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Calibration Range (calibration + range)
Kinds of Calibration Range Selected AbstractsSimultaneous quantification of 2,,2,-difluorodeoxycytidine and 2,,2,-difluorodeoxyuridine nucleosides and nucleotides in white blood cells using porous graphitic carbon chromatography coupled with tandem mass spectrometryRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 19 2009Robert S. Jansen A novel assay for the simultaneous quantification of the widely used anticancer agent 2,,2,-difluorodeoxycytidine (gemcitabine; dFdC), its deaminated metabolite 2,,2,-difluorodeoxyuridine (dFdU) and their mono-, di- and triphosphates (dFdCMP, dFdCDP, dFdCTP, dFdUMP, dFdUDP and dFdUTP) in peripheral blood mononuclear cells (PBMCs) is described. Separation of all eight compounds was achieved within 15,min using a porous graphitic carbon column (Hypercarb) with a gradient from 0 to 25,mM ammonium bicarbonate in acetonitrile/water (15:85, v/v). Calibration ranges in PBMC lysate from 4.29 to 429, 29.0 to 2900, 31.4 to 3140 and 36.9 to 3690,nM for dFdC, dFdCMP, dFdCDP and dFdCTP and from 42.1 to 4210, 25.4 to 2540, 43.2 to 4320 and 52.7 to 5270,nM for dFdU, dFdUMP, dFdUDP and dFdUTP, respectively, were validated. Accuracies were within 82.3,119% at the lower limit of quantification (LLOQ) and the precisions were less than 20.0%. At the other tested levels accuracies were within 91.4,114% and precisions less than 14.9%. Mixtures of 13C,15N2 -labeled dFdC and dFdU nucleotides were synthesized and used as internal standards. Whole blood samples showed extensive ongoing dFdC metabolism when stored at room temperature, but not on ice-water, which made the addition of enzyme inhibitors unnecessary. Stock solutions and samples were stable under all analytically relevant conditions. The method was successfully applied to clinical samples. Copyright © 2009 John Wiley & Sons, Ltd. [source] Barrel Plating Rhodium Electrode: Application to Flow Injection Analysis of HydrazineELECTROANALYSIS, Issue 14 2005Jun-Wei Sue Abstract We introduce here the application of barrel plating technology for mass production of disposable-type electrodes. Easy for mass production, barrel plating rhodium electrode (Rh-BPE) is for the first time demonstrated for analytical application. Hydrazine was chosen as a model analyte to elucidate the electrocatalytic and analytical ability of the Rh-BPE system in pH,7 phosphate buffer solution. Flow injection analysis (FIA) of hydrazine showed a linear calibration range of 25,1000,ppb with a slope and a regression coefficient of 5,nA/ppb and 0.9946, respectively. Twenty-two replicate injections of 25,ppb hydrazine showed a relative standard deviation of 3.17% indicating a detection limit (S/N=3) of 2.5,ppb. The system can be continuously operated for 1 day without any alteration in the FIA signals and is tolerable to the interference of oxalic acid, gelatine, Triton X-100, and albumin for even up to 100 times excess in concentration with respect to 400,ppb hydrazine. Since the fabrication cost of the electrode is cheap, it is thus disposable in nature. Furthermore, barrel plating technique can be extendable to other transition metals for application in many fields of research interest. [source] On-line biosensors for simultaneous determination of glucose, choline, and glutamate integrated with a microseparation systemELECTROPHORESIS, Issue 18 2003Guoyue Shi Abstract An effective microseparation system integrated with ring-disc electrodes and two microfluidic devices was fabricated for in vivo determination using a microdialysis pump. The major interference of ascorbic acid (AA) was excluded by direct oxidation with ascorbate oxidase. Glucose, glutamate, and choline were successfully determined simultaneously through the biosensors modified with a bilayer of osmium-poly(4-vinylpyridine)gel-horseradish peroxidase (Os-gel-HRP)/glucose oxidase (GOD), glutamate oxidase (GlutaOD) or choline oxidase (ChOD). To stabilize the biosensors, 0.2% polyethylenimine (PEI) was mixed with the oxidases. The cathodic currents of glucose, glutamate, and choline biosensors started to increase after the standard solutions were injected into the microseparation system. The on-line biosensors show a wide calibration