Matthias Unger

General approach for the analysis of various alkaloid classes using capillary electrophoresis and capillary electrophoresis-mass spectrometry

Abstract The analysis of various alkaloid classes employing capillary electrophoresis (CE) and on-line combined CE-mass spectrometry (CE-MS) is described. A CE method is presented for the analysis of alkaloids without derivatisation or purification. The separation of four different groups of alkaloids consisting of monoterpenoid indole alkaloids, protoberberines/benzophenanthridines, β-carboline alkaloids, and isoquinolines from poppy by free zone capillary electrophoresis has been obtained using a 1:1 mixture of 100 mmol 1 −1 ammonium acetate (pH 3.1) and acetonitrile. The influence of alkaloid structure on the electrophoretic mobility is discussed. The CE-MS reconstructed total ion current (RIC) of the indole- and the opium-type standard alkaloids shows a decreased signal-to-noise ratio compared to CE using only UV detection. As expected the single-ion traces (or individual mass traces) of the [M+H] + ions show higher signal-to-noise ratios than the RIC. The electrospray MS data of the alkaloids are dominated by the protonated molecules and the Na + -, and K + -adducts. They display the typical pattern resulting from cluster formation or doubly charged species.

High-performance liquid chromatographic, capillary electrophoretic and capillary electrophoretic-electrospray ionisation mass spectrometric analysis of selected alkaloid groups

Systems for efficient separation of selected alkaloid groups by high performance liquid chromatography (HPLC), capillary electrophoresis (CE) and capillary electrophoresis coupled with electrospray ionisation mass spectrometry (CE-ESI-MS) are described. The optimized HPLC system was applied for the separation of 23 standard indole alkaloids as well as for qualitative and quantitative analyses of crude alkaloid extracts of Rauvolfia serpentina X Rhazya stricta hybrid cell cultures. The developed conditions for CE analysis proved to be efficient for separation of mixtures of standard indole and beta-carboline alkaloids. The described buffer system is also applicable in the combination of CE with electrospray ionisation mass spectrometry. This analytical technique allowed the separation and identification of components of standard indole alkaloid mixture as well as crude extracts of R. serpentina roots, R. serpentina cell suspension cultures and cortex of Aspidosperma quebracho-blanco. The influence of buffer composition and analyte structures on separation is discussed.

ANTHRAQUINONES FROM OPHIORRHIZA PUMILA TISSUE AND CELL CULTURES

We have succeeded in initiating and establishing systems of tissue and cell cultures of Ophiorrhiza pumila. Examination of the constituents of the methanol extract of the cultured calli revealed the presence of 11 anthraquinones including two new ones whose structures have been rigorously proved using advanced spectroscopic methods. These findings demonstrated a remarkable difference in the constituents between the wild plants and the callus tissue or cultured cells; the former is devoid of anthraquinones and contains a variety of camptothecin-related alkaloids whereas the latter contains a significant amount of anthraquinones and shows no indication of the presence of alkaloids after several sub-culturings or final establishment of well growing cell suspensions.

Automated, fast, and sensitive quantification of drugs in human plasma by LC/LC-MS: quantification of 6 protease inhibitors and 3 nonnucleoside transcriptase inhibitors.

An analytic assay based on automated sample preparation and liquid chromatography (LC) coupled with electrospray mass spectrometry (ESI-MS) was developed for the quantification of 6 protease inhibitors (PIs) and 3 nonnucleoside reverse transcriptase inhibitors (NNRTIs). The 6 PIs, amprenavir, indinavir, ritonavir, lopinavir, nelfinavir, and saquinavir, as well as the three NNRTIs, nevirapine, efavirenz, and delavirdine, require a succinct analysis technique for therapeutic drug monitoring in HIV/AIDS patients. After protein precipitation, samples were loaded on a C8, 10 × 4-mm extraction column, washed, and, after activation of the column-switching valve, backflushed onto the 30 × 2.1 mm C8 analytic column. [M+H]+ ions were detected in the selected ion mode. A nonlinear fit (y−1 = a + b/x, all r2 > 0.999) for amprenavir, indinavir, ritonavir, lopinavir, nelfinavir, and saquinavir and a linear fit (y = ax + b, all r2 > 0.999) for nevirapine, efavirenz, and delavirdine led to best regression. Absolute recoveries were as follows: PIs > 81%; NNRTIs > 76%. Interday and intraday precision were <12.5% for the PIs and <11.7% for the NNRTIs. Interday and intraday accuracy were <12.2% for the PIs and <14.9% for the NNRTIs. Limits of quantification were 20, 40, 50, 40, 40, 20, and 100 μg/L for amprenavir, indinavir, ritonavir, lopinavir, nelfinavir, saquinavir, and the NNRTIs, respectively. The assay allows fast analysis of patient samples for therapeutic drug monitoring (TDM) and has successfully been used for TDM and pharmacokinetic drug–drug interactions studies.

