Simultaneous gas chromatographic determination of pirimiphos-methyl and malathion residues in peanuts,peanut hulls,peanut meats,and peanut oil

A gas chromatographic method developed for the simultaneous determination of residues of pirimiphos-methyl (O-[2-(dimethyl-amino)-6-methyl-4-pyrimidinyl] O,O-dimethyl phosphorothioate) and malathion (diethyl mercaptosuccinate S-ester with O,O-dimethyl phosphorodithioate), in whole peanuts, peanut hulls, peanut meats, and peanut oil gives good recoveries and has a limit of detection for pirimiphos-methyl and malathion of 0.04 ppm. Quantitative analysis for the two insecticides with electronic integration was reliable to 0.1 ppm.     

Solvent deuterium kinetic isotope effects for the methanolyses of neutral C=O, P=O and P=S esters catalyzed by a triazacyclododecane : Zn(2+)-methoxide complex

The methanolyses of several organophosphate/phosphonate/phosphorothioate esters (O,O-diethyl O-(4-nitrophenyl) phosphate, paraoxon, ; O,O-diethyl S-(3,5-dichlorophenyl) phosphorothioate, ; O-ethyl O-(2-nitro-4-chlorophenyl) methylphosphonate, ; O,O-dimethyl O-(3-methyl-4-nitrophenyl) phosphorothioate, fenitrothion, ; O-ethyl S-(3,5-dichlorophenyl) methylphosphonothioate ) and a carboxylate ester (p-nitrophenyl acetate, ) catalyzed by methoxide and the Zn(2+)((-)OCH(3)) complex of 1,5,9-triazacyclododecane ( : Zn(2+)((-)OCH(3))) were studied in methanol and d(1)-methanol at 25 degrees C. In the case of the methoxide reactions inverse skie's were observed for the series with values ranging from 2 to 1.1, except for where the k(D)/k(H) = 0.90 +/- 0.02. The inverse k(D)/k(H) values are consistent with a direct nucleophilic methoxide attack involving desolvation of the nucleophile with varying extents of resolvation of the TS. With the : Zn(2+)((-)OCH(3)) complex all the skie values are k(D)/k(H) = 1.0 +/- 0.1 except for where the value is 0.79 +/- 0.06. Arguments are presented that the fractionation factors associated with complex : Zn(2+)((-)OCH(3)) are indistinguishable from unity. The skie's for all the complex-catalyzed methanolyses are interpreted as being consistent with an intramolecular nucleophilic attack of the Zn(2+)-coordinated methoxide within a pre-equilibrium metal : substrate complex.     

Preparation and identification of generic antigens and broad-spectrum monoclonal antibodies for detection of organophosphorus pesticides

A simple, rapid, and high-sensitivity assay was developed to detect the multiresidue of organophosphorus pesticides (OPs) in the environment and food. Two separate generic haptens (Hapten A and B) with same O,O-dimethyl phosphorothioate group and aromatic ring and different spacer arms were synthesized and conjugated to bovine serum albumin (BSA) for immunogens and to ovalbumin (OVA) for coating antigens to study the effect of hapten and coating antigen heterology on immunoassay sensitivity. A broad-spectrum monoclonal antibody (MAb) was produced and a competitive ELISA developed using Hapten B-BSA as the immunogen and Hapten A-OVA as the coating antigen for the multiresidue determination of OPs, including parathionmethyl, fenitrothion, fenthion, chlorthion, and fenchlorphos. Several assay conditions, including organic solvent, pH, ionic strength, and incubation time, were studied sequentially to optimize the immunoassay. Using the optimal assay, 50% inhibition concentration values were estimated to be 34.5, 47.5, 79.8, 125.2, and 373.1 ng/mL for parathionmethyl, fenitrothion, fenthion, chlorthion, and fenchlorphos, respectively. The results indicated that the MAb showed specificity to all the above five OPs, and the assay could be developed for multiresidue determinations.     