range (10,7,10,5 mol/L) with a detection limit of 10,8 mol/L at the working potential of ,50 mV. The variations of glucose, glutamate, and choline were determined simultaneously in a free moving rat when we perfused the medial frontal cortex with 100 ,mol/L N -methyl- D -aspartate (NMDA) solution, which is the agonist of the NMDA receptor. [source] Selective detection of superoxide anion radicals generated from macrophages by using a novel fluorescent probeFEBS JOURNAL, Issue 7 2007Jing Jing Gao Quantitation of superoxide radical (O,2,·) production at the site of radical generation remains challenging. A simple method to detect nanomolar to micromolar levels of superoxide radical in aqueous solution has been developed and optimized. This method is based on the efficient trapping of O2,· using a novel fluorescent probe (2-chloro-1,3-dibenzothiazolinecyclohexene), coupled with a spectra character-signaling increase event. A high-specificity and high-sensitivity fluorescent probe was synthesized in-house and used to image O2,· in living cells. Better selectivity for O2,· over competing cellular reactive oxygen species and some biological compounds illustrates the advantages of our method. Under optimal conditions, the linear calibration range for superoxide anion radicals was 5.03 × 10,9,3.33 × 10,6 m. The detection limit was 1.68 × 10,9 m. Fluorescence images of probe-stained macrophages stimulated with 4,-phorbol 12-myristate 13-acetate were obtained successfully using a confocal laser scanning microscope. [source] Conjugated Polyelectrolytes as Light-Up Macromolecular Probes for Heparin SensingADVANCED FUNCTIONAL MATERIALS, Issue 2 2009Kan-Yi Pu Abstract Two cationic poly(fluorene- alt -benzothiadiazole)s with different side chains are designed and synthesized. Both polymers show low fluorescence in aqueous solution due to the charge-transfer character of the polymer's excited states. Fluorescence turn-on biosensors for heparin detection and quantification are developed, taking advantage of complexation-induced aggregation, which increases the polymer fluorescence in aqueous solution. It is found that good polymer water-solubility is beneficial to the sensitivity and fluorescence contrast of the heparin turn-on sensor as a result of the low fluorescence background. Moreover, stronger complexation between the polymer/heparin leads to a substantially larger fluorescence increase in the presence of heparin relative to that in the presence of its analog, hyaluronic acid (HA), allowing discrimination of heparin from HA. Heparin quantification with a practical calibration range covering the whole therapeutic dosing levels (0.2,8 U mL,1) is realized based on the polymer with good water-solubility. This investigation provides a new insight for designing conjugated polymers with a light-up signature for biomolecular sensing. [source] Raman spectroscopy for spinline crystallinity measurements.JOURNAL OF APPLIED POLYMER SCIENCE, Issue 5 2008Abstract Online Raman spectra, obtained at different points along the spinline during the melt spinning of polypropylene homopolymer (hPP) fibers, are presented. The percentage crystallinity corresponding to each spectrum was determined from the normalized intensity of the 809-cm,1 Raman band. A calibration curve for propylene crystallinity was established offline with compression-molded films and fibers spun under different processing conditions. Several hPPs and propylene,ethylene copolymers (with 5,15% ethylene) were used to cover a wide calibration range for propylene crystallinity (9.5,60.9%) with an R2 value of 0.989. This calibration curve was subsequently used to predict the polypropylene crystallinity in the spinline as a function of distance from the spinneret. Under identical conditions of quench and throughput, at a fixed point along the spinline, the overall crystallinity developed in the fiber was found to increase with an increase in the spinning speed. As the spinning speed increased, the point of the onset of crystallization moved closer to the spinneret. The rise in crystallinity was more gradual, at 750 m/min as opposed to 1500 m/min. Increasing the throughput at constant spinning speed was found to decrease the rate of crystallization because of a decrease in the spinline stress. At