Direct determination of estriol 3- and 16-glucuronides in pregnancy urine by column-switching high-performance liquid chromatography with fluorescence detection

An HPLC method for the direct and simultaneous determination of estriol 3- and 16-glucuronides in pregnancy urine is described. The method is based on direct derivatization of the glucuronic acid moiety in estriol glucuronides in urine with 6,7-dimethoxy-1-methyl-2(1H)-quinoxalinone-3-propionylcarboxylic acid hydrazide. The derivatization reaction proceeds in aqueous solution (or urine sample) in the presence of pyridine and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide at 37°C. The resulting fluorescent derivatives were separated by column-switching chromatography using a first column (YMC-Pack C₄) for clean-up of the derivatives and a second column (YMC Pack Ph) for the complete separation of the derivatives. The derivatives were detected spectrofluorimetrically at 445 nm with excitation at 367 nm. The detection limits (signal-to-noise ratio=3) for estriol 3- and 16-glucuronides were 150 and 180 fmol in a 5 μl of urine (14 and 17 ng ml⁻¹ urine), respectively. The present method is highly sensitive and simple without any clean-up such as conventional solid-phase extraction.     

Automated analysis of a novel anti-epileptic compound, CGP 33 101, and its metabolite, CGP 47 292, in body fluids by high-performance liquid chromatography and liquid-solid extraction

Automated procedures for the determination of CGP 33 101 in plasma and the simultaneous determination of CGP 33 101 and its carboxylic acid metabolite, CGP 47 292, in urine are described. Plasma was diluted with water and urine with a pH 2 buffer prior to extraction. The compounds were automatically extracted on reversed-phase extraction columns and injected onto an HPLC system by the automatic sample preparation with extraction columns (ASPEC) automate. A Supelcosil LC-18 (5 μm) column was used for chromatography. The mobile phase was a mixture of an aqueous solution of potassium dihydrogen phosphate, acetonitrile and methanol for the assay in plasma, and of an aqueous solution of tetrabutylammonium hydrogen sulfate, tripotassium phosphate and phosphoric acid and of acetonitrile for the assay in urine. The compounds were detected at 230 nm. The limit of quantitation was 0.11 μml/l (25 ng/mol) for the assay of CGP 33 101 in plasma, 11 μmol/l (2.5 μg/ml) for its assay in urine and 21 μmol/l (5 μg/ml) for the assay of CGP 47 292 in urine.     

Quantification of local water and biomass in wild type PA01 biofilms by Confocal Raman Microspectroscopy

Confocal Raman Microspectroscopy (CRM) can be used as a tool for the in situ evaluation of the chemical composition of living, fully submerged, unstained biofilms. In this study the estimation of the local water content in Pseudomonas aeruginosa PA01 biofilms is given as an example. The ratio of the area of the O-H stretching vibration band at 3450 cm(-1), (water), to that of the C-H stretching bands at 2950 cm(-1) (biomass), was used to estimate the relative biofilm water content. The quantification of biofilm water and biomass was based on calibration curves generated from protein solutions. Water/biomass ratios (W:BR) equivalent to that of a 30% (w/v) protein solution were observed within some biofilm colonies.     

On-line biological sample cleanup for electrospray mass spectrometry using sol-gel columns

Using a slight overpressure, a urine sample is loaded onto a monolithic photopolymerized sol-gel column that has been derivatized with hydrophobic carbon chains and then the complex urine matrix is washed with aqueous solution. A buffer containing organic solvent is used to elute the adsorbed peptides by an applied voltage and the sample is then introduced into a mass spectrometer by sheath flow electrospray. The importance of desalting this type of sample is demonstrated by an experiment that shows that the signal intensity of a test solution with neurotensin, sprayed directly into the mass spectrometer, decreased from 4.5x10(4) cps to no detectible signal when just 10% urine is added to the sample solution. We suggest that this procedure may find general application for desalting biological samples prior to mass spectrometric analysis.     