Optimization and validation of a method of analysis for fenitrothion and its main metabolites in forestry air samples using sorbent tubes with thermal desorption cold trap injection and gas chromatography-mass spectrometry

An analytical methodology using thermal-desorption cold trap (TCT) and GC-MS was developed for the determination of the insecticide fenitrothion and its main metabolites, 3-methyl-4-nitrophenol and fenitrooxon, in forestry atmospheres. The sampled atmosphere was pumped through a glass tube containing 100 mg of Tenax adsorbent at a flow rate of 50 ml min(-1). Adsorption/thermal desorption and breakthrough experiments were performed to test the ability to quantitatively trap the compounds. The detection limits of method for these compounds ranged between 1.6 and 2.1 ng m(-3). This methodology was developed to evaluate the persistence of fenitrothion in forest atmospheres after treatment. Spray application at 21.5 mg active ingredient m(-2) resulted in atmosphere levels of the insecticide of 78.3 ng m(-3) (after 2 h of application). Within 2-4 days following treatment, the presence of fenitrooxon fell to 50-55%. During this period residues of metabolites began to appear, disappearing 19 days later.     

Determination of 7-iodo-1,3-dihydro-1-methyl-5(2'-fluorophenyl)-2h-1,4-benzodiazepin-2-one (Ro 7-9957) and its major biotransformation products in blood and urine by electron capture-gas-liquid chromatography.

A sensitive and specific electron capture-gas chromatographic assay was developed for the determination of 7-iodo-1,3-dihydro-1-methyl-5(2'-fluorophenyl)-2H-1,4-benzodiazepin-2-one (I) and its major metabolites in blood and urine. The overall recovery of I and its N-desmethyl metabolite (II) from blood is apparently quantitative. The recovery of the major urinary metabolite, the N-desmethyl-3-hydroxy analog (IV), and the minor metabolites, the N-desmethyl analog (II) and the N-methyl-3-hydroxy analog (III) added to urine as authentic reference standards ranged from 80 to 85%. The sensitivity limits of detection are of the order of 2-3 ng of I and 4-5 ng of II per ml of blood or urine. The method was applied to the determination of blood levels and the urinary excretion pattern in a dog following oral and intravenous administration of a 1-mg/kg dose (total 13 mg), and in man following the intravenous administration of single 5- and 10-mg doses. The N-desmethyl metabolite II was more predominant in dog blood than was the orally or intravenously administered I, but II was barely measurable in human blood.     

Determination of diazinon, chlorpyrifos, and their metabolites in rat plasma and urine by high-performance liquid chromatography

This study reports a simple and rapid high-performance liquid chromatographic (HPLC) method for the determination of the insecticide diazinon (O,O-diethyl-O[2-isopropyl-6-methylpyridimidinyl] phosphorothioate), its metabolites diazoxon (O,O-diethyl-O-2-isopropyl-6-methylpyridimidinyl phosphate) and 2-isopropyl-6-methyl-4-pyrimidinol, the insecticide chlorpyrifos (O,O-diethyl-O[3,5,6-trichloro-2-pyridinyl] phosphorothioate) and its metabolites chlorpyrifos-oxon (O,O-diethyl-O[3,5,6-trichloro-2-pyridinyl] phosphate), and TCP (3,5,6-trichloro-2-pyridinol) in rat plasma and urine samples. The method is based on using C18 Sep-Pak cartridges for solid-phase extraction and HPLC with a reversed-phase C18 column and programmed UV detection ranging between 254 and 280 nm. The compounds are separated using a gradient of 1% to 80% acetonitrile in water (pH 3.0) at a flow rate ranging between 1 and 1.5 mL/min in a period of 16 min. The limits of detection ranged between 50 and 150 ng/mL, and the limits of quantitation were 100 to 200 ng/mL. The average percentage recovery of five spiked plasma samples were 86.3 +/- 8.6, 77.4 +/- 7.0, 82.1 +/- 8.2, 81.8 +/- 8.7, 73.1 +/- 7.4, and 80.3 +/- 8.0 and from urine were 81.8 +/- 7.6, 76.6 +/- 7.1, 81.5 +/- 7.9, 81.8 +/- 7.1, 73.7 +/- 8.6, and 80.7 +/- 7.7 for diazinon, diazoxon, 2-isopropyl-6-methyl-4-pyrimidinol, chlorpyrifos, chlorpyrifos-oxon, and TCP, respectively. The relationship between the peak area and concentration was linear over a range of 200 to 2,000 ng/mL. This method was applied in order to analyze these chemicals and metabolites following dermal administration in rats.     

