Simultaneous quantification of cyclophosphamide, 4-hydroxycyclophosphamide, N,N',N"-triethylenethiophosphoramide (thiotepa) and N,N',N"-triethylenephosphoramide (tepa) in human plasma by high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry

The alkylating agents cyclophosphamide (CP) and N, N', N"-triethylenethiophosphoramide (thiotepa) are often co-administered in high-dose chemotherapy regimens. Since these regimens can be complicated by the occurrence of severe and sometimes life-threatening toxicities, pharmacokinetically guided administration of these compounds, to reduce variability in exposure, may lead to improved tolerability. For rapid dose adaptations during a chemotherapy course, we have developed and validated an assay, using liquid chromatography coupled with electrospray tandem mass spectrometry (LC/MS/MS), for the routine quantification of CP, thiotepa and their respective active metabolites 4-hydroxycyclophosphamide (4OHCP) and N, N', N"-triethylenephosphoramide (tepa) in plasma. Because of the instability of 4OHCP in plasma, the compound is derivatized with semicarbazide (SCZ) immediately after sample collection and quantified as 4OHCP-SCZ. Sample pretreatment consisted of protein precipitation with a mixture of methanol and acetronitrile using 100 microl of plasma. Chromatographic separation was performed on an Zorbax Extend C18 column (150 x 2.1 mm i.d., particle size 5 microm), with a quick gradient using 1 mM ammonia solution and acetonitrile, at a flow-rate of 0.4 ml min(-1). The analytical run time was 10 min. The triple quadrupole mass spectrometer was operating in the positive ion mode and multiple reaction monitoring was used for drug quantification. The method was validated over the concentration ranges 200-40,000 ng ml(-1) for CP, 50-5000 ng ml(-1) for 4OHCP-SCZ and 5-2500 ng ml(-1) for thiotepa and tepa, using 100 microl of human plasma. These dynamic concentration ranges proved to be relevant in daily practice. Hexamethylphosphoramide was used as an internal standard. The coefficients of variation were <12% for both intra-day and inter-day precisions for each compound. Mean accuracies were also between the designated limits (+/- 15%). This robust and rapid LC/MS/MS assay is now successfully applied for routine therapeutic drug monitoring of CP, thiotepa and their metabolites in our hospital.     

Development and validation of an assay for the quantitative determination of cladribine nucleotides in MDCKII cells and culture medium using weak anion-exchange liquid chromatography coupled with tandem mass spectrometry

The development and validation of an assay for the quantitative analysis of cladribine mono-, di- and triphosphate (2-chloro, 2'-deoxyadenosine 5'-mono-, di- and triphosphate or 2CdAMP, 2CdADP and 2CdATP) in culture medium (Optimem) and cell lysate is described. Cladribine mono- and diphosphate reference compounds were obtained by thermal degradation of cladribine triphosphate. The reference compounds were characterized using ion-pairing reversed-phase high-performance liquid chromatography with ultraviolet detection. The bioanalytical assay for 2CdAMP, 2CdADP and 2CdATP is based on weak anion-exchange liquid chromatography coupled with tandem mass spectrometry in the positive ion mode (WAXLC/MS/MS). A fused-silica electrospray capillary was used instead of a stainless steel electrospray capillary to minimize adsorption of analytes and thus decrease variation in the analyte signals. Dynamic ranges of 1.11-27.7, 0.550-55.0 and 1.31-52.3 nM for 2CdAMP, 2CdADP and 2CdATP, respectively, were validated in culture medium and cell lysate. Optimem samples required stabilization with 30% methanol to prevent conversion of 2CdATP into 2CdAMP and 2CdADP. All intra- and interday accuracies and precisions were within +/-20%. The stability of the compounds was assessed under various analytically relevant conditions. The method was successfully used to investigate cladribine nucleotide transport in vitro in Madin-Darby canine kidney II (MDCKII) cells.     

Validated assay for the simultaneous determination of the anti-cancer agent gemcitabine and its metabolite 2',2'-difluorodeoxyuridine in human plasma by high-performance liquid chromatography with tandem mass spectrometry

A sensitive and specific high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) assay for the quantitative determination of gemcitabine (dFdC) and its metabolite 2',2'-difluorodeoxyuridine (dFdU) is presented. A 200-microL aliquot of human plasma was spiked with a mixture of internal standards, didanosine, lamivudine and fludarabine, and extracted using solid-phase extraction. Dried extracts were reconstituted in 1 mM ammonium acetate/acetonitrile (97:3, v/v) and 10-microL volumes were injected onto the HPLC system. Separation was achieved on a 150 x 2.1 mm C18 bonded phase endcapped with polar groups (Synergi Hydro-RP column) using an eluent composed of 1 mM ammonium acetate (pH 6.8)/acetonitrile (94:6, v/v). Detection was performed by positive ion electrospray ionization followed by MS/MS. The assay quantifies a range from 0.5 to 1000 ng/mL for gemcitabine and from 5 to 10,000 ng/mL for dFdU using 200 microL of human plasma sample. Validation results demonstrate that gemcitabine and dFdU concentrations can be accurately and precisely quantified in human plasma. This assay is used to support clinical pharmacologic studies with gemcitabine.     

