Synthesis of hydroxylated intermediate metabolites of dimetacrin

To prove the chemical structure of metabolites of the antidepressant drug (dimetacrin²) (Istonil®³), 2- and 3-hydroxy-9,9-dimethyl-10-dimethylaminopropyl-acridane and 2-hydroxy-monometacrin⁴ were synthesized using substituted alkoxy diphenylamines (V) as intermediates. The methoxy compounds on treatment with strong acids cyclized either to the corresponding acridanes or to their O-demethylated derivatives, VI (X ? OCH₃ or OH), depending on concentration and temperature.     

The synthesis of possible hydroxylated metabolites of 2-chlorophenothiazine derivatives

The synthesis of 7-hydroxy, 8-hydroxy and 7,8-dihydroxy prochlorperazines (3a) and per-phenazines (3b) is reported. The parent ehlorophenothiazines were prepared analogously to previously reported chlorpromazine (3e) metabolite syntheses. The prochlorperazine side chain was introduced in one step using 1-(3-chloropropyl)-4-methylpiperazine and the perphenazine side chain in two steps, first reaction with 1-bromo-3-chloropropane followed by reaction with 1-piperazineethanol. The methoxymethyleneoxy O-proteetive groups were removed under mild conditions using methanolic hydrogen chloride. The preparations of 7-hydroxydesmethylprochlorperazine and 7-hydroxychlorpromazine quaternary methyl iodide are also reported.     

Synthesis and identification of hydroxylated metabolites of the anti-estrogenic agent cyclofenil

The detection of metabolites of the anti-estrogenic substance cyclofenil, listed on the World Anti-Doping Agency (WADA) Prohibited List since 2004 is described. Target substances are hydroxylated metabolites, bearing an aliphatic hydroxyl group either in the 2-, 3- or 4-position of the aliphatic ring, in addition to the phenolic functions on the aromatic rings. Structural identification used NMR as well as high-resolution mass spectrometry after nano-electrospray ionisation (ESI). Unambiguous detection of all three synthesised cyclofenil metabolites M1-M3 was done using gas chromatography for separation and electron ionisation mass spectrometry for detection of the per-silylated compounds in comparison with a reference urine deriving from an excretion study within the WADA 2007 Educational Programme.     

Syntheses of some hydroxylated metabolites of psychotropic trifluoromethyl analogs of chlorpromazine

The syntheses of 7-hydroxy derivatives of trifluoperazine, nor,-trifluoperazine, fluphenazine, triflupromazine and nor,-triflupromazine are described. These were prepared as analytical standards for the identification of these metabolites in biological materials.     

Atropisomeric determination of chiral hydroxylated metabolites of polychlorinated biphenyls using HPLC-MS

Background

Polychlorinated biphenyls (PCBs) are a group of environmental persistent organic pollutants, which can be metabolized into a series of metabolites, including hydroxylated metabolites (OH-PCBs) in biota. Nineteen of 209 PCB congeners can form chiral stable isomers. However, atropisomeric determination of the hydroxylated metabolites of these chiral PCBs has never been reported by LC methods. In this work, a novel HPLC-MS method was developed to detect five chiral OH-PCBs (4OH-PCB91, 5OH-PCB91, 4OH-PCB95, 5OH-PCB95 and 5OH-PCB149) using HPLC-MS without a derivatization step.

Results

The influences of column-type, column temperature, flow rate and ratio of the mobile phase on the atropisomeric separation were investigated in detail. In the final method, calibration curves, based on peak areas against concentration, were linear in a range of 1–100 ng mL^-1 of five chiral OH-PCBs with correlation coefficients ranging from 0.9996 to 0.9999 for all atropisomers of OH-PCBs. The relative standard deviations measured at the 10.0 ng mL^-1 level for atropisomers of five chiral OH-PCBs were in the range of 0.60-7.55% (n?=?5). Calculated detection limits (S/N?=?3) of five chiral OH-PCBs were between 0.31 and 0.60 ng mL^-1 for all OH-PCB atropisomers.

Conclusion

This HPLC-MS method was developed to detect chiral OH-PCBs and further successfully applied to measure OH-PCB atropisomer levels and enantiomeric fractions (EFs) in rat liver microsomal samples. The results from LC-MS method were highly consistent with those from GC-ECD method. It is the first time to report these OH-PCB atropisomers detected in microsomes by HPLC-MS. The proposed method might be applied also to detect chiral OH-PCBs in environmental samples and for metabolites of PCBs in vivo.