a fixed distance from the spinneret under identical conditions of quench and spinning speed, fibers spun at a higher throughput showed less overall crystallinity. The onset and rate of crystallization was found to be faster in the lower melt index H502-25RG resin as compared to the 5D49 resin under the spinning conditions explored. The experimental data presented here were used to validate fundamental fiber-spinning models (see part II of this series of articles). The validated models and experimental observations can be used to guide the fiber spinning of isotactic polypropylene for rapid product development. © 2008 Wiley Periodicals, Inc. J Appl Polym Sci, 2008 [source] PRELIMINARY EVALUATION OF THE APPLICATION OF THE FTIR SPECTROSCOPY TO CONTROL THE GEOGRAPHIC ORIGIN AND QUALITY OF VIRGIN OLIVE OILSJOURNAL OF FOOD QUALITY, Issue 4 2007ALESSANDRA BENDINI ABSTRACT A rapid Fourier transform infrared (FTIR) attenuated total reflectance spectroscopic method was applied to determine qualitative parameters such as free fatty acid (FFA) content and the peroxide value (POV) in virgin olive oils. Calibration models were constructed using partial least squares regression on a large number of virgin olive oil samples. The best results (R2 = 0.955, root mean square error in cross validation [RMSECV] = 0.15) to evaluate FFA content expressed in oleic acid % (w/w) were obtained considering a calibration range from 0.2 to 9.2% of FFA relative to 190 samples. For POV determination, the result obtained, built on 80 olive oil samples with a calibration range from 11.1 to 49.7 meq O2/kg of oil, was not satisfactory (R2 = 0.855, RMSECV = 3.96). We also investigated the capability of FTIR spectroscopy, in combination with multivariate analysis, to distinguish virgin olive oils based on geographic origin. The spectra of 84 monovarietal virgin olive oil samples from eight Italian regions were collected and elaborated by principal component analysis (PCA), considering the fingerprint region. The results were satisfactory and could successfully discriminate the majority of samples coming from the Emilia Romagna, Sardinian and Sicilian regions. Moreover, the explained variance from this PCA was higher than 96%. PRACTICAL APPLICATIONS The verification of the declared origin or the determination of the origin of an unidentified virgin olive oil is a challenging problem. In this work, we have studied the applicability of Fourier transform infrared coupled with multivariate statistical analysis to discriminate the geographic origin of virgin olive oil samples from different Italian regions. [source] Quantification of fudosteine in human plasma by high-performance liquid chromatography-electrospray ionization mass spectrometry employing precolumn derivatization with 9-fluorenylmethyl chloroformateJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 5 2006Fengguo Xu Abstract This paper describes a novel method for the sensitive and selective determination of fudosteine in human plasma. The method involves a derivatization step with 9-fluorenylmethyl chloroformate (FMOC-Cl) in borate buffer and detection based on high-performance liquid chromatography-electrospray ionization mass spectrometry (LC/ESI/MS). After acetonitrile-induced protein precipitation of plasma samples, fudosteine was derivatized with FMOC-Cl, then extracted by ethyl acetate, evaporated, reconstituted and injected using an LC/ESI/MS instrument. Separation was achieved using an ODS column and isocratic elution. Excellent linearity was obtained for the entire calibration range from 0.05 to 20 µg/ml. Validation assays of the lower limit of quantification (LLOQ) as well as for the intra- and inter-batch precision and accuracy met the international acceptance criteria for bioanalytical method validation. Using the developed analytical method, fudosteine could be detected for the first time in human plasma with a low limit of detection (LLOD) of 0.03 µg/ml. The proposed method has been successfully applied to study the pharmacokinetics of fudosteine in healthy Chinese volunteers after single and multiple oral administration. Copyright © 2006 John Wiley & Sons, Ltd. [source] On-line 2D-LC-ESI/MS/MS determination of rifaximin in rat serumBIOMEDICAL CHROMATOGRAPHY, Issue 11 2009R. Nageswara Rao