chromatography (HSCCC), the collected fractions were generally analyzed by high-performance liquid chromatography (HPLC) to determine the relative purities of each fraction. Our paper reports for the first time a preparative isolation-purity detection hyphenated system: online coupling of HSCCC with high-performance liquid chromatography-diode array detection (HSCCC-HPLC-DAD). The introduction of online purity analysis in HSCCC has dramatically improved the efficiency of this technique by overcoming the drawbacks of post analysis in HSCCC isolation. The effluent from the outlet of HSCCC was splitted into two parts: one was collected, while the other was introduced directly into an HPLC-DAD system for purity analysis through a switch valve. Therefore, the purities of the obtained fractions from HSCCC were monitored, and fractions with high purities were collected. This strategy has been successfully demonstrated with the preparative isolation and purification of hyperoside from Hypericum perforatum (St. Jone's Wort); a model of TBE-300A HSCCC was used to isolate and separate hyperoside from H. perforatum with a two-phase solvent system composed of ethyl acetate-ethanol-water at the volume ratio of 5:1:5 (v/v) using online detection technique. The isolation was done in less than 3.5 h, and a total of 83.0-mg hyperoside at over 99.0% purity was yielded from 300 mg of the partially purified extract. This new strategy possesses general utility in the preparation of bioactive compounds from traditional Chinese medicine (TCM).

Chromatography, High Pressure Liquid↗
PubMed2006

The combination of gas chromatography-olfactometry and multidimensional gas chromatography for the characterisation of essential oils.

A research area of great interest to the flavour industry is the analysis of odour active compounds in essential oils. In this paper, a methodology is presented for the identification of character-impact odorants in essential oil samples using (a) gas chromatography-olfactometry (GC-O); (b) comprehensive two-dimensional gas chromatography (GC x GC) combined to time-of-flight mass spectrometry (TOFMS) and (c) heart-cut multidimensional gas chromatography-olfactometry (MDGC-O). The specific advantages and limitations of each technique are discussed. The advantage of combining these techniques in a strategy to identify character-impact odorants is demonstrated using examples from coriander leaf (Coriandrum sativum) and hop (Humulus lupulus) essential oils. In particular, resolution of co-eluting regions of compounds and evaluation of their individual odour activity is discussed. In coriander leaf, only E-2-dodecenal was found to contribute to a co-eluting odour region, E-2-dodecen-1-ol and 1-dodecanol being present below detection threshold. Using MDGC on a hop essential oil sample, eight significant peaks were resolved from an 18 s heart-cut where a potent odorant was perceived during GC-O.

Chromatography, Gas↗
PubMed2001

Comparison of hydrophobic charge induction chromatography with affinity chromatography on protein A for harvest and purification of antibodies.

Efficient harvest and recovery of high-purity monoclonal antibodies was achieved using hydrophobic charge induction chromatography (HCIC). Both simple and complex feedstocks were studied, including protein-free cell culture supernatant and the clarified/concentrated milk of transgenic goats. Viral clearance studies demonstrated a 4-log reduction of MVM virus (minute virus of mice), along with substantial reduction of DNA content. Sorbent characterization studies confirmed that HCIC is based on the pH-dependent behavior of a dual-mode, ionizable ligand. Binding, based on hydrophobic interaction, was achieved under near-physiological conditions, and in the absence of lyotropic salt. Desorption was accomplished under mild conditions--pH 4.0. At this pH, both ligand and antibody carry a net positive charge, and desorption occurs on the basis of electrostatic charge repulsion. pH-based control of chromatographic function was demonstrated. Chromatography on this antibody-selective HCIC sorbent was evaluated as a cost-effective, process-compatible alternative to affinity chromatography protein A sorbents.

Antibodies, Monoclonal↗
PubMed2000

Speciation of aluminium in forest soil extracts by size exclusion chromatography with UV and ICP-AES detection and cation exchange fast protein liquid chromatography with ETAAS detection.

