Biomedical subjects

C W Huck

Publications and source records attributed to C W Huck.

15 recordsLinked to original sources

PubMed2006

Role of carbon nano-materials in the analysis of biological materials by laser desorption/ionization-mass spectrometry.

At present, carbon nano-materials are being utilized in various procedures, especially in laser desorption/ionization-mass spectrometry (LDI-MS) for analyzing a range of analytes, which include peptides, proteins, metabolites, and polymers. Matrix-oriented LDI-MS techniques are very well established, with weak organic acids as energy-absorbing substances. Carbon materials, such as nano-tubes and fullerenes are being successfully applied in the small-mass range, where routine matrices have strong background signals. In addition, the role of carbon nano-materials is very well established in the fractionation and purification fields. Modified diamond powder and surfaces are utilized in binding peptides and proteins from complex biological fluids and analyzed by matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS). Polylysine-coated diamond is used for solid-phase extraction to pre-concentrate DNA oligonucleotides. Graphite is useful for desalting, pre-concentration, and as energy-absorbing material (matrix) in desorption/ionization. Carbon nano-tubes in their different derivatized forms are used as matrix materials for the analysis of a range of analytes, such as carbohydrates, amino acids, peptides, proteins, and some environmental samples by LDI-MS. Fullerenes are modified in different ways to bind serum entities analyzed through MALDI/TOF-MS and are subsequently utilized in their identifications. In addition, the fullerenes are a promising matrix in LDI-MS, but improvements are needed.

Biological Products↗

PubMed2006

Silica particles encapsulated poly(styrene-divinylbenzene) monolithic stationary phases for micro-high performance liquid chromatography.

In the paper we demonstrate a new approach for the preparation and application of continuous silica bed columns that involve encapsulation (entrapment) of functionalized silica microparticles, which can be used as packing material in micro high performance liquid chromatography (micro-HPLC) and capillary electrochromatography (CEC). Like traditional packed columns, these capillaries possess characterized silica particles that offer high phase ratio and narrow pore size distribution leading to high retention and separation efficiency, respectively. More importantly, immobilization of the microparticles stabilizes the separation bed and eliminates the need for retaining frits. The developed capillary columns were fabricated in exactly the same way as a packed capillary column (slurry packing) but with an additional entrapment step. This immobilization of the packed bed was achieved by in situ polymerization of styrene and divinylbenzene in presence of decanol as a porogen and azobisisobutyronitrile as thermal initiator. Silica particles with different particle sizes and pore sizes ranging from 60 to 4000 A were studied. In addition different modified silica was used, including C-18 reversed phase, anion exchange and chiral stationary phases. Efficient separation of polyphenolic compounds, peptides, proteins and even DNA mutation were achieved using the developed technique depending on the properties of the silica particles used (particles pore size). For example, using 3 microm ProntoSIL C-18 particles with 300 A pore size, separation efficiencies in the range of 120,000-200,000 plates/m were obtained for protein separation, in a 6 cm x 200 microm i.d. capillary column. Using encapsulated silica C-18 with 1000 A pore size, separation of DNA homo and hetero duplexes were achieved under denaturing HPLC conditions for mutation detection. In addition, nucleotides were separated using anion exchange material encapsulated with poly(styrene-divinylbenzene) (PS/DVB), which indicated that the chromatographic properties of the silica packing material were still active after polymerization. The prepared capillary columns were found to be stable and could easily be operated continuously up to a pressure of 350 bar without column damage and capillary can be cut to any desired length.

Chromatography, High Pressure Liquid↗

PubMed2006

Near infrared spectroscopy, cluster and multivariate analysis hyphenated to thin layer chromatography for the analysis of amino acids.

A method based on near-infrared spectroscopy (NIRS) was developed for the rapid and non-destructive determination and quantification of solid and dissolved amino acids. The statistical results obtained after optimisation of measurement conditions were evaluated on the basis of statistical parameters, Q-value (quality of calibrations), R(2), standard error of estimation (SEE), standard error of prediction (SEP), BIAS applying cluster and different multivariate analytical procedures. Experimental optimisation comprised the selection of the highest suitable optical thin-layer (0.5, 1.0, 1.5, 2.0, 2.5, 3.0 mm), sample temperature (10-30 degrees C), measurement option (light fibre, 0.5 mm optical thin-layer; boiling point tube; different types of cuvettes) and sample concentration in the range between 100 and 500 ppm. Applying the optimised conditions and a 115-QS Suprasil cuvette (V = 400 microl), the established qualitative model enabled to distinguish between different dissolved amino acids with a Q-value of 0.9555. Solid amino acids were investigated in the transflectance mode, allowing to differentiate them with a Q-value of 0.9155. For the qualitative and quantitative analysis of amino acids in complex matrices NIRS was established as a detection system directly onto the plate after prior separation on cellulose based thin-layer chromatography (TLC) sheets employing n-butanol, acetic acid and distilled water at a ratio of 8:4:2 (v/v/v) as an optimised mobile phase. Due to the prior separation step, the established calibration curve was found to be more stable than the one calculated from the dissolved amino acids. The found lower limit of detection was 0.01 mg/ml. Finally, this optimised TLC-NIRS method was successfully applied for the qualitative and quantitative analysis of L-lysine in apple juice. NIRS is shown not only to offer a fast, non-destructive detection tool but also to provide an easy-to-use alternative to more complicated detection methods such as mass spectrometry (MS) for qualitative and quantitative TLC analysis of amino acids in crude samples.