Abstract A highly sensitive and selective on-line two-dimensional reversed-phase liquid chromatography/electrospray ionization,tandem mass spectrometry (2D-LC-ESI/MS/MS) method was developed and validated to determine rifaximin in rat serum by direct injection. The 2D-LC-ESI/MS/MS system consisted of a restricted access media column for trapping proteins as the first dimension and a Waters C18 column as second dimension using 0.1% aqueous acetic acid:acetonitrile as mobile phase in a gradient elution mode. Rifampacin was used as an internal standard. The linear dynamic range was 0.5,10 ng/mL (r2 > 0.998). Acceptable precision and accuracy were obtained over the calibration range. The assay was successfully used in analysis of rat serum to support pharmacokinetic studies. Copyright © 2009 John Wiley & Sons, Ltd. [source] Automated analysis of fluvoxamine in rat plasma using a column-switching system and ion-pair high-performance liquid chromatographyBIOMEDICAL CHROMATOGRAPHY, Issue 12 2008Shicheng Liu Abstract We have established a robust, fully automated analytical method for the analysis of fluvoxamine in rat plasma using a column-switching ion-pair high-performance chromatography system. The plasma sample was injected onto a precolumn packed with Shim-pack MAYI-ODS (50 µm), where the drug was automatically purified and enriched by on-line solid-phase extraction. After elution of the plasma proteins, the analyte was back-flushed from the precolumn and then separated isocratically on a reversed-phase C18 column (L-column ODS) with a mobile phase (acetonitrile,0.1% phosphoric acid, 36:64, v/v) containing 2 mm sodium 1-octanesulfonate. The analyte was monitored by a UV detector at a wavelength of 254 nm. The calibration line for fluvoxamine showed good linearity in the range of 5,5000 ng/mL (r > 0.999) with the limit of quantification of 5 ng/mL (RSD = 6.51%). Accuracy ranged from ,2.94 to 4.82%, and the within- and between-day precision of the assay was better than 8% across the calibration range. The analytical sensitivity and accuracy of this assay is suitable for characterization of the pharmacokinetics of orally-administered fluvoxamine in rats. Copyright © 2008 John Wiley & Sons, Ltd. [source] Optimization and validation of RP-HPLC-UV method with solid-phase extraction for determination of buparvaquone in human and rabbit plasma: application to pharmacokinetic studyBIOMEDICAL CHROMATOGRAPHY, Issue 5 2008Gantala Venkatesh Abstract A simple, sensitive and specific reversed-phase high-performance liquid chromatographic method with UV detection at 251 nm was developed for quantitation of buparvaquone (BPQ) in human and rabbit plasma. The method utilizes 250 µL of plasma and sample preparation involves protein precipitation followed by solid-phase extraction. The method was validated on a C18 column with mobile phase consisting of ammonium acetate buffer (0.02 m, pH 3.0) and acetonitrile in the ratio of 18:82 (v/v) at a flow rate of 1.1 mL/min. The calibration curves were linear (correlation coefficient ,0.998) in the selected range. The method is specific and sensitive with limit of quantitation of 50 ng/mL for BPQ. The validated method was found to be accurate and precise in the working calibration range. Stability studies were carried out at different storage conditions and BPQ was found to be stable. Partial validation studies were carried out using rabbit plasma and intra- and inter-day precision and accuracy were within 7%. This method is simple, reliable and can be routinely used for preclinical pharmacokinetic studies for BPQ. Copyright © 2007 John Wiley & Sons, Ltd. [source] The effect of alkylamine additives on the sensitivity of detection for paclitaxel and docetaxel and analysis in plasma of paclitaxel by liquid chromatography,tandem mass spectrometryBIOMEDICAL CHROMATOGRAPHY, Issue 8 2006Songmei Gao Abstract The formation of multiple molecular ions, especially due to sodium adduct ion formation, is commonly observed in electrospray mass spectrometry and may make reproducible and sensitive quantitation difficult. The objective of this work was to investigate the underlying mechanism involved in the suppression of multiple molecular ion formation and to improve the sensitivity of detection for the two anti-neoplastic