A simultaneous determination method for 5‐fluorouracil and its metabolites in human plasma with linear range adjusted by in‐source collision‐induced dissociation using hydrophilic interaction liquid chromatography–electrospray ionization–tandem mass spectrometry

We applied a new technique for quantitative linear range shift using in‐source collision‐induced dissociation (CID) to complex biological fluids to demonstrate its utility. The technique was used in a simultaneous quantitative determination method of 5‐fluorouracil (5‐FU), an anticancer drug for various solid tumors, and its metabolites in human plasma by liquid chromatography–electrospray ionization–tandem mass spectrometry (LC/ESI‐MS/MS). To control adverse effects after administration of 5‐FU, it is important to monitor the plasma concentration of 5‐FU and its metabolites; however, no simultaneous determination method has yet been reported because of vastly different physical and chemical properties of compounds. We developed a new analytical method for simultaneously determining 5‐FU and its metabolites in human plasma by LC/ESI‐MS/MS coupled with the technique for quantitative linear range shift using in‐source CID. Hydrophilic interaction liquid chromatography using a stationary phase with zwitterionic functional groups, phosphorylcholine, was suitable for separation of 5‐FU from its nucleoside and interfering endogenous materials. The addition of glycerin into acetonitrile‐rich eluent after LC separation improved the ESI‐MS response of high polar analytes. Based on the validation results, linear range shifts by in‐source CID is the reliable technique even with complex biological samples such as plasma. Copyright © 2016 John Wiley & Sons Ltd.     

A new high-performance liquid chromatographic/electrospray ionization tandem mass spectrometric method for the simultaneous determination of cyclophosphamide, methotrexate and 5-fluorouracil as markers of surface contamination for occupational exposure monitoring

A new high-performance liquid chromatographic/electrospray ionization tandem mass spectrometric (HPLC/ESI-MS/MS) method was developed for the simultaneous quantification of 5-fluorouracil (5FU), methotrexate (MTX) and cyclophosphamide (CP) in environmental samples. These compounds, commonly used in the treatment of cancer, are recognized as genotoxic. In order to estimate the occupational exposure of hospital personnel handling these drugs, wipe samples were taken from the working surfaces and directly analyzed (with trophosphamide as internal standard) using a reversed-phase capillary column and MS/MS detection. This is the first HPLC/MS/MS method for the simultaneous determination of 5FU, MTX and CP. The present method offers high sensitivity, with detection limits of 1.1 microg l(-1) for MTX and CP and 33.3 microg l(-1) for 5FU, avoiding any sample preconcentration procedure. Rapidity, specificity, high accuracy (mean values between 92.4 and 99.9%) and precision (mean RSD values between 3.4 and 12.1%) make the method suitable for the routine determination of these three antineoplastic drugs.     

LC‐MS/MS method for simultaneous analysis of uracil, 5,6‐dihydrouracil, 5‐fluorouracil and 5‐fluoro‐5,6‐dihydrouracil in human plasma for therapeutic drug monitoring and toxicity prediction in cancer patients

The chemotherapeutic drug 5‐fluorouracil (5‐FU) is widely used for treating solid tumors. Response to 5‐FU treatment is variable with 10–30% of patients experiencing serious toxicity partly explained by reduced activity of dihydropyrimidine dehydrogenase (DPD). DPD converts endogenous uracil (U) into 5,6‐dihydrouracil (UH₂), and analogously, 5‐FU into 5‐fluoro‐5,6‐dihydrouracil (5‐FUH₂). Combined quantification of U and UH₂ with 5‐FU and 5‐FUH₂ may provide a pre‐therapeutic assessment of DPD activity and further guide drug dosing during therapy. Here, we report the development of a liquid chromatography–tandem mass spectrometry assay for simultaneous quantification of U, UH₂, 5‐FU and 5‐FUH₂ in human plasma. Samples were prepared by liquid–liquid extraction with 10:1 ethyl acetate‐2‐propanol (v/v). The evaporated samples were reconstituted in 0.1% formic acid and 10 μL aliquots were injected into the HPLC system. Analyte separation was achieved on an Atlantis dC₁₈ column with a mobile phase consisting of 1.0 mm ammonium acetate, 0.5 mm formic acid and 3.3% methanol. Positively ionized analytes were detected by multiple reaction monitoring. The analytical response was linear in the range 0.01–10 μm for U, 0.1–10 μm for UH₂, 0.1–75 μm for 5‐FU and 0.75–75 μm for 5‐FUH₂, covering the expected concentration ranges in plasma. The method was validated following the FDA guidelines and applied to clinical samples obtained from ten 5‐FU‐treated colorectal cancer patients. The present method merges the analysis of 5‐FU pharmacokinetics and DPD activity into a single assay representing a valuable tool to improve the efficacy and safety of 5‐FU‐based chemotherapy. Copyright © 2012 John Wiley & Sons, Ltd.     