Analysis of antibiotics in urine and wipe samples from environmental and biological monitoring--comparison of HPLC with UV-, single MS- and tandem MS-detection

Results of the simultaneous determination of the structurally different antibiotics cefazoline, cefotiame, cefuroxime, chloramphenicol, ciprofloxacin, ofloxacin, sulfamethoxazole and trimethoprim from environmental and biological monitoring using high-performance liquid chromatography with UV, single mass and tandem mass spectrometry were compared. For sample enrichment and clean-up a SPE method using bakerbond C18 cartridges was developed. Mean recovery rates were above 70%. Because of the complex urine matrix, only the wipe samples could be analyzed by UV-detection. However, UV-detection and single MS-detection are useful for control measurements after spillage, e.g. (LOD=1-2 ng/cm(2)). Samples from biological monitoring of occupational uptake should be analyzed by LC-MS/MS. The limits of detection (LOD) in urine ranged from 0.4 to 70 microg/L for LC-MS and 0.01 to 0.9 microg/L for LC-MS/MS detection. The limits of detection in wipe samples ranged from 0.003 to 0.13 ng/cm(2).     

Microbial fuel cells demonstrate high coulombic efficiency applicable for water remediation

Microbial fuel cells (MFCs) convert biomass into electricity by the metabolic activity of microorganisms and are also used for remediation and water treatment. Power output was compared for a dual chambered membrane MFC using either E. coli or two Yamuna river samples, Yamuna (before the Sangam region)--slow flow (sample 1) and Sangam region--fast flow (sample 2). E. coli and the two river water samples 1 and 2 gave a maximum voltage of 779, 463 and 415 mV respectively. Using E. coli the maximum power density obtained with a 100 omega resistor was 220.66 mW/cm2 and the highest power generated 6068.41 mW. The results demonstrate E. coli, river sample 1 and river sample 2 have a comparable coulombic efficiency of 85.2, 71 and 77% respectively when using 0.4% sucrose as substrate. The decrease in chemical oxidative demand of all river water samples using MFC technology demonstrates efficient remediation of inland water.     

Continuous adsorption and recovery of Cr(VI) in different types of reactors

This study reports the results of experiments on continuous adsorption and desorption of Cr(VI) ions by a chemically modified and polysulfone-immobilized biomass of the fungus Rhizopus nigricans. A fixed quantity of polymer-entrapped biomass beads corresponding to 2 g of dry biomass powder was employed in packed bed, fluidized bed, and stirred tank reactor for monitoring the continuous removal and recovery of Cr(VI) ions from aqueous solution and synthetic chrome plating effluent. Parameters such as flow rate (5, 10 and 15 mL/min), inlet concentration of Cr(VI) ions (50, 100, 150 and 250 mg/L) and the depth of biosorbent packing (22.8, 11.2 and 4.9 cm) were evaluated for the packed bed reactor. The breakthrough time and the adsorption rates in the packed bed column were found to decrease with increasing flow rate and higher Cr inlet concentrations and to increase with higher depths of sorbent packing. To have a comparative analysis of Cr adsorption efficiency in different types of reactors, the fluidized bed reactor and stirred tank reactor were operated using the same quantities of biosorbent material. For the fluidized bed reactor, Cr(VI) solution of 100 mg/L was pumped at 5 mL/min and fluidized by compressed air at a flow rate of 0.5 kg/cm.(2) The stirred tank reactor had a working volume of 200 mL capacity and the inlet/outlet flow rate was 5 mL/min. The maximum removal efficiency (mg Cr/g biomass) was obtained for the stirred tank reactor (159.26), followed by the fluidized reactor (153.04) and packed bed reactor (123.33). In comparison to the adsorption rate from pure chromate solution, approximately 16% reduction was monitored for synthetic chrome plating effluent in the packed bed. Continuous desorption of bound Cr ions from the reactors was effective with 0.01 N Na(2)CO(3) and nearly 80-94% recoveries have been obtained for all the reactors.     

Simultaneous optical and electrical recording of single gramicidin channels

We report here an approach for simultaneous fluorescence imaging and electrical recording of single ion channels in planar bilayer membranes. As a test case, fluorescently labeled (Cy3 and Cy5) gramicidin derivatives were imaged at the single-molecule level using far-field illumination and cooled CCD camera detection. Gramicidin monomers were observed to diffuse in the plane of the membrane with a diffusion coefficient of 3.3 x 10(-8) cm(2)s(-1). Simultaneous electrical recording detected gramicidin homodimer (Cy3/Cy3, Cy5/Cy5) and heterodimer (Cy3/Cy5) channels. Heterodimer formation was observed optically by the appearance of a fluorescence resonance energy transfer (FRET) signal (irradiation of Cy3, detection of Cy5). The number of FRET signals was significantly smaller than the number of Cy3 signals (Cy3 monomers plus Cy3 homodimers) as expected. The number of FRET signals increased with increasing channel activity. In numerous cases the appearance of a FRET signal was observed to correlate with a channel opening event detected electrically. The heterodimers also diffused in the plane of the membrane with a diffusion coefficient of 3.0 x 10(-8) cm(2)s(-1). These experiments demonstrate the feasibility of simultaneous optical and electrical detection of structural changes in single ion channels as well as suggesting strategies for improving the reliability of such measurements.     