agents paclitaxel and docetaxel. The results showed that alkylamine additives could significantly improve the detection of paclitaxel and docetaxel by suppression of multiple molecular ions through preferential formation of a predominant alkylamine adduct ion. Possible binding sites, binding interactions and binding competition were investigated for the sodium adduct and alkylamine adduct ions using various experimental techniques. The formation of a predominant amine adduct ion may be due to increased surface activity in the droplet. The optimal alkylamine for both analytes was octylamine, which increased peak heights of paclitaxel and docetaxel 4.8 and 3.7-fold (n = 3), respectively. The precision of the signals for the analytes was also improved 5.7-fold. A quantitative assay in plasma for paclitaxel was partially validated for the calibration range 1.0,1000 ng/mL (r = 0.9977) when using 0.05% octylamine as a reconstitution solution additive. The limit of detection (LOD) and limit of quantitation (LOQ) were 0.5 and 0.9 ng/mL, respectively. Acceptable precision, accuracy, specificity and sample stability were demonstrated for this assay. This approach may prove useful for other analytes with similar binding sites. Copyright © 2005 John Wiley & Sons, Ltd. [source] Therapeutic monitoring of imipramine and desipramine by micellar liquid chromatography with direct injection and electrochemical detectionBIOMEDICAL CHROMATOGRAPHY, Issue 5 2005Devasish Bose Abstract A micellar liquid chromatographic (MLC) procedure was developed for the clinical monitoring of imipramine and its active metabolite, desipramine. The determination of these highly hydrophobic substances was carried out after direct injection of the serum samples using a mobile phase composed of 0.15 m SDS,6% (v/v) pentanol buffered at pH 7, pumped at 1.5 mL/min into a C18 column (250 × 4.6 mm), and electrochemical detection at 650 mV. Using this MLC method, calibration was linear (r > 0.995) and the limits of detection (ng/mL) were 0.34 and 0.24 for imipramine and desipramine, respectively. Repeatabilities and intermediate precision were tested at three different concentrations in the calibration range and a CV (%) below 2.2 was obtained. In this MLC procedure, the serum is determined without treatment, thus allowing repeated serial injections without changes in retention factors, and reducing the time and consumables required to carry out the pretreatment process. The assay method can be applied to the routine determination of serum imipramine and its metabolite in therapeutic drug monitoring. Copyright © 2004 John Wiley & Sons, Ltd. [source] Direct Simultaneous Determination of ,- and ,-Naphthol Isomers at GC-Electrode Modified with CNTs Network Joined by Pt Nanoparticles Through Derivative VoltammetryELECTROANALYSIS, Issue 5 2006Xiao-Gang Wang Abstract The semi-derivative technique was adopted to improve the resolution and surfactant was added to sample solution to enhance the sensitivity, , - and , -naphthol isomers could be determined directly and simultaneously at glassy carbon electrode modified with carbon nanotubes network joined by Pt nanoparticles. In 0.1,mol,L,1 HAc-NaAc buffer solution (pH,5.8), the linear calibration ranges were 1.0×10,6 to 8.0×10,4 mol,L,1 for both , - and , -naphthols, with detection limits of 5.0×10,7 for , - and 6.0×10,7,mol,L,1 for , -naphthol. The amount of naphthol isomers in artificial wastewater has been tested with above method, and the recovery was from 98% to 103%. [source] Assay of vitamin B in urine by capillary electrochromatography with methacrylate-based monolithic columnELECTROPHORESIS, Issue 19 2010Xiaoyi Wei Abstract A novel and simple method for the separation of major vitamin B analytes, such as thiamine, riboflavin, nicotinamide, vitamin B4, pyridoxine, has been developed by CEC using the monolithic column. It has been found that the baseline separation of the five analytes could be achieved with 5.0,mM phosphate buffer at pH 4.0. Compared with the open-tubular capillary and the bared capillary columns, the poly(butylmethacrylate-co-ethylene glycol dimethacrylate) monolithic capillary could exhibit the best resolution in the analysis. Then the method was validated and the linear calibration ranges were obtained with