Effect of Laser Phototherapy on Enzymatic Activity of Salivary Glands of Hamsters Treated with 5‐Fluorouracil

The chemotherapeutic agent 5‐Fluorouracil (5‐FU) can induce salivary gland hypofunction (SGH); however, previous studies did not reach final conclusions on the influence of this drug on glandular tissue. Thus, the aim of this study was to investigate the effect of 5‐FU on submandibular (SMs) and sublingual glands (SLs), as well as, the effect of laser phototherapy (LPT) on SGH induced by 5‐FU. Eighty‐five hamsters were divided into three groups: control (C), chemotherapy (CT) and laser (L), and the SGH was induced by two injections of 5‐FU in groups CT and L. The irradiation was performed using a diode (λ780 nm/20 mW/5 J cm^?2/0.2 J and 10 s per point/spot size of 0.04 cm²) and applied daily. On the euthanasia day, SMs and SLs were removed and biochemical analyses were carried out. The lactate dehydrogenase activity was increased in group CT when compared with group C for SLs and SMs (P < 0.05). In addition, the peroxidase and catalase activities were increased and superoxide dismutase was decreased by 5‐FU (P < 0.05). However, LPT appears to be a protective mechanism against oxidative stress, tending to alter the activity of these antioxidant enzymes, suggesting LPT as a promising therapy to modulate the 5‐FU harmful effect.     

Effects of American ginseng on pharmacokinetics of 5‐fluorouracil in rats

The pharmacokinetics of 5‐fluorouracil (5‐FU) in combination with or without American ginseng (seven‐consecutive days oral dose) in rats were evaluated using liquid chromatography–electrospray ionization–mass spectrometry (LC‐MS). Chromatographic separation was performed on a reverse LC column within a total run time of 6.5 min, which allowed for a relatively quick analysis. The limit of quantification for 5‐FU was 15 ng/mL and this method was linear over 15–50,000 ng/mL. This method supported stabilizing determination of the plasma concentration of 5‐FU over a period of 24 h. Precision both interday and intraday (coefficient of variation) was within 14% and accuracy (relative error) ranged from ?5 to 14%. In view of the observed pharmacokinetic parameters, including maximum concentration, time to maximum concentration, area under the concentration–time curve (AUC), mean residence time, elimination half‐life and clearance, our results showed no significant differences in all of the pharmacokinetic parameters between the ginseng co‐treated group and 5‐FU alone group. Some increase in AUC was observed in 5‐FU plus ginseng group; however, the difference did not reach statistical significance compared with 5‐FU alone. It appeared that American ginseng administration did not significantly alter the kinetics of 5‐FU. More studies are still needed to confirm our results. Copyright © 2014 John Wiley & Sons, Ltd.     