Electricity generation and treatment of paper recycling wastewater using a microbial fuel cell

Increased interest in sustainable agriculture and bio-based industries requires that we find more energy-efficient methods for treating cellulose-containing wastewaters. We examined the effectiveness of simultaneous electricity production and treatment of a paper recycling plant wastewater using microbial fuel cells. Treatment efficiency was limited by wastewater conductivity. When a 50 mM phosphate buffer solution (PBS, 5.9 mS/cm) was added to the wastewater, power densities reached 501 +/- 20 mW/m(2), with a coulombic efficiency of 16 +/- 2%. There was efficient removal of soluble organic matter, with 73 +/- 1% removed based on soluble chemical oxygen demand (SCOD) and only slightly greater total removal (76 +/- 4%) based on total COD (TCOD) over a 500-h batch cycle. Cellulose was nearly completely removed (96 +/- 1%) during treatment. Further increasing the conductivity (100 mM PBS) increased power to 672 +/- 27 mW/m(2). In contrast, only 144 +/- 7 mW/m(2) was produced using an unamended wastewater (0.8 mS/cm) with TCOD, SCOD, and cellulose removals of 29 +/- 1%, 51 +/- 2%, and 16 +/- 1% (350-h batch cycle). These results demonstrate limitations to treatment efficiencies with actual wastewaters caused by solution conductivity compared to laboratory experiments under more optimal conditions.     

Simultaneous determination of ascorbic acid, aminothiols, and methionine in biological matrices using ion-pairing RP-HPLC coupled with electrochemical detector

A novel highly sensitive ion-pairing reversed-phase high performance liquid-chromatography/electrochemical detection method for simultaneous determination of l-ascorbic acid, aminothiols, and methionine in biological matrices was developed, optimized, and validated. Reduced forms of the analytes were extracted from the sample matrices with 10% meta-phosphoric acid solution((aqueous)). To determine the total vitamin C, the total aminothiols, and the total methionine, samples were treated with tris(2-carboxyethyl)phosphine solution in 0.05% trifluoroacetic acid solution((aqueous)) subsequent to deproteination to reduce the oxidized forms of these compounds. Various analytes were separated on a C(18) (250 × 4.6 mm, 5 μm) analytical column using methanol-0.05% trifluoroacetic acid solution((aqueous)) (05/95, v/v), containing 0.1mM 1-octane sulphonic acid as the ion-pairing agent) as the isocratic mobile phase pumped at a flow rate of 1.5 mL min(-1) at room temperature. The column eluents were monitored at a voltage of 0.85 V. These analytes were efficiently resolved in less than 20 min using n-acetyl cysteine as the internal standard. The present method was specific for the analysis of these analytes and demonstrated acceptable values for linearity (r(2)>0.999 in the range of 0.2-10,000 ng mL(-1) for all the analytes), recovery (>96%), precision (%RSD ≤ 2.0), and sensitivity (on column limit of detection: 250-400 fg and limit of quantification: 0.8-1.25 pg), indicating that the proposed method could be efficiently used for determination of these analytes in the context of clinical research.     

Quantitative determination of circulating and urinary asymmetric dimethylarginine (ADMA) in humans by gas chromatography-tandem mass spectrometry as methyl ester tri(N-pentafluoropropionyl) derivative

