Sep-Pak Plus Research Articles

Validation of Viral Inactivation Protocols for Therapeutic Blood Products against Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-COV-2).

Therapeutic blood products including convalescent plasma/serum and immunoglobulins concentrated from convalescent plasma, such as intravenous immunoglobulins or hyperimmune globulins, and monoclonal antibodies are passive immunotherapy options for novel coronavirus disease 2019 (COVID-19). They have been shown to improve the clinical status and biological and radiological parameters in some groups of COVID-19 patients. However, blood products are still potential sources of virus transmission in recipients. The use of pathogen reduction technology (PRT) should increase the safety of the products. The purpose of this study was to determine the impact of solvent/detergents (S/D) procedures on SARS-COV-2 infectivity elimination in the plasma of donors but also on COVID-19 convalescent serum (CCS) capacity to neutralize SARS-COV-2 infectivity. In this investigation, S/D treatment for all experiments was performed at a shortened process time (30 min). We first evaluated the impact of S/D treatments (1% TnBP/1% TritonX-45 and 1% TnBP/1% TritonX-100) on the inactivation of SARS-COV-2 pseudoparticles (SARS-COV-2pp)-spiked human plasma followed by S/D agent removal using a Sep-Pak Plus C18 cartridge. Both treatments were able to completely inactivate SARS-COV-2pp infectivity to an undetectable level. Moreover, the neutralizing activity of CCS against SARS-COV-2pp was preserved after S/D treatments. Our data suggested that viral inactivation methods using such S/D treatments could be useful in the implementation of viral inactivation/elimination processes of therapeutic blood products against SARS-COV-2.

An Efficient Automated Radiosynthesis and Bioactivity Confirmation of VMAT2 Tracer [18F]FP-(+)-DTBZ.

The aim of this study was to optimize the radiolabeling method of [18F]fluoropropyl-(+)-dihydrotetrabenazine ([18F]FP-(+)-DTBZ) to fulfill the demand of preclinical and clinical application. Optimized labeling conditions were performed by altering the molar ratio of precursor to base (P/B), base species, solvents, reaction temperature, reaction time, and precursor concentration through manual radiosynthesis of [18F]FP-(+)-DTBZ. The conditions with the highest radiochemical yield (RCY) were applied to automated radiosynthesis, and the crude product was purified with a Sep-Pak Plus C18 cartridge. Quality control and stability of [18F]FP-(+)-DTBZ were carried out by HPLC. In vitro cellular uptake and blocking assays were conducted in human neuroblastoma cell line SH-SY5Y. In vivo imaging with small animal positron emission tomography (microPET) was performed with Sprague-Dawley rats. Under the optimized conditions (P/K2CO3 = 1:8, heating at 120°C for 3min in dimethyl sulfoxide), an RCY of 88.7% was obtained with 1.0mg precursor. The optimized reaction conditions were successfully applied to an automated module and gave a high activity yield (AY) of 30-55% in about 40min with a > 99.0% radiochemical purity (RCP) and a > 44.4GBq/μmol molar activity (Am). Stability test displayed that the RCP retained > 98.0% in 8h in saline and in phosphate buffer saline (PBS, pH7.4). In vitro cellular uptake assay showed accumulation of [18F]FP-(+)-DTBZ in SH-SY5Y cells, which could be significantly inhibited by vesicular monoamine transporter 2 (VMAT2) inhibitor DTBZ. MicroPET images of rat brain displayed that the striatum showed the highest uptake with a standardized uptake value (SUV) of 3.91 ± 0.30 at ~ 70min. Co-injection with DTBZ (1.0mg/kg) resulted in a 75% decrease of the striatal SUV, confirming the specificity of [18F]FP-(+)-DTBZ to VMAT2. We obtained an optimized radiolabeling method of [18F]FP-(+)-DTBZ and successfully applied it to a commercial available module. The automated synthesis gave a high AY and RCP of [18F]FP-(+)-DTBZ with high and specific binding to VMAT2, facilitating its routine application for VMAT2 tracing.