Assay of urinary alpha-fluoro-beta-alanine by gas chromatography-mass spectrometry for the biological monitoring of occupational exposure to 5-fluorouracil in oncology nurses and pharmacy technicians

The validation of an analytical method for the measurement of the unnatural amino acid alpha-fluoro-beta-alanine (AFBA), the main metabolite of the antineoplastic drug 5-fluorouracil (5FU), in urine for the biological monitoring of the exposure of hospital workers to the drug when preparing the therapeutical doses and administering to cancer patients is described. The method employed a two-step extractive derivatization of the analyte from urine to the N-trifluoroacety-n-butyl ester derivative and detection by selected-ion monitoring gas chromatography-mass spectrometry of structurally specific fragments. The limit of detection was 20 ng/mL with quantification accuracy better than +/-20% and precision (CV%) better than +/-20% in the range 0.020-10 microg/mL. Norleucine was used as the internal standard and the sample-to-sample analysis time was less than 15 min. The validated method has been applied to the biological monitoring of some hospital workers potentially exposed to 5FU and to matched control subjects. On a total number of 65 analyzed urine samples from control and exposed subjects, only three, obtained from exposed subjects, were found to be positive, with values of 20, 30 and 1150 ng/mL, respectively.     

Studies of anticancer drug cytotoxicity based on long‐term HepG2 spheroid culture in a microfluidic system

Cell‐on‐a‐chip systems have become promising devices to study the effectiveness of new anticancer drugs recently. Several microdevices for liver cancer culture and evaluation of the drug cytotoxicity have been reported. However, there are still no proven reports about high‐throughput and simple methods for the evaluation of drug cytotoxicity on liver cancer cells. The paper presents the results of the effects of the anticancer drug (5‐fluorouracil, 5‐FU) on the HepG2 spheroids as a model of liver cancer. The experiments were based on the long‐term 3D spheroid culture in the microfluidic system and monitoring of the effect of 5‐FU at two selected concentrations (0.5 mM and 1.0 mM). Our investigations have shown that the initial size of the spheroids has influence on the drug effect. With the increase of the spheroids diameter, the drug resistance (for the two tested 5‐FU concentrations) decreases. This phenomenon was observed both through cells metabolism analysis, as well as changes in spheroids sizes. In our research, we have shown that the lower 5‐FU (0.5 mM) concentration causes higher decrease in HepG2 spheroids viability. Moreover, due to the microsystem construction, we observe the drug resistance effect (10th day of culture) regardless of the initial size of the created spheroids and the drug concentration.     

Interaction of glycyrrhetinic acid, furosemide and hydrochlorothiazide with bovine serum albumin and their displacement interactions: capillary electrophoresis and fluorescence quenching study

Licorice is the most widely used crude drug in traditional Chinese medicine. Glycyrrhetinic acid (GA) is the metabolite of glycyrrhizic acid, which is the main bioactive ingredient of licorice. In this work, capillary electrophoresis-frontal analysis (CE-FA) was applied to study the binding of bovine serum albumin with GA and two diuretics: furosemide (FU) and hydrochlorothiazide (HZ). The binding parameters of GA were determined by Scatchard analysis, which showed that there are two kinds of binding sites in bovine serum albumin for GA. However, the results showed that the CE-FA method was not suitable for the interaction study of FU and HZ. Therefore, utracentrifugation-CE was used to probe the binding characteristic of these two drugs and the results showed only one kind of binding site for them under the studied conditions. Displacement interactions between these drugs were also investigated by utracentrifugation-CE method and the results showed that GA hardly displaces HZ while it can slightly displace FU and FU can slightly displace HZ. For comparison, the binding of these drugs was also studied by the fluorescence quenching method and the data were processed by the Stern-Volmer quenching equation. Results showed that the binding constants were basically consistent for two methods for all drugs studied. The number of binding sites on one protein molecule was well consistent for FU and HZ while it was quite different for GA.     