Amino Acids↗

PubMed2005

Characterisation of the thiol-disulphide chemistry of desmopressin by LC, mu-LC, LC-ESI-MS and Maldi-Tof.

To date, the majority of therapeutic peptides and proteins have to be administered via parenteral routes, which are painful and inconvenient. In order to gain sufficient high blood concentrations after oral application, various barriers in the gastrointestinal tract have to be overcome. Apart from a poor membrane uptake and intense enzymatic degradation, this study will demonstrate that thiol-disulphide reactions are an underestimated essential part of the presystemic metabolism. Glutathione, integrative part of the antioxidant defence system in the gastrointestinal tract, may play an important role in the inactivation of orally given peptides and proteins. In order to verify this hypothesis, desmopressin which bears a single disulphide bond was used as model peptide drug. Desmopressin was incubated with GSH in various concentrations, and the extent of thiol/disulphide exchange reactions between the peptide drug and GSH was investigated in dependence on pH and ratio of reactants determined as a function of time via HPLC, LC-MS and Maldi-Tof-MS analyses. Results showed that desmopressin is degraded by 1% reduced glutathione at pH 4 and pH 5.5. In presence of 0.01%, 0.1% and 1% of reduced glutathione 6.1%, 19.4% and 52.1% of desmopressin, respectively, were degraded. The masses of the conjugates after deconvolution measured by liquid chromatography and electrospray ionisation mass spectrometric detection were m/z 1069.67, m/z 1376.50, m/z 1683.40 and m/z 2138. These molecular masses, confirmed by Maldi-Tof-MS analysis, correspond with the masses of conjugates expected in theory. Under defined conditions, these results reveal that thiol-disulphide exchange reactions have a considerable impact on the alteration of peptide drugs and proteins.

Chromatography, High Pressure Liquid↗

PubMed2005

Sample pretreatment and determination of non steroidal anti-inflammatory drugs (NSAIDs) in pharmaceutical formulations and biological samples (blood, plasma, erythrocytes) by HPLC-UV-MS and micro-HPLC.

The article discusses the qualitative and quantitative determination of non-steroidal anti-inflammatory drugs like salicin, salicylic acid, tenoxicam, ketorolac, piroxicam, tolmetin, naproxen, flurbiprofen, diclofenac and ibuprofen by reversed phase high performance liquid chromatography (RP-HPLC) and micro-HPLC (micro-HPLC) hyphenated with UV-absorbance and mass spectrometric detection. Both detection methods delivered calibration plots with good linearity (r(2) > 0.9800), limits of detection in the low nanogram range and recovery rates between 94 and 104 %. For the analysis of biological samples such as blood, plasma and erythrocytes liquid-liquid extraction (LLE) and solid phase extraction (SPE) on the basis of new synthesized glycidylmethacrylate/divinylbenzene copolymer (GMA/DVB) particles and commercially available material on the basis of poly(divinylbenzene-co-N-vinylpyrrolidone) copolymer were investigated. Finally the use of a micro-HPLC system with separation columns in the range of 8 cm x 200 microm I.D. for the determination of non-steroidal anti-inflammatory drugs (NSAIDs) is presented, emphasizing on the type of column and sample amount needed.

Anti-Inflammatory Agents, Non-Steroidal↗

PubMed2004

Simultaneous determination of gentisic, salicyluric and salicylic acid in human plasma using solid-phase extraction, liquid chromatography and electrospray ionization mass spectrometry.

