Simultaneous determination of four anthracyclines and three metabolites in human serum by liquid chromatography–electrospray mass spectrometry

A sensitive and very specific method, using liquid chromatography–electrospray mass spectrometry (LC–ES-MS), was developed for the determination of epirubicin, doxorubicin, daunorubicin, idarubicin and the respective active metabolites of the last three, namely doxorubicinol, daunorubicinol and idarubicinol in human serum, using aclarubicin as internal standard. Once thawed, 0.5-ml serum samples underwent an automated solid-phase extraction, using C₁₈ Bond Elut cartridges (Varian) and a Zymark Rapid-Trace robot. After elution of the compounds with chloroform–2-propanol (4:1, v/v) and evaporation, the residue was reconstituted with a mixture of 5 mM ammonium formate buffer (pH 4.5)–acetonitrile (60:40, v/v). The chromatographic separation was performed using a Symmetry C₁₈, 3.5 μm (150×1 mm I.D.) reversed-phase column, and a mixture of 5 mM ammonium formate buffer (pH 3)–acetonitrile (70:30, v/v) as mobile phase, delivered at 50 μl/min. The compounds were detected in the selected ion monitoring mode using, as quantitation ions, m/z 291 for idarubicin and idarubicinol, m/z 321 for daunorubicin and daunorubicinol, m/z 361 for epirubicin and doxorubicin, m/z 363 for doxorubicinol and m/z 812 for aclarubicin (I.S.). Extraction recovery was between 71 and 105% depending on compounds and concentration. The limit of detection was 0.5 ng/ml for daunorubicin and idarubicinol, 1 ng/ml for doxorubicin, epirubicin and idarubicin, 2 ng/ml for daunorubicinol and 2.5 ng/ml for doxorubicinol. The limit of quantitation (LOQ) was 2.5 ng/ml for doxorubicin, epirubicin and daunorubicinol, and 5 ng/ml for daunorubicin, idarubicin, doxorubicinol and idarubicinol. Linearity was verified from these LOQs up to 2000 ng/ml for the parent drugs (r≥0.992) and 200 ng/ml for the active metabolites (r≥0.985). Above LOQ, the within-day and between-day precision relative standard deviation values were all less than 15%. This assay was applied successfully to the analysis of human serum samples collected in patients administered doxorubicin or daunorubicin intravenously. This method is rapid, reliable, allows an easy sample preparation owing to the automated extraction and a high selectivity owing to MS detection.     

Short Note: Streptomyces peucetius converts anthracycline intermediates efficiently in culture media containing oil cake as carbon source

Alternative media constituents like carbon sources and buffers were evaluated for the large scale production of daunorubicin. Streptomyces peucetius cultivated on the media containing sesamum oil cake as carbon source with HEPES or phosphate buffer showed good yield of the antibiotic and the intermediates were also converted into the final product more efficiently.     

Cell separation by countercurrent centrifugal elutriation: recent developments

Countercurrent centrifugal elutriation (CCE) is a cell separation technique that separates particles predominantly according to their size, and to some degree according to their specific density, without a need for antibodies or ligands tagging cell surfaces. The principles of this technique have been known for half a century. Still, numerous recent publications confirmed that CCE is a valuable supplement to current cell separation technology. It is mainly applied when homogeneous populations of cells, which mirror an in vivo situation, are required for answering scientific questions or for clinical transplantation, while antibodies or ligands suitable for cell isolation are not available. Currently, new technical developments are expanding its application toward fractionation of healthy and malignant tissue cells and the preparation of dendritic cells for immunotherapy.     

Advances in cell separation: recent developments in counterflow centrifugal elutriation and continuous flow cell separation

Cell separation by counterflow centrifugal elutriation (CCE) or free flow electrophoresis (FFE) is performed at lower frequency than cell cloning and antibody-dependent, magnetic or fluorescence-activated cell sorting. Nevertheless, numerous recent publications confirmed that these physical cell separation methods that do not include cell labeling or cell transformation steps, may be most useful for some applications. CCE and FFE have proved to be valuable tools, if homogeneous populations of normal healthy untransformed cells are required for answering scientific questions or for clinical transplantation and cells cannot be labeled by antibodies, because suitable antibodies are not available or because antibody binding to a cell surface would induce the cell reaction which should be investigated on purified cells or because antibodies bound to the surface hamper the use of the isolated cells. In addition, the methods are helpful for studying the biological reasons for, or effects of, changes in cell size and cellular negative surface charge density. Although the value of the methods was confirmed in recent years by a considerable number of important scientific results, activities to further develop and improve the instruments have, unfortunately, declined.     