Asymmetric dimethylarginine (ADMA; N(G),N(G)-dimethyl-L-arginine) is the most important endogenous inhibitor of nitric oxide synthase and a potential risk factor for cardiovascular diseases. This article describes a gas chromatographic-tandem mass spectrometric (GC-tandem MS) method for the accurate quantification of ADMA in human plasma or serum and urine using de novo synthesized [2H(3)]-methyl ester ADMA (d(3)Me-ADMA) as the internal standard. Aliquots (100 microl) of plasma/serum ultrafiltrate or native urine and of aqueous solutions of synthetic ADMA (1 microM for plasma and serum; 20 microM for urine) are evaporated to dryness. The residue from plasma/serum ultrafiltrate or urine is treated with a 100 microl aliquot of 2M HCl in methanol, whereas the residue of the ADMA solution is treated with a 100 microl aliquot of 2M HCl in tetradeuterated methanol. Methyl esters are prepared by heating for 60 min at 80 degrees C. After cooling to room temperature, the plasma or urine sample is combined with the d(3)Me-ADMA sample, the mixture is evaporated to dryness, the residue treated with a solution of pentafluoropropionic (PFP) anhydride in ethyl acetate (1:4, v/v) and the sample is incubated for 30 min at 65 degrees C. Solvent and reagents are evaporated under a stream of nitrogen gas, the residue is treated with a 200 microl aliquot of 0.4M borate buffer, pH 8.5, and toluene (0.2 ml for plasma, 1 ml for urine). Reaction products are extracted by vortexing for 1 min, the toluene phase is decanted, and a 1 microl aliquot is injected into the GC-tandem MS instrument. Quantitation is performed by selected reaction monitoring (SRM) of the common product ion at m/z 378 which is produced by collision-induced dissociation of the ions at m/z 634 for endogenous ADMA and m/z 637 for d(3)Me-ADMA. In plasma and urine of healthy humans ADMA was measured at concentrations of 0.39+/-0.06 microM (n=12) and 3.4+/-1.1 micromol/mmol creatinine (n=9), respectively. The limits of detection and quantitation of the method are approximately 10 amol and 320 pM of d(3)Me-ADMA, respectively.     

Unified gas chromatographic-mass spectrometric method for quantitating tyrosine metabolites in urine and plasma

Tyrosine and many of its catabolites play significant roles in the in the toxicity associated with acquired and congenital forms of hypertyrosinemia. We now report a specific and sensitive GC/MS method for the simultaneous determination of tyrosine metabolites maleylacetone (MA), fumarylacetone (FA), succinylacetone (SA), fumarate and acetoacetate in urine and plasma. Tyrosine metabolites and an internal standard, 2-oxohexanoic acid (OHA), in urine or plasma samples were derivatized to their methyl esters with a 12% boron trifluoride-methanol complex (12%BF3-MeOH). The reaction mixture was extracted with methylene chloride and analyzed by GC/MS, using a selected ion monitoring (SIM) mode. The detection limits were in the range of 0.08-0.4 ng and the quantitation limits were 0.2-2 ng. Most of the intraday and interday coefficients of variation for three concentrations (low, medium and high) of the analytes were below 10%. Sensitivity and selectivity are superior to existing HPLC or enzymatic methods and derivatization of samples is simpler than the traditional silylation of organic acids used for analysis by GC/MS or derivatization to oximes, followed by silylation in the case of the ketoacids, such as SA. Furthermore, the current procedure can be performed in aqueous solution, which results in a high percentage yield without appreciable analyte degradation or formation of side products. Thus far, the method has been successfully applied in the analysis of over 5000 urine and plasma samples from humans and rodents.     

Photoelectropolymerization of aniline in a dye-sensitized solar cell.

A dye-sensitized solar cell was constructed using a porphyrin photosensitizer and, in place of the usual iodide redox system, a solution in aniline solvent containing lithium perchlorate electrolyte, camphorsulfonic acid, and poly(ethylene oxide) copolymer. Irradiation generated polyaniline within the cell, initially following a proposed photoelectropolymerization mechanism, and eventually operating as a solar cell with polyaniline as the hole transport medium. Overall energy conversion efficiency was 0.8% at moderate light intensities (14.6 mW cm(-2)) but lower at higher light intensities due to conductivity limitations.     

pH- and thermal-dependent conformational transition of PGAIPG, a repeated hexapeptide sequence from tropoelastin