Aluminium speciation was studied in forest soil extracts by size exclusion chromatography (SE) with UV and inductively coupled plasma-atomic emission spectrometric (ICP-AES) detection and cation exchange fast protein liquid chromatography (FPLC) with ETAAS detection. Size exclusion chromatography was performed on a Superdex HR75 10/30 column. Isocratic clution with 0.15 mol dm(-3) NaCl in TRIS-HCl buffer (pH = 5.5) was applied over 100 min at a flow rate of 0.35 cm(3) min(-1). The chromatographic run was followed at 278 nm and separated Al species also determined 'off line' in 0.875 cm3 fractions by ICP-AES. The analytical procedure enabled speciation of high molecular weight Al complexes. Cation exchange FPLC was performed on a Mono S HR 5/5 column. Aqueous 8 mol dm(-3) NH4NO3 linear gradient elution was applied over 10 min at a flow rate of 1 cm3 min(-1). Separated Al species were collected in 0.5 cm3 fractions and Al determined 'off line' by ETAAS. The analytical procedure enabled speciation of some positively charged monomeric Al species. Negatively charged species were eluted with the solvent front. The combination of the two analytical techniques was successfully employed in speciation of Al in forest soil extracts. Water was used as an extracting solution. It was found experimentally that 80-95% of Al in aqueous extracts of forest soils exists in monomeric Al forms. Water soluble Al (30-40%) is bound to high molecular weight complexes with humic substances. The remaining monomeric Al in the low molecular weight fraction exists as AIF2+, Al-oxalate and Al-citrate species.

Aluminum↗
PubMed1988

Determination of drug enantiomers in biological samples by coupled column liquid chromatography and liquid chromatography-mass spectrometry.

Liquid chromatography-mass spectrometry (LC-MS) and coupled column chromatography can be used to overcome problems likely to occur in direct separation and determination of drug enantiomers in biological samples. This is exemplified here with the direct separation and determination of terbutaline in human plasma at the nmol/l level. A beta-cyclodextrin column with an aqueous mobile phase was used for chiral separation. For coupled column chromatography, the concentration of each enantiomer was calculated from the enantiomeric area ratio and the racemate concentration. A deuterium-labelled internal standard was used in the LC-MS experiments.

Chromatography, Gas↗
PubMed2002

Surface-enhanced laser desorption-ionization retentate chromatography mass spectrometry (SELDI-RC-MS): a new method for rapid development of process chromatography conditions.

Protein biochip arrays carrying functional groups typical of those employed for chromatographic sorbents have been developed. When components of a protein mixture are deposited upon an array's functionalized surface, an interaction occurs between the array's surface and solubilized proteins, resulting in adsorption of certain species. The application of gradient wash conditions to the surface of these arrays produces a step-wise elution of retained compounds akin to that accomplished while utilizing columns for liquid chromatography (LC) separations. In retentate chromatography-mass spectrometry (RC-MS), the "retentate" components that remain following a wash are desorbed and ionized when a nitrogen laser is fired at discrete spots on the array after treatment with a laser energy-absorbing matrix solution. Ionized components are analyzed using a time-of-flight mass spectrometer (TOF MS). The present study demonstrates that protein biochips can be used to identify conditions of pH and ionic strength that support selective retention-elution of target proteins and impurity components from ion-exchange surfaces. Such conditions give corresponding behavior when using process-compatible chromatographic sorbents under elution chromatography conditions. The RC-MS principle was applied to the separation of an Fab antibody fragment expressed in Escherichia coli as well as to the separation of recombinant endostatin as expressed in supernatant of Pichia pastoris cultures. Determined optimal array binding and elution conditions in terms of ionic strength and pH were directly applied to regular chromatographic columns in step-wise elution mode. Analysis of collected LC fractions showed favorable correlation to results predicted by the RC-MS method.

Chromatography, Liquid↗
PubMed1989

Protein aggregation in high-performance liquid chromatography: hydrophobic interaction chromatography of beta-lactoglobulin A.

Aggregation of beta-lactoglobulin A under acidic buffer conditions was studied in hydrophobic interaction chromatography. At high ammonium sulfate concentrations, pH 4.5 and 4 degrees C, UV chromatograms revealed a maximum of three peaks for beta-lactoglobulin A concentrations greater than 5 mg/mL, suggesting three distinct aggregate species. The size of the smallest aggregate (tetramer) and its stoichiometric relationship to the other two aggregates (octamer and dodecamer) were determined from the chromatographic data and a simple mass balance model. These stoichiometries agreed with those determined in a separate study by on-line low-angle laser light scattering. In addition, the association constants describing the formation of octamer from two tetramer molecules and the formation of dodecamer from the octameric and tetrameric species were found to be (2.4 +/- 0.5) X 10(4) M-1 and (3.3 +/- 0.8) X 10(3) M-1, respectively. Analysis of the beta-lactoglobulin A system is based on a model in which aggregates form in solution upon injection before adsorbing to the column matrix. The column retains those species formed in solution and induces little change in the relative amounts of each species. These results illustrate another example by which multiple peaks can arise in high-performance liquid chromatography, beyond the previously described studies of protein conformational changes during chromatography.

Chromatography, High Pressure Liquid↗
PubMed1975

Statistical theory of chromatography: new outlooks for affinity chromatography.

We have developed further the statistical approach to chromatography initiated by Giddings and Eyring, and applied it to affinity chromatography. By means of a convenient expression of moments the convergence towards the Laplace-Gauss distribution has been established. The Gaussian character is not preserved if other causes of dispersion are taken into account, but expressions of moments can be obtained in a generalized form. A simple procedure is deduced for expressing the fundamental constants of the model in terms of purely experimental quantities. Thus, affinity chromatography can be used to determine rate constants of association and dissociation in a range considered as the domain of the stopped-flow methods.

Analysis of Variance↗
PubMed1981

Studies on the specific interaction of concanavalin A and saccharides by affinity chromatography. Application of quantitative affinity chromatography to a multivalent system.

Quantitative affinity chromatography of concanavalin A (Con A) was studied in order to clarify its mechanism of saccharide binding. Another aim of this work was to confirm the validity of frontal affinity chromatography, which we have developed for the study of specific interactions, for multivalent systems. Since Cona A exists as either a dimer or a tetramer, it is a suitable multivalent test system. Immobilized p-aminophenyl-beta-D-glucopyranoside (AP beta-Glc Sepharose) was prepared and the interactions of homogeneous preparations of Con A with this affinity adsorbent were analyzed. Experiments were carried out under conditions where Con a exists as the dimer (5 degrees C, pH 7.9, I=0.19). The intrinsic dissociation constant of Con A for the immobilized ligand (Kd) could be determined from the extent of retardation. alpha-Con A (intact Con A) had a stronger affinity than beta-Con A. Changes in pH and ionic strength had different effects on the affinity of alpha- and beta-Con A. The dissociation constants of soluble saccharides and their derivatives (counter ligands) for Con A (K1) could be determined from their ability to diminish the elution volume of Con A. K1 values of various monosaccharides for beta-Con A including those having very weak affinity, were determined. Further, the K1 values of a series of glucobioses were determined and compared. It was found that the binding mode of the reducing terminal glucose residue must be taken into account. The results are discussed in relation to their configurations. Frontal affinity chromatography proved to be very useful as a tool to analyze specific interactions in multivalent systems.

Carbohydrates↗
PubMed1987

Disposable reversed-phase chromatography columns for improved detection of carboxylic acids in body fluids by electron-capture gas-liquid chromatography.

Disposable reversed-phase chromatography columns were tested for their effectiveness in removing unreacted trichloroethanol (TCE) from derivatized samples for gas-liquid chromatography analysis. Derivatized acidic chloroform extracts of saponified whole cells of Mycobacterium species, spent culture media, and derivatized acidic chloroform extracts of serum and cerebrospinal fluids from patients with tuberculous meningitis were tested. Samples were added to preconditioned reversed-phase chromatography columns, and various solvents and solvent mixtures were tested to determine maximum recovery of the TCE derivatives. With this procedure, we were able to quickly remove the TCE reagent and efficiently recover TCE-derivatized carboxylic acids. Use of these columns improved the reagent cleanup procedure, simplified the derivatization step, permitted increased detection of trace components, such as tuberculostearic acid, in body fluids, and improved the selectivity of the procedure for detection of carboxylic acids.

Body Fluids↗
PubMed1991

[Retinol Acetate Reference Standard for Thin-layer Chromatography (Control 901) and Retinol Palmitate Reference Standard for Thin-layer Chromatography (Control 901) of National Institute of Hygienic Sciences].

The raw materials of retinol acetate and retinol palmitate were examined for the preparation of the "Retinol Acetate Reference Standard for Thin-layer Chromatography" and "Retinol Palmitate Reference Standard for Thin-layer Chromatography", respectively. Analytical data obtained were as follows: thin-layer chromatography, no impurities were detected in retinol acetate and one impurities was detected in retinol palmitate; The Rf values of retinol acetate and retinol palmitate were consistent with those of Reference Standards (Control 713), respectively; ultraviolet spectrum, lambda max = 326 approximately 327 nm; relative extinction, within the range reported in JPXI; weight variation of capsules, retinol acetate 224.0 +/- 15.5 mg (RSD 6.9%), retinol palmitate 222.0 +/- 13.8 mg (RSD 6.2%); assay, retinol acetate 57000 I.U./g, retinol palmitate 57000 I.U./g. Based on the above results, these raw materials were authorized to be the Reference Standards of the National Institute of Hygienic Sciences.

Chromatography, Thin Layer↗
PubMed1981

[Chromatography and rechromatography in high-performance liquid chromatography of peptide mixtures: the complete primary structure of an immunoglobulin L-chain of kappa-type, subgroup I (Bence-Jones Protein Den) (author's transl)].

In continuation of our work on the separation of enzymatic hydrolysates by high-pressure liquid chromatography with reverse-phases we present two new buffer systems: firstly 0.005M potassium phosphate, pH 6.0 and secondly aqueous trifluoroacetic acid, pH 2.15. As organic solvent acetonitrile is always used. The excellent properties are demonstrated by the separation of the tryptic peptides of Bence-Jones protein Den (Mr = 23000). Like ammoniumacetate these buffers are well suited for the first separation of peptide mixtures but can be used rather effectively in rechromatographies. If peaks are not or not fully resolved during the first chromatography they can be separated by a rechromatography in one of the other systems. This was most successful since in both cases the same high resolving technique is employed. From the primary separation 14 peptides could be isolated in analytically pure form. The remaining fragments were completely purified after one rechromatography. The amino acid analysis yielded also integer numbers and by a modified Edman degradation the primary structure could be determined. As with protein Wes (Kratzin, H., Yang, C.Y., Krusche, T.U. & Hilschmann, N. (1980) Hoppe-Seyler's Z. Physiol. Chem. 361, 1591--1598) all the peptides were recovered after high-pressure liquid chromatography, none was missing. From these data and from homology reasons the complete amino acid sequence of protein Den could be established. It contains 214 residues and belongs to subgroup I of the kappa-chains. The valine residue in position 191 indicates that it belongs to allotype Inv b+.

Amino Acid Sequence↗
PubMed1990

The determination of total and unbound midazolam in human plasma. A comparison of high performance liquid chromatography, gas chromatography and gas chromatography/mass spectrometry.

Midazolam concentrations in patients' plasma was determined after extraction with high performance liquid chromatography (HPLC), gas chromatography (GC) and gas chromatography/mass spectrometry (GC/MS). GC was selected for routine plasma assays in terms of selectivity, simplicity, precision, accuracy and sensitivity (0.02 microgram/mL); HPLC analysis was less sensitive (0.1 microgram/mL) than GC; GC/MS was used for analysis validation. Plasma protein binding of midazolam was determined by GC in patients' plasma after in vitro incubation with midazolam, ultrafiltration and extraction; 5% of the drug was unbound to plasma proteins. Midazolam distribution in lipoprotein fractions separated by ultracentrifugation of plasma obtained from patients on prolonged midazolam treatment was also assayed by GC.

