Enantiomeric separation of some piperidine-2,6-dione drugs using chiralcel OJ-R column

A newly developed reversed phase cellulose tris(4-methyl benzoate) known as Chiralcel OJ-R was used to investigate the chiral recognition and enantiomeric separation of eight racemic piperidine-2,6-dione compounds—namely, aminoglutethimide and its major metabolite acetylaminoglutethimide, glutethimide, cyclohexylamino-glutethimide, pyridoglutethimide, thalidomide, phenglutarimide, and 3-phenylacetyl-amino-2,6-piperidinedione (antineoplaston A-10). Chiral separation of these compounds was achieved under varying ratios of the mobile phase, except for phenglutarimide and 3-phenylacetylamino-2,6-piperidinedione, for which separation was unsuccessful. Possible chiral recognition mechanisms are also presented. Chirality 9:10–12, 1997. © 1997 Wiley-Liss, Inc.     

Beta-oxidation of 2-ethyl-5-carboxypentyl phthalate in rodent liver

[7-14C]-2-Ethyl-5-carboxypentyl phthalate was isolated and purified from urine of rats given [7-14C]-di-(2-ethylhexyl) phthalate. This metabolite was shown to serve as a precursor for 2-ethyl-3-carboxypropyl phthalate in vivo. 2-Ethyl-5-carboxypentyl phthalate was oxidized to 2-ethyl-3-carboxypropyl phthalate in liver slices from control or, much more rapidly, from clofibrate-pretreated rats. Inhibition by KCN in liver slices from untreated rats, and strong inhibition by acrylate, suggested that formation of 2-ethyl-3-carboxypropyl phthalate involved mitochondrial beta-oxidation. The strong enhancement of the production of this compound by clofibrate (a very weak inducer for mitochondrial dehydrogenases), and strong inhibition by chlorpromazine suggested that peroxisomes may also be able to oxidize 2-ethyl-5-carboxypentyl phthalate. We were able to detect beta-oxidation of 2-ethyl-5-carboxypentyl phthalate to 2-ethyl-3-carboxypropyl phthalate using purified mitochondria, but strong phthalate monoester hydrolase activity observed during incubation of the former compound with purified peroxisomes made it impossible to determine whether 2-ethyl-3-carboxypropyl phthalate could be produced in the latter organelle or not. 2-Ethyl-5-carboxypentyl phthalate was such an inefficient substrate for beta-oxidation compared to palmitic acid that it is unlikely that it contributes significantly to the production of H2O2 in rats chronically exposed to di-(2-ethylhexyl) phthalate. Normal fatty acids are most likely to serve as the dominant substrates for peroxisomal beta-oxidase.     

Characterization of a novel diclofenac metabolite in human urine by capillary gas chromatography-negative chemical ionization mass spectrometry

A sensitive analytical method was developed to characterize diclofenac metabolites in small amounts of body fluids. Desalted and lyophilized urine samples were extracted with supercritical carbon dioxide directly or after acidic hydrolysis. The extracts were derivatized with N-tert.-butyldimethylsilyl-N-methyltrifluoroacetamide. The derivatives were separated by capillary gas chromatography and identified by negative chemical ionization mass spectrometry. Full mass spectra were obtained at a level of 1·10⁻⁹ g/ml. With direct extraction, the metabolites could be analysed in one step as open-chained acids and as (cyclic) oxindoles. By acidic hydrolysis the conjugates were transformed to the oxindoles. With both methods, a new main metabolite, [2-[(2,6-dichloro-4-hydroxy-3-methoxyphenyl)amino]phenyl]acetic acid, was identified. The mechanism of its formation is discussed.     

Determination of the enantiomers of metoprolol and its major acidic metabolite in human urine by high-performance liquid chromatography with fluorescence detection

Enantiomers of metoprolol and its acidic metabolite H 117/04 were determined in human urine by high-performance liquid chromatography (HPLC) with fluorometric detection after chiral derivatization. The carboxyl functional group of the major metabolite was blocked by esterification after solid-phase extraction, which helped to quantitate this compound from interfering substances. The assay method was validated. The recovery of (−)- and (+)-metoprolol from urine was 86.3–90.5%; and the recovery of the (−)- and (+)-acidic metabolite H 117/04 from urine was 74.4–83.9% at different concentrations.     

Identification of 4-methoxybenzoyl-N-glycine in urine by gas chromatography/mass spectrometry.