The secondary structure of PGAIPG (Pro-Gly-Ala-IIe-Pro-Gly), a repeated hexapeptide of tropoelastin, in buffer solution of different pH was determined by using attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy. The thermal-dependent structural change of PGAIPG in aqueous solution or in solid state was also examined by thermal FTIR microspectroscopy. The conformation of PGAIPG in aqueous solution exhibited a pH-dependent structural characterization. A predominant peak at 1614 cm(-1) (aggregated beta-sheet) with a shoulder near 1560 cm(-1) (beta-sheet) appeared in pH 5.5-8.5 buffer solutions. A new broad shoulder at 1651 cm(-1) (random coil and/or alpha-helix) with 1614 cm(-1) was observed in the pH 4.5 buffer solution. However, the broad shoulder at 1651 cm(-1) was converted to a maximum peak at 1679 cm(-1) (beta-turn/antiparallel beta-sheet) when the pH shifted from 4.5 to 3.5, but the original pronounced peak at 1614 cm(-1) became a shoulder. Once the pH was lowered to 2.5, the IR spectrum of PGAIPG was dominated by major absorption at 1679 cm(-1) with a minor peak at 1552 cm(-1) (alpha-helix/random coil). The result indicates that the pH was a predominant factor to transform PGAIPG structure from aggregated beta-sheet (pH 8.5) to beta-turn/intermolecular antiparallel beta-sheet (pH 2.5). Moreover, a partial conformation of PGAIPG with minor alpha-helix/random coil structures was also explored in the lower pH buffer solution. There was no thermal-dependent structural change for solid-state PGAIPG. The thermal-induced formation of aggregated beta-sheet for PGAIPG in aqueous solution was found from 28 to 30 degrees C, however, which might be correlated with the formation of an opaque gel that turned from clear solution. The formation of aggregated beta-sheet structure for PGAIPG beyond 30 degrees C might be due to the intermolecular hydrogen bonded interaction between the hydrophobic PGAIPG fragments induced by coacervation.     

Nucleation of calcium oxalate crystals on an imprinted polymer surface from pure aqueous solution and urine

Calcium oxalate (CaOx) is the most common component of human kidney stones. Heterogeneous nucleation is regarded as the key mechanism in this process. In this study, we have used an imprinted 6-methacrylamidohexanoic acid/divinylbenzene co-polymer as a biomimetic surface to nucleate CaOx crystal formation. The polymer was imprinted with either calcium oxalate monohydrate (COM) or dihydrate (COD) template crystals. These were washed out of the polymer, which was then immersed in various test solutions. The test solutions were an aqueous solution of calcium chloride and sodium oxalate, artificial urine and a sample of real urine. Crystals that formed on the polymer surface were characterized by X-ray powder diffraction, Fourier transform infrared spectroscopy, atomic absorption spectroscopy and scanning electron microscopy. Results showed that in the aqueous solution the COM-imprinted polymer induced the nucleation of COM. The COD-imprinted polymer induced only trace amounts of COD crystallization, together with larger quantities of COM. In artificial and real urines, COM also specifically precipitated on the COM-imprinted surface. The results show that, at least to some extent, the imprinted polymers direct formation of their morphologically matched crystals. In the case of COD, however, it appears that either rapid hydrate transformation of COD to COM occurs, or the more stable COM polymorph is directly co-precipitated by the polymer. Our results support the hypothesis that heterogeneous nucleation plays a key role in CaOx stone formation and that the imprinted polymer model could provide an additional and superior diagnostic tool for stone researchers to assess stone-risk in urine.Abbreviations COD calcium oxalate dihydrate - COM calcium oxalate monohydrate - COT calcium oxalate trihydrate - dvb divinylbenzene - 6-maaha 6-methylacrylamidohexanoic acid     

Model of ionic transport for bovine ciliary epithelium: effects of acetazolamide and HCO

The possible existence of transepithelial bicarbonate transport across the isolated bovine ciliary body was investigated by employing a chamber that allows for the measurement of unidirectional, radiolabeled fluxes of CO2 + HCO. No net flux of HCO was detected. However, acetazolamide (0.1 mM) reduced the simultaneously measured short-circuit current (I(sc)). In other experiments in which (36)Cl- was used, a net Cl- flux of 1.12 microeq. h(-1). cm(-2) (30 microA/cm(2)) in the blood-to-aqueous direction was detected. Acetazolamide, as well as removal of HCO from the aqueous bathing solution, inhibited the net Cl- flux and I(sc). Because such removal should increase HCO diffusion toward the aqueous compartment and increase the I(sc), this paradoxical effect could result from cell acidification and partial closure of Cl- channels. The acetazolamide effect on Cl- fluxes can be explained by a reduction of cellular H+ and HCO (generated from metabolic CO2 production), which exchange with Na+ and Cl- via Na+/H+ and Cl-/HCO exchangers, contributing to the net Cl- transport. The fact that the net Cl- flux is about three times larger than the I(sc) is explained with a vectorial model in which there is a secretion of Na+ and K+ into the aqueous humor that partially subtracts from the net Cl- flux. These transport characteristics of the bovine ciliary epithelium suggest how acetazolamide reduces intraocular pressure in the absence of HCO transport as a driving force for fluid secretion.     

