Christian W. Huck

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.

Analysis of protein phosphorylation by monolithic extraction columns based on poly(divinylbenzene) containing embedded titanium dioxide and zirconium dioxide nano‐powders

The potential of an organic monolith with incorporated titanium dioxide (TiO2) and zirconium dioxide (ZrO2) nanoparticles was evaluated for the selective enrichment of phosphorylated peptides from tryptic digests. A pipette tip was fitted with a monolith based on divinylbenzene (DVB) of highly porous structure, which allows sample to pass through the monolithic bed. The enrichment of phosphopeptides was enhanced by increasing the pipetting cycles during the sample preparation and a higher recovery could be achieved with adequate buffer systems. A complete automated process was developed for enrichment of phosphopeptides leading to high reproducibility and resulting in a robust method designed to minimize analytical variance while providing high sensitivity at high sample throughput. The effect of particle size on the selectivity of phosphopeptides was investigated by comparative studies with nano‐ and microscale TiO2 and ZrO2 powders. Eleven phosphopeptides from α‐casein digest could be recovered by an optimized mixture of microscale TiO2/ZrO2 particles, whereas nine additional phosphopeptides could be retained by the same mixture of nano‐structured material. When compared to conventional immobilized metal‐ion affinity chromatography and commercial phosphorylation‐enrichment kits, higher selectivity was observed in case of self fabricated tips. About 20 phosphopeptides could be retained from α‐casein and five from β‐casein digests by using TiO2 and ZrO2 based extraction tips. Further selectivity for phosphopeptides was demonstrated by enriching a digest of in vitro phosphorylated extracellular signal regulated kinase 1 (ERK1). Two phosphorylated peptides of ERK1 could be identified by MALDI‐MS/MS measurements and a following MASCOT database search.

Disruption of vascular endothelial homeostasis by tobacco smoke: impact on atherosclerosis

The World Health Organization (WHO) predicts that by 2020 tobacco will become the largest single health problem worldwide and will cause an estimated 8.4 million deaths annually (http://www5.who.int/tobacco/). Although the impact of smoking on human health is well defined from the medical point of view, surprisingly little is known about the mechanisms by which tobacco smoke mediates its disastrous effects. Here, we demonstrate that tobacco smoke dramatically changes vascular endothelial cell and tissue morphology, leading to a loss of endothelial barrier function within minutes. Long‐term exposure of endothelial cells to tobacco smoke extracts induces necrosis that may trigger a pro‐inflammatory status of the vessel wall. Pre‐incubation of the extracts without cells for 6 h at 37°C led to a complete loss of activity. Further, the endothelium could be rescued by changing to fresh medium even at times when the extracts had lost their activity. Finally, we show that N‐acetyl cysteine and statins inhibit the adverse tobacco smoke effects.

Nanostructured Diamond-Like Carbon on Digital Versatile Disc as a Matrix-Free Target for Laser Desorption/Ionization Mass Spectrometry

A nanostructured diamond-like carbon (DLC) coated digital versatile disk (DVD) target is presented as a matrix-free sample support for application in laser desorption/ionization mass spectrometry (LDI-MS). A large number of vacancies, defects, relative sp(2) carbon content, and nanogrooves of DLC films support the LDI phenomenon. The observed absorptivity of DLC is in the range of 305-330 nm (nitrogen laser, 337 nm). The universal applicability is demonstrated through different analytes like amino acids, carbohydrates, lipids, peptides, and other metabolites. Carbohydrates and amino acids are analyzed as sodium and potassium adducts. Peptides are detectable in their protonated forms, which avoid the extra need of additives for ionization. A bovine serum albumin (BSA) digest is analyzed to demonstrate the performance for peptide mixtures, coupled with the material-enhanced laser desorption/ionization (MELDI) approach. The detection limit of the described matrix-free target is investigated to be 10 fmol/microL for [Glu(1)]-fibrinopeptide B (m/z 1570.6) and 1 fmol/microL for L-sorbose (Na(+) adduct). The device does not require any chemical functionalization in contrast to other matrix-free systems. The inertness of DLC provides longer lifetimes without any deterioration in the detection sensitivity. Broad applicability allows high performance analysis in metabolomics and peptidomics. Furthermore the DLC coated DVD (1.4 GB) sample support is used as a storage device for measured and processed data together with sampling on a single device.