Release Behavior of 5-Fluorouracil from Chitosan-Gel Nanospheres Immobilizing 5-Fluorouracil Coated with Polysaccharides and Their Cell Specific Cytotoxicity

Abstract

Small-sized chitosan-gel nanospheres, CNSs (average diameter 250 nm), containing 5-fluorouracil (5FU) or immobilizing 5FU derivatives (aminopentyl-carbamoyl-5FU or aminopentyl-ester-methylene-5FU) were prepared by the glutaraldehyde crosslinking technique and the emulsion method. When chitosan was crosslinked with glutaraldehyde, these 5FU derivatives were simultaneously immobilized to CNSs by means of Schiff's base formation. The CNSs were coated with anionic polysaccharides, such as 6-O-carboxymethyl-N-acetyl-α-l,4-polygalactosamine/Na (CM-NAPGA/Na), 6-O-carboxymethyl-chitin/Na (CM-chitin/Na), and sodium hyaluronate, through formation of a polyelectrolyte complex membrane to give CNS/polyanion, i.e., CNS/G, CNS/C, and CNS/H, respectively. The polyelectrolyte complex of polysaccharide was employed to achieve the controlled release and effective targeting of 5FU by the CNSs. The release rate of 5FU from the CNSs could be controlled by immobilization of 5FU, degree of deacetylation of chitosan used and coating with polysaccharides. Since very few galactosamine residues are known to be able to cross-react with ligands for galactose, the galactosamine residues on the surface of CNS/Gs are expected to act as the targeting moieties for hepatocyte. The CNS/G showed the lectinmediated aggregation phenomenon by the addition of APA lectin. Moreover, CNS/G had the highest cytotoxic activity among the three kinds of CNS/polyanion and CNS in HLE human hepatoma cell culture system in vitro.     

Synthesis and antitumor activity of 6-O-carboxymethyl chitin fixing 5-fluorouracils through pentamethylene, monomethylene spacer groups via amide, ester bonds.

In order to provide the water-soluble and biodegradable macromolecular prodrug of 5-fluorouracil (5FU), the fixation of 5FUs to 6-O-carboxymethyl chitin(CM-chitin) through pentamethylene, monomethylene spacer groups via amide, ester bonds was carried out. The obtained CM-chitin/5FU conjugate showed the slow release of 5FU and exhibited remarkable antitumor activity against P388 lymphocytic leukemia in mice by intraperitoneal(i.p.) implantation/i.p. injection.     

Affibody‐Conjugated RALA Polymers Delivering Oligomeric 5‐Fluorodeoxyuridine for Targeted Therapy of HER2 Overexpressing Gastric Cancer

Affibody‐conjugated RALA (affi‐RA) are designed for delivering oligomeric 5‐fluorodeoxyuridine (FUdR, metabolite of 5‐FU) strand to raise the selectivity of 5‐fluorouracil (5‐FU), decrease its toxicity and improve its suboptimal therapeutic efficacy. The nanodrugs, FUdR@affi‐RA, are spontaneously assembled by electrostatic interaction between positively charged affi‐RA and negatively charged FUdR₁₅‐strands (15 consecutive FUdR). FUdR@affi‐RA exhibits excellent stability under simulated physiological conditions. Compared with free FUdR, FUdR@affi‐RA shows excellent targeting and higher cytotoxicity in human epidermal growth factor receptor 2 (HER2) overexpressing gastric cancer N87 cells. Moreover, the anticancer mechanism studies reveal that FUdR@affi‐RA enhances the expression and activity of apoptosis‐associated proteins in the Bcl‐2/Bax‐caspase 8,9‐caspase 3 apoptotic pathway induced by FUdR. This study indicates that the fusion vector, affi‐RA, presents a promising delivery system platform for nucleoside analogue drugs and provides a new strategy for the development of therapeutics of cancer treatment.     

Determination of 5-fluorouracil in serum microsamples by a high-performance liquid chromatographic method and its application to pharmacokinetic studies in rats