Combining poly (methacrylic acid-co-ethylene glycol dimethacrylate) monolith microextraction and on-line pre-concentration-capillary electrophoresis for analysis of ephedrine and pseudoephedrine in human plasma and urine

A method based on poly (methacrylic acid-co-ethylene glycol dimethacrylate) (MAA-EGDMA) monolith microextraction (PMME) and field-enhanced sample injection (FESI) pre-concentration technique was proposed for sensitive capillary electrophoresis-ultraviolet (CE-UV) analysis of ephedrine (E) and pseudoephedrine (PE) in human plasma and urine. The PMME device consisted of a regular plastic syringe (1 mL), a poly (MAA-EGDMA) monolithic capillary (2 cm x 530 microm I.D.) and a plastic pinhead connecting the former two components seamlessly. The extraction was achieved by driving the sample solution through the monolithic capillary tube using a syringe pump, for the desorption step, an aliquot of organic solvent, which normally provided an excellent medium to ensure direct compatibility for FESI in CE, was injected via the monolithic capillary and collected into a vial for subsequent analysis by CZE. The best separation was achieved using a buffer composed of 0.1M phosphate electrolyte (pH 2.5) and 10% acetonitrile (v/v). The combination of both pre-concentration procedures allowed the detection limits of the analytes down to 5.3 ng/mL and 8.0 ng/mL in human plasma and urine, respectively. Excellent method of reproducibility was found over a linear range 50-5000 ng/mL in plasma and urine sample. Plasma and urine samples from volunteers receiving pseudoephedrine have also been successfully analysed.     

黄土性土壤固化对黑麦草生长和根系活力的影响

采用盆栽试验,研究了安塞黄绵土不同容重、不同固化剂(路邦EN-1固化剂)掺量对黑麦草生长和根系活力的影响.结果表明:黄绵土容重在1.2～1.4 9·cm-3范围内,随土壤容重的增加,黑麦草叶绿素含量、根系活力、根冠比、根生物量和植株生物量均降低;各土壤容重条件下,黑麦草叶绿素含量、根系活力、根冠比、根生物量和植株生物量均高于对照,且随着固化剂掺量的增加均呈先增加后降低的趋势.土壤容重和固化剂掺量交互作用对根生物量和总生物量的影响均显著(P＜0.05).总体来看,土壤容重1.3 g·cm-3、固化剂掺量0.15％处理下,各指标值均最高.     

Determination of urinary triclosan by stir bar sorptive extraction and thermal desorption-gas chromatography-mass spectrometry

We have developed an analytical method for the determination of urinary 5-chloro-2-(2,4-dichlorophenoxy)phenol (triclosan), which utilizes stir bar sorptive extraction (SBSE) and thermal desorption (TD)-gas chromatography-mass spectrometry (GC-MS). Human urine sample is de-conjugated by treatment with beta-glucuronidase and sulfatase. A stir bar coated with polydimethylsiloxane (PDMS) is added to the urine sample in a vial and the sample is stirred for 60 min at room temperature (25 degrees C). Then, the PDMS stir bar is subjected to TD-GC-MS. The detection limit of triclosan is 0.05 ng mL(-1). The method shows linearity over the calibration range (0.1-10 ng mL(-1)) and the correlation coefficient (r) is higher than 0.993 for triclosan standard solution. The average recoveries of triclosan in human urine sample are 102.8-113.1% (RSD: 2.4-6.7%). This simple, sensitive, and selective analytical method may be used in the determination of trace amounts of triclosan in human urine samples.     

Gas chromatographic-negative-ion chemical ionization mass spectrometric determination of a new dihydropyridine calcium antagonist (MPC-1304) and its metabolites in human plasma and urine

A gas chromatographic-negative-ion chemical ionization mass spectrometric method was developed for the determination of a new calcium antagonist, (±)-methyl 2-oxopropyl 1,4-dihydro-2,6-dimethyl-4-(2-nitrophenyl)-3,5-pyridinedicar☐ylate, and its metabolites in plasma and urine. The sample was extracted with n-hexane-diethyl ether. The dried organic layer was subjected to acetylation: the aqueous layer was acidified and extracted with ethyl acetate, and after the ethyl acetate extract was dried the resulting residue was subjected to methylation. Aliquots of each reactant solution were injected into the gas chromatograph-mass spectrometer, equipped with a chemical ionization source and negative-ion monitoring mode, and analysed by the elected-ion monitoring method using deuterium-labelled internal standards. Detection was limited to 0.02–0.05 ng/ml of plasma and urine for each metabolite. A precise and sensitive assay for the determination of a new dihydropyridine calcium antagonist and its metabolites in plasma and urine was thus established.