Increased Inhibition of Angiotensin Converting Enzyme (ACE) Obtained from Indonesian Buffalo Meat Protein Using SEP-PAK Plus C18

Increased Inhibition of Angiotensin Converting Enzyme (ACE) Obtained from Indonesian Buffalo Meat Protein Using SEP-PAK Plus C18

Controlled water deficit modifies the phenolic composition and sensory properties in Cabernet Sauvignon wines

Abstract This study investigates the chemical composition and sensory properties of wines from Cabernet Sauvignon vines grown under controlled water deficit in two consecutive seasons. The wines were made from fruit of grapevines that were maintained under three water status levels (i.e. T1, Ψstem = −0.8 MPa; T2, Ψstem = −0.9 MPa; T3, Ψstem = −1.0 MPa) from veraison until harvest. Our results suggest that wine phenolic composition was affected by controlled water deficit, where T3 wines exhibited a higher concentration of total phenols, total anthocyanins and chroma (C*) in both seasons. These results coincide with the principal component analysis that indicated a substantial separation between years and deficit irrigation. We found that irrigation treatments only produce differences in concentration, but not in anthocyanin composition in both years. Separation of proanthocyanidins fractions by solid phase extraction using Sep-Pak Plus tC18 cartridges showed only a change in the concentration of the monomeric fraction in 2014 season, but not in the proportion of the different proanthocyanidins fractions in both seasons. Finally, the sensory composition of wines showed differences that depend on the season and resulted in more red fruits, more fullness perception in mouth and more color intensity in wines from less irrigated treatments.

An Automated Multidose Synthesis of the Potentiometric PET Probe 4-[18F]Fluorobenzyl-Triphenylphosphonium ([18F]FBnTP).

The aim of this study was the automated synthesis of the mitochondrial membrane potential sensor 4-[18F]fluorobenzyl-triphenylphosphonium ([18F]FBnTP) on a commercially available synthesizer in activity yields (AY) that allow for imaging of multiple patients. A three-pot, four-step synthesis was implemented on the ELIXYS FLEX/CHEM radiosynthesizer (Sofie Biosciences) and optimized for radiochemical yield (RCY), radiochemical purity (RCP) as well as chemical purity during several production runs (n=24). The compound was purified by solid-phase extraction (SPE) with a Sep-Pak Plus Accell CM cartridge, thereby avoiding HPLC purification. Under optimized conditions, AY of 1.4-2.2GBq of [18F]FBnTP were obtained from 9.4 to 12.0GBq [18F]fluoride in 90-92min (RCY=28.6±5.1% with n=3). Molar activities ranged from 80 to 99GBq/μmol at the end of synthesis. RCP of final formulations was > 99% at the end of synthesis and > 95% after 8h. With starting activities of 23.2-33.0GBq, RCY decreased to 16.1±0.4% (n=3). The main cause of the decline in RCY when high amounts of [18F]fluoride are used is radiolytic decomposition of [18F]FBnTP during SPE purification. In initial attempts, the probe was synthesized with RCY < 0.6% when starting activities up to 44.6GBq were used. Rapid radiolysis of the intermediate 4-[18F]fluorobenzaldehyde and the final product [18F]FBnTP during purification was identified as the main cause for low yields in high-activity runs. Radiolytic decomposition was hindered by the addition of radical scavengers during synthesis, purification, and formulation, thereby improving AY and RCP. The formulated probe in injectable form was synthesized without the use of HPLC and passed all applicable quality control tests.

TnBP⁄Triton X-45 treatment of plasma for transfusion efficiently inactivates hepatitis C virus.