correlation coefficients more than 0.997. The precision and the recovery were also investigated and showed a good result. Furthermore, the proposed method was successfully applied to assay the concentration of vitamin B analytes and the metabolic situation in human urine samples. [source] Analysis of low content drug tablets by transmission near infrared spectroscopy: Selection of calibration ranges according to multivariate detection and quantitation limits of PLS modelsJOURNAL OF PHARMACEUTICAL SCIENCES, Issue 12 2008Manel Alcalą Abstract The content uniformity of low dose products is a major concern in the development of pharmaceutical formulations. Near infrared spectroscopy may be used to support the design and optimization of potent drug manufacturing processes through the analysis of blends and tablets in a relatively short time. A strategy for the selection of concentration ranges in the development of multivariate calibration is presented, evaluating the detection and quantitation limits of the obtained multivariate models. The strategy has been applied to the determination of an active principle in pharmaceutical tablets of low concentration (0,5%, w/w), using Fourier Transform Near Infrared (FT-NIR) transmission spectroscopy. The quantitation and detection limits decreased as the upper concentration level of the calibration models was reduced. The results obtained show that the selection of concentration ranges is a critical aspect during model design. The selection of wide concentration ranges with high levels is not recommended for the determination of analytes at minor levels (<1%, w/w), even when the concentration of interest is within the range of the model. © 2008 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 97:5318,5327, 2008 [source] Determination of avoparcin in animal tissues and milk using LC-ESI-MS/MS and tandem-SPEJOURNAL OF SEPARATION SCIENCE, JSS, Issue 22 2008Koichi Inoue Abstract A highly sensitive and selective method using LC-ESI-MS/MS and tandem-SPE was developed to detect trace amounts of avoparcin (AV) antibiotics in animal tissues and milk. Data acquisition using MS/MS was achieved by applying multiple reaction monitoring of the product ions of [M + 3H]3+ and the major product ions of AV-, and -, at m/z 637 , 86/113/130 and m/z 649 , 86/113/130 in ESI(+) mode. The calculated instrumental LODs were 3 ng/mL. The sample preparation was described that the extraction using 5% TFA and the tandem-SPE with an ion-exchange (SAX) and InertSep C18-A cartridge clean-up enable us to determine AV in samples. Ion suppression was decreased by concentration rates of each sample solution. These SPE concentration levels could be used to detect quantities of 5 ppb (milk), 10 ppb (beef), and 25 ppb (chicken muscle and liver). The matrix matching calibration graphs obtained for both AV-, (r >0.996) and -, (r >0.998) from animal tissues and milk were linear over the calibration ranges. AV recovery from samples was higher than 73.3% and the RSD was less than 12.0% (n = 5). [source] Separation and determination of five major opium alkaloids with mixed mode of hydrophilic/cation-exchange monolith by pressurized capillary electrochromatographyJOURNAL OF SEPARATION SCIENCE, JSS, Issue 17 2007Xucong Lin Abstract A method for the separation and determination of five major opium alkaloids (narcotine, papaverine, thebaine, codeine, and morphine) in pericarpium papaveris by pressurized CEC (pCEC) with monolithic column has been developed. Under the optimum condition, linear calibration ranges of narcotine, papaverine, thebaine, codeine, and morphine were obtained as 2,85, 2,85, 5,75, 10,65, and 10,65 ,g/mL, respectively. LODs of these analytes were 1.5,6.0 ,g/mL. The RSD (n = 7) of the migration time and peak area were 1.94,5.24 and 4.05,8.21%, respectively. The proposed method was successfully applied to the analysis of pericarpium papaveris samples. Average recoveries of 79.0,95.9% at different fortified levels of alkaloids were achieved with RSD less than 4.6%. Meanwhile, the mechanism of the separation of the alkaloids on the monolithic column was also discussed. The result showed that the separation of alkaloids was mainly based on the mixed mode of hydrophilic interaction (HI) and cation exchange. [source] Determination