Phytoanalysis: a challenge in phytomics

This article is concerned with fast analytical methods for qualitative and quantitative determination of plant ingredients and phytopharmaceutical products. The emphasis is not only on conventional techniques, such as HPLC or CE, and the influence of the stationary phase or the detection method chosen, but also on the development of new stationary phases and the use of non-standard analytical techniques, such as micro-liquid chromatography (μLC), capillary electrochromatography (CEC) or near-infrared (NIR) spectroscopy, in phytomics.

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 μ-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.

Protein profiling for cancer biomarker discovery using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and infrared imaging: A review

Cancer biomarker refers to a substance or process that is indicative of the presence of cancer in the body. A biomarker might be either a molecule secreted by a tumor or it can be a specific response of the body to the presence of cancer. Cancer biomarker-based diagnostics have applications for establishing disease predisposition, early detection, cancer staging, therapy selection, identifying whether or not a cancer is metastatic, therapy monitoring, assessing prognosis, and advances in the adjuvant setting. Full adoption of cancer biomarkers in the clinic has to date been slow, and only a limited number of cancer biomarker products are currently in routine use. Among proteomic technologies, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS) is a technique that has allowed rapid progress in cancer biology. Different further developed methods including e.g. SELDI (surface-enhanced laser desorption/ionization) and MELDI (material-enhanced laser desorption/ionization) are simple and high-throughput techniques that analyze with high sensitivity and specificity intact proteins expressed in complex biological mixtures, such as serum, urine, and tissues. The combination of mass spectrometry (MS) with infrared (IR) spectroscopic imaging is an attempt to combine different technologies in systems analytics. Both MALDI-TOF and infrared tissue imaging enable studying proteins distribution in tissue samples with a resolution down to 50 and 5 μm, respectively. In this review, we summarize recent applications and the synergistic combination of these new technologies to proteomic profiling for cancer biomarker discovery.

Evaluation of extraction methods for the simultaneous analysis of simple and macrocyclic trichothecenes

The article is concerned with the simultaneous determination of simple and macrocyclic trichothecenes using high-performance liquid chromatography (LC) coupled to UV and mass spectrometric (MS) detection. Emphasis is put on the liquid-liquid extraction (LLE) and solid phase extraction (SPE) procedure from plant material such as wheat, comparing SPE cartridges packed with different stationary phases based on silica and polymer sorbents. In this coherence a polymeric material on the basis of poly(glycidyl methacrylate-divinylbenzene) (GMA-DVB) is developed with special regard on synthesis procedures to enhance the extraction recovery of trichothecenes in a broad polarity range. Evaluation of extraction techniques showed that the introduced material is competitive with commercially available high quality SPE materials. Percentage recovery is 82% for polar compounds, 89% for medium polar compounds and 98% for lipophilic compounds.

Analysis of vitamin E in food and phytopharmaceutical preparations by HPLC and HPLC-APCI-MS-MS

SummaryThe analysis of α, β, γ, δ-tocopherols, trienols, α-tocopheryl acetate and nicotinate (vitamin E) in complex matrices was carried out using a new liquid chromatographic (HPLC) method giving better separation efficiency, selectivity and sensitivity than that described in the literature. The use of normal-phase (NP)-HPLC on silica gel with issoctane-diisopropylether-1,4-dioxane as optimized mobilepphase yielded higher resolution than conventional reversed-phase (RP)-HPLC using methanol mobile phase. Identification of peaks was by UV-absorbance at 295 nm, diode array, or fluorescence detection (λex = 295 nm,λex = 330 nm). The latter was found to be more selective and ten times more sensitive than UV-absorbance detection. A quadrupole, ion-trap mass spectrometer with an atmospheric-pressure ionization (APCl) interface was used to detect vitamin E constituents in the femtomole range. With collision-induced dissociation (CID) in the ion source, which gave characteristic fragmentation, the identity of the investigated compounds could be confirmed. Plots of peak area versus amount injected allowed quantitation of α, β, γ, δ-tocopherols and-trienols, α-tocopheryl acetate and nicotinate in real samples such as peanut, almond, spinach, spelt grain bran, latex and tablets. The method described offers fast identification and quantitation of vitamin E constituents of complex biological origin.

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. Johns 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. Johns 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.