Acetaminophen stimulates the peroxidative metabolism of anthracyclines

Acetaminophen, a common analgesic and antipyretic drug, is frequently administered to individuals undergoing anthracycline chemotherapy. Here, the effect of acetaminophen on the metabolism of daunorubicin and doxorubicin by isolated enzymes lactoperoxidase and myeloperoxidase, and by myeloperoxidase-containing human leukemia HL-60 cells was investigated using spectrophotometric and EPR techniques. We report that at pharmacological concentrations acetaminophen strongly stimulates oxidation of the anthracyclines by lactoperoxidase and myeloperoxidase systems, which results in irreversibly altered (colorless) products. The initial rate and efficacy of daunorubicin oxidation depends on pH. While at pH approximately 7 the oxidation is rapid and extensive, almost no oxidation occurs at pH approximately 5. In the absence of daunorubicin, oxidation of acetaminophen by lactoperoxidase/hydrogen peroxide is only weakly dependent on pH, however, at pH 7.4 it strongly depends on [daunorubicin]. Ascorbate and reduced glutathione strongly inhibited oxidation of anthracyclines by lactoperoxidase and HL-60 systems. Using EPR, a daunorubicin-derived radical was detected in a daunorubicin/acetaminophen/peroxidase/hydrogen peroxide system as a narrow single line (0.175 mT) with g = 2.0047. When daunorubicin was omitted, only an acetaminophen-melanin EPR signal was detected (g = 2.0043, line width approximately 0.5 mT). Similar results were obtained with doxorubicin. We suggest that the stimulation by acetaminophen is primarily due to its preferential oxidation by peroxidases to the corresponding phenoxyl radical, which subsequently reacts with daunorubicin (doxorubicin). Because biological properties of oxidatively transformed anthracyclines will certainly be different from those of their parent compounds, the possible acetaminophen-enhanced degradation of the anthracyclines in vivo is likely to interfere with anticancer and/or cardiotoxic activities of these agents.     

Simultaneous quantitation of plasma doxorubicin and prochlorperazine content by high-performance liquid chromatography

A high-performance liquid chromatographic method has been developed and tested for simultaneous extraction, elution and determination of doxorubicin and prochlorperazine content in human plasma samples. The procedure consists of extraction through a conditioned C₁₈ solid-phase extraction cartridge, elution from a Spherisorb C₈ reversed-phase column by an isocratic mobile phase (60% acetonitrile, 15% methanol and 25% buffer) followed by detection with electrochemical and fluorescence detectors. Recovery of doxorubicin and prochlorperazine from pooled human plasma samples (n=3) containing 100 ng/ml of the two drugs was 77.8±3.5% and 89.1±6.0%, respectively. The lower limits of quantitation for doxorubicin and prochlorperazine in plasma samples were 6.25 ng/ml and 10 ng/ml, respectively. A linear calibration curve was obtained for up to 2 μg/ml of doxorubicin and prochlorperazine. This combination method may be of particular value in clinical studies where phenothiazines such as prochlorperazine are used to enhance retention of doxorubicin in drug resistant tumor cells.     

Asymmetric cell division: recent developments and their implications for tumour biology

The ability of cells to divide asymmetrically is essential for generating diverse cell types during development. The past 10 years have seen tremendous progress in our understanding of this important biological process. We have learned that localized phosphorylation events are responsible for the asymmetric segregation of cell fate determinants in mitosis and that centrosomes and microtubules play important parts in this process. The relevance of asymmetric cell division for stem cell biology has added a new dimension to the field, and exciting connections between asymmetric cell division and tumorigenesis have begun to emerge.     

Nanovaccines: recent developments in vaccination

In the past 100 years, vaccination has contributed immensely to public health by preventing a number of infectious diseases. Attenuated, killed or part of the microorganism is employed to stimulate the immune system against it. Progress in biotechnology has provided protective immunity through DNA vaccines. In recent years, nanovaccine is a novel approach to the methodology of vaccination. Nanomaterials are delivered in the form of microspheres, nanobeads or micro-nanoprojections. Painless, effective and safe needle-free routes such as the intranasal or the oral route, or patches of microprojections to the skin are some of the approaches which are in the experimental stage at present but may have a great future ahead in nanovaccination.     

Auxin receptors: recent developments

Rapid advances have been made in the study of auxin binding proteins (ABPs) in the last five years. In particular, an ABP in maize membranes has been cloned, sequenced and both monoclonal and polyclonal antibodies to this ABP have been developed. Structural and functional analysis has begun and there is good electrophysiological evidence that ABP in the plasma membrane functions as a receptor, probably involved in auxin-induced cell expansion. The role of the large amount of ABP in the endoplasmic reticulum is less clear, as is the relationship to soluble ABPs. At present there is only some circumstantial evidence relating any ABP to cell division. Receptors for synthetic inhibitors of auxin transport (phytotropins) are also of interest in relation to auxin action, but are less well characterised. Identification of new naturally-occurring phytotropins could lead to novel plant growth regulators.     