Characterization of In Vivo Metabolites of a Potential Anti-obesity Compound,the 3-Methyl-1H-Purine-2,6-Dione Derivative C-11, Employing Ultra-High Performance Liquid Chromatography Coupled with Quadrupole Time-of-Flight Mass Spectrometry

C-11 (2-((7-Ethyl-3-methyl-8-(4-(2-(methyl(pyridin-2-yl)-amino)-ethoxy)phenyl)-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-1-yl)methyl)benzonitrile-one hydrochloride), which is based on the structure of rosiglitazone, was first synthesized in our laboratory and shown to be a promising anti-obesity drug candidate in our previous pharmacological study. Considering the importance of metabolic fate in vivo in the further development of drug candidates during early drug discovery, it is essential to characterize the metabolism of C-11 in vivo. In this work, a method based on ultra-high performance liquid chromatography combined with quadrupole time-of-flight mass spectrometry (UPLC/Q-TOF-MS) was successfully developed to investigate the in vivo metabolic profile of C-11 in rats. Rat urine, feces, and plasma samples were collected from male Sprague–Dawley rats after intravenous administration of C-11 in a single dose of 30 mg kg⁻¹ body weight. Besides the parent drug, a total of 25 metabolites (including 18 phase I and 7 phase II metabolites) were detected and tentatively identified by comparing their mass spectrometry profiles with those of C-11. This enabled the metabolic pathways of C-11 to be proposed for the first time. Our results revealed that N-depyridinylation, N-demethylation, hydroxylation, glucuronidation, and sulfate conjugation are the predominant metabolic pathways of C-11 in rats. The present study provides systematic information on the metabolism of C-11 in vivo, which should lead to a better understanding of its safety and mechanism of action.

     

Analysis of Fenitrothion and Metabolites in Stored Wheat

Abstract

A simple and rapid method for the analysis of fenitrothion and its metabolites, fenitrooxon, S-methyl fenitrothion, demethyl fenitrothion, demethyl S-methyl fenitrothion, 3-methyl-4-nitrophenol, and dimethyl phosphorothioic acid in stored wheat has been developed. Simultaneous analysis of the extract was conducted using FPD-GLC after derivatization with diazoethane except for 3-methyl-4-nitrophenol which was analyzed directly by EC-GLC. Recoveries of all compounds from wheat fortified at the levels from 0.1 to 5.0 ppm were greater than 90%.

The developed method was used to quantitatively determine major metabolites found in grain treated with fenitrothion and stored at 20°C for 12 months. Demethyl fenitrothion, 3-methyl-4-nitrophenol, and dimethyl phosphorothioic acid were the major breakdown products of fenitrothion found in stored wheat. Confirmation of these metabolites was carried out by chemical derivatization plus FPD-GLC and by TLC.     

Thermochemistry of Heteroatomic Compounds XXI: Heat Capacities of Some Derivatives of Methylphosphonic and Dithiophosphorus Acids

The heat capacities of 2,4-dimethyl-2-oxo-(I)-, 2,5,5-trimethyl-2-oxo(II)-1,3,2-dioxaphosphorinanes, methylphosphonic acid (III), O,O-dimethyl(IV)-, O,O-diethyl(V)-, O,O-di-isopropyl(VI)esters of dithiophosphorus acid, 4,5-dimethyl-2-thiono-2-mercapto-1,3,2-dioxaphospholane(VII), and 4-methyl-2-thiono-2-mercapto-1,3,2-dioxaphosphorinane(VIII) were determined using the scanning calorimetry.     

Determination of a new calcium antagonist, sesamodil fumarate (SD-3211), and its metabolite in plasma by liquid chromatography with electrochemical detection

A sensitive and selective high-performance liquid chromatographic method is described for the determination of a novel calcium antagonist, (+)-(R)-3,4-dihydro-2-[5-methoxy-2-[3-[N-methyl-N-[2-[(3,4- methylenedioxy)phenoxy]ethyl]amino]propoxy]phenyl]-4-methyl-3-oxo-2H- 1,4-benzothiazine hydrogen fumarate (sesamodil fumarate; JAN, SD-3211, I), and its N-desmethylated metabolite (II) in plasma. Compounds I and II and an internal standard were isolated from plasma by solid-phase and liquid-liquid extraction. The extract was chromatographed on a reversed-phase C18 column, and the compounds of interest were detected by dual coulometric electrodes operated in an oxidative screen mode. The limit of determination for both I and II was at least 0.4 ng/ml in plasma. The utility of the assay was demonstrated by determining plasma levels of I and II in five dogs administered an oral dose of 60 mg of the drug.     