Risk of transmission of hepatitis C virus (HCV) by clinical plasma remains high in countries with a high prevalence of hepatitis C, justifying the implementation of viral inactivation treatments. In this study, we assessed the extent of inactivation of HCV during minipool solvent/detergent (SD; 1% TnBP / 1% Triton X-45) treatment of human plasma. Luciferase-tagged infectious cell culture-derived HCV (HCVcc) particles were used to spike human plasma prior to treatment by SD at 31 ± 0.5°C for 30 min. Samples were taken before and after SD treatment and filtered on a Sep-Pak Plus C18 cartridge to remove the SD agents. Risk of cytotoxicity was assessed by XTT cell viability assay. Viral infectivity was analyzed based on the luciferase signals, 50% tissue culture infectious dose viral titer, and immunofluorescence staining for HCV NS5A protein. Total protein, cholesterol, and triglyceride contents were determined before and after SD treatment and C18 cartridge filtration. Binding analysis, using patient-derived HCV clinical isolates, was also examined to validate the efficacy of the inactivation by SD. SD treatment effectively inactivated HCVcc within 30 min, as demonstrated by the baseline level of reporter signals, total loss of viral infectivity, and absence of viral protein NS5A. SD specifically targeted HCV particles to render them inactive, with essentially no effect on plasma protein content and hemostatic function. More importantly, the efficacy of the SD inactivation method was confirmed against various genotypes of patient-derived HCV clinical isolates and against HCVcc infection of primary human hepatocytes. Therefore, treatment by 1% TnBP / 1% Triton X-45 at 31°C is highly efficient to inactivate HCV in plasma for transfusion, showing its capacity to enhance the safety of therapeutic plasma products. We propose that the methodology used here to study HCV infectivity can be valuable in the validation of viral inactivation and removal processes of human plasma-derived products.

Determination method of ultra-high-intensity sweetener, advantame, in processed foods by HPLC and LC-MS/MS

A simple method using HPLC and LC-MS/MS was developed for the determination of ultra-high-intensity sweetener, advantame, in processed foods. Advantame was extracted by dialysis, and cleaned up on a Sep-Pak Plus C18 cartridge, then determined by HPLC and LC-MS/MS. The recoveries from 5 kinds of processed foods fortified at the levels of 0.001 g/kg and 0.01 g/kg were 64.1-89.9% (RSD 0.9-6.9%) by HPLC and 68.8-99.9% (RSD 0.8-4.9%) by LC-MS/MS. The quantitation limit was 0.0004 g/kg by HPLC and 0.00004 g/kg by LC-MS/MS.

Identificación de las proteínas secretadas por el hongo Ustilago maydis (DeCandole) Corda (Basidiomiceto) cultivado en condiciones in vitro

Introducción: Ustilago maydis es un hongo basidiomiceto que infecta al maíz y teozintle produciendo una enfermedad conocida como carbón común o huitlacoche. Actualmente no existen reportes acerca del secretoma del hongo cultivado bajo condiciones in vitro. Un estudio de esta naturaleza permitiría caracterizar los genes involucrados en varios procesos importantes, entre los que se tienen aquellos relacionados con la nutrición, la patogenicidad y la diferenciación del hongo. El objetivo de esta investigación fue identificar las proteínas secretadas al medio de cultivo por las formas de levadura o micelio de este hongo cultivado en dos condiciones de pH.Método: Se generaron las formas de micelio o levadura de Ustilago maydis (cepa FB2¾a2b2) a través del cultivo en medios mínimos con pH 3 y 7 respectivamente y se determinó su cinética de crecimiento. Las proteínas secretadas al medio se concentraron en una columna de fase reversa Sep-Pak Plus C18 y se eluyeron con una solución de acetonitrilo (60 %) + ácido trifluoroacético (0.1 %), seguida de su liofilización parcial, y precipitación con ácido tricloroacético-acetona. Posteriormente las muestras fueron sometidas a electroforesis en poliacrilamida (SDS-PAGE) y los geles teñidos con azul de Coomassie. Las bandas de proteína se cortaron del gel y se digirieron con tripsina. Las mezclas de péptidos fueron inyectados para su análisis en un espectrómetro de masas y el espectro MS/MS obtenido fue procesado en Masslynx 4.0 antes de someterlo al programa MASCOT (Matrix Science) para realizar las búsquedas no-redundantes en la base de datos del National Center for Biotechnology Information.Resultados: El crecimiento de U. maydis a pH 7 fue mayor que a pH 3 (D.O. a 600 nm= 1.35 y 0.85, respectivamente) a las 30 h de incubación. El proceso dimórfico de levadura a micelio a pH 3 se inició a las 8 h después de iniciados los cultivos. A las 30 h de cultivo se observó que el 100 % de las células tenían una morfología micelial. A este tiempo, se colectó el sobrenadante y se sometió al proceso descrito anteriormente, obteniéndose de manera reproducible 8 bandas de proteína del medio mínimo del cultivo a pH 7 y 2 del medio mínimo del cultivo de pH 3. Las proteínas se analizaron, pero solo fue posible identificar 5, todas ellas provenientes del medio de pH 7, cuyas masas teóricas oscilan entre 31 y 68 kDa y presentan valores de punto isoeléctrico calculados en un rango de4.72 a10.13. Cuatro de las 5 proteínas fueron identificadas como de secreción de U. maydis. Tres de las 5 proteínas son de función desconocida, una está relacionada con el precursor de la spherulin 4 y la última parece ser una proteína GPI (glycosyl phophotidylinositol ) relacionada con una glucanasa.Discusión o Conclusión: Los resultados aquí presentados son los primeros datos descritos de proteínas secretadas al medio en condiciones in vitro por la forma de levadura de U. maydis Con este trabajo se establecieron las bases para el estudio posterior del secretoma del hongo.

