Identification of potassium dehydroandrographolidi succinas and its major metabolites in rat urine by liquid chromatography-tandem mass spectrometry

A rapid, sensitive and specific liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed for identification of potassium dehydroandrographolidi succinas and its metabolites in rat urine. Five male rats were administrated a single dose (100 mg/kg) of potassium dehydroandrographolidi succinas by i.v. injection. The urine were sampled from 0 to 24 h and purified by using Oasis? HLB extraction cartridge, then the purified urine samples were separated on a reversed-phase C18 column with a linear gradient and detected by an on-line MS detector. Identification and structural elucidation of the metabolites were performed by comparing their changes in molecular mass (Δm) and MS/MS spectra with those of the parent drug. Seven metabolites and the parent drug were found in rat urine. All these metabolites were reported for the first time.     

Liquid chromatography-tandem mass spectrometry analysis of anisodamine and its phase I and II metabolites in rat urine

A sensitive and specific method for the analysis of anisodamine and its metabolites in rat urine by liquid chromatography-electrospray ionization tandem mass spectrometry (LC-MS/MS) was developed. Various extraction techniques (free fraction, acid hydrolyses and enzyme hydrolyses) and their comparison were carried out for investigation of the metabolism of anisodamine. After extraction procedure the pretreated samples were injected on a reversed-phase C18 column with mobile phase (0.2 ml/min) of methanol/0.01% triethylamine solution (adjusted to pH 3.5 with formic acid) (60:40, v/v) and detected by MS/MS. Identification and structural elucidation of the metabolites were performed by comparing their changes in molecular masses (DeltaM), retention-times and full scan MS(n) spectra with those of the parent drug. At least 11 metabolites (N-demethyl-6beta-hydroxytropine, 6beta-hydroxytropine, tropic acid, N-demethylanisodamine, hydroxyanisodamine, anisodamine N-oxide, hydroxyanisodamine N-oxide, glucuronide conjugated N-demethylanisodamine, sulfate conjugated and glucuronide conjugated anisodamine, sulfate conjugated hydroxyanisodamine) and the parent drug were found in rat urine after the administration of a single oral dose 25mg/kg of anisodamine. Hydroxyanisodamine, anisodamine N-oxide and the parent drug were detected in rat urine for up 95 h after ingestion of anisodamine.     

Metabolism and toxicological detection of the designer drug 4-iodo-2,5-dimethoxy-amphetamine (DOI) in rat urine using gas chromatography-mass spectrometry

The amphetamine-derived designer drug 4-iodo-2,5-dimethoxy-amphetamine (DOI) is an upcoming substance on the illicit drug market. In the current study, the identification of its metabolites in rat urine and their toxicological detection in the authors' systematic toxicological analysis (STA) procedure were examined. DOI is extensively metabolized by O-demethylation and beside small amounts of parent compound it was found to be excreted mainly in form of metabolites. The STA procedure using full-scan GC-MS allowed proving an intake of a common drug users' dose of DOI by detection of the two O-demethyl metabolite isomers in rat urine. Assuming similar metabolism, the described STA procedure should be suitable for proof of an intake of DOI in human urine.     

Mass spectrometric identification of urinary and plasma metabolites of 9-hydroxy-19,20-bis-nor-prostanoic acid (rosaprostol)

9-Hydroxy-19,20-bis-nor-prostanoic acid (Rosaprostol) is an antiulcer compound with antisecretory and cytoprotective action. We studied the metabolites of Rosaprostol found in human plasma and in human and rat urine. Sixteen different metabolites were tentatively identified on the basis of their mass spectra. Two presumed metabolites were synthesized. To clarify the identities of some of them, deuterated Rosaprostol was administered to rats and mass spectra of the deuterated and protonated metabolites were examined. Rosaprostol is metabolized following three metabolic pathways leading, combined, to oxidized compounds with a lower number of carbons than the parent drug.     

Analysis of benzphetamine and its metabolites in rat urine by liquid chromatography–electrospray ionization mass spectrometry

An analytical method to identify and determine benzphetamine (BMA) and its five metabolites in urine was developed by liquid chromatography–electrospray ionization mass spectrometry (LC–ESI–MS) using the solid-phase extraction column Bond Elut SCX. Deuterium-labeled compounds, used as internal standards, were separated chromatographically from each corresponding unlabeled compound in the alkaline mobile phase with an alkaline-resistant ODS column. This method was applied to the identification and determination of BMA and its metabolites in rat urine collected after oral administration of BMA. Under the selected ion monitoring mode, the limit of quantitation (signal-to-noise ratio 10) for BMA, N-benzylamphetamine (BAM), p-hydroxybenzphetamine (p-HBMA), p-hydroxy-N-benzylamphetamine (p-HBAM), methamphetamine (MA) and amphetamine (AM) was 700 pg, 300 pg, 500 pg, 1.4 ng, 6 ng and 10 ng in 1 ml of urine, respectively. This analytical method for p-HBMA, structurally closer to the unchanged drug of all the metabolites, was very sensitive, making this a viable metabolite for discriminating the ingestion of BMA longer than the parent drug or other metabolites in rat.     

