Microcoulometric determination of total organochlorine pesticide and polychlorinated biphenyl residues in grey seal (Halichoerus grypus) blubber.

The determination of iron(III) with thiocyanate by means of sample injection into a chamber in a closed loop system of circulating reagent is described. Restitution of the absorbance to baseline by ligand exchange and by a redox process in the presence of iodide is discussed, together with some kinetic aspects of the decolorization reaction responsible for the return to the baseline. The iron content of the samples is deduced from the height of the generated transient signal, which is directly proportional to the iron(III) content. As many as 350 determinations/h can be processed when ligand exchange is used and 120/h when iodide is used for baseline restitution. Sample injection techniques of this type are particularly useful in industrial processes and quality control, and environmental monitoring, since they result in a decrease in operating cost and time.     

Structure elucidation of aplidine metabolites formed in vitro by human liver microsomes using triple quadrupole mass spectrometry

The cyclic depsipeptide aplidine is a new anti-cancer drug of marine origin. Four metabolites of this compound were found after incubation with pooled human microsomes using gradient high-performance liquid chromatography with ultraviolet detection. After chromatographic isolation, the metabolites have been identified using nano-electrospray triple quadrupole mass spectrometry. A highly specific sodium-ion interaction with the cyclic structure opens the depsipeptide ring, and cleavage of the amino acid residues gives sequence information when activated by collision-induced dissociation in the second quadrupole. The aplidine molecule could undergo the following metabolic reactions: hydroxylation at the isopropyl group (metabolites apli-h 1 and apli-h 2); C-dealkylation at the N(Me)-leucine group (metabolite apli-da); hydroxylation at the isopropyl group and C-dealkylation at the N(Me)-leucine group (metabolite apli-da/h), and C-demethylation at the threonine group (metabolite apli-dm). The identification of these metabolites formed in vitro may greatly aid the elucidation of the metabolic pathways of aplidine in humans.     

High performance liquid chromatographic determination of theophylline metabolites in human liver microsomes

A high performance liquid chromatographic (HPLC) technique for the determination of three metabolites of theophylline, 3-methylxanthine (3-MX), 1-methylxanthine (1-MX) and 1,3-dimethyluric acid (1,3-DMU) in human liver microsomes is described. The analytes were extracted from human liver microsomes with methylene chloride/isopropanol and stepwise gradient elution was employed for the resolution of peaks. The limits of quantitation were 15 ng/mL for 3-MX, 20 ng/mL for 1-MX and 20 ng/mL for 1,3-DMU. The calibration range was linear for the three metabolites and the calibration ranges were 15-250 ng/mL for 3-MX, 20-250 ng/mL for 1-MX and 250-4000 ng/mL for 1,3-DMU. The absolute recovery ranged from 63-84% for 3-MX, 65-79% for 1-MX and 77-89% for 1,3-DMU over the calibration curve range. Accuracy for all three metabolites was within +/- 10% and adequate selectivity was demonstrated by the lack of interfering peaks in blank chromatograms. The within-run and interday precision were within 10% RSD for all three metabolites tested at two concentrations. The advantage of this method over previous methods is that the use of quaternary ammonium ion pair reagents in the mobile phase has been obviated. Also, unlike a previous radiometric HPLC method, the need for radiolabelled theophylline has also been eliminated. The method was used to characterize theophylline metabolism in human liver microsomes for immunoinhibition studies and to investigate the interaction of theophylline with selected quinolone antibiotics.     

A new method for the purification of cytochrome-P450 from human liver microsomes.