A high-performance liquid chromatographic method is described for the quantitative determination of 5-fluorouracil (5FU) in only 30 microL of serum. A reversed-phase C(18) column is used for the separation and analysis of 5FU. The mobile phase consists of methanol-20mmol/L phosphate buffer, pH 6.80 (5:95, v/v). The calibration curve for 5FU is linear over the range of 0.05-10 microg/mL. The extraction recoveries of 5FU and p-aminobenzoic acid (as an internal standard) are > 91.28% and 81.98%, respectively. The intra-day and inter-day coefficients of variation of 5FU are less than 6.29% and 9.57% at four different concentrations, respectively. The method is simple, sensitive, and reliable. It is superior to previous methods in that the sample volume needed is relatively small (just 30 microL of serum). Therefore, this method can be utilized to determine 5FU in rats for study in various fields, especially in pharmacokinetics, controlled-release, and combination therapy with 5FU, etc. In this study, it is successfully applied to pharmacokinetic studies of 5FU after administrations of an intravenous bolus dose (25 mg/kg) and two oral doses (25 and 75 mg/kg) in rats.     

FTIR and circular dichroism spectroscopic study of interaction of 5-fluorouracil with DNA

5-Fluorouracil (5FU) is an anticancer chemotherapeutic drug which exerts cytotoxic effect by inhibiting cellular DNA replication. In the present study, we explore the binding of 5FU with DNA and resulting structural and conformational changes on DNA duplex. UV-visible, Fourier transform infrared (FTIR) and circular dichroism (CD) spectroscopic techniques were employed to explore these interactions. A constant concentration of calf thymus DNA was incubated with varying concentrations of 5FU. UV-visible and FTIR spectroscopic results revealed that intercalation is the primary mode of interaction between 5FU and nitrogenous bases of the nucleic acid. The binding constant was found to be 9.7×10(4); which is indicative of moderate type of interaction between 5FU and DNA duplex. It was also observed that 5FU intercalates slightly more between AT base pairs compared to GC pairs. FTIR and circular dichroism spectroscopic results revealed that 5FU disturbs native B-conformation of DNA though, DNA remains in its B conformation even at higher concentrations of 5FU.     

Syntheses of 5-Fluorouracil-Terminated Monomethqxypqly(Ethylene Glycol)S,Their Hydrolysis Behavior,and Their Antitumor Activities

The covalent attachments of 5-fluorouracil (5FU) units to poly(ethylene glycol) monomethoxy ether (MeO-PEG) attached through ester, amide, and ether bonds were carried out; three types of linkages were obtained through which 5FU units were attached to MeO-PEG. For the investigation of the release of the 5FU units, the homogeneous hydrolysis was investigated in vitro in the presence and absence of enzymes. Although the rate of release of l-β-carboxyethyl-5FU or 1-β-hydroxyethyl-5FU from the compounds was fast, the release of 5FU itself was slow. In addition, the antitumor activity of these three types of MeO-PEG-bound 5FU unit was tested in vivo by preliminary screening by the National Cancer Institute or by the Japanese Foundation for Cancer Research.     

Chemosensitization of HT29 and HT29-5FU Cell Lines by a Combination of a Multi-Tyrosine Kinase Inhibitor and 5FU Downregulates ABCC1 and Inhibits PIK3CA in Light of Their Importance in Saudi Colorectal Cancer