A method is developed for the simultaneous extraction of gentisic (GA), salicyluric (SUA) and salicylic acid (SA) in human plasma from Willow Bark extract, by solid phase extraction (SPE) using Waters Oasis HLB (divinylbenzene-n-vinylpyrrolidone copolymer) cartridges. Also, a method is optimized comprising of reversed-phase (RP) high-performance liquid chromatography (HPLC) in connection with electrospray ionization mass spectrometry (ESI-MS), fluorescence detection (FLD) and photo diode array detection (DAD) to identify and quantify GA, SUA and SA in the SPE effluents. An improved sensitivity regarding the lower detection limit (LOD) of < 7 ng/ml, the limit of quantitation (LOQ) of 20 ng/ml and short analysis times of < 15 min is required. The validated SPE method shows linearity in the range of 9.0-58.2 ng/ml for GA, 9.4-191.5 ng/ml for SUA and 12.8-1101.6 ng/ml for SA. The correlation coefficient values are > 0.9994 and 0.99 for fluorescence detection (FLD) and electrospray ionization mass spectrometry (ESI-MS), respectively. The recoveries are from 91.3-102.1% for gentisic acid (GA), 86.8-100.5% for salicyluric acid (SUA) and 75.8-81.4% for salicylic acid (SA) depending on the starting concentrations. RP-LC-ESI-MS/MS studies using collision induced dissociation (CID) confirm that the investigated analytes are not artifacts and facilitate further specific identification in addition to the determination of the parent ion mass even in the presence of co-eluting peaks. The established method is also used to analyze gentisic (GA), salicyluric (SUA) and salicylic acid (SA), not only after intake of Willow Bark capsules (Assalix, BNO 1455) but also as naturally occurring constituents in human plasma after the intake of salicylic acid containing foods.

Calibration↗

PubMed2004

Structural elucidation of catechin and epicatechin in sorrel leaf extracts using liquid-chromatography coupled to diode array-, fluorescence-, and mass spectrometric detection.

Flavonoids are natural compounds in medicinal herbs and posses several biological activities important in plant drug design. Especially strong antioxidant effects play an important role against radical oxidative stress causing pathological processes, such as arteriosclerosis or cancer. The aim of this work was to investigate unknown analytes found in sorrel leaf (Rumex acetosa) extracts in order to discover new leading compounds to enable quality control in phytopharmaceuticals made thereof. Therefore compounds of interest were separated after methanolic extraction by reversed-phase liquid chromatography (RP-LC) employing silica-C18 as stationary phase using gradient elution with water (10 mM H3PO4) and acetonitrile as mobile phase. Structural elucidation was carried out by diode array, fluorescence, and mass spectroscopic detection. Photodiode-array detection allowed the extraction of UV-absorbance spectra from the peaks of interest. Absorbance maxima were found at 203 and 279 nm with a shoulder at 230 nm. Additionally fluorescence emission and excitation spectra were recorded from the analytes using a fluorescence detector (FLD) after stop flow (lambdaex = 279 nm, lambdaem = 307 nm). The chromatographic reversed-phase system was coupled to an ion-trap mass spectrometer (LC-MS) by using an electrospray ionization interface (ESI). After optimization processes the separation was carried out using an ammonium acetate buffer at pH 5.5. Mass spectrometric detection turned out to be more sensitive in negative mode. Collisionally induced dissociation (CID) was used to obtain fragment ions of structural relevance (LC-MS/MS). Finally, compounds of interest coming from sorrel leaf (Rumex acetosa) eluting at low acetonitrile concentrations were confirmed to be catechin and epicatechin.

Catechin↗

PubMed2002

Simultaneous determination of hypericin and hyperforin in human plasma and serum using liquid-liquid extraction, high-performance liquid chromatography and liquid chromatography-tandem mass spectrometry.

A method for the simultaneous extraction of hypericin and hyperforin from a St. John's Wort extract, which is used in case of moderate depressions and skin injuries, from human plasma and serum by liquid-liquid extraction (LLE) with n-hexane-ethylacetate (70:30, w/w) was developed. A reversed-phase high-performance liquid chromatographic (RP-HPLC) method with UV, fluorescence (FLD) and mass spectrometric (MS) detection using electrospray ionization (ESI) was used to identify and quantify hypericin and hyperforin in the extracts from blood samples. Linearity was obtained in the ranges 8.4-28.7 ng/ml (hypericin) and 21.6-242.6 ng/ml (hyperforin). Recoveries were between 32.2 and 35.6% for hypericin and 100.1 and 89.9% for hyperforin. Intra-day accuracy and precision for this method ranged between 3.2 and 4.3% and 2.6 and 2.8%, respectively. After validation of the LLE, the method was tested on real plasma samples which were obtained by ingestion of St. John's Wort extract capsules. Blood samples were taken 2, 4, and 6 h after ingestion. Finally, this method proved to be highly suitable for clinical and pharmacologically relevant studies.

Anthracenes↗

PubMed2002

Fast analysis of flavonoids in plant extracts by liquid chromatography-ultraviolet absorbance detection on poly(carboxylic acid)-coated silica and electrospray ionization tandem mass spectrometric detection.