Extracts and kavalactones of Piper methysticum G. FORST (kava-kava) inhibit P-glycoprotein in vitro

Root extracts from kava-kava (Piper methysticum G. Forst) are clinically used for the treatment of anxiety and restlessness. Due to reported cases of liver toxicity, kava-kava extracts were withdrawn from the market in several countries in 2002. Because the efflux transporter P-glycoprotein (P-gp) is involved in the absorption, distribution, and excretion of many drugs and often participates in drug-drug interactions, we studied the effect of a crude kava extract and the main kavalactones kavain, dihydrokavain, methysticin, dihydromethysticin, yangonin, and desmethoxyyangonin on the P-gp-mediated efflux of calcein-acetoxymethylester in the P-gp-overexpressing cell line P388/dx and the corresponding cell line P388. The crude extract and the kavalactones showed a moderate to potent inhibitory activity with f2 (concentration needed to double baseline fluorescence) values of 170 μg/ml and 17 to 90 μM, respectively. The f2 value of yangonin could not be determined due to its higher lipophilicity. In conclusion, our results for the first time demonstrate P-gp-inhibitory activity of kava-kava and its components in vitro.

Improved detection of alkaloids in crude extracts applying capillary electrophoresis with field amplified sample injection

Abstract A simple and effective method for the sensitive detection of alkaloids in crude plant extracts applying capillary electrophoresis with field amplified sample injection (FASI) is described. This method was compared with normal pressure injection for the determination of alkaloids in methanolic extracts from roots of Berberis vulgaris L. (Berberidaceae) and Hydrastis canadensis L. (Ranunculaceae) using a 1:1 mixture of 200 mM ammonium acetate at pH 3.1 and methanol. By introducing a short plug of 70% methanol (v/v) before electrokinetic injection with 16 kV for 8 s the concentration sensitivity was 1000-times higher compared to hydrodynamic injection for 1 s. No difference between both injection methods for selectivity and resolution of the obtained electropherograms was found. The influence of voltage and injection time on the introduced sample amount was investigated using a mixture of berberine and chelidonine as model substances.

Capillary Electrophoresis of Natural Products: Current Applications and Recent Advances

Capillary electrophoresis (CE) was introduced as a new analytical technique in the 1970s and rapidly proved to be a powerful tool for the separation and detection of various classes of natural and synthetic compounds. Since the availability of commercially manufactured high-performance instruments, CE represents an interesting alternative to high-pressure liquid chromatography (HPLC), mainly because of its speed and high separation efficiency. In this overview a short description of the basic and widely used CE methods will be given and the applicability of these methods for the analysis of natural products will be discussed. Due to the growing number of publications dealing with CE or CE/MS of secondary plant metabolites, an exhaustive overview of all current applications cannot be given in this contribution. Therefore, the focus of this mini-review will be on the advances and new aspects of recently published CE methods in natural products analysis.

Capillary zone electrophoresis of alkaloids influence of structure on electrophoretic mobility

Abstract A comprehensive discussion of important aspects for the analysis of alkaloids by capillary zone electrophoresis (CZE) is given. The influence of structure on the electrophoretic mobility (EM) of indole alkaloids was investigated using a running buffer which is generally applicable to the CZE analysis of alkaloids. The EM, which at the applied conditions was mostly dependent on the size and shape of the solvated analyte ions, was additionally affected by the presence of hydrogen bonds or ion–dipole interactions between protonated and unprotonated alkaloids of the same species. This could be derived from the existence of alkaloidal dimer cluster ions [2M+H]+ when mass spectrometry was used for detection.

Analysis of Rauwolfia Alkaloids Employing Capillary Electrophoresis-Mass Spectrometry

Abstract Capillary electrophoresis-mass spectrometry (CE-MS) was applied to analyse extracts of roots and cell suspension cultures from Rauwolfia serpentina. Most of the alkaloids known to be present in the respective plant material have been resolved via CE and assigned according to their electrospray mass spectra.

Modelling antibiotic and cytotoxic isoquinoline effects in Staphylococcus aureus, Staphylococcus epidermidis and mammalian cells

Isoquinolines (IQs) are natural substances with an antibiotic potential we aim to optimize. Specifically, IQ-238 is a synthetic analog of the novel-type N,C-coupled naphthylisoquinoline (NIQ) alkaloid ancisheynine. Recently, we developed and tested other IQs such as IQ-143. By utilizing genome-wide gene expression data, metabolic network modelling and Voronoi tessalation based data analysis - as well as cytotoxicity measurements, chemical properties calculations and principal component analysis of the NIQs - we show that IQ-238 has strong antibiotic potential for staphylococci and low cytotoxicity against murine or human cells. Compared to IQ-143, systemic effects are less pronounced. Most enzyme activity changes due to IQ-238 are located in the carbohydrate metabolism. Validation includes metabolite measurements on biological replicates. IQ-238 delineates key properties and a chemical space for a good therapeutic window. The combination of analysis methods allows suggestions for further lead development and yields an in-depth look at staphylococcal adaptation and network changes after antibiosis. Results are compared to eukaryotic host cells.