Methodology for determining major constituents of ayahuasca and their metabolites in blood

There is an increasing interest in potential medical applications of ayahuasca, a South American psychotropic plant tea with a long cultural history of indigenous medical and religious use. Clinical research into ayahuasca will require specific, sensitive and comprehensive methods for the characterization and quantitation of these compounds and their metabolites in blood. A combination of two analytical techniques (high-performance liquid chromatography with ultraviolet and/or fluorescence detection and gas chromatography with nitrogen-phosphorus detection) has been used for the analysis of some of the constituents of ayahuasca in blood following its oral consumption. We report here a single methodology for the direct analysis of 14 of the major alkaloid components of ayahuasca, including several known and potential metabolites of N,N-dimethyltryptamine and the harmala alkaloids in blood. The method uses 96-well plate/protein precipitation/filtration for plasma samples, and analysis by HPLC-ion trap-ion trap-mass spectrometry using heated electrospray ionization to reduce matrix effects. The method expands the list of compounds capable of being monitored in blood following ayahuasca administration while providing a simplified approach to their analysis. The method has adequate sensitivity, specificity and reproducibility to make it useful for clinical research with ayahuasca.     

Simple high-performance liquid chromatographic method for the concurrent determination of the amine metabolites vanillylmandelic acid, 3-methoxy-4-hydroxyphenylglycol, 5-hydroxyindoleacetic acid, dihydroxyphenylacetic acid and homovanillic acid in urine using electrochemical detection

A simple method for the concurrent analysis of the noradrenaline metabolites vanillylmandelic acid and 3-methoxy-4-hydroxyphenylglycol, the dopamine metabolites dihydroxyphenylacetic acid and homovanillic acid, and the serotonin metabolite 5-hydroxyindoleacetic acid in human urine is described. Following organic extraction of the metabolites from acidified urine, they are separated by single-step gradient elution high-performance liquid chromatography on a reversed-phase column. Detection and quantification are achieved with an electrochemical detector using a carbon-paste electrode; samples can be injected at 40-min intervals. Optimisation of analytical parameters is described, and examples of the application of the method in the fields of clinical chemistry and clinical neuroscience are given. This provides a convenient method for the concurrent study of the metabolism of three major biogenic amines, and is readily adaptable for studies on cerebrospinal fluid and brain tissue.     

Direct analysis of psychoactive tryptamine and harmala alkaloids in the Amazonian botanical medicine ayahuasca by liquid chromatography–electrospray ionization-tandem mass spectrometry

A direct injection/liquid chromatography–electrospray ionization-tandem mass spectrometry procedure has been developed for the simultaneous quantitation of 11 compounds potentially found in the increasingly popular Amazonian botanical medicine and religious sacrament ayahuasca. The method utilizes a deuterated internal standard for quantitation and affords rapid detection of the alkaloids by a simple dilution assay, requiring no extraction procedures. Further, the method demonstrates a high degree of specificity for the compounds in question, as well as low limits of detection and quantitation despite using samples for analysis that had been diluted up to 200:1. This approach also appears to eliminate potential matrix effects. Method bias for each compound, examined over a range of concentrations, was also determined as was inter- and intra-assay variation. Its application to the analysis of three different ayahuasca preparations is also described. This method should prove useful in the study of ayahuasca in clinical and ethnobotanical research as well as in forensic examinations of ayahuasca preparations.     

Liquid chromatography-mass spectrometry in metabolic research. I. Metabolites of benzbromarone in human plasma and urine.

Seven benzbromarone metabolites were identified in human plasma and urine by electron-impact mass spectrometry after semipreparative high-performance liquid chromatographic fractionation and/or by liquid chromatography-mass spectrometry using a thermospray interface. The major metabolite in plasma and urine was a hydroxybenzofuranoyl species; the 1-hydroxyethyl entity was identified as a minor metabolite. Five urinary metabolites occurred in trace amounts, all of them carrying OH and/or C = O groups in different positions. The hydroxybenzofuranoyl metabolite has often been mistaken for benzarone in previous studies.     

In vivo metabolism of lidocaine in the rat. Isolation of urinary metabolites as pentafluorobenzoyl derivatives and their identification by combined gas chromatography-mass spectrometry

The metabolism of a large dose (40 mg/kg intraperitoneally) of lidocaine (LIDO) in mature male Sprague-Dawley rats is described. Pentafluorobenzoyl chloride was used to derivatize the hydrolyzed urinary metabolites prior to extraction and analysis as pentafluorobenzoyl-derivatives by combined gas chromatography-mass spectrometry. