Biomedical subjects

G K Bonn

Publications and source records attributed to G K Bonn.

18 recordsLinked to original sources

PubMed2001

Mutation detection by capillary denaturing high-performance liquid chromatography using monolithic columns.

The high resolving power of the chromatographic separation of single- and double-stranded nucleic acids in 200 microm i.d. monolithic poly(styrene-divinylbenzene) capillary columns was utilized for mutation screening in polymerase chain reaction amplified polymorphic loci. Recognition of mutations is based on the separation of homo- and heteroduplex species by ion-pair reversed-phase high-performance liquid chromatography (IP-RP-HPLC) under partially denaturing conditions, resulting in characteristic peak patterns both for homozygous and heterozygous samples. Six different single nucleotide substitutions and combinations thereof were confidently identified in 413 bp amplicons from six heterozygous individuals each of which yielded a different unique chromatographic profile. Alternatively, mutations were identified in short, 62 bp PCR products upon their complete on-line denaturation at 75 degrees C taking advantage of the ability of IP-RP-HPLC to resolve single-stranded nucleic acids of identical length that differ in a single nucleotide. Separations in monolithic capillary columns can be readily hyphenated to electrospray ionization mass spectrometry and promise increased sample throughput by operating in arrays similar to those already used in capillary electrophoresis.

Alleles↗

PubMed2001

Determination of flavonoids and stilbenes in red wine and related biological products by HPLC and HPLC-ESI-MS-MS.

To investigate probable health benefits of flavonoids and stilbenes in red wine a new reversed-phase (RP) high-performance liquid-chromatographic (HPLC) method with enhanced separation efficiency and improved selectivity, sensitivity, and speed has been established for determination of the flavonoids quercetin, myricetin and kaempferol and the stilbenes cis- and trans-resveratrol, in a single run . UV-absorbance, fluorescence (FLD), and mass-spectrometric (MS) detection were also evaluated. UV-absorbance detection at 320 nm for stilbenes and 377 nm for flavonoids enables their determination up to the nanogram range with a linearity of R2>0.9999 (linear range 50 ng mL(-1)-50 microg mL(-1)). Calculated values of average recoveries were between 95 and 105% for all analytes. For resveratrol, fluorescence detection was highly selective and twice as sensitive as UV detection, and linearity was satisfactory (R2>0.9996; linear range see UV detection). For the detection of the hydrophilic glycosidic compounds piceid and rutin, which are coeluted with other hydrophilic ingredients, the validated RP HPLC system was coupled to a quadrupole ion-trap mass-spectrometer (MS) via an electrospray interface (ESI) with 25% ammonia solution as sheath liquid. MS detection was, highly linear (R2>0.9878; linear range 50 ng mL(-1)-50 microg mL(-1)) for all investigated analytes and the limits of detection were in the low nanogram range. Compared with UV detection MS detection resulted in a 200% increase in signal intensity for myricetin and 400% increases for quercetin and kaempferol, but equal signal intensity for resveratrol. Calculated values of average recoveries were 102% for myricetin and 79% for piceid. Collision induced dissociation (CID) was also used to obtain characteristic fragmentation fingerprints to facilitate qualitative and quantitative analysis even in complex matrices. Finally, this hyphenated HPLC-ESI-MS method was highly suitable and an essential improvement compared with UV- and fluorescence detection.

Chromatography, High Pressure Liquid↗

PubMed2000

Recent developments in polymer-based sorbents for solid-phase extraction.

A review with 136 references on the principles and recent developments in the solid-phase extraction based on polymer sorbents is presented. New polymer-based materials, chromatographic modes, experimental configurations are described and their advantages for a rapid sample preparation of certain classes of compounds with different functional groups are discussed and compared to silica-based sorbents.

Chromatography, Liquid↗

PubMed2000

Isolation and characterization of methoxylated flavones in the flowers of Primula veris by liquid chromatography and mass spectrometry.

Characterization of six flavones, which were named substances G1, G2, G3, G4, G5 and G6 according to their R(F) values in normal-phase thin-layer chromatography, is reported. The pure flavones were purified after maceration with methanol by normal-phase solid-phase extraction, normal-phase medium-pressure liquid chromatography, normal-phase preparative thin-layer chromatography and preparative reversed-phase high-performance liquid chromatography (RP-HPLC). The collected fractions of several isolation steps were analyzed by normal-phase (NP) and RP-HPLC. Detection and identification of the substances G was accomplished by UV detection at 213-216 nm, diode array UV detection, or fluorescence detection (lambda(ex)=330 nm; lambda(cm)=440 nm). The molecular mass, the elementary composition, and the structure of the six components was determined by electron-impact high-resolution mass spectrometry (EI-HRMS). Substance G4 was identified as 3',4',5'-trimethoxyflavone. The substances G1-G6 were shown to be mono-, di- tri- and pentamethoxyflavones. HPLC-electrospray ionization tandem mass spectrometry (ESI-MS-MS) of the flavones was carried out employing a 150x2 mm I.D. column packed with a 3 microm/100 A octadecylsilica stationary phase and a mobile phase comprising 1.0% acetic acid in water-acetonitrile (50:50). Comparative RP-HPLC-ESI-MS of the raw methanol extract and the isolated substances G1-G6 proved that the isolated compounds were pure and were not artifacts. Finally, RP-HPLC-ESI-MS-MS was used to identify substances G1-G6 in phytopharmaceutical drugs.

Chromatography, High Pressure Liquid↗

PubMed2000

Development and evaluation of a new method for the determination of the carotenoid content in selected vegetables by HPLC and HPLC-MS-MS.

Epidemologic studies have shown inverse correlation between the consumption of carotenoid-rich vegetables and the incidence of cancer. Therefore, analytical techniques for the quantitative determination of carotenoids in complex sample matrices are important. The most used method is reversed-phase (RP)-high-performance liquid chromatography (HPLC). In this study, seventeen mobile-phase systems described in the literature and six RP-HPLC columns with differences in particle size and porosity are evaluated. Derived from these results, a new mobile-phase (acetonitrile, methanol, chloroform, and n-heptane) including solvent modifiers is presented, which allows an improved and more efficient separation of carotenoids. From all columns tested, the best chromatographic parameters are found using a silica C18 column (250 x 2 mm, 5 microm, 100 A). As was found, absorbance detection at 450 nm allows the determination of the carotenoids down to the picogram range with good linearity (R2 > 0.98). For the identification and quantitation of carotenoids in complex sample matrices (containing additionally other ultraviolet-absorbing compounds), the optimized RP chromatographic system is coupled to a mass spectrometer (MS) using an atmospheric pressure ionization interface. The calibration plots show high linearity (R2 > 0.99), and the detection limit is found in the lower nanogram range. Furthermore, collision-induced dissociation in the ion source allows for the identification of carotenoids by their characteristic fragmentation pathways. In this study, a total of nine species of vegetables commonly consumed in Central Europe are analyzed for their contents of carotenoids (namely lutein, zeaxanthin, beta-cryptoxanthin, and beta-carotene) by RP-HPLC and RP-HPLC-MS-MS. It is found that good sources for lutein are spinach, kale, and broccoli, and sources for beta-carotene are broccoli, spinach, kale, carrots, and tomatoes. This new method is an improvement for the identification and quantitation of carotenoids in complex biological tissues.

Carotenoids↗

PubMed1998

Determination of airborne, volatile amines from polyurethane foams by sorption onto a high-capacity cation-exchange resin based on poly(succinic acid).

A high-capacity carboxylic acid-functionalized resin prepared by ring-opening metathesis polymerization based on cross-linked endo,endo-poly(norborn-2-ene-5,6-dicarboxylic acid) was used for the sampling of volatile, airborne amines from polyurethane (PU) foams. Six tertiary amines which represent commonly used promotors for the formation of PUs from diisocyanates and polyols, namely pentamethyldiethylenetriamine, diazabicyclooctane, N-methylmorpholine, N-ethylmorphine, 1,4-dimethylpiperazine and N,N-dimethylethanolamine, were sorbed onto the new resin. The sorption behavior of the new material was investigated in terms of loading capacities, the influence of concentration, flow-rate as well as of the amount of resin. Breakthrough curves were recorded from each single component as well as of mixtures thereof. Finally, the resin was used for the sampling of amines evaporating from PU foams applied in buildings. Further information about time dependent concentration profiles were obtained using a combination of GC-MS and Fourier transform IR spectroscopy.

Adsorption↗

PubMed1998

Capillary electrophoresis of peptides and proteins in fused-silica capillaries coated with derivatized polystyrene nanoparticles.

High-resolution capillary electrophoretic separation of proteins and peptides was achieved by coating the inner wall of 75 microm ID fused-silica capillaries with 40-140 nm polystyrene particles which have been derivatized with alpha-omega-diamines such as ethylenediamine or 1,10-diaminodecane. A stable and irreversibly adsorbed coating was obtained upon deprotonation of the capillary surface with aqueous sodium hydroxide and subsequent flushing with a suspension of the positively charged particles. At pH 3.1, the detrimental adsorption of proteins to the capillary inner wall was suppressed efficiently because of electrostatic repulsion of the positively charged proteins from the positively charged coating which enabled protein separations with maximum efficiencies of 400000 plates per meter. A substantial improvement of separation efficiency in particle-coated capillaries was observed after in-column derivatization of amino functionalities with 2,3-epoxy-l-propanol, resulting in a more hydrophilic coating. Five basic and four acidic proteins could be separated in less than 7 min with efficiencies up to 1900000 theoretical plates per meter. Finally, coated capillaries were applied to the high-resolution analysis of protein glycoforms and bioactive peptides.

Animals↗

PubMed1994

High-resolution liquid chromatography of fluorescent dye-labeled nucleic acids.

Using 100 mM of triethylammonium acetate as ion-pairing reagent, phosphodiester oligonucleotides labeled fluorescently at their 5' terminus could be separated successfully on alkylated nonporous 2.3-microns poly(styrene-divinylbenzene) particles by means of high-resolution liquid chromatography. Applying excitation wavelengths of 490, 520, 550, and 575 nm, respectively, optimum sensitivity was achieved for the fluorophores 5-carboxyfluorescein, 2',7'-dimethoxy-4',5'-dichloro-6-carboxyfluorescein, N,N,N',N'-tetramethyl-6-carboxyrhodamine, and 6-carboxy-X-rhodamine (FAM, JOE, TAMRA, and ROX, respectively) at emission wavelengths of 520, 550, 580, and 605 nm, respectively. With calibration curves being linear over at least three orders of magnitude, the lower detection limits were 0.5, 2, 2, and 3 fmol, respectively. Depending on the type of fluorescent dye attached, retention times increased in the order JOE < FAM < TAMRA < ROX. Subsequently, fluorescent oligonucleotides were employed to prime polymerase chain reactions (PCR). Again the fluorophores were found to increase the retention times of double-stranded nucleic acids, but to a lesser degree than those of single-stranded oligonucleotides. Using a single FAM label attached to one of the two PCR primers, the sensitivity of fluorescence detection was found to be approximately 1 fmol or 30-70 times higher than that of uv absorbance detection depending on the length of the PCR product. Since the technique allows the separation of PCR products differing only 4 to 8 base pairs in length within a size range of 50 to 200 base pairs, it may be employed for the quantitative assessment of competitive PCR.

Base Sequence↗

PubMed1994

Capillary zone electrophoresis and micellar electrokinetic chromatography of 4-aminobenzonitrile carbohydrate derivatives.

Aldoses, ketoses and uronic acids were derivatized successfully within 15 min at a temperature of 90 degrees C by reductive amination with 4-aminobenzonitrile. Subsequently, the derivatives were separated as their borate complexes by capillary zone electrophoresis, using 175 mM borate buffer, pH 10.5, as carrier. The electrophoretic mobilities were determined by the complex stability, which was found to depend on the number of hydroxyl groups on any given carbohydrate derivative, the presence of substituents, and most strongly on the configuration of the vicinal hydroxyl groups at C-3 and C-4 in aldoses and uronic acids, and with regard to ketoses on those at C-4 and C-5. Time of analysis could be reduced considerably by the use of micellar electrokinetic chromatography, which separated 4-aminobenzonitrile sugar derivatives on the basis of their differential partitioning into an electroendosmotically driven aqueous phase and into sodium dodecyl sulfate micelles. Optimum resolution was achieved with a Tris-phosphate buffer, pH 7.5, containing 100 mM of sodium dodecyl sulfate. The method made it possible to resolve several carbohydrates which had not been resolved successfully by means of capillary zone electrophoresis, such as glucose and fructose. Moreover, separation selectivity could be adjusted by varying the capillary temperature. Finally, on-column UV monitoring at 285 nm allowed the detection of glucose with a lower mass detection limit of 1 fmol and a concentration sensitivity of 0.3 microM.

Borates↗

PubMed1994

High-performance liquid chromatography for routine analysis of hepatitis C virus cDNA/PCR products.

Ion-pair reversed-phase high-performance liquid chromatography on alkylated nonporous polystyrene-divinylbenzene particles with a mean diameter of 2.1 microns was used to analyze PCR products according to their chain length within a few minutes. The simple and reliable procedure allows the simultaneous separation and isolation of DNA fragments differing in chain length by 1%-5% up to a size of 500 base pairs with recovery rates exceeding 97%. A greater than 70-fold increase in sensitivity could be achieved through the use of a fluorescein-labeled primer, which allowed the determination of a 127-bp hepatitis C virus cDNA/PCR product with a lower mass detection limit of 2 fmol. Calibration curves showed excellent linearity over a range of at least 4 magnitudes. Finally, the stationary phase allowed the routine analysis of hundreds of PCR products with high reproducibility of both retention times and peak areas.

Base Sequence↗

PubMed1993

High-resolution liquid chromatography of oligonucleotides on nonporous alkylated styrene-divinylbenzene copolymers.

Phosphodiester-oligonucleotides were separated by ion-pair reversed-phase HPLC on nonporous polystyrene-based particles having a diameter of 2.1 +/- 0.12 microns. With unmodified poly(styrene-divinylbenzene) beads it was not possible to resolve oligonucleotides efficiently. However, upon addition of polyvinyl alcohol during polymerization baseline resolution of phosphorylated oligodeoxyadenylic acids with a chain length of up to 30 bases was obtained, with triethylammonium acetate serving as ion-pairing reagent. An even higher separation efficiency was achieved by Friedel-Crafts alkylation of poly(styrene-divinylbenzene) particles. At a column temperature of 50 degrees C, the number of theoretical plates exceeded 6 x 10(5) per meter. The maximum loading capacity for a 50 x 4.6-mm i.d. column still resulting in the highest resolution was 0.1 microgram. Calibration curves showed excellent linearity over a range of at least 4 magnitudes, with a lower mass detection limit of 0.16 ng. Recoveries ranged from 96.7 to 100.8%. The same stationary phase also allowed the separation of phosphorylated from dephosphorylated oligonucleotides, the former ones being eluted earlier.

Alkylation↗

PubMed1993

High-resolution liquid chromatography of DNA fragments on non-porous poly(styrene-divinylbenzene) particles.

DNA restriction fragments and PCR products were separated by means of ion-pair reversed-phase high-performance liquid chromatography on alkylated non-porous poly(styrene-divinylbenzene) particles with a mean diameter of 2.1 microns. Optimum resolution was obtained by using an acetonitrile gradient in 100 mM of triethylammonium acetate and a column temperature of 50 degrees C. This allowed the separation of DNA fragments differing in chain length by 1-5% up to a size of 500 base pairs. PCR products could be analyzed directly in less than two minutes with a concentration sensitivity of at least 300 ng/ml. Compared with anion-exchange chromatography or gel electrophoresis no desaltation of the purified DNA molecules is required because the volatile buffer system can be readily evaporated. Subsequently, the method was used for the semiquantitative evaluation of the expression of multidrug resistance genes in mononuclear white blood cells.

Base Sequence↗

PubMed1992

Comparative study of capillary zone electrophoresis and high-performance liquid chromatography in the analysis of oligonucleotides and DNA.

Capillary zone electrophoresis and high-performance liquid chromatography were compared with regard to the separation of oligonucleotides and double-stranded DNA. Both anion-exchange and reversed-phase high-performance liquid chromatography on non-porous particles are considered to be superior to capillary electrophoresis in terms of speed and selectivity in the analysis of oligonucleotides up to 30 bases in length. Moreover, reversed-phase chromatography allows the simultaneous purification of detritylated oligonucleotides with recoveries > 90%. Compared with anion-exchange chromatography, there is no need for a subsequent desaltation step because the volatile buffer system can be readily evaporated. With regard to dsDNA, however, the resolving power of capillary electrophoresis cannot be matched by anion-exchange chromatography at present. Moreover, the combined use of hydroxyethylcellulose and ethidium bromide not only yielded a separation efficiency equal to that achieved by means of gel-filled capillaries but also avoids some of their limitations such as the destruction of the gel matrix at high current densities and the bias involved in electrokinetic injection.

Base Sequence↗

PubMed1992

High-performance liquid chromatographic separation of detritylated oligonucleotides on highly cross-linked poly-(styrene-divinylbenzene) particles.

Detritylated oligonucleotides were separated by reversed-phase high-performance liquid chromatography on highly cross-linked polystyrene-based particles having a mean particle diameter of 2.3 microns. The addition of poly(vinyl alcohol) during polymerization, which resulted in the presence of hydroxyl groups on the surface of the poly(styrene-divinylbenzene) beads, was necessary to obtain baseline resolution of phosphorylated oligodeoxyadenylic acids with a chain length of up to 30 bases. The impact of temperature was investigated and optimum resolution was achieved at 40 degrees C. At pH 7.0, the retention times of oligonucleotides were found to depend on the ratio of bases and to increase in the order of C less than G less than A less than T. Under the same conditions, it was possible to separate phosphorylated from dephosphorylated oligonucleotides, the former being eluted earlier. Recoveries ranged from 92 to 100%.

Base Sequence↗

PubMed1992

Thin-layer electrophoresis of hydroxyethyl starches on a modified silica gel support.

The recent developed thin-layer electrophoresis on modified silanized silica gel was applied to the separation of hydroxyethyl starches (HES) and glycogen and of HES with different degrees of substitution. This method permits a rapid qualitative and semi-quantitative determination of HES in animal tissues such as liver, lung, heart and kidney after their disintegration with alkali and precipitation with ethanol.

Animals↗

PubMed1991

[Analysis of polycyclic aromatic hydrocarbons in lipid-containing biological matrices--study of contamination of breast milk by benzo(a)pyrene along transit routes through Tyrol].

41 samples of human milk from the region of Tyrol were investigated with regard to benzo(a)pyrene content. A complicated analytical preparatory method had to be worked out. The percentage recovery of benzo(a)pyrene was 85-90. Analysis was carried out by means of capillary gas chromatography and flame ionization detection or mass spectrometry. The limit of benzo(a)pyrene detection is 0.1-1 microgram/kg (single ion monitoring). None of the investigated samples of milk showed the presence of benzo(a)pyrene.

Air Pollutants↗

PubMedDate not supplied

High-resolution capillary electrophoretic analysis of DNA in free solution.

Capillary electrophoretic separations of double-stranded DNA fragments in the size range of 20-2200 base pairs were achieved in less than 20 min with the use of a Tris-borate buffer containing hydroxyethylcellulose. Analyses were carried out in both uncoated and phenylmethyl-coated capillaries of fused silica with internal diameters of 50 and 100 microns, respectively, and an effective column length of 50 cm. The addition of ethidium bromide resulted in an improved resolution of double-stranded DNA fragments, thereby permitting even the separation of fragments differing only 1-2 base pairs in length. Moreover, resolution was found to be linearly proportional to the size of the cation used to adjust ionic strength Cs+ greater than RB+ greater than K+ greater than Na+ greater than Li+. However, the analysis times also increased with increasing cation size due to a decrease in electroosmotic flow. Elution order was verified by spiking restriction digests with slab gel electrophoretically purified components. Subsequently, the described system was applied to the detection and quantitation of an mRNA transcript of the androgen receptor, which had been amplified by polymerase chain reaction and purified by size-exclusion chromatography to avoid peak broadening due to conductivity differences between sample and running buffer. Since the actual amount of DNA introduced into the capillary cannot be defined, molar ratio-peak area ratios of the polymerase chain reaction product to various restriction fragments of known concentration were used to determine the amount of amplified DNA. The coefficient of variation was as small as 3.4% and the results were in good agreement with a spectrophotometric assay.

Base Sequence↗

PubMedDate not supplied

Capillary electrophoretic analysis of flavonoids.

Combining the effects of electrophoresis and electroendosmosis, flavonoids were separated in less than ten minutes in a fused silica capillary tube with a borate buffer adjusted to pH 10. An increase in the concentration of borate from 0.1 to 0.2 M resulted in longer migration times due to a decrease in electroosmotic flow, but also in improved selectivity and higher resolution of flavonoids. The calibration curve of rutin showed a detection limit of 0.02 mg/mL and linearity over its pharmaceutical concentration range. Using an internal standard of known concentration, the content of rutin in a methanolic extract of Sambuci flos could be determined with a coefficient of variation as small as 3.8% by the molar ratio-peak area ratio method.

Boric Acids↗