PROSITE: recent developments.

PROSITE is a compilation of sites and patterns found in protein sequences; it can be used as a method of determining the function of uncharacterized proteins translated from genomic or cDNA sequences.     

Cancer gene and immunotherapy: recent developments

Gene and immunotherapeutic approaches to treat human malignant tumors are reviewed. Special attention is given to the different strategies of cancer gene therapy and to recent aspects of cytokine-supported tumor immunotherapy or tumor-specific vaccination. The limitations of these therapy approaches are critically discussed especially with respect to immune escape mechanisms.     

Mitochondrial biogenesis: recent developments and insights

Biosynthesis of a functional mitochondrion requires the coordinate expression of genes in both mitochondrial and nuclear DNAs. In yeast, three mitochondrial genes are split and RNA splicing plays a pivotal role in their expression. The recent finding that some introns are capable of self-splicing activity in vitro has permitted analysis of the mechanisms involved in RNA catalysis and may eventually shed light on the evolution of splicing mechanisms in general. Most mitochondrial proteins are encoded by nuclear genes, synthesized in the cytoplasm and imported by the organelle. The availability of cloned genes coding for several constituent subunits of the ubiquinol-cytochrome c reductase, which are imported by mitochondria, has allowed study of selected steps in the addressing of proteins to mitochondria and their intercompartmental sorting within the organelle. Recent developments are discussed.     

Plasmid rolling-circle replication: recent developments

It is now well established that a large majority of small, multicopy plasmids of Gram-positive bacteria use the rolling-circle (RC) mechanism for their replication. Furthermore, the host range of RC plasmids now includes Gram-negative organisms as well as archaea. RC plasmids can be broadly classified into at least five families, individual members of which are spread among widely different bacteria. There is significant homology in the basic replicons of plasmids belonging to a particular family, and there is compelling evidence that such plasmids have evolved from common ancestors. Major advances have recently been made in our understanding of plasmid RC replication, including the characterization of the biochemical activities of the plasmid initiator proteins and their interaction with the double-strand origin, the domain structure of the initiator proteins and the molecular basis for the function of single-strand origins in plasmid lagging strand synthesis. Over the past several years, there has been a 'renaissance' in studies on RC replication as a result of the discovery that many plasmids replicate by this mechanism, and studies in the next few years are likely to reveal new and novel mechanisms used by RC plasmids for their regulated replication.     

Phytochemical biopesticides: some recent developments

Synthetic pesticides in general, are highly toxic, persistent and their harmful residues contaminate crops, food commodities and pollute soils and groundwater. They adversely affect non-target organisms like pollinators, fish, birds, animals, and their excessive use results in increased resistance in pests. Phytochemical biopesticides on the other hand are less toxic, least persistent, environment friendly and safe to humans and non target organisms. Several phytochemical biopesticides like azadirachtin, nicotine, pyrethrins, rotenone, veratrum, annonins, rocaglamides, isobutylamides etc. have been successfully commercilalized in the past. In this review pesticidal products based on Madhuca indica (Mahua), Sapindus mukorossi (soapnut), Curcuma longa (turmeric), Pongamia glabra (karanja), Eupatorium adenophorum (Crofton weed), Tagetes erecta (marigold), Rheum emodi (Himalayan Rhubarb) and essential oil bearing plants have been discussed. Natural insecticide synergists derived from Anethum sowa and their semisynthetic derivatives have been used to prolong efficacy and counter resistance in insect pests.     

Bacterial endophytes: recent developments and applications.

Endophytic bacteria have been found in virtually every plant studied, where they colonize the internal tissues of their host plant and can form a range of different relationships including symbiotic, mutualistic, commensalistic and trophobiotic. Most endophytes appear to originate from the rhizosphere or phyllosphere; however, some may be transmitted through the seed. Endophytic bacteria can promote plant growth and yield and can act as biocontrol agents. Endophytes can also be beneficial to their host by producing a range of natural products that could be harnessed for potential use in medicine, agriculture or industry. In addition, it has been shown that they have the potential to remove soil contaminants by enhancing phytoremediation and may play a role in soil fertility through phosphate solubilization and nitrogen fixation. There is increasing interest in developing the potential biotechnological applications of endophytes for improving phytoremediation and the sustainable production of nonfood crops for biomass and biofuel production.