An unusual metabolite was detected in the urine of two children with neurological dysfunctions of unclear aetiology by using gas chromatography/mass spectrometry (GC/MS). On the basis of the analysis of its fragmentation pathways, synthesis of tentative compound and its GC/MS analysis it was stated that the unknown metabolite is 4-methoxybenzoyl-N-glycine.     

3-Ethyl-2,7-dimethyl octane, a testosterone dependent unique urinary sex pheromone in male mouse (Mus musculus)

A previous investigation revealed that urine from normal male mice contained five unique volatile constituents; namely: 3-cyclohexene-1-methanol (I); 3-amino triazole (II); 4-ethyl phenol (III); 3-ethyl-2,7-dimethyl octane (IV); 1-iodoundecane (V). The present study was designed to find out whether the production of these male specific urinary compounds was androgen-dependent. Urine of castrated and castrated plus testosterone-treated male mice was analyzed using gas chromatography linked mass spectrometry (GC-MS). Even though castrated male urine contained 10 detectable compounds, the five male specific compounds present in intact males were absent in castrated male mice urine. Only 3-ethyl-2,7-dimethyl octane (IV) reappeared following testosterone treatment into castrated males. Our earlier bioassay revealed that this compound was involved in attracting females. The present study concluded that this compound was a male specific volatile cue that acted as a releaser pheromone and its production was under the control of androgen.     

β-Oxidation of 2-ethyl-5-carboxypentyl phthalate in rodent liver

[7-¹⁴C]2-Ethyl-5-carboxypentyl phthalate was isolated and purified from urine of rats given [7-¹⁴C]-di-(2-ethylhexyl) phthalate. This metabolite was shown to serve as a precursor for 2-ethyl-3-carboxypropylphthalate in vivo. 2-Ethyl-5-carboxypentyl phthalate was oxidized to 2-ethyl-3-carboxypropyl phthalate in liver slices from control or, much more rapidly, from clofibrate-pretreated rats. Inhibition by KCN in liver slices from untreated rats, and strong inhibition by acrylate, suggested that formation of 2-ethyl-3-carboxy-propyl phthalate involved mitochondria β-oxidation. The strong enhancement of the product of this compound by clofibrate (a very weak inducer for mitochondrial dehydrogenases), and strong inhibition by chlorpromazine suggested that peroxisomes may also be able to oxidize 2-ethyl-5-carboxypentyl phthalate. We were able to detect β-oxidation of 2-ethyl-5-carboxypentyl phthalate to 2-ethyl-3-carboxypropyl phthalate using purified mitochondria, but strong phthalate monoester hydrolase activity observed during incubation of the former compound with purified peroxisomes made it impossible to determine whether 2-ethyl-3-carboxypropyl phthalate could be produced in the latter organelle or not. 2-Ethyl-5-carboxypentyl phthalate was such an inefficient substrate for β-oxidation compared to palmitic acid that it is unlikely that it contributes significantly to the production of H₂O₂ in rats chronically exposed to di-(2-ethylhexyl) phthalate. Normal fatty acids are most likely to serve as the dominat substrates for peroxisomal β-oxidase.     

ISolation and identification of a sulfur-containing metabolite of spironolactone from human urine

In the urine of normal subjects Who were given an oral dose of 500 mg spironolactone (3-(3-oxo-7α-acetylthio-17β-hydroxy-4-androsten-17α-yl)-propionic acid γ-lactone; Aldactone^R) together with 100, uCi H-20, 21 spironolactone, a so far unknown major metabolite has been detected by thin layer chromatography. The metabolite then could be isolated by means of counter-current-distribution. According to masspectral and magnetic resonance data, the metabolite has been assigned the structure of 3-(3-oxo-7α-niethyl sulfonyl-6β, 17β-dihydroxy-4-androsten-17α-yl)-propionic acid γ-lactone. By oxidation of the corresponding methylsulfinyl compound — another already known metabolite of spironolactone-with m-chloroperbenzoic acid, a compound has been isolated which proved to be identical with the new metabolite according to TIC, MS and NMR.     

Stereospecific gas chromatographic/mass spectrometric assay of the chiral labetalol metabolite 3-amino-1-phenylbutane.