PURIFICATION OF ANGIOTENSIN CONVERTING ENZYME INHIBITORY PEPTIDE DERIVED FROM KACANG GOAT MEAT PROTEIN HYDROLYSATE

The objective of this study was to identify the Angiotensin Converting Enzyme (ACE) inhibitorypeptide derived from Kacang goat meat protein hydrolysate. Kacang goat meat loin section washydrolyzed with pepsin, trypsin and chymotrypsin. Protein hydrolysate of Kacang goat meat was thentested the protein concentration and ACE inhibitory activity. ACE inhibitory peptide of the proteinhydrolysate was purified through several steps of purification by column SEP-PAK Plus C18 Cartridgeand RP-HPLC using a Cosmosil column 5PE-SM, 4.6 x 250 mm. The sequence of amino acid of ACEinhibitory peptide was identified by amino acid sequencer. The results showed that amino acidssequence of ACE inhibitory peptide derived from protein hydrolysate of Kacang goat meat was leu-thrglu-ala-pro-leu-asn-pro-lys-ala-arg- asn-glu-lys. It had a molecular weight (MW) of 1581 and occurredat the position of 20th to 33rd residues of b-actin of goat meat protein (Capra hircus). The ACE inhibitoryactivity (IC50) of the peptide was 190 mg/mL or 120 mM.

DNA Damage Processing by Human 8-Oxoguanine-DNA Glycosylase Mutants with the Occluded Active Site

8-Oxoguanine-DNA glycosylase (OGG1) removes premutagenic lesion 8-oxoguanine (8-oxo-G) from DNA and then nicks the nascent abasic (apurinic/apyrimidinic) site by β-elimination. Although the structure of OGG1 bound to damaged DNA is known, the dynamic aspects of 8-oxo-G recognition are not well understood. To comprehend the mechanisms of substrate recognition and processing, we have constructed OGG1 mutants with the active site occluded by replacement of Cys-253, which forms a wall of the base-binding pocket, with bulky leucine or isoleucine. The conformational dynamics of OGG1 mutants were characterized by single-turnover kinetics and stopped-flow kinetics with fluorescent detection. Additionally, the conformational mobility of wild type and the mutant OGG1 substrate complex was assessed using molecular dynamics simulations. Although pocket occlusion distorted the active site and greatly decreased the catalytic activity of OGG1, it did not fully prevent processing of 8-oxo-G and apurinic/apyrimidinic sites. Both mutants were notably stimulated in the presence of free 8-bromoguanine, indicating that this base can bind to the distorted OGG1 and facilitate β-elimination. The results agree with the concept of enzyme plasticity, suggesting that the active site of OGG1 is flexible enough to compensate partially for distortions caused by mutation.