A procedure for the solubilization and purification of cytochrome-P450 (cyt-P450) from human liver microsomes is described. Successive treatment of microsomes with protease XXVII and 3-(3-cholamidopropyl)dimethylammoniopropanesulphonic acid gave a solubilized cyt-P450 in more than 80% yield and with a three-fold increase in specific activity. With this treatment it was possible to eliminate 80% of cytochrome-b5 and 75% of NADPH cyt-P450 reductase. The solubilized cyt-P450 was filtered on a Sephacryl-200 column and then subjected to high performance liquid chromatography with a Mono-P column (chromatofocussing). The recovery of separated cyt-P450 was about 50% with a specific activity of 11.5 nmol cyt-P450/mg protein. Also with this technique it was possible to determine the isoelectric points of cyt-P450. These results allowed us to confirm the usefulness of our method, for the study the cyt-P450 from surgical biopsies.     

Liquid chromatographic determination of 6-thiopurine metabolites formed in vitro in electrochemical and enzymatic oxidative activation

A micellar liquid chromatographic method is described which was developed for the separation of the oxidation metabolites of 6-thiopurine formed in vitro by electrochemical and enzymatic activation. Electrochemical activation was carried out with an electrochemical cell on-line with the chromatograph. In the potential range 0.4–0.8 V vs. Pd, intermediate purine-6-sulfenic acid could be detected together with purine-6-sulfinic acid and 6-thiopurine disulfide. At potentials > 0.8 V, purine-6-sulfonic acid was detected and the oxidation of 6-thiopurine was completed. Intermediates and products formed in the horseradish peroxidase-catalyzed oxidation of 6-thiopurine were also studied. Enzymatic activation with horseradish peroxidase was similar to electrochemical oxidation at <0.8 V. Detection of sulfenic acid in the enzymatic oxidation supports earlier results which indicated that this metabolite may have biological significance. The results also provide some insight into the enzymatic oxidation pathway.     

Ultra-performance liquid chromatography/tandem mass spectrometric determination of testosterone and its metabolites in in vitro samples

This paper describes the development and partial validation of a fast, sensitive and specific ultra-performance liquid chromatography/tandem mass spectrometric (UPLC/MS/MS) method for the determination of testosterone (T) and its four metabolites, 6beta-OH-T, 16alpha-OH-T, 16beta-OH-T and 2alpha-OH-T, in in vitro samples. The analytical method involves direct dilution of samples with acetonitrile containing an internal standard, followed by separation of testosterone and the four metabolites on an Acquity UPLCtrade mark C(18) column and detected by selected reaction monitoring (SRM) in positive ionization mode using turbo ionspray ionization. The parent compound and its metabolites investigated were well separated (Rs >1.5) with a run time of 4 min under a gradient condition. The method was partially validated. The linear concentration range was 0.01 to 5 microM for all the compounds of interest. Inter-assay mean bias and relative standard deviation (RSD) were in the range of -12% to 8% and 4.1% to 8.5%, respectively. Intra-assay mean bias and RSD were in the range of -8.0% to 5.2% and 3.4% to 9.6%, respectively. The lower limit of quantitation for this assay was 0.01 microM. The differences in LC/MS performance were investigated by conducting a comparison of UPLC with another method previously optimized for HPLC-based separation and quantification of testosterone and its metabolites.     

A method for the structural determination of cannabichromene metabolites by mass spectrometry

Ready identification of hydroxy metabolites of cannabichromene (CBC) by mass spectrometry of their trimethylsilyl derivatives is prevented by the dominant fragmentation to give a substituted chromenyl ion; this suppresses ions diagnostic of the position of metabolic hydroxylation. To overcome this difficulty, metabolites were hydrogenated over a rhodium/alumina catalyst to reduce the double bond responsible for chromenyl ion formation and to redirect the fragmentation to the site of metabolic attack. This resulted in the production of abundant diagnostic fragment ions enabling all monohydroxy-CBCs to be readily identified.     