We previously identified 3-amino-1-phenylbutane (APB) as an oxidative N-dealkylated, metabolite of the antihypertensive agent labetalol. Labetalol has two asymmetric centers and is used clinically as a mixture of the four possible stereoisomers; APB has one asymmetric center. We now report an enantiospecific gas chromatographic/mass spectrometric assay for APB in urine. After adding the internal standard 1-methyl-2-phenoxyethylamine and alkalinizing, the urine samples were extracted with ether. The extracts were derivatized with the optically active acid chloride prepared from (S)-alpha-methoxy-alpha-trifluoromethylphenylacetic acid. The derivatives were separated by capillary gas chromatography and detected by electron capture negative ion chemical ionization mass spectrometry with selected ion monitoring. The derivative of the R enantiomer eluted first, and the [M--32]- ions were monitored for both the drug and the internal standard. The method was linear in the 0.05-2.5 micrograms enantiomer-1 ml-1 range and had inter-assay and intra-assay coefficients of variation of less than 6%. The assay was used in the analysis of urine samples from a patient in labetalol therapy and no interference was found. Further studies are needed to elucidate the oxidative metabolism of labetalol and its stereochemical aspects.     

Abnormal deoxyribose metabolites in the urine of a child with a possible new inborn error of metabolism

The urinary extract of a child investigated because of strabismus was found to contain large amounts of a compound which was identified using gas chromatography/mass spectrometry as 2-deoxyerythropentono-1,4-lactone. This lactone has not been observed previously in urinary extracts. When ion-exchange chromatography was used to isolate the organic acids from urine, the major peaks obtained by gas chromatography were shown to be 2-deoxyerythropentonic acid, 2-deoxyerythropentono-1,5-lactone and 2-deoxyerythropentono-1,4,lactone. Another abnormal metabolite, 2-deoxyribitol, was also excreted by the patient although this compound could not be detected in the urine of normal children. It is proposed that these unusual compounds accumulate in the urine of this child as a result of a defect in the catabolism of 2-deoxyribose.     

Biotransformation of urapidil: isolation and identification of metabolites in mouse, rat, dog and man

The identification of biotransformation products of the new antihypertensive drug urapidil in mouse, rat, dog and man has been performed by means of high-performance liquid chromatographic and mass spectrometric techniques. In urine, three metabolites were found in addition to the unchanged drug. The para-hydroxylated product (1) (6-(3-[4-(o-methoxy-p-hydroxyphenyl)piperazinyl]-propylamino)-1, 3-dimethyl-uracil), the O-demethylated compound (2) (6-(3-[4-(o-hydroxyphenyl)piperazinyl]-propylamino)-1, 3-dimethyluracil) and the uracil-N-dealkylated compound (3) (6-(3-[4-(o-methoxyphenyl)piperazinyl]-propylamino)-1-methyluracil). In urine of dog, the metabolite with the N-oxide structure (5) was also identified, but only in trace amounts (6-(3-[4-(o-methoxyphenyl)piperazinyl-N-oxide]-propylamino)-1, 3-dimethyluracil).     

The occurrence of substituted 3-methyl-3-hydroxyglutaric acids in urine in propionic acidaemia and in beta-ketothiolase deficiency

THe urine of two patients with propionic acidaemia contained 3-ethyl-3-hydroxyglutaric acid which has a propionyl residue in place of one of the acetyl residues of the normal metabolite 3-methyl-3-hydroxyglutaric acid. Related compounds, 2,3-dimethyl-3-hydroxyglutaric acid, 2-methyl-3-ethyl-3-hydroxyglutaric acid and 2,3,4-trimethyl-3-hydroxyglutaric acid, could not be detected in propionic acidaemia urine, but 2,3-dimethyl-3-hydroxyglutaric acid was excreted by a patient with beta-ketothiolase deficiency.     

Transformation of cinoxacin by Beauveria bassiana

The ability of the fungus Beauveria bassiana ATCC 7159 to transform the antibacterial agent cinoxacin was investigated. Cultures in sucrose-peptone broth were dosed with cinoxacin, grown for 20 days, and then extracted with ethyl acetate. Two metabolites were detected and purified by high-performance liquid chromatography. The major metabolite was identified by mass and proton nuclear magnetic resonance spectra as 1-ethyl-1,4-dihydro-3-(hydroxymethyl)[1,3]dioxolo[4,5-g]cinnolin-4-one and the minor metabolite was identified as 1-ethyl-1,4-dihydro-6,7-dihydroxy-3-(hydroxymethyl)cinnolin-4-one. B. bassiana also reduced quinoline-3-carboxylic acid to 3-(hydroxymethyl)quinoline.     