High-performance liquid chromatographic determination of the 1,4-diketo and 1,4-diketo-2,3-dihydroxy metabolites of lonapalene in rat urine

Individual high-performance liquid chromatographic (HPLC) methods have been developed for the determination of two major metabolites of lonapalene in rat urine. The highly unstable and polar 1,4-diketo-2,3-dihydroxy metabolite (II) is extracted from urine by two extraction columns (phenyl followed by silica), further purified by means of HPLC with a fully end-capped C18 HPLC column and quantified by an ultraviolet detector at 280 nm. Ascorbic acid is used as an antioxidant during extraction and overnight injection of II. Urine samples for total II (free plus conjugated) determination are incubated with arylsulfatase and beta-glucuronadase prior to extraction. The 1,4-diketo metabolite (III) is extracted from urine with a C18 extraction column, further purified with a C18 HPLC column, and quantified by an ultraviolet detector at 260 nm. The detection limit for both metabolites is 100 ng/ml of urine (signal-to-noise = 2.5). The methods were used to analyze urine samples from a long-term toxicology study of lonapalene in rats and to determine the linearity of dose-concentration relationships for both metabolites.     

Solid-phase microextraction coupled with high performance liquid chromatography using on-line diode-array and electrochemical detection for the determination of fenitrothion and its main metabolites in environmental water samples

The aim of this study was to develop a methodology for the analysis of the insecticide fenitrothion and its two main environmental metabolites, fenitrooxon and 3-methyl-4-nitrophenol. For this purpose, a solid-phase microextraction (SPME) method coupled to high performance liquid chromatography (LC) was optimized. Two on-line detectors, diode array (DAD) and direct current amperometrical (DCAD) were used in order to determine sensitivity and selectivity. The effects of the extraction parameters, including exposure and desorption time, pH, temperature, salt concentration and desorption mode on the extraction efficiency were studied. A satisfactory reproducibility for extractions from samples at 20 ppb-level with RSD < 12.5% (n = 10) was obtained. The calibration graphs were linear in the range of 10-1000 microg l(-1) and detection limits for the target compounds were between 1.2 and 11.8 microg l(-1) depending on which detector was used. The method was applied for determining fenitrothion and both its metabolites in river waters which run through forest areas near to aerial application of the pesticide.     

Gas‐phase copper and silver complexes with phosphorothioate and phosphorodithioate pesticides investigated using electrospray ionization mass spectrometry

Efforts to improve agricultural productivity have led to a growing dependency on organophosphorus pesticides. Phosphorothioate and phosphorodithioate pesticides are organophosphorus pesticide subclasses with widespread application for the control of insects feeding on vegetables and fruits. However, even low doses of these pesticides can cause neurological problems in humans; thus, their determination and monitoring in agricultural foodstuffs is important for human health. Phosphorothioate and phosphorodithioate pesticides may be poorly ionized during electrospray, adversely affecting limits of detection. These pesticides can form complexes with Cu²⁺ and Ag⁺, however, potentially improving ionization. In the present work, we used electrospray ionization/mass spectrometry (ESI/MS) to study fenitrothion, parathion, diazinon, and malathion coordination complexes with silver and copper ions. Stable 1 : 1 and 1 : 2 metal/pesticide complexes were detected. Mass spectra acquired from pesticide solutions containing Ag⁺ or Cu²⁺ showed a significant increase in signal‐to‐background ratio over those acquired from solutions containing only the pesticides, with Ag⁺ improving detection more effectively than Cu²⁺. Addition of Ag⁺ to a pesticide solution improved the limit of detection by ten times. The relative affinity of each pesticide for Ag⁺ was related to complex stability, following the order diazinon > malathion > fenitrothion > parathion. The formation of Ag⁺–pesticide complexes can significantly improve the detection of phosphorothioate and phosphorodithioate pesticides using ESI/MS. The technique could potentially be used in reactive desorption electrospray ionization/mass spectrometry to detect phosphorothioate and phosphorodithioate pesticides on fruit and vegetable skins. Copyright © 2015 John Wiley & Sons, Ltd.     