Blood Proteins↗
PubMed2006

Advantages of ultra performance liquid chromatography over high-performance liquid chromatography: comparison of different analytical approaches during analysis of diclofenac gel.

Miniaturization embracing instrumentation, column particle size, and column dimensions is one of the major current trends in separation techniques. This leads to shortening of analysis time and great savings in solvent consumption. Ultra performance liquid chromatography (UPLC) is one of the new developments in liquid chromatography. An ultra-high pressure system allows using of small particle-packed columns with small diameter, which has a positive effect on both system efficiency and analysis time. An analytical method for determination of the active substance diclofenac, the degradation product 1-(2,6-dichlorphenyl)-2-indolinone, and the preservatives methylparaben and propylparaben was used for testing and comparing LC systems. Various octadecylsilica-based analytical columns were examined. Acquity UPLC BEH C18 (2.1 x 50 mm, 1.7 microm) and (2.1 x 100 mm, 1.7 microm) were tested for UPLC. The following analytical columns were used in a test for HPLC: Purospher RP 18e (125 x 4.0 mm, 5 microm), Zorbax Eclipse XDB C18 (75 x 4.6 mm, 3.5 microm), Zorbax Eclipse SB C18 (50 x 4.6 mm, 1.8 microm), as was a monolithic column (Chromolith Performance RP-18e (100 x 4.6 mm). Results of a System Suitability Test (SST) were calculated and compared for each chromatographic peak. System efficiency and analysis duration were compared with regard to solvent consumption and system maintenance

Anti-Inflammatory Agents, Non-Steroidal↗
PubMed2004

Characterisation of organic compounds in aerosol particles from a Finnish forest by on-line coupled supercritical fluid extraction-liquid chromatography-gas chromatography-mass spectrometry.

During the European Union project Quantification of Aerosol Nucleation in the European Boundary Layer (QUEST), which began in spring 2003, atmospheric aerosol particles were collected in a Finnish Scots pine forest using a high-volume sampler. The organic compounds in the filter samples were then analysed by on-line coupled supercritical fluid extraction-liquid chromatography-gas chromatography-mass spectrometry (SFE-LC-GC-MS). The sample was first extracted by SFE. During LC the extracts were fractionated into three fractions according to polarity. The final separation was carried out by GC-MS. A fraction volume as high as 840 microL was transferred to the GC, using the partial concurrent eluent evaporation technique. The same instrumentation, with an in-situ SFE derivatisation method, was used to analyse organic acids. Major compounds such as n-alkanes and PAH were analysed quantitatively. Their concentrations were lower than those usually observed in urban areas or in other forest areas in Europe. The wind direction was one of the most important factors affecting changes in the daily concentrations of these compounds. Scots pine needles were analysed with the same system to obtain reference data for identification of biogenic compounds in aerosol particles. Other organic compounds found in this study included hopanes, steranes, n-alkanals, n-alkan-2-ones, oxy-PAH, and alkyl-PAH; some biogenic products, including oxidation products of monoterpenes, were also identified.

Aerosols↗
PubMed2006

Fractionation of selenium-containing proteins in serum by multiaffinity liquid chromatography before size-exclusion chromatography-ICPMS.

Immunoaffinity chromatography has been investigated for fractionation of serum into selenoalbumin and true selenoproteins. Among several albumin-depletion kits tested, a multiaffinity column specifically binding albumin and five other major serum proteins provided the best results. It extracted ca 95% of both albumin and selenoalbumin, which enabled interference-free determination of glutathione peroxidase, selenoprotein P, and selenoalbumin by size-exclusion chromatography combined with inductively coupled plasma mass spectrometry (SEC-ICPMS). The efficiency of the multiaffinity column did not vary over a period of 18 months. The purity of fractions separated by immunoaffinity LC was confirmed by elution-volume matching with standards in SEC-ICPMS and by selenopeptide mapping in capillary HPLC-ICPMS. Quantification of the selenium distribution among the different proteins in human serum from a control group and from a person on a selenium-rich diet revealed that 67% of the supplemented selenium was incorporated into albumin, 30% into glutathione peroxidase, and 3% into selenoprotein P.

Albumins↗