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.     

Liquid chromatography-electrospray ionization ion trap mass spectrometry for analysis of mesocarb and its metabolites in human urine

A method is described for the determination of metabolites of mesocarb in human urine by combining gradient liquid chromatography and electrospray ionization (ESI)-ion trap mass spectrometry. Seven metabolites (two isomers of hydroxymesocarb, p-hydroxymesocarb, two isomers of dihydroxymesocarb and two isomers of trihydroxymesocarb) and parent drug were detected in human urine after the administration of a single oral dose 10 mg of mesocarb (Sydnocarb, two tablets of 5 mg). Various extraction techniques (free fraction, enzyme hydrolyses and acid hydrolyses) and their comparison were carried out for investigation of the metabolism of mesocarb. After extraction procedure the residue was dissolved in methanol and injected into the column HPLC (Zorbax SB-C18 (Narrow-Bore 2.1 x 150 mm i.d., 5 microm particles)) with mobile phase (0.2 ml/min) of methanol/0.2 mM ammonium acetate. Conformation of the results and identification of all metabolites are performed by LC-MS and LC-MS/MS. The major metabolites of mesocarb in urine of the human were p-hydroxylated derivative of the phenylcarbamoyl group of the parent drug (p-hydrohymesocarb) and dihydroxylated derivative of mesocarb (two isomers of dihydroxymesocarb). This analytical method for dihydrohymesocarb was very sensitive for discriminating the ingestion of mesocarb longer than the parent drug or other metabolites in human urine. The dihydroxymesocarb was detected in urine until 168-192 h after administration of the drug.     

Detection of fenspiride and identification of in vivo metabolites in horse body fluids by capillary gas chromatography-mass spectrometry: administration, biotransformation and urinary excretion after a single oral dose

Studies related to the in vivo biotransforrmation and urinary excretion of fenspiride hydrochloride in the horse are described. After oral administration, the drug is metabolised by both phase I functionalisation and phase II conjugation pathways. Following enzymatic deconjugation, fenspiride and its phase I metabolites were isolated from post-administration biofluids using bonded co-polymeric mixed mode solid-phase extraction cartridges to isolate the basic compounds. Following trimethylsilylation (TMS), the parent drug and metabolites were identified by capillary gas chromatography-mass spectrometry (GC-MS). Fenspiride (A) and seven metabolites (B-->G) arising from oxidation on both the aromatic and heterocyclic substructures were detected in urine. The positive ion electron ionisation mass spectra of the TMS derivatives of fenspiride and its metabolites provided useful information on its metabolism. Positive ion methane chemical ionisation-GC-MS of the derivatives provided both derivatised molecular mass and structural information. Unchanged fenspiride can be detected in post-administration plasma and urine samples for up to 24 h. Maximum urinary levels of 100-200 ng ml(-1) were observed between 3 and 5 h after administration. After enzymatic deconjugation, the major phenolic metabolite (G) can be detected in urine for up to 72 h. This metabolite is the analyte of choice in the GC-MS screening of post-race equine urine samples for detection of fenspiride use. However, a distinct difference was observed in the urinary excretion of this metabolite between the thoroughbred horses used in UK study and the quarterbred and standardbred horses used for the USA administrations.     

Structural identification of the metabolites for strictosamide in rats bile by an ion trap-TOF mass spectrometer and mass defect filter technique

We report herein, a facile metabolite identification workflow on the antimicrobial strictosamide, which is derived from accurate mass measurement by a hybrid ion trap-TOF mass spectrometer. In step 1, the parent drug and metabolites in rat bile were separated on an HPLC column followed by ion trap-TOF mass spectrometer analysis after a single oral dose of 50mg/kg strictosamide. In step 2, mass defect filter technique, which enables high-resolution mass spectrometers to be utilized for detecting drug metabolites based on well-defined mass defect ranges, was used to find metabolites in the mass spectrum. In step 3, the differences of accurate masses and their mass fragmentation pattern among the parent drug and metabolites used to assign structures for the metabolites successfully. As a result, five metabolites of strictosamide were found in rat bile, and all the metabolites were reported for the first time.     