Stabilization and determination of a PPAR agonist in human urine using automated 96-well liquid-liquid extraction and liquid chromatography/tandem mass spectrometry

Two stability challenges were encountered during development of an urine assay for a proliferator-activated receptor (PPAR) agonist, I (2-{[5,7-dipropyl-3-(trifluoromethyl)-1,2-benzisoxazol-6-yl]oxy}-2-methyl propionic acid), indicated for the treatment of Type II diabetes. First, the analyte was lost in urine samples due to adsorption on container surface which is a common problem during clinical sample handling. Secondly, the acylglucuronide metabolite (III), a major metabolite of I, displayed limited stability and effected the quantitation of parent drug due to the release of I through hydrolysis. Therefore, a clinical collection procedure was carefully established to stabilize I and its acylglucuronide metabolite, III, in human urine. The metabolite was not quantitated with this method. The urine samples are treated with bovine serum albumin (BSA) equal to 1.75% of the urine volume and formic acid equal to 1% of urine volume. Compound (I) and internal standard (II) were extracted from urine with 1 mL ethyl acetate using a fully automated liquid-liquid extraction in 96-well plate format. The analytes are separated by reverse phase high-performance liquid chromatography (HPLC) with tandem mass spectrometry in multiple-reaction-monitoring (MRM) mode used for detection. The urine method has a lower limit of quantitation (LLOQ) of 0.05 ng/mL with a linearity range of 0.05-20 ng/mL using 0.05 mL of urine. The method was validated and used to assay urine clinical samples.     

Identification of YH439 and its metabolites in rat urine by gas chromatography–mass spectrometry and liquid chromatography–mass spectrometry

YH439 is a potential drug candidate for the treatment of various hepatic disorders. YH439 and its three metabolites have been identified in rat urine by liquid chromatography–mass spectrometry (LC–MS) and by gas chromatography (GC)–MS. Identification of YH439 and its metabolites was established by comparing their GC retention times and mass spectra with those of the synthesized authentic standards. Both electron impact- and positive chemical ionization MS have been evaluated. The metabolism study was performed in the rat using oral administration of the drug. A major metabolite (YH438) was identified as the N-dealkylation product of YH439. Other identified metabolites were caused by the loss of the methyl thiazolyl amine group (metabolite II) from YH439, the isopropyl hydrogen malonate group (metabolite IV) and the decarboxylated product (metabolite III) of metabolite II.     

Detection of new exemestane metabolites by liquid chromatography interfaced to electrospray-tandem mass spectrometry

Exemestane is an irreversible aromatase inhibitor used for anticancer therapy. Unfortunately, this drug is also misused in sports to avoid some adverse effects caused by steroids administration. For this reason exemestane has been included in World Anti-Doping Agency prohibited list. Usually, doping control laboratories monitor prohibited substances through their metabolites, because parent compounds are readily metabolized. Thus metabolism studies of these substances are very important. Metabolism of exemestane in humans is not clearly reported and this drug is detected indirectly through analysis of its only known metabolite: 17β-hydroxyexemestane using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) and gas chromatography coupled to mass spectrometry (GC-MS). This drug is extensively metabolized to several unknown oxidized metabolites. For this purpose LC-MS/MS has been used to propose new urinary exemestane metabolites, mainly oxidized in C6-exomethylene and simultaneously reduced in 17-keto group. Urine samples from four volunteers obtained after administration of a 25mg dose of exemestane were analyzed separately by LC-MS/MS. Urine samples of each volunteer were hydrolyzed followed by liquid-liquid extraction and injected into a LC-MS/MS system. Three unreported metabolites were detected in all urine samples by LC-MS/MS. The postulated structures of the detected metabolites were based on molecular formulae composition obtained through high accuracy mass determination by liquid chromatography coupled to hybrid quadrupole-time of flight mass spectrometry (LC-QTOF MS) (all mass errors below 2ppm), electrospray (ESI) product ion spectra and chromatographic behavior.     