Characterization of in vitro metabolites of rutaecarpine in rat liver microsomes using liquid chromatography/tandem mass spectrometry

Following incubation of rutaecarpine, a new cyclooxygenase-2 inhibitor, with rat liver microsomes, the structures of the metabolites were characterized by liquid chromatography with tandem mass spectrometry. Nine metabolites corresponding to mono- or dihydroxylated rutaecarpine were formed. Characteristic product ions for the identification of rutaecarpine metabolites were observed at m/z 136, 158 and 286. The loss of water led to the fragment ion at m/z 286, indicating the hydroxylation of the aliphatic ring. The fragment ion at m/z 136 indicated the hydroxylated form of the phenyl group of the quinazolinone moiety, while that at m/z 158 indicated the hydroxylated form of the aromatic ring of the indole moiety.     

A comprehensive gas chromatography coupled to high resolution mass spectrometry based method for the determination of polybrominated diphenyl ethers and their hydroxylated and methoxylated metabolites in environmental samples

We report here an efficient and comprehensive analytical methodology based on gas chromatography with high resolution mass spectrometry (GC–HRMS) to simultaneously determine PBDEs from mono to deca brominated and hydroxy (OH-) and methoxy (MeO-) PBDE metabolites in environmental samples, particularly, sediment, fish tissue and milk. Among a number of extraction and clean-up methods tested, pressurized liquid extraction followed by gel permeation chromatography and florisil clean-up proved to be simple, robust and optimized so that all target analytes (parent compounds and metabolites) were collected in a single fraction. Extracts were analyzed by GC–HRMS to identify PBDEs. Following, the same extracts were derivatized and re-analyzed by GC–HRMS to determine 11 target and 35 non-target OH- and MeO-PBDEs. Monitoring of the M⁺ for MeO-PBDEs and the [M−CH₂CO]⁺ ions for derivatized OH-PBDEs at 10,000 resolution permitted unequivocal identification of the PBDE metabolites in the environmental matrices examined. The method was validated in terms of accuracy, precision, detection limits and long-term stability. The analytical precision obtained with this method was between 0.3 and 17%, and the limits of quantification were lower than 3.28 pg/g dry weight, 20.5 and 41.4 pg/g lipid weight in sediment, milk and fish, respectively. The method was applied to determine PBDEs and target and non-target metabolites in all three matrices.     

Multi-residue method for the determination of chlorinated phenol metabolites in urine

Electron-capture-gas chromatographic (EC-GC) methods for the determination of chlorinated phenol metabolites of hexachlorobenzene (HCB) and pentachlorophenol (PCP) in urine are presented. After extraction the sample was reacted with diazomethane to produce the methyl ether of each metabolite prior to determination by EC-GC. An acid alumina column was used for cleanup and separation of methylated phenols into groups. Average recoveries of greater than 80% were obtained from urine fortified with known amounts of the phenol metabolites under investigation. A level of 1 ppb¹ was established as minimum detection limit for each phenol metabolite. Previously unreported urinary metabolites of HCB and PCP were found as a result of a rat feeding study. Levels of chlorinated phenol residues from (a) human general population and (b) a worker occupationally exposed to PCP are also included.     

Characterization of in vitro metabolites of deoxypodophyllotoxin in human and rat liver microsomes using liquid chromatography/tandem mass spectrometry

The in vitro metabolism of deoxypodophyllotoxin (DPT), a medicinal herbal product isolated from Anthriscus sylvestris (Apiaceae), was investigated in rats and human microsomes and human recombinant cDNA-expressed CYPs. The incubation of DPT with pooled human microsomes in the presence of NADPH generated five metabolites while its incubation with dexamethasone (Dex)-induced rat liver resulted in seven metabolites (M1-M7) with major metabolic reactions including mono-hydroxylation, O-demethylation and demethylenation. Reasonable structures of the seven metabolites of DPT could be proposed, based on the electrospray tandem mass spectra. Chemical inhibition by ketoconazole and metabolism studies with human recombinant cDNA-expressed CYPs indicated that CYP 3A4 and 2C19 are the major CYP isozymes in the metabolism of DPT in human liver microsomes.     