Nucleophilic degradation of fenitrothion insecticide and performance of nucleophiles: a computational study

Ab initio and density functional theory (DFT) calculations have been performed to understand the destruction chemistry of an important organophosphorus insecticide O,O-dimethyl O-(3-methyl-4-nitrophenyl) phosphorothioate, fenitrothion (FN), toward nucleophilic attack. Breaking of the P-OAr linkages through nucleophilic attack is considered to be the major degradation pathway for FN. One simple nucleophile, hydroxide (OH(-)), and two different α-nucleophiles, hydroperoxide (OOH(-)) and hydroxylamine anion (NH(2)O(-)), have been considered for this study. Nucleophilic attack at the two different centers, S(N)2@P and S(N)2@C, has been monitored, and the computed reaction energetics confirms that the S(N)2@P reactions are favorable over the S(N)2@C reactions for all the nucleophiles. All electronic structure calculations for the reaction are performed at DFT-B3LYP/6-31+G(d) level of theory followed by a refinement of energy at ab initio MP2/6-311++G(2d,2p) level. The effect of aqueous polarization on both the S(N)2 reactions is taken into account employing the conductor-like screening model (COSMO) as well as polarization continuum model (PCM) at B3LYP/6-31+G(d) level of theory. Relative performance of the two α-nucleophiles, OOH(-) and NH(2)O(-), at the P center has further been clarified using natural bond orbital (NBO), conceptual DFT, and atoms in molecules (AIM) approaches. The strength of the intermolecular hydrogen bonding in the transition states and topological properties of the electron density distribution for -X-H···S (X = O, N) intermolecular hydrogen bonds are the subject of NBO and AIM analysis, respectively. Our calculated reaction energetics and electronic properties suggest that the relative order of nucleophilicity for the nucleophiles is OOH(-) > NH(2)O(-) > OH(-) for the S(N)2@P, whereas for the S(N)2@C the order, which gets little altered, is NH(2)O(-) > OOH(-) > OH(-).     

High performance liquid chromatographic determination of diazinon, permethrin, DEET (N, N-diethyl-m-toluamide), and their metabolites in rat plasma and urine

A rapid method was developed for the analysis of the insecticide (A) diazinon (O,O-diethyl O-2-isopropyl-6-methylpyridimidinyl) phosphorothioate, its metabolites (B) diazoxon (O,O-diethyl O-2-isopropyl-6-methylpyridimidinyl) phosphate, and (C) 2-isopropyl-6-methyl-4-pyrimidinol, the insecticide (D) permethrin [3-(2,2-dichloro-ethenyl)-2,2-dimethylcyclopropanecarboxylic acid (3-phenoxyphenyl)methylester], its metabolites (E) m-phenoxybenzyl alcohol, and (F) m-phenoxybenzoic acid, the insect repellent (G) DEET (N,N-diethyl-m-toluamide), and its metabolites (H) m-toluamide and (I) m-toluic acid in rat plasma and urine. The method is based on using C18 Sep-Pak cartridges (Waters Corporation, Milford, Mass., U.S.A.) for solid phase extraction and high performance liquid chromatography with a reversed phase C18 column, and absorbance detection at 230 nm for compounds A, B, and C, and at 210 nm for compounds D-I. The compounds were separated using a gradient from 1% to 99% acetonitrile in water (pH 3.0) at a flow rate ranging between 1 and 1.7 mL/min in a period of 17 min. The limits of detection were ranged between 20 and 100 ng/mL, while limits of quantification were 80-200 ng/mL. The relationship between peak areas and concentration was linear over a range of 100-1000 ng/mL. This method was applied to determine the above insecticides and their metabolites following dermal administration in rats.     