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Simultaneous analysis of prednisone, prednisolone and their major hydroxylated metabolites in urine by high-performance liquid chromatography

A high-performance liquid chromatographic technique for the simultaneous determination of prednisone, prednisolone and their major hydroxylated metabolites, viz., 20 beta-hydroxyprednisone, 6 beta-, 20 alpha- and 20 beta-hydroxyprednisolone, in human urine is presented. The retention times were 6.5, 11.4, 18.1, 24.2, 31.6 and 35.3 min, respectively. The technique employs betamethasone as the internal standard. Samples are extracted with ethyl acetate using a diatomaceous earth extraction column, and the extract was dried and injected onto a silica gel column with ultraviolet detection at 254 nm. The calibration curve is linear within the studied range 50-1500 ng/ml for prednisolone and 50-750 ng ml for the other steroids. The intra-day and inter-day coefficients of variation are less than 10% for prednisone and prednisolone but higher for the metabolites. The assay was used to study the excretion rate profile of each of these steroids in the urine of a normal male subject receiving a 49.3-mg intravenous dose of prednisolone. The results indicate that prednisone, 6 beta-, 20 alpha- and 20 beta-hydroxyprednisolone may be the major unconjugated metabolites of prednisolone while 20 beta-hydroxyprednisone may be a minor metabolite.     

New electronically enriched boronobutadienes for the synthesis of hydroxylated cyclohexenes via tandem [4+2]/allylboration

This communication describes the preparation and behavior of ether-substituted 1-boronobutadienes 1 and 2 in the Vaultier tandem [4+2]/allylboration three-component reaction involving electron-poor dienophiles and aldehydes. Whereas diene 1 failed to undergo the second, allylboration step of this process, diene 2 reacted with maleimides to give products 12, and even reacted with moderately activated dienophiles such as acrylates and acrylamide derivative 14 to provide a new stereoselective approach to the synthesis of hydroxyalkylated cyclohexene derivatives.     

An assay of sulphasalazine,sulphapyridine and its acetylated and hydroxylated metabolites in biological fluids by liquid chromatography

Summary An HPLC assay was developed for the determination of sulphasalazine (salicylazosulphapyridine), and its main metabolites; sulphapyridine, acetylsulphapyridine, hydroxysulphapyridine and acetylhydroxysulphapyridine in human serum, synovial fluid and urine. Sep-Pak C₁₈ cartridge was used for the extraction of the investigated compounds and the added sulphadimidine internal standard. The methylene chloride-chloroform-methanol eluate was evaporated and the dissolved dry residue was injected onto a Hypersil ODS (100×2.1 mm) 5 m microbore, analytical column. A linear gradient was used, the mobile phase was ternary. The assay is sufficiently sensitive for routine therapeutic drug monitoring and phenotyping in patients treated with sulphasalazine.     

Simultaneous determination of tanshinone IIA and its three hydroxylated metabolites by liquid chromatography/tandem mass spectrometry

A rapid and sensitive method based on liquid chromatography/tandem mass spectrometry (LC/MS/MS) for the simultaneous determination of tanshinone IIA and its three hydroxylated metabolites, tanshinone IIB, hydroxytanshinone IIA and przewaquinone A, in a rat liver microsome was developed and fully validated. A single step of liquid-liquid extraction with ethyl acetate was utilized in this method. Chromatographic separation of the sample matrix from the analytes and the internal standard diazepam was performed using a Shim-pack VP-ODS analytical column. Detection was performed on a triple quadrupole tandem mass spectrometer equipped with an electrospray ionization source and operated in selected reaction monitoring (SRM) mode. The method was linear in the concentration range of 1-500 ng/mL for all analytes. The intra- and inter-day precisions (RSD %) were within 15% and deviations of the assay accuracies were within 15.0% for all analytes. The analytes proved to be stable during sample storage, preparation and analyses. This validated method was successfully applied to the enzyme kinetic study of tanshinone IIA in liver microsome. The elimination of tanshinone IIA and formation of tanshinone IIB and hydroxytanshinone IIA in the liver microsome all exhibited a sigmoidal kinetics profile. The formation of przewaquinone A shows a typical hyperbolic profile. In addition, this method has now been applied in the analysis of other bio-samples including plasma, urine, bile and feces.     