of cellular redox status by stable isotope dilution liquid chromatography/mass spectrometry analysis of glutathione and glutathione disulfideRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 4 2008Peijuan Zhu Oxidation of glutathione (GSH) to glutathione disulfide (GSSG) occurs during cellular oxidative stress. The redox potential of the 2GSH/GSSG couple, which is determined by the Nernst equation, provides a means to assess cellular redox status. It is difficult to accurately quantify GSH and GSSG due to the ease with which GSH is oxidized to GSSG during sample preparation. To overcome this problem, a stable isotope dilution liquid chromatography/multiple reaction monitoring mass spectrometry (LC/MRM-MS) method has been developed using 4-fluoro-7-sulfamoylbenzofurazan (ABD-F) derivatization. ABD-F derivatization of the GSH thiol group was rapid, quantitative, and occurred at room temperature. The LC/MRM-MS method, which requires no sample clean-up, was validated within the calibration ranges of 5 to 400,nmol/mL in cell lysates for GSH and 0.5 to 40,nmol/mL in cell lysates for GSSG. Calibration curves prepared by adding known concentrations of GSH and GSSG to cell lysates were parallel to the standard curve prepared in buffers. GSH and GSSG concentrations were determined in two monocyte/macrophage RAW 267.4 cell lines with or without 15-LOX-1 expression (R15LO and RMock cells, respectively) after treatment with the bifunctional electrophile 4-oxo-2(E)-nonenal (ONE). R15LO cells synthesized much higher concentrations of the lipid hydroperoxide, 15(S)-hydroperoxyeicosatetraenoic acid (15-HPETE), which undergoes homolytic decomposition to ONE. GSH was depleted by ONE treatment in both RMock and R15LO cells, leading to significant increases in their redox potentials. However, R15LO cells had higher GSH concentrations (most likely through increased GSH biosynthesis) and had increased resistance to ONE-mediated GSH depletion than RMock cells. Consequently, R15LO cells had lower reduction potentials at all concentrations of ONE. GSSG concentrations were higher in R15LO cells after ONE treatment when compared with the ONE-treated RMock cells. This suggests that increased expression of 15(S)-HPETE modulates the activity of cellular GSH reductases or the transporters involved in removal of GSSG. Copyright © 2008 John Wiley & Sons, Ltd. [source] Development and validation of an on-line two-dimensional reversed-phase liquid chromatography,tandem mass spectrometry method for the simultaneous determination of prostaglandins E2 and F2, and 13,14-dihydro-15-keto prostaglandin F2, levels in human plasmaBIOMEDICAL CHROMATOGRAPHY, Issue 3 2009Junji Komaba Abstract We developed and validated an on-line reverse-phase two-dimensional LC/MS/MS (2D-LC/MS/MS) system for simultaneous determination of the levels of prostaglandin (PG) E2 as well as PGF2, and its metabolite 13,14-dihydro-15-keto PGF2, (F2, -M) in human plasma. Analytes were extracted by a three-step solid-phase extraction. Samples were then analyzed by on-line 2D-LC/MS/MS with electrospray ionization in negative mode. The 2D-LC system is composed of two reverse-phase analytical columns with a trapping column linking the two analytical columns. While an acidic buffer was used for both separation dimensions, differing organic solvents were employed for each dimension: methanol for the first and acetonitrile for the second to increase resolving power. The 2D-LC/MS/MS method was highly selective and sensitive with a significantly lower limit of quantitation (0.5 pg/mL for PGE2 and 2.5 pg/mL for PGF2, and F2, -M, respectively). Linearity of the 2D-LC/MS/MS system was demonstrated for the calibration ranges of 0.5,50 pg/mL for PGE2 and 2.5,500 pg/mL for PGF2, and F2, -M, respectively. Acceptable precision and accuracy were obtained throughout the calibration curve ranges. This highly selective and sensitive method was successfully utilized to determine the endogenous levels of PGE2, PGF2,, and F2, -M in plasma samples from six (four male and two female) normal volunteers. The mean concentrations for each analyte were 0.755 pg/mL for PGE2, 5.70 pg/mL for PGF2, and 9.48 pg/mL for F2, -M. Copyright © 2008 John Wiley & Sons, Ltd. 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