Metabolism and pharmacokinetic studies of propionylpromazine in horses

The propionylpromazine concentrations in plasma after intramuscular administration to horses were determined using gas chromatography with nitrogen-phosphorus detection. After hydrolysis by β-glucuronidase/arylsulphatase, the parent drug and three metabolites were detected in urine. The metabolites were identified as 2-(1-hydroxypropyl)promazine, 2-(1-propenyl)promazine and 7-hydroxypropionylpromazine by gas chromatography-mass spectrometry. No N-demethylated or sulphoxidated metabolites of propionylpromazine were observed in the horse urine.     

Liquid chromatography-mass spectrometry assay for quantitation of ifosfamide and its N-deschloroethylated metabolites in rat microsomal medium

A specific and sensitive quantitative assay has been developed using high performance liquid chromatography-electrospray ionization mass spectrometry (HPLC-ESI-MS) for the simultaneous quantitation of the antitumor drug ifosfamide (IFM) and its two metabolites, N2-deschloroethylifosfamide (N2-DCE-IFM) and N3-deschloroethylifosfamide (N3-DCE-IFM) in microsomal medium. The analytes and the internal standard (cyclophosphamide) were isolated by ethylacetate extraction from rat liver microsomes. They were analysed on a Nucleosil C18 HD column (125 mm x 4 mm, 5 microm) using a step gradient with the mobile phase (2 mM ammonium formate and methanol). The HPLC-ESI-MS method used selected ion monitoring of ions m/z 199.1 Th and m/z 261.1 Th and was validated in the concentrations ranges of 100-5000 ng/mL for IFM and 50-2500 ng/mL for its N-deschloroethylated metabolites (DCE-IFM) with good accuracy and precision (CV less than 15%). The low limits of quantitation (LLOQ) were found at 50 ng/mL for N-deschloroethylated metabolites and at 100 ng/mL for the parent drug (IFM). The method was applied for the determination of ifosfamide and its N-deschloroethylated metabolites in rat microsomal incubations.     

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.     

Studies on the toxicological detection of the designer drug 4-bromo-2,5-dimethoxy-beta-phenethylamine (2C-B) in rat urine using gas chromatography-mass spectrometry

The phenethylamine-derived designer drug 4-bromo-2,5-dimethoxy-beta-phenethylamine (2C-B) is known to be extensively metabolized in various species including humans. In rat urine, 2C-B was found to be excreted mainly via its metabolites. In the current study, the toxicological detection of these metabolites in the authors' systematic toxicological analysis (STA) procedure was examined. The STA procedure using full-scan GC-MS allowed proving an intake of a common drug abusers' dose of 2C-B by detection of the O-demethyl deaminohydroxy and two isomers of the O-demethyl metabolites in rat urine. Assuming similar metabolism, the described STA procedure should be suitable for proof of an intake of 2C-B in human urine.     

Study on the stereoselective excretion of tetrahydropalmatine enantiomers in rats and identification of in vivo metabolites by liquid chromatography‐tandem mass spectrometry

The objective of this work was to study the stereoselectivity in excretion of tetrahydropalmatine (THP) enantiomers by rats and identify the metabolites of racemic THP (rac‐THP) in rat urine. Urine and bile samples were collected at various time intervals after a single oral dose of rac‐THP. The concentrations of THP enantiomers in rat urine and bile were determined using a modification of an achiral–chiral high‐performance liquid chromatographic (HPLC) method that had been previously published. The cumulative urinary excretion over 96 h of (?)‐THP and (+)‐THP was found to be 55.49 ± 36.9 μg and 18.33 ± 9.7 μg, respectively. The cumulative biliary excretion over 24 h of (?)‐THP and (+)‐THP was 19.19 ± 14.6 μg and 12.53 ± 10.4 μg, respectively. The enantiomeric (?/+) concentration ratios of THP changed from 2.80 to 5.15 in urine, and from 1.36 to 1.80 in bile. The mean cumulative amount of (?)‐THP was significantly higher than that of (+)‐THP both in urine and bile samples. However, the enantiomeric (?/+) concentration ratios in rat urine and bile were significantly lower than those ratios in rat plasma. These findings suggested the excretion of THP enantiomers was stereoselective rather than a reflection of chiral pharmacokinetic aspects in plasma and (?)‐THP was preferentially excreted in rat urine and bile. Three O‐demethylation metabolites and the parent drug rac‐THP were detected by liquid chromatography‐tandem mass spectrometry in rat urine. One metabolite was obtained by preparative HPLC and identified as 10‐O‐demethyl‐THP. Chirality, 2010. © 2009 Wiley‐Liss, Inc.     