Monitoring of urinary acrolein concentration in patients receiving cyclophosphamide and ifosphamide

Acrolein, the metabolite of cyclophosphamide and ifosphamide, irritates mucous membranes and is considered pathogenetically important in hemorrhagic cystitis. Increasing fluid intake or administering sodium 2-mercaptoethanesulfonate (mesna), a thiol compound, can reduce the risk of this complication. We measured urinary acrolein concentrations using headspace-solid-phase microextraction gas chromatography and mass spectrometry (headspace-SPME-GC-MS) in 19 patients receiving cyclophosphamide and ifosphamide (36 occasions). Peak acrolein concentrations occurred at 1-12h (mean +/- S.D., 5.0+/-2.7) after starting therapy, ranging from 0.3 to 406.8 nM (39.7+/-76.7), with varying patterns over time. Maintaining high urine volume was important for preventing increases in urinary acrolein concentration, as urinary acrolein concentration tended to rise as urine volume decreased. Urinalysis detected occult blood in three cases, but the patients had no clinical symptoms of hemorrhagic cystitis. In clinical trials involving cyclophosphamide and ifosphamide, monitoring of urinary acrolein concentration could indicate when to take heightened preventive measures against hemorrhagic cystitis.     

Determination of acyclovir and its metabolite 9-carboxymethoxymethylguanine in serum and urine using solid-phase extraction and high-performance liquid chromatography

A reversed-phase ion-pair high-performance liquid chromatography method for the determination of acyclovir and its metabolite 9-carboxymethoxymethylguanine is described. The samples are purified by reversed-phase solid-phase extraction. The components are separated on a C₁₈ column with a mobile phase containing 18% acetonitrile, 5 mM dodecyl sulphate and 30 mM phosphate buffer, pH 2.1, and measured by fluorescence detection using an excitation wavelength of 285 nm and an emission wavelenght of 380 nm. Detection limits are 0.12 μM (plasma)) and 0.60 μM (urine) for acyclovir, and 0.26 μM (plasma) and 1.3 μM (urine) for metabolite. Correlation coefficients that were better than 0.998 were obtained normally. This analytical method, which enables simultaneous measurement of parent compound and metabolite, has been used in kinetics studies and for therapeutic drug monitoring in different patient groups with variable degrees of renal dysfunction.     

Characterisation of metabolites of 3-ethyl-3-(4-pyridyl)-piperidine-2,6-dione, a potential breast cancer drug

The identification of metabolites from the pyridylglutarimide 3-ethyl-3-(4-pyridyl)piperidine-2,6-dione (PG, Rogletimide) was achieved using liquid chromatography—mass spectrometry with a thermospray interface (LC—TSP—MS). The urinary metabolites include PG N-oxide, the products of 4- and 5-hydroxylation in the piperidine residue (4- and 5-hydroxy-PG) and a γ-butyrolactone derived via terminal hydroxylation in the ethyl residue. In addition to the above metabolites, several products of glutarimide ring-opening could be detected in the plasma extracts after multiple-dose treatment. Thus LC—TSP—MS is potentially a simple and rapid technique in studies of drug metabolism for the important glutarimide class of drug.     

Simultaneous analysis of the di(2-ethylhexyl)phthalate metabolites 2-ethylhexanoic acid, 2-ethyl-3-hydroxyhexanoic acid and 2-ethyl-3-oxohexanoic acid in urine by gas chromatography–mass spectrometry

A gas chromatographic–mass spectrometric method was developed for the quantitative analysis of the three Di(2-ethylhexyl)phthalate (DEHP) metabolites, 2-ethylhexanoic acid, 2-ethyl-3-hydroxyhexanoic acid and 2-ethyl-3-oxohexanoic acid in urine. After oximation with O-(2,3,4,5,6-pentafluorobenzyl)-hydroxylamine hydrochloride and sample clean-up with Chromosorb P filled glass tubes, all three organic acids were converted to their tert.-butyldimethylsilyl derivatives. Quantitation was done with trans-cinnamic acid as internal standard and GC–MS analysis in the selected ion monitoring mode (SIM). Calibration curves for all three acids in the range from 20 to 1000 μg/l showed correlation coefficients from 0.9972 to 0.9986. The relative standard deviation (RSD) values determined in the observed concentration range were between 1.3 and 8.9% for all three acids. Here we report for the first time the identification of 2-ethyl-3-hydroxyhexanoic acid and 2-ethyl-3-oxohexanoic acid in human urine next to the known DEHP metabolite 2-ethylhexanoic acid. In 28 urine samples from healthy persons we found all three acids with mean concentrations of 56.1±13.5 μg/l for 2-ethylhexanoic acid, 104.8± 80.6 μg/l for 2-ethyl-3-hydroxyhexanoic acid and 482.2± 389.5 μg/l for 2-ethyl-3-oxohexanoic acid.