Mechanistic and computational study of a palladacycle-catalyzed decomposition of a series of neutral phosphorothioate triesters in methanol

The methanolytic cleavage of a series of O,O-dimethyl O-aryl phosphorothioates (1a?g) catalyzed by a C,N-palladacycle, (2-[N,N-dimethylamino(methyl)phenyl]-C1,N)(pyridine) palladium(II) triflate (3), at 25 °C and sspH 11.7 in methanol is reported, along with data for the methanolytic cleavage of 1a?g. The methoxide reaction gives a linear log k2?OMe vs sspKa (phenol leaving group) Br?nsted plot having a gradient of βlg = ?0.47 ± 0.03, suggesting about 34% cleavage of the P?OAr bond in the transition state. On the other hand, the 3-catalyzed cleavage of 1 gives a Br?nsted plot with a downward break at sspKa (phenol) 13, signifying a change in the rate-limiting step in the catalyzed reaction, with the two wings having βlg values of 0.0 ± 0.03 and ?1.93 ± 0.06. The rate-limiting step for good substrates with low leaving group sspKa values is proposed to be substrate/pyridine exchange on the palladacycle, while for substrates with poor leaving groups, the rate-limiting step is a chemical one with extensive cleavage of the P?OAr bond. DFT calculations support this process and also identify two intermediates, namely, one where substrate/pyridine interchange has occurred to give the palladacycle coordinated to substrate through the S═P linkage and to methoxide (6) and another where intramolecular methoxide attack has occurred on the P═S unit to give a five-coordinate phosphorane (7) doubly coordinated to Pd via the S? and through a bridging methoxide linked to P and Pd. Attempts to identify the existence of the phosphorane by 31P NMR in a d4-methanol solution containing 10 mM each of 3, trimethyl phosphorothioate (a very slow cleaving substrate), and methoxide proved unsuccessful, instead showing that the phosphorothioate was slowly converted to trimethyl phosphate, with the palladacycle decomposing to Pd0 and free pyridine. These results provide the first reported example where a palladacycle-promoted solvolysis reaction exhibits a break in the Br?nsted plot signifying at least one intermediate, while the DFT calculations provide further insight into a more complex mechanism involving two intermediates.     

Detection and Identification of Dizocilpine and its Major Urinary Metabolites in the Horse: A Preliminary Report

Dizocilpine ([+]-10,11-dihydro-5-methyl-5H-dibenzo[a,d]cyclohepten-5,10-imine), is a potent and selective NMDA (N-methyl-D-aspartate) receptor antagonist, which acts by blocking receptor ion channels. Dizocilpine is pharmacologically related to ketamine and phencyclidine; as such, it has the potential to affect behavior and performance in horses, with particular efficacy at lower concentrations. We now report development of a sensitive method for the detection of dizocilpine and preliminary characterization of its urinary metabolites in the horse. Dizocilpine (MW 221) readily produces a protonated species [M+H]⁺ in formic acid, and yields a m/z 205 product ion in Multiple Reaction Monitoring (MRM), allowing highly sensitive detection of parent drug. The 17 AMU loss most likely represents an unusual loss of CH₅ from the exocyclic methyl group. No unchanged dizocilpine was identified in unhydrolysed urine, and the presence of hydroxymethyl and carboxydizocilpine glucuronide metabolites were supported by observation of m/z 414→238 and 428→235 transitions. Urine samples from horses dosed with dizocilpine (0.0132 and 0.0656 mg kg^?1, iv) were hydrolysed with glucuronidase and were found to contain dizocilpine and OH-dizocilpine. Tentatively identified phase I post-hydrolysis compounds include dizocilpine itself, an hydroxymethyl metabolite, two ring-hydroxylated metabolites, a di-hydroxy metabolite, and a carboxy-dizocilpine metabolite. Corresponding Phase II glucuronidated metabolites were also identified as well as a number of combination metabolites and a posssible n-glucuronide metabolite for a total of at least six identifiable urinary glucuronide metabolites. Among the phase I metabolites, the hydroxymethyl metabolite apparently predominated, especially at the 0.0132 mg kg^?1 dose. The goal of this research was to identify a target analyte for dizocilpine in post-administration equine urine, so that work may begin on development of a forensically validated qualitative method for this target analyte. Given the likelihood that the doses of dizocilpine used in attempts to influence the behavior or performance of horses, either alone or in combination with other agents, are expected to be in the order of 0.02 mg kg^?1 or less, these results suggest selection of the phase I hydroxymethyl metabolite of dizocilpine as the optimal target analyte for regulatory control of dizocilpine in performance horses.     

Determination of water soluble imidazo-1,4-benzodiazepines in blood by electron- capture gas--liquid chromatography and in urine by differential pulse polaragraphy.