Synthesis and Radiosynthesis of a Novel PET Fluorobenzyl Piperazine for Melanoma Tumour Imaging; [18F]MEL054

2-{2-[4-(4-[18F]-Fluorobenzyl)piperazin-1-yl]-2-oxoethyl}isoindolin-1-one ([18F]MEL054), is a new potent indolinone-based melanin binder designed to target melanotic tumours. [18F]MEL054 was prepared by an automated two-step radiosynthesis, comprising of the preparation of 4-[18F]fluorobenzaldehyde from 4-formyl-N,N,N-trimethylanilinium triflate, followed by reductive alkylation with 2-(2-oxo-2-piperazin-1-ylethyl)isoindolin-1-one. 4-[18F]Fluorobenzaldehyde was prepared on a GE TRACERlab FXFN module in 68 ± 8 % radiochemical yield (RCY, non-decay corrected), purified by a Sep-Pak Plus C18 cartridge and eluted into the reactor of an in-house modified Nuclear Interface [18F]FDG synthesis module for the subsequent reductive alkylation reaction. HPLC purification produced [18F]MEL054 in a collected RCY of 34 ± 9 % (non-decay corrected), the total preparation time (including Sep-Pak Plus C18 and HPLC purification) did not exceed 105 min. The radiochemical purity of [18F]MEL054 was greater than 99 % with a specific radioactivity of 71–119 GBq μmol–1 and [18F]MEL054 remained stable in saline solution (&gt;98 %) after 3 h.

Trace analysis of sulfonylurea herbicides in water samples by solid-phase extraction and liquid chromatography-tandem mass spectrometry

A sensitive method for the simultaneous determination of 30 sulfonylurea herbicides in tap and leaching waters has been developed. Liquid chromatography tandem-mass spectrometry (LC-MS2) in electrospray ionization positive mode was used for the separation, identification and quantification of these compounds. The procedure involves a preconcentration step based on solid-phase extraction with a silica-based bonded C18 cartridge (Sep-Pak Plus) and a N-vinyl-pyrrolidone polymer cartridge (Oasis HLB). The best results were obtained with Oasis HLB using methanol as elution solvent. Average recoveries of 30 analytes from water samples were in the range of 79–115% with a relative standard deviation of <6.1%. The limits of quantification (LOQs) obtained in tap and leaching water samples were in the range of 0.1–5.9 and 0.4–5.8ngL−1, respectively. The proposed method was used to determine sulfonylurea herbicide levels in leaching water samples taken from three lysimeters located in an experimental greenhouse.

Simple synthesis of carbon-11-labeled chromen-4-one derivatives as new potential PET agents for imaging of DNA-dependent protein kinase (DNA-PK) in cancer

Carbon-11-labeled chromen-4-one derivatives were synthesized as new potential PET agents for imaging of DNA repair enzyme DNA-dependent protein kinase (DNA-PK) in cancer. The target tracers, X-[11C]methoxy-2-morpholino-4H-chromen-4-ones (X=8, 7, 6, 5; [11C]4a–d), were prepared from their corresponding precursors, X-hydroxy-2-morpholino-4H-chromen-4-ones (X=8, 7, 6, 5; 5a–d), with [11C]CH3OTf through O-[11C]methylation and isolated by a simplified solid-phase extraction (SPE) method using a C-18 Sep-Pak Plus cartridge. The radiochemical yields decay corrected to end of bombardment (EOB), from [11C]CO2, were 40–60%. The specific activity at end of synthesis (EOS) was 185–370GBq/μmol.