A new volumetric method for the determination of uranium(VI)

Summary A new volumetric method for the estimation of uranium(VI) salt based on its photoreduction in the presence of diethyl ether has been developed. The recommended procedure consists of exposing uranium(VI) solution in about 1 N sulphuric acid solution with an excess of saturated aqueous ether solution in a glass vessel to the light from a Phillips repro lamp or sun light for 1 hour. The uranium(IV) salt formed is estimated by titration with a standard solution of sodium vanadate.The reduction does not proceed to uranium(III) stage under any conditions of exposure. Fluoride, phosphate, arsenate and perchlorate are not found to interfere either with the photochemical reduction or with the subsequent oxidimetric titration. But chloride and bromide ions markedly inhibit the photochemical reaction.     

A rapid gas chromatographic method for the determination of diazepam and metabolites in body fluids

A rapid method is described for the extraction of diazepam and its metabolites from plasma and urine. The procedure is applicable to subsequent analysis by electron capture gas chromatography, and has been used for the analysis of clinical samples. The detection limit for diazepam is about 0.01 μg/ml, using a 2-ml sample. Quantification of lower levels of benzodiazepines requires a sample clean-up procedure, and the method is not suitable for this purpose.     

A new volumetric method for the determination of molybdenum(VI)

Summary A new volumetric method has been developed for the determination of molybdenum(VI). The method consists in the reduction of molybdenum(VI) by heating with a slight excess of hydrazine sulphate in 1 to 2 M hydrochloric acid medium for ten minutes on a water bath. The mixture is cooled and the molybdenum(V) obtained determined by titration with a standard solution of ceric sulphate at an overall acidity of 4 N hydrochloric acid, using diphenyl benzidine as indicator and adding 5 ml of syrupy phosphoric acid for 50 ml of the mixture. Alternately the molybdenum(V) can be titrated with a standard solution of ceric sulphate at an overall acidity of 3 N hydrochloric acid using ferroin as indicator and adding 5 ml of syrupy phosphoric acid for 50 ml of the titration mixture. The molybdenum(V) can also be titrated with a standard solution of sodium vanadate in 8 N sulphuric acid medium, using N-phenyl anthranilic acid as indicator. Alternately, the titration with sodium vanadate can be made with diphenyl benzidine as indicator in 4 N acid medium, adding 5 ml of syrupy phosphoric acid and 1 ml of 1.0 M oxalic acid to catalyse the indicator action. The method now proposed is much more convenient than the methods currently available. It is simple because it does not require any costly chemicals or complicated apparatus. Furthermore, it has the advantages of great rapidity and excellent precision.     

A new method for the construction of the hydroxylated tropane skeleton: enantioselective synthesis of (-)-Bao Gong Teng A

An efficient and highly diastereoselective method for the construction of the hydroxylated tropane skeleton is described. The method features a new intramolecular reductive coupling reaction of N-acyl N,O-acetal with aldehyde, cooperatively mediated by BF(3)·OEt(2) and SmI(2). On the basis of this method, a new enantioselective total synthesis of (-)-Bao Gong Teng A has been accomplished.     

Mass spectrometric determination of appearance energies for ions formed from CoF(4) and CoF(3) molecules

Knudsen cell mass spectrometry was applied to the evaluation of the ionization efficiency curves for the ions originating from CoF(4) molecules. Cobalt tetrafluoride was obtained in the gas phase over the CoF(3)(s)-TbF(4)(s) system in the temperature range from 640 to 690 K. From the ionization efficiency curves the appearance energies of the ions formed from the CoF(4) molecules were determined by means of Vogt's deconvolution method. Clausius-Clapeyron plots for the ions from CoF(4) molecules were measured. Evaporation of pure CoF(3)(s) was carried out, and the appearance energies of the ions formed from CoF(3) molecules were determined. The ionization energies for CoF(4) and CoF(3) molecules were found to be (14.3 +/- 0.2) and (13.3 +/- 0.1) eV, respectively.