Analysis of phenprocoumon and its hydroxylated and conjugated metabolites in human urine by high-performance liquid chromatography after solid-phase extraction

The anticoagulant phenprocoumon is mainly metabolized in humans to hydroxylated metabolites and their glucuronides. A method is described for the determination of phenprocoumon, 4'-hydroxyphenprocoumon, 6-hydroxyphenprocoumon, 7-hydroxyphenprocoumon, and their glucuronide and sulphate conjugates in human urine. Reversed-phase high-performance liquid chromatography is performed after selective extraction with disposable quaternary amine columns of untreated, and beta-glucuronidase- or sulphatase-treated urine samples. Urinary excretion data are presented for total, glucuronidated, sulphated and free phenprocoumon, 4'-hydroxyphenprocoumon, 6-hydroxyphenprocoumon and 7-hydroxyphenprocoumon in twelve patients after an average daily dosage of 1.3-4.2 mg phenprocoumon.     

Determination of dithiocarbamates and metabolites in plants by liquid chromatography-mass spectrometry

A quantitative matrix solid-phase dispersion and liquid chromatography-atmospheric pressure chemical ionization mass spectrometry (LC-APCI-MS) method is outlined for the simultaneous analysis of dithiocarbamates (DTCs) and their degradation products in plants. Compounds analyzed are dazomet, disulfiram, thiram and the metabolites ethylenthiourea and propylenthiourea. The performance of two different sample preparation protocols, the proposed one and other based on solid-phase extraction, as well as, of both atmospheric pressure ionization sources, APCI and electrospray, were compared. The effect of several parameters on the extraction, separation and detection was studied. Dithiocarbamates and metabolites were dispersed with carbograph, eluted with a mixture of dichloromethane-methanol, and then, identified by monitoring the base peak of the spectra corresponding to [M + H]+. The method was validated for avocados, cherries, lemons, nuts, oat, oranges, peaches, rice and tomatoes. Average recoveries varied from 33 to 109%, and relative standard deviation were between 4 and 21% with limits of quantification ranged from 0.25 to 2.5 mg kg(-1), except for thiram and disulfiram, which were not recovered from fruits with high acid content. The procedure was applied to the determination of DTCs and their metabolites in fruits, vegetables and cereals taken from different markets of Valencia, Spain.     

Determination of yohimbine and its two hydroxylated metabolites in humans by high-performance liquid chromatography and mass spectral analysis.

The existence of at least two metabolites of yohimbine (YO) in humans is demonstrated. Combined high-performance liquid chromatographic (HPLC), NMR and mass spectral analyses permitted them to be identified as hydroxylated metabolites at the C-10 and C-11 positions. A normal-phase HPLC method allowing the simultaneous determination of YO and its main metabolite, 11-hydroxyyohimbine (11-OHYO), in biological samples is described. This assay was performed using a LiChrosorb Si 60 column and a mobile phase consisting of 0.02 M sodium acetate (pH 5)-methanol (5:95, v/v) at a flow-rate of 1 ml/min. Detection was achieved by a fluorimetric method (excitation at 280 nm and emission at 320 nm). The extraction yields of YO, 10-OHYO and 11-OHYO from plasma were 91.8, 45.3 and 17.8%, respectively, and their respective within-day reproducibilities were 3.8, 1.4 and 5.9%. The between-day reproducibility for YO at the concentrations of 1 and 10 ng/ml were 8.9 and 6.4%, respectively. The accuracy of the method for YO at concentrations of 1 and 10 ng/ml were 5.1 and 2.3%, respectively. The limits of determination of YO, 10-OHYO and 11-OHYO were 0.1, 0.5 and 1 ng/ml, respectively. The method was used in bioavailability study of YO following oral and intravenous administration in humans.     

Determination of clomipramine and its hydroxylated and demethylated metabolites in plasma and urine by liquid chromatography with electrochemical detection

A procedure for the determination of clomipramine and its 8-hydroxy, demethyl, 8-hydroxydemethyl and didemethyl metabolites in plasma and urine by high-performance liquid chromatography with electrochemical detection is described. A 1-ml plasma or urine sample is made alkaline with a carbonate buffer (pH 9.8) and extracted with 20% ethyl acetate in n-heptane. After back-extraction into an acid phosphate buffer (pH 2.4), an aliquot is injected into a 5-microns ion-paired reversed-phase column and eluted with a mobile phase containing a phosphate buffer with tetramethylammonium chloride-acetonitrile (57:43). The detection is coulometric with a first cell at +0.40 V, a second at +0.73 V and a guard cell set at 0.75 V for oxidation of the mobile phase. The method provides recoveries in the general range of 80-110% and a day-to-day precision of 3.7-8.8%, depending on the compound. The minimum quantifiable level for all compounds was 0.2 ng/ml with a 20-microliters injection. Steady-state plasma concentration data and urinary levels are reported for 24 depressed patients receiving daily either 75-150 mg orally or 50-75 mg by infusion.     