An automated SPE-based high-yield synthesis of [11C]acetate and [11C]palmitate: no liquid–liquid extraction, solvent evaporation or distillation required

An automated method is described for the rapid and high-yield synthesis of two of the most commonly used radioactive fatty acids: [(11)C]acetate and [(11)C]palmitate. Reaction of [(11)C]CO(2) with the respective Grignard reagents in diethyl ether solution proceeded for 2 min at 40°C. Quenching of the reaction and liberation of nonreacted [(11)C]CO(2) occurred upon addition of a fourfold molar excess of aqueous 0.1 M HCl (acetate) or nonaqueous HCl/Et(2)O (palmitate). Labeled products were then purified by adsorption to an Alumina-N Sep-Pak Plus cartridge and eluted with either aqueous NaH(2)PO(4) solution (acetate) or 100% ethanol (palmitate). High-performance liquid chromatography analysis confirmed that the radiochemical purity of each product was >98%, and decay-corrected radiochemical yields averaged 33% for [(11)C]palmitate and 40% for [(11)C]acetate. The method requires no liquid-liquid extraction, solvent evaporation or distillation capabilities and can be readily adapted to existing radiosynthesis modules.

Synthesis of Long-Chain [18F]Deoxyfluoropoly(ethylene glycol) Methyl Ethers and Their Noninvasive Pharmacokinetic Analysis by Positron Emission Tomography

[¹⁸F]DeoxyfluoroPEG methyl ethers with an average molecular weight of 2 kDa ([¹⁸F]1a) and 10 kDa ([¹⁸F]1b) were synthesized by the fluorination of the tosylates 3a,b with [¹⁸F]nBu₄NF at 80°C for 20 min followed by flash filtration through a Sep-Pak Plus Alumina-N cartridge. After the intravenous administration of [¹⁸F]1a and [¹⁸F]1b to rats, their pharmacokinetics was analyzed by noninvasive, real-time, whole-living-body monitoring using positron imaging technology. The effect of PEG's molecular weight on their blood circulation and organ clearance were quantitatively visualized for the first time.

Biochemical Conservation and Evolution of Germacrene A Oxidase in Asteraceae

Sesquiterpene lactones are characteristic natural products in Asteraceae, which constitutes approximately 8% of all plant species. Despite their physiological and pharmaceutical importance, the biochemistry and evolution of sesquiterpene lactones remain unexplored. Here we show that germacrene A oxidase (GAO), evolutionarily conserved in all major subfamilies of Asteraceae, catalyzes three consecutive oxidations of germacrene A to yield germacrene A acid. Furthermore, it is also capable of oxidizing non-natural substrate amorphadiene. Co-expression of lettuce GAO with germacrene synthase in engineered yeast synthesized aberrant products, costic acids and ilicic acid, in an acidic condition. However, cultivation in a neutral condition allowed the de novo synthesis of a single novel compound that was identified as germacrene A acid by gas and liquid chromatography and NMR analyses. To trace the evolutionary lineage of GAO in Asteraceae, homologous genes were further isolated from the representative species of three major subfamilies of Asteraceae (sunflower, chicory, and costus from Asteroideae, Cichorioideae, and Carduoideae, respectively) and also from the phylogenetically basal species, Barnadesia spinosa, from Barnadesioideae. The recombinant GAOs from these genes clearly showed germacrene A oxidase activities, suggesting that GAO activity is widely conserved in Asteraceae including the basal lineage. All GAOs could catalyze the three-step oxidation of non-natural substrate amorphadiene to artemisinic acid, whereas amorphadiene oxidase diverged from GAO displayed negligible activity for germacrene A oxidation. The observed amorphadiene oxidase activity in GAOs suggests that the catalytic plasticity is embedded in ancestral GAO enzymes that may contribute to the chemical and catalytic diversity in nature.

Structural Identification and Antioxidant Properties of Major Anthocyanin Extracted from Omija (Schizandra chinensis) Fruit