Influence of thermal history on rheological properties of various bitumen

This paper focuses on the influence of thermal history on the rheological properties of unmodified and polymer modified bitumen (PMB), measured at elevated service temperatures, and contributes to the development of test methods for measuring binder properties, which can be used as indicators for asphalt rutting. It was found that the storing and preparation conditions prior to the rheological measurement can have a large influence, especially in the range of long loading times or low frequencies. For elastomer modification, the homogenization and sample pouring temperature and the corresponding change in microstructure, as revealed by fluorescence microscopy, have a large impact on the rheological measurements. For binders with semi-crystalline modifiers, the storage conditions between sample preparation and testing have the largest impact on the rheological behaviour. This can be related to variations in crystallinity, as shown by calorimetry. The main conclusion from this study is that sample preparation and handling is extremely important for the rheological properties of PMBs. Reproducibility can only be achieved when these conditions are controlled more accurately, especially in binder specification tests for rutting susceptibility.     

Early induction of binucleated cells by ultraviolet A (UVA) radiation: a possible role of microfilaments.

The effects of UVA (365 nm) radiation on the cellular distribution of F-actin and formation of binucleated cells have been studied using 3T3 Swiss albino mouse fibroblasts and V79 Chinese hamster fibroblasts. Ultraviolet A at biologically relevant fluences was found to disintegrate the actin filaments in the cells shortly (5 min) after irradiation, concomitant with the formation of cells with two nuclei. In 76-100% of the bi- and multinucleated cells the distribution of F-actin was clearly altered. Cells in GI phase of the cell cycle were most probably involved in the formation of binucleated cells. The disintegration of F-actin was presumably not due to depolymerization of F-actin to G-actin, as the amount of F-actin in the cells was unaltered after UVA exposure but rather due to direct breakage of the actin filaments. Ultraviolet B (297/302 nm) had no effect on the cellular distribution of microfilaments, not even at highly lethal fluences.     

Spin–orbit and correlation effects in platinum hydride (PtH)

The low-lying electronic states of PtH were studied by all-electron one- and two-component variational calculations on the multireference CI levels. The orbital optimization is performed within a one-component formalism, whereas the further refinement of the wave functions follows two different schemes: The most demanding approach introduces spin–orbit coupling in the CI optimization step, giving a simultaneous treatment of electron correlation and spin–orbit coupling. The second, considerably less demanding approach, corresponds almost to a perturbational treatment, introducing spin–orbit coupling as a final step after the CI optimization by diagonalizing the resulting Hamiltonian matrix over CI states. © 1998 John Wiley & Sons, Inc. Int J Quant Chem 68: 53–64, 1998     

Dual influence of lithium chloride on the anionic propagation of polystyryllithium in ethereal solvents

The addition of lithium chloride (LiCl) to a solution of polystyryllithium (PStLi) in tetrahydropyran (THP) reduces the rate of propagation of PStLi at a low concentration of the latter but accelerates it at higher concentrations of PStLi. Moreover, the addition of LiCl, which is dimeric in ethereal solutions, increases the conductance of PStLi solutions in tetrahydrofuran (THF) and THP to a much greater extent than expected from the separate conductances of PStLi and LiCl, which is itself even less dissociated than PStLi. These phenomena are fully explained by the dual action of LiCl. Below a certain concentration of PStLi, the dissociation, not of LiCl as such, as claimed before, but of its solvated dimer into free Li⁺ ions and ClLiCl⁻ triple ions provides Li⁺ ions that repress the ionic dissociation of PStLi by a common ion effect. This, in turn, diminishes the concentration of free polystyryl anions, which are the dominating species responsible for the propagation of PStLi, resulting in retardation. However, at higher concentrations of PStLi, Li⁺ ions produced by its dissociation are scavenged by the scavenging action of LiCl dimers, producing quintuple cations. This reduces the concentration of free Li⁺ ions and, therefore, increases the concentration of the reactive free polystyryl anions, resulting in an acceleration of the propagation. © 2002 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 40: 2148–2157, 2002