Oral absorption and excretion of icaritin, an aglycone and also active metabolite of prenylflavonoids from the Chinese medicine Herba Epimedii in rats

Icaritin (ICT) is a main aglycone and also active intestinal metabolite of prenylflavonoids from the Chinese medicine Herba Epimedii. In the present study, the oral absorption and excretion of this compound was investigated using rats for exploring its fate in the body, so as to better understanding its in vivo pharmacological activities. The free (parent) and total (parent plus conjugated metabolites) ICT concentrations in rat plasma, urine and bile, after intravenous (i.v.) and oral administration both at 5mg/kg, were determined before and after enzymatic hydrolysis with β-glucuronidase/sulphatase, respectively, by a HPLC-UV method. The results showed that free ICT plasma concentration after i.v. dose was rapidly decreased with average t(1/2, λ) of 0.43h, while the total ICT concentration was decreased slowly with t(1/2, λ) of 6.86h. The area under the curve of ICT conjugated metabolites was about 11-fold higher than that of free ICT. The majority of ICT in the body was excreted from the bile with 68.05% of dose over 8h after i.v. dosing, in which only 0.15% was in parent form. While very little amount of ICT was excreted from the urine with 3.01% of dose over 24h, in which the parent form was 0.62%. After oral administration, very little amount of parent ICT was detected only in 0.5, 1 or 2h plasma samples with the concentration less than LOQ, however, its total plasma concentration after enzymatic hydrolysis treatment was at relative high level with average maximum concentration of 0.49μg/ml achieved at 1h post dose. The oral bioavailability of ICT was 35% of dose, estimated by its total plasma drug concentrations. It is concluded that ICT can be easily absorbed into the body, and then rapidly conversed to its conjugated metabolites, and finally removed from the body mainly by biliary excretion.     

New designer drug p-methoxymethamphetamine: studies on its metabolism and toxicological detection in urine using gas chromatography-mass spectrometry

Studies are described on the metabolism and the toxicological analysis of the new designer drug rac-p-methoxymethamphetamine (PMMA) in rat urine using gas chromatography-mass spectrometry (GC-MS). The identified metabolites indicated that PMMA was extensively metabolized mainly by O-demethylation to pholedrine and to a minor extent to p-methoxyamphetamine (PMA), 1-hydroxypholedrine diastereomers (one being oxilofrine), 4'-hydroxy-3'-methoxymethamphetamine and 4'-hydroxy-3'-methoxyamphetamine. The authors' systematic toxicological analysis (STA) procedure using full-scan GC-MS after acid hydrolysis, liquid-liquid extraction and microwave-assisted acetylation allowed the detection of the main metabolites of PMMA in rat urine after a dose corresponding to that of drug users. Therefore, this procedure should be suitable for detection of PMMA intake in human urine via its metabolites. However, it must be considered that pholedrine and oxilofrine are also in therapeutic use. Differentiation of PMMA, PMA and/or pholedrine intake is discussed.     

Toxicological detection of the new designer drug 1-(4-methoxyphenyl)piperazine and its metabolites in urine and differentiation from an intake of structurally related medicaments using gas chromatography-mass spectrometry

Studies are described on the toxicological analysis of the piperazine-derived designer drug 1-(4-methoxyphenyl)piperazine (MeOPP) in rat urine using gas chromatography-mass spectrometry (GC-MS). The authors' systematic toxicological analysis (STA) procedure using full-scan GC-MS after acid hydrolysis, liquid-liquid extraction and microwave-assisted acetylation allowed the detection of MeOPP and its metabolites 1-(4-hydroxy phenyl)piperazine and 4-hydroxyaniline in rat urine after administration of a single dose corresponding to doses commonly taken by drug users. Therefore, this procedure should also be suitable for detection of a MeOPP intake in human urine. However, the metabolites of MeOPP are not unique and can be produced from other drugs. Therefore, differentiation of use of this designer drug from use of the medicaments dropropizine, oxypertine or others, which are metabolized to the MeOPP isomer 1-(2-methoxyphenyl)piperazine, is discussed.     