Omija (Schizandra chinensis) is used as an ingredient in traditional medicine in East Asia. It is consumed as tea and wine and display pinkish-red color and beneficial physiological activity. However, the origin of Omija's unique color and bioactivity has not been studied extensively and its application is very limited. Thus, it was required to determine the chemical structure of major phenolic compounds of Omija fruit and evaluate their antioxidant activity. The colorants extracted from a domestic Omija cultivar were concentrated by a Sep-pak(R) Plus C(18) cartridge. A major high-performance liquid chromatography (HPLC) peak of anthocyan represented 94.1% of total absorbable compounds at 520 nm, which was further identified by LC-ESI-MS. The mass-to-charge ratio (m/z) of the major anthocyan was determined to be 727. Highly pure anthocyan fraction with a semipreparative HPLC was acid-hydrolyzed, and the sugar moieties linked to anthocyan (cyanidin) were characterized by thin layer chromatography (TLC) and high-performance anion exchange chromatography (HPAEC) analyses. The linkage patterns of sugars and core cyanidin structure were determined by (1)H- and (13)C-NMR analyses. Antioxidant activity of the extract and the purified anthocyanin was evaluated by 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) methods. As a result, the structure of the purified colorant was identified as Cya-3-O-xylrut. At the same molar level of the samples tested, the purified Cya-3-O-xylrut (31.2% and 39.2%) had substantially greater antioxidant activity than l-ascorbic acid (17.1% and 10.1%) from DPPH and ABTS methods, respectively. In this study, Omija colorant mostly consisted of Cya-3-O-xylrut explained 86% (DPPH) and 98% (ABTS) of total antioxidant activity derived from water extract from Omija.

Cucurbitacin D isolated from Trichosanthes kirilowii induces apoptosis in human hepatocellular carcinoma cells in vitro

The aim of the present study is to examine the effects of the anti-tumor component isolated from Trichosanthes kirilowii on human hepatocellular carcinoma cells. Using Sephadex G-25 column chromatography, Sep-Pak Plus C18 cartridge and high-performance liquid chromatography (HPLC), we isolated the active component from trichosanthes extract. By fast atom bombardment mass spectrometric analysis, the molecular mass of the active fraction was determined, the active components identified, and their mechanisms of action were analyzed by cell growth assay, cell cycle analysis, TUNEL staining and Western blot analysis. We found that the anti-tumor components isolated from the extract of trichosanthes (EOT) are cucurbitacin D and dihydrocucurbitacin D, and suggest that cucurbitacin D induces apoptosis through caspase-3 and phosphorylation of JNK in hepatocellular carcinoma cells. These results suggest that cucurbitacin D isolated from Trichosanthes kirilowii could be a valuable candidate for anti-tumor drug.

Determination of Hexaconazole in Surface Water Samples from River and the Sea by Liquid Chromatography-Electrospray Tandem Mass Spectrometry

A simple and selective method was developed for determining the concentration of hexaconazole in river and sea water samples by using liquid chromatography/tandem mass spectrometry with an electrospray ionization interface in the positive ion mode and selective reaction monitoring mode. Trace amounts of hexaconazole were collected in a Sep-Pak Plus tC18 cartridge that was eluted with methanol. The detection limit for hexaconazole was 6 ng/l. The recovery of a standard aqueous solution containing 1 microg/l was 96%. The recovery of hexaconazole in the river and sea water samples was 95% and 90%, respectively. Hexaconazole was not detected in the sea water samples. Trace peaks of hexaconazole were found in the river water samples, the concentration being less than 6 ng/l in all cases. The biological degradation of hexaconazole was tested by using river water. No degradation of hexaconazole was apparent in river water incubated at 20 degrees C for 3 weeks.

ＧＣ／ＭＳによる魚介類中の農薬一斉分析法の検討

A simple pretreatment of sample and comprehensive analysis method has been developed for the GC/MS determination of 29 pesticides in fish and shellfish. For the pretreatment, the pesticides in the sample were extracted with acetonitrile and the lipid in the sample extract was eliminated by partitioning between hexane and acetonitrile. After Sep-Pak Plus Florisil or Florisil PR column clean-up, the sample extract was analyzed by GC/MS (SIM). Recovery tests were performed by use of Sep-Pak Plus Florisil and Florisil PR for fish and shellfish. On the whole, recoveries (%) and CV (%) by Florisil PR were better than those by Sep-pak Plus Florisil in both of Japanese smelt (fish) and corbicula (shellfish). However, Sep-Pak Plus Florisil had better results in pyrokiron, fenthion, etc., so selection of clean-up procedure in agreement with the purpose was recommended. The quantification limits were 0.002∼0.005μg/g for most of the 29 pesticides.