Günther Stecher

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.

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 three flavonoids by CE-uV and CE-ESI-MS. Determination of naringenin from a phytomedicine

Complex extracts of plant constituents often require very effective separation techniques to allow the identification of different compounds. Capillary electrophoresis (CE) coupled to mass spectrometry (MS) detection can provide structure-selective information about the analytes in such matrices and has turned out to be an attractive alternative to HPLC methods. Therefore, a CE method has been established for the analysis of a flavonoid mixture consisting of 5-methoxyflavone, biochanin A, hesperetin, and naringenin obtained from plant extracts. The method uses a fused-silica capillary and a buffer system consisting of 40 mM NH 4 OAc, 15% ACN (pH 9.5). After validation of the CE-method in combination with a quadrupole mass spectrometer (with an electrospray interface (ESI) and 0.1% triethylamine in 2-propanol/water (80/20 v/v) as sheath liquid in the negative ion mode), MS detection showed a sensitivity for hesperetin and naringenin similar to that of UV detection. Detection limits were at 0.4-0.6 mg/L for all analytes using UV, 0.5 mg/L for hesperetin and naringenin, and 2 mg/L for biochanin A using MS detection. Employing external calibration allowed the reliable quantitation of naringenin in a phytomedicine containing five different herbal drugs. In addition, collision-induced dissociation (CID) in the ion source of the MS was used to confirm the identity of the peaks via their characteristic fragments.

Attenuation of nucleoside and anti-cancer nucleoside analog drug uptake in prostate cancer cells by Cimicifuga racemosa extract BNO-1055

This study aimed to investigate the mechanisms underlying the anti-proliferative effects of the ethanolic Cimicifuga racemosa extract BNO-1055 on prostate cells and evaluate its therapeutic potential. BNO-1055 dose-dependently attenuated cellular uptake and incorporation of thymidine and BrdU and significantly inhibited cell growth after long-time exposure. Similar results were obtained using saponin-enriched sub-fractions of BNO-1055. These inhibitory effects of BNO-1055 could be mimicked using pharmacological inhibitors and isoform-specific siRNAs targeting the equilibrative nucleoside transporters ENT1 and ENT2. Moreover, BNO-1055 attenuated the uptake of clinically relevant nucleoside analogs, e.g. the anti-cancer drugs gemcitabine and fludarabine. Consistent with inhibition of the salvage nucleoside uptake pathway BNO-1055 potentiated the cytotoxicity of the de novo nucleotide synthesis inhibitor 5-FU without significantly altering its uptake. Collectively, these data show for the first time that the anti-proliferative effects of BNO-1055 result from hindered nucleoside uptake due to impaired ENT activity and demonstrate the potential therapeutic use of BNO-1055 for modulation of nucleoside transport.

High performance separation technologies and spectroscopic tools for plant extract characterization in phytomics

The review is concerned with fast analytical methods for the qualitative and quantitative determination of plant constituents and phytopharmaceutical products. Emphasis is put on the determination of leading compounds, the role of the stationary phase in the analysis of sugars, phenolic compounds, proteins or DNA-mutations and the use of non-standard analytical tools such as micro high performance liquid chromatography (μ-LC) or near infrared reflectance spectroscopy (NIR) next to conventional high performance liquid chromatography (HPLC). Finally the importance of spectroscopic methods such as mass spectrometry (MS) in phytomics will be discussed giving examples for HPLC-MS and μ-HPLC-MS hyphenation.