Colorectal cancer (CRC) remains one of the main causes of death worldwide and in Saudi Arabia. The toxicity and the development of resistance against 5 fluorouracil 5FU pose increasing therapeutic difficulties, which necessitates the development of personalized drugs and drug combinations. Objectives: First, to determine the most important kinases and kinase pathways, and the amount of ABC transporters and KRAS in samples taken from Saudi CRC patients. Second, to investigate the chemosensitizing effect of LY294002 and HAA₂₀₂₀ and their combinations with 5FU on HT29, HT29-5FU, HCT116, and HCT116-5FU CRC cells, their effect on the three ABC transporters, cell cycle, and apoptosis, in light of the important kinase pathways resulting from the first part of this study. Methods: The PamChip^® peptide micro-array profiling was used to determine the level of kinase and targets in the Saudi CRC samples. Next, RT-PCR, MTT cytotoxicity, Western blotting, perturbation of cell cycle, annexin V, and immunofluorescence assays were used to investigate the effect on CRC, MRC5, and HUVEC cells. Results: The kinase activity profiling highlighted the importance of the PI3K/AKT, MAPK, and the growth factors pathways in the Saudi CRC samples. PIK3CA was the most overexpressed, and it was associated with increased level of mutated KRAS and the three ABC transporters, especially ABCC1 in the Saudi samples. Next, combining HAA₂₀₂₀ with 5FU exhibited the best synergistic and resistance-reversal effect in the four CRC cells, and the highest selectivity indices compared to MRC5 and HUVEC normal cells. Additionally, HAA₂₀₂₀ with 5FU exerted significant inhibition of ABCC1 in the four CRC cells, and inhibition of PIK3CA/AKT/MAPK7/ERK in HT29 and HT29-5FU cells. The combination also inhibited EGFR, increased the preG₁/S cell cycle phases, apoptosis, and caspase 8 in HT29 cells, while it increased the G₁ phase, p21/p27, and apoptosis in HT29-5FU cells. Conclusion: We have combined the PamChip kinase profiling of Saudi CRC samples with in vitro drug combination studies in four CRC cells, highlighting the importance of targeting PIK3CA and ABCC1 for Saudi CRC patients, especially given that the overexpression of PIK3CA mutations was previously linked with the lack of activity for the anti-EGFRs as first line treatment for CRC patients. The combination of HAA₂₀₂₀ and 5FU has selectively sensitized the four CRC cells to 5FU and could be further studied.     

Properties of small molecular drug loading and diffusion in a fluorinated PEG hydrogel studied by 1H molecular diffusion NMR and 19F spin diffusion NMR

R(f)-PEG (fluoroalkyl double-ended poly(ethylene glycol)) hydrogel is potentially useful as a drug delivery depot due to its advanced properties of sol-gel two-phase coexistence and low surface erosion. In this study, (1)H molecular diffusion nuclear magnetic resonance (NMR) and (19)F spin diffusion NMR were used to probe the drug loading and diffusion properties of the R(f)-PEG hydrogel for small anticancer drugs, 5-fluorouracil (FU) and its hydrophobic analog, 1,3-dimethyl-5-fluorouracil (DMFU). It was found that FU has a larger apparent diffusion coefficient than that of DMFU, and the diffusion of the latter was more hindered. The result of (19)F spin diffusion NMR for the corresponding freeze-dried samples indicates that a larger portion of DMFU resided in the R(f) core/IPDU intermediate-layer region (where IPDU refers to isophorone diurethane, as a linker to interconnect the R(f) group and the PEG chain) than that of FU while the opposite is true in the PEG-water phase. To understand the experimental data, a diffusion model was proposed to include: (1) hindered diffusion of the drug molecules in the R(f) core/IPDU-intermediate-layer region; (2) relatively free diffusion of the drug molecules in the PEG-water phase (or region); and (3) diffusive exchange of the probe molecules between the above two regions. This study also shows that molecular diffusion NMR combined with spin diffusion NMR is useful in studying the drug loading and diffusion properties in hydrogels for the purpose of drug delivery applications.     

Characterization of novel pH-sensitive polymeric micelles prepared by the self-assembly of amphiphilic block copolymer with poly-4-vinylpyridine block synthesized by mechanochemical solid-state polymerization

We fabricated novel pH-sensitive polymeric micelles consisting of amphiphilic block copolymer containing pyridyl groups as side chains in the hydrophobic block. The number average particle diameter of the polymeric micelles at pH 7 was approximately 200 nm. A decrease in pH resulted in deformation of the polymeric micelles over a very narrow pH range (between pH 5.7 and 5.6). Interestingly, micellization and demicellization occurred reversibly in this narrow pH range. Polymeric micelles incorporating 5-fluorouracil (5FU) were also prepared. Decreasing the pH of this polymeric micelle solution from 7 to 5.5 resulted in the rapid release of 5FU at pH 5.6; the drug was completely released within 30 min. These results suggest that deformation of the polymeric micelles caused the rapid release of 5FU.     