A sensitive and specific electron-capture gas--liquid chromatographic (GLC--ECD) assay was developed for the determination of 8-chloro-6-(2'-fluorophenyl)-1-methyl-4H-imidazo(1,5a)(1,4)benzodiazepine (I) or 8-chloro-1,4-dimethyl-6-(2'-fluorophenyl)-4H-imidazo (1,5a)(1,4)benzodiazepine (II) in blood. The assay for both compounds involves extraction into benzene--methylene chloride (9:1) from blood buffered to pH 12.6 The overall recovery of I and II from blood is 86% +- 5.0 (S.D.) and the sensitivity limit of detection is of the order of 2 to 3 ng of I or II per milliltre of blood. The major urinary metabolite of I is 8-chloro-6-(2'-fluorophenyl)-1-hydroxymethyl-4H-imidazo(1,5a)(1,4)benzodiazepine, (IA) present as a glucuronide conjugate while 8-chloro-6-(2'-fluorophenyl)-4-hydroxyl-1-methyl-4H-imidazo(1,5a)(1,4)benzodiazepine, (IB) and 8-chloro-6-(2'-fluorophenyl)-4-hydroxy-1-hydroxymethyl-4H-imidazo(1,5a)(1,4) benzodiazepine, (IC) are minor metabolites. The major metabolite IA is extracted into benzene--methylene chloride (9:1) from urine buffered to pH 11.0 (after incubation with glucuronidase--sulfatase as pH 5.0), and analyzed by differential pulse polarography (DPP) in 0.1 M phosphate buffer PH 3). The overall recovery of IA is 84 +- 3.0% (S.D.) with a sensitivity limit of 50 ng per millilitre of urine. The metabolites of compound II have not as yet been elucidated. The GLC--ECD and DPP assays were applied to the determination of blood levels and urinary excretion in dogs following single 10 mg/kg intravenous and oral doses of I and following single 6 mg/kg intravenous and 10 mg/kg oral doses of II. Blood levels of compound I were also evaluated in man following intravenous infusion of single 10 mg doses.     

Biosorption of copper(II) from aqueous solutions by Spirogyra species

Batch studies were conducted to investigate the kinetics and isotherms of Cu(II) biosorption on the biomass of green alga Spirogyra species. It is observed that the biosorption capacity of the biomass strongly depends on pH and algal dose. The maximum biosorption capacity of 133.3 mg Cu(II)/g of dry weight of biomass was observed at an optimum pH of 5 in 120 min with an algal dose of 20 g/L. Desorption studies were conducted with 133.3 mg/g of Cu(II) loaded biomass using different desorption agents including HCl, EDTA, H2SO4, NaCl, and H2O. The maximum desorption of 95.3% was obtained with HCl in 15 min. The results indicate that with the advantages of high metal biosorption capacity and satisfactory recovery of Cu(II), Spirogyra can be used as an efficient and economic biosorbent material for the removal and recovery of toxic heavy metals from polluted water.     

Different quantitation approaches for xenobiotics in human urine samples by liquid chromatography/electrospray tandem mass spectrometry

The potential of liquid chromatography combined with tandem mass spectrometry (LC/MS/MS) for the determination of pesticide metabolites in human urine at the sub-ppb level is explored. Metabolites from two organophosphorous pesticides, 4-nitrophenol (from parathion and parathion-methyl) and 3-methyl-4-nitrophenol (from fenitrothion), are taken as model analytes to conduct this study. After direct injection of the urine sample (10 microL), different approaches were evaluated in order to achieve correct quantitation of analytes using an electrospray ionisation (ESI) interface. Thus, the feasibility of using external calibration was checked versus the use of different isotope-labeled internal standards. The advantages of applying coupled-column liquid chromatography (LC/LC) as an efficient clean-up without any type of sample manipulation are also discussed. The combination of LC/LC with ESI-MS/MS allows the direct analysis of free metabolites in urine, as the automated clean-up performed by the coupled-column technique is sufficient for the removal of interferences that suppress the ionisation of analytes in the ESI source. Using this procedure with external calibration, good precision and recoveries, and detection limits below 1 ng/mL are reached with analysis run times of around 8 min. The hyphenated technique LC/LC/ESI-MS/MS is proved to be a powerful analytical tool, allowing the rapid, sensitive and selective determination of 4-nitrophenol and 3-methyl-4-nitrophenol in human urine without any sample treatment.