Thick film biosensors for metabolites in undiluted whole blood and plasma samples

The new electrochemical thick film biosensors from Roche Diagnostics are presented. Following considerations about the principal requirements that biosensors have to fulfil to be useful for diagnostic purposes, the basic design of these thick film biosensors is shown. In this paper, the new generation of biosensors for glucose, lactate and urea are presented, as well as data from a new biosensor for creatinine. All biosensors are designed for multiple use, at minimum 500 samples or 1 week in-use (depending on type of enzyme used), for determinations in undiluted whole blood or plasma, with extra electrodes to compensate for interferences. The sensors are integrated in a disposable cassette requiring 38 microtl sample volume. The analytical ranges of the sensors scope well with the normal and pathological concentrations of metabolites in human blood, e.g. for glucose 0.5-40.0 mmol/L. Both biosensors and interference-compensating electrodes are developed to have a cycle time of 90 s maximum. Method comparison diagrams show excellent correlation of results obtained by biosensors compared to results achieved by reference methods. In addition, the possibility of urea and creatinine determinations in diluted urine is presented.     

Quantitation of the enantiomers of rimantadine and its hydroxylated metabolites in human plasma by gas chromatography/mass spectrometry.

A gas chromatographic/mass spectrometric procedure has been developed for the quantitation in human plasma of the enantiomers of rimantadine and its three hydroxylated metabolites. The assay utilized derivatization of all analytes with the optically active reagent S-alpha-methyl-alpha-methoxy(pentafluorophenyl)acetic acid, selective ion monitoring, methane negative ion chemical ionization mass spectrometry and stable isotope dilution techniques. This method has been used to measure plasma concentrations of the enantiomers of rimantadine, m-hydroxyrimantadine and p-hydroxyrimantadine (equatorial and axial epimers) in the ranges 2.5-250, 2.5-50, 1.25-62.5 and 1.25-62.5 ng/mL, respectively, in six subjects given a single 200 mg dose of racemic rimantadine. Although there are no significant differences in the concentration-time profiles of R- and S-rimantadine, large stereospecific differences in the disposition of their metabolites are observed.     

Analysis of hydroxylated polybrominated diphenyl ether metabolites by liquid chromatography/atmospheric pressure chemical ionization tandem mass spectrometry

Hydroxylated polybrominated diphenyl ether (OH‐PBDEs) metabolites have the potential to cause endocrine disruption as well as other health effects. Currently, gas chromatography/mass spectrometry (GC/MS) after derivatization is used for the analysis of OH‐PBDEs. However, there is a need for the direct analysis of OH‐PBDEs at relatively low concentrations in environmental and biological samples. Liquid chromatography with atmospheric pressure chemical ionization tandem mass spectrometry (LC/APCI‐MS/MS) was evaluated for the analysis of nine OH‐PBDEs, ranging from tri‐ to hexabrominated. Separation of the nine isomeric metabolites was achieved with reversed‐phase liquid chromatography, followed by detection by APCI‐MS in negative mode. Notably, a significant decrease in ionization was observed in 6‐hydroxyl‐substituted PBDE metabolites in the presence of an ortho‐substituted bromine, relative to the other hydroxylated metabolites. This is probably due to the formation of dioxins in the source as a result of the high‐temperature conditions, which prevented ionization by hydrogen abstraction. The MS/MS experiments also provided evidence of the neutral losses of HBr and Br₂, indicating the possible use of neutral loss scanning and selected reaction monitoring (SRM) for the screening of brominated metabolites in samples. The applicability of LC/APCI‐MS/MS was demonstrated for the analysis of metabolites of BDEs 47 and 99 formed in human liver microsomes. The LC/APCI‐MS/MS method was able to detect metabolites that had previously been identified by GC/MS following derivatization. Copyright © 2010 John Wiley & Sons, Ltd.