Mass spectrometric identification and characterization of new fluoxymesterone metabolites in human urine by liquid chromatography time-of-flight tandem mass spectrometry

In this study fluoxymesterone urinary profiles were investigated by liquid chromatography quadrupole time-of-flight tandem mass spectrometry (LC-QTOFMS) with accurate mass measurement. Twelve metabolites including the parent drug were detected in two fluoxymesterone positive control urine samples. Three parameters were employed for evaluation of the accuracy of the chemical formulae in positive full scan experiment, which contained error between actual and calculated mass weights of prontonated and isotopic molecules together with abundance match between prontonated and isotopic molecules. The 13 analytes were determined with mass accuracy less than 1.1 ppm and isotopic abundance match more than 94 marks. Based on the ionization, CID fragmentation, the accurate mass of the product ion and comparison of the accurate mass weight and retention time with reference standard, fluoxymesterone and its 12 metabolites containing three unreported ones were detected. The chemical structures of three unreported metabolites were identified as: 9-fluro-17β-ol-17-methyl-11-en-5α-androstan-3-one (F13), 9-fluro-17β-ol-17-methyl-11-en-5β-androstan-3-one (F8) and 9-fluro-17β-ol-17-methyl-5-androstan-3,6,11-trione, and meanwhile a dihydroxylated metabolite (F12), 6,16-dihydroxylated fluoxymesterone, was also detected in human urine, which was previously reported to be available only in equine urine.     

Metabolic profiling of a potential antifungal drug, 3-(4-bromophenyl)-5-acetoxymethyl-2,5-dihydrofuran-2-one, in mouse urine using high-performance liquid chromatography with UV photodiode-array and mass spectrometric detection

3-(4-bromophenyl)-5-acetyloxymethyl-2,5-dihydrofuran-2-one (LNO-18-22) is a representative member of a novel group of potential antifungal drugs, derived from a natural 3,5-disubstituted butenolide, (-)incrustoporine, as a lead structure. This lipophilic compound is characterized by high in vitro antifungal activity and low acute toxicity. For the purpose of in vivo studies, a new bioanalytical high-performance liquid chromatographic method with UV photodiode-array and mass spectrometric detection (HPLC-PDA-MS), involving a direct injection of diluted mouse urine was developed and used in the evaluation of the metabolic profiling of this drug candidate. The separation of LNO-18-22 and its phase I metabolites was performed in 37 min on a 125 mmx4 mm chromatographic column with Purospher RP-18e using an acetonitrile-water gradient elution. Scan mode of UV detection (195-380 nm) was employed for the identification of the parent compound and its biotransformation products in the biomatrix. Finally, the identity of LNO-18-22 and its metabolites was confirmed using HPLC-MS analyses of the eluate. These experiments demonstrated the power of a comprehensive analytical approach based on the combination of xenobiochemical methods and the results from tandem HPLC-PDA-MS (chromatographic behaviour, UV and MS spectra of native metabolites versus synthetic standards). The chemical structures of five phase I LNO-18-22 metabolites and one phase II metabolite were elucidated in the mouse urine, with two of these metabolites having very unexpected structures.     

Metabolism of [14C]methamphetamine in man, the guinea pig and the rat

1. The metabolites of (+/-)-2-methylamino-1-phenyl[1-(14)C]propane ([(14)C]methamphetamine) in urine were examined in man, rat and guinea pig. 2. In two male human subjects receiving the drug orally (20mg per person) about 90% of the (14)C was excreted in the urine in 4 days. The urine of the first day was examined for metabolites, and the main metabolites were the unchanged drug (22% of the dose) and 4-hydroxymethamphetamine (15%). Minor metabolites were hippuric acid, norephedrine, 4-hydroxyamphetamine, 4-hydroxynorephedrine and an acid-labile precursor of benzyl methyl ketone. 3. In the rat some 82% of the dose of (14)C (45mg/kg) was excreted in the urine and 2-3% in the faeces in 3-4 days. In 2 days the main metabolites in the urine were 4-hydroxymethamphetamine (31% of dose), 4-hydroxynorephedrine (16%) and unchanged drug (11%). Minor metabolites were amphetamine, 4-hydroxyamphetamine and benzoic acid. 4. The guinea pig was injected intraperitoneally with the drug at two doses, 10 and 45mg/kg. In both cases nearly 90% of the (14)C was excreted, mainly in the urine after the lower dose, but in the urine (69%) and faeces (18%) after the higher dose. The main metabolites in the guinea pig were benzoic acid and its conjugates. Minor metabolites were unchanged drug, amphetamine, norephedrine, an acid-labile precursor of benzyl methyl ketone and an unknown weakly acidic metabolite. The output of norephedrine was dose-dependent, being about 19% on the higher dose and about 1% on the lower dose. 5. Marked species differences in the metabolism of methamphetamine were observed. The main reaction in the rat was aromatic hydroxylation, in the guinea pig demethylation and deamination, whereas in man much of the drug, possibly one-half, was excreted unchanged.