A highly hydrophilic poly(7-oxonorbornene-5,6-dicarboxylic acid-block-norbornene) [=poly-(ONDCA-b-NBE)]-coated silica was investigated for the liquid chromatographic (LC) determination of flavonoids in plant extracts of complex biological origin using UV absorbance and mass spectrometric (MS) detection. Compared to the most commonly used octadecyl derivatized silica this sorbent allowed fast separations even at extreme pH values. Furthermore, UV absorbance and MS detection were evaluated. As we found, UV detection at 254 nm allows the determination of flavonoids down to the ng range with a linearity of R2>0.9906. For the more selective characterization the validated LC system was coupled to a quadrupole ion trap mass spectrometer via an electrospray ionization (ESI) interface. MS detection showed high linearity (R2>0.9904) for all investigated flavonoids. Due to the relatively high flow-rate of 1 ml/min the limits of detection were found in the lower-microg range. Collision induced dissociation was applied to obtain characteristic fragmentation fingerprints. Finally, the validated LC-ESI-MS-MS method demonstrated that this poly-(ONDCA-b-NBE) stationary phase allows fast characterization and quantitation in onion, elderflower blossom, lime blossom, St. John's Wort and red wine.

Calibration↗

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↗

PubMed1999

Histidine77, glutamic acid81, glutamic acid123, threonine126, asparagine194, and tryptophan197 of the human emopamil binding protein are required for in vivo sterol delta 8-delta 7 isomerization.

The human emopamil binding protein (hEBP) exhibits sterol Delta8-Delta7 isomerase activity (EC 5.3.3.5) upon heterologous expression in a sterol Delta8-Delta7 isomerization-deficient erg2-3 yeast strain. Ala scanning mutagenesis was used to identify residues in the four putative transmembrane alpha-helices of hEBP that are required for catalytic activity. Isomerization was assayed in vivo by spectrophotometric quantification of Delta5,7-sterols. Out of 64 Ala mutants of hEBP only H77A-, E81A-, E123A-, T126A-, N194A-, and W197A-expressing yeast strains contained 10% or less of wild-type (wt) Delta5,7-sterols. All substitutions of these six residues with functionally or structurally similar amino acid residues failed to fully restore catalytic activity. Mutants E81D, T126S, N194Q, and W197F, but not H77N and E123D, still bound the enzyme inhibitor 3H-ifenprodil. Changed equilibrium and kinetic binding properties of the mutant enzymes confirmed our previous suggestion that residues required for catalytic activity are also involved in inhibitor binding [Moebius et al. (1996) Biochemistry 35, 16871-16878]. His77, Glu81, Glu123, Thr126, Asn194, and Trp197 are localized in the cytoplasmic halves of the transmembrane segments 2-4 and are proposed to line the catalytic cleft. Ala mutants of Trp102, Tyr105, Asp109, Arg111, and Tyr112 in a conserved cytoplasmic domain (WKEYXKGDSRY) between transmembrane segments 2 and 3 contained less than 10% of wt Delta5,7-sterols, implying that this region also could be functionally important. The in vivo complementation of enzyme-deficient yeast strains with mutated cDNAs is a simple and sensitive method to rapidly analyze the functional consequences of mutations in sterol modifying enzymes.

Alanine↗

PubMedDate not supplied

Evaluation of detection methods for the reversed-phase HPLC determination of 3',4',5'-trimethoxyflavone in different phytopharmaceutical products and in human serum.

Quantitative determination of the major compound, 3',4',5'-trimethoxyflavone (1), in plant extracts, in tablets of Flos and of Radix Primulae veris and in human serum has been accomplished using reversed-phase HPLC with UV, fluorescence and mass spectrometric (MS) detection. Compared to UV detection, fluorescence detection showed greater selectivity, was 10-fold more sensitive and allowed the determination of 1 in human serum after sample pre-treatment by solid-phase extraction. MS detection of 1 using electrospray ionisation (ESI) interface could be improved by substituting trifluoroacetic acid with the more polar and less conductive additive acetic acid, giving rise to a 230-fold improvement in analyte detectability at the cost of an increase of only 45% in the peak width of the eluting peak at its half height. Further optimisation of the acetic acid concentration showed the highest signal intensity at 1.25% for HPLC-atmospheric pressure chemical ionisation (APCI)-MS and at 0.75% for HPLC-ESI-MS. The optimised MS method proved to be extremely selective, 50 times more sensitive than UV detection and 5 times more sensitive than fluorescence detection. Furthermore, fragment-ion spectra produced by collision induced dissociation-MS have been used as "fingerprints" for identifying compounds in the highly complex mixtures examine.

Chromatography, High Pressure Liquid↗