A Designed 5‐Fluorouracil‐Based Bridged Silsesquioxane as an Autonomous Acid‐Triggered Drug‐Delivery System

Two new prodrugs, bearing two and three 5‐fluorouracil (5‐FU) units, respectively, have been synthesized and were shown to efficiently treat human breast cancer cells. In addition to 5‐FU, they were intended to form complexes through H‐bonds to an organo‐bridged silane prior to hydrolysis‐condensation through sol–gel processes to construct acid‐responsive bridged silsesquioxanes (BS). Whereas 5‐FU itself and the prodrug bearing two 5‐FU units completely leached out from the corresponding materials, the prodrug bearing three 5‐FU units was successfully maintained in the resulting BS. Solid‐state NMR (²⁹Si and ¹³C) spectroscopy show that the organic fragments of the organo‐bridged silane are retained in the hybrid through covalent bonding and the ¹H NMR spectroscopic analysis provides evidence for the hydrogen‐bonding interactions between the prodrug bearing three 5‐FU units and the triazine‐based hybrid matrix. The complex in the BS is not affected under neutral medium and operates under acidic conditions even under pH as high as 5 to deliver the drug as demonstrated by HPLC analysis and confirmed by FTIR and ¹³C NMR spectroscopic studies. Such functional BS are promising materials as carriers to avoid the side effects of the anticancer drug 5‐FU thanks to a controlled and targeted drug delivery.     

Trace determination of tamoxifen and 5-fluorouracil in hospital and urban wastewaters

Tamoxifen and 5-fluorouracil are widely used in cancer therapy. They are highly toxic (teratogenic, mutagenic, etc.), as are most of the anticancer drugs. Two methods were set up to analyse these drugs in wastewaters to evaluate the potential for environmental contamination by cytostatic agents. Liquid–liquid extraction followed by purification on OASIS® MCX cartridge and gas chromatography with mass spectrometry detection (GC-MS) was used for the analysis of tamoxifen. 5-Fluorouracil was extracted with an ENV+?(Isolute) cartridge (solid-phase extraction), derivatized with pentafluorobenzyl bromide (PFBBr) and detected by GC-MS. Both methods showed good recoveries (>70%), repeatability (RSD<10%) and limits of detection (LOD 6–15?ng/L). Wastewaters from a residential area, a hospital, and sewage-treatment plants (STPs) were analysed using the analytical methods developed in this study. Tamoxifen was detected in wastewaters of the hospital, residential area, and influent of STPs, but not in treated wastewaters. 5-Fluorouracil in all wastewaters was below the LOD of the analytical method.     

Blood group B galactosyltransferase: insights into substrate binding from NMR experiments

The biosynthesis of human blood group B antigens is accomplished by a highly specific galactosyltransferase (GTB). On the basis of NMR experiments, we propose a "molecular tweezers mechanism" that accounts for the exquisite stereoselectivity of donor substrate selection. Transferred NOE experiments for the first time reveal the bioactive conformation of the donor substrate UDP-galactose (UDP-Gal) and of its enzymatically inactive analogue, UDP-glucose (UDP-Glc). Both bind to GTB in a folded conformation that is sparsely populated in solution, whereas acceptor ligands bind in a conformation that predominates in solution. The bound conformations of UDP-Gal and UDP-Glc are identical within experimental error. Therefore, GTB must discriminate between the two activated sugars on the basis of a hitherto unknown transition state that can only be formed in the case of UDP-Gal. A full relaxation and exchange matrix analysis of STD NMR experiments reveals that acceptor substrates dissociate significantly faster (k(off) > 100 Hz) from the binding pocket than donor substrates (k(off) approximately 10 Hz). STD NMR experiments also directly show that proper recognition of the hexopyranose rings of the UDP sugars requires bivalent metal cations. At the same time, this analysis furnishes the complete three-dimensional structure of the enzyme with its bound donor substrate UDP-Gal on the basis of a prior crystal structure analysis. We propose that, upon acceptor binding, GTB uses the Asp 302 and Glu 303 side chains as "molecular tweezers" to promote bound UDP-Gal but not UDP-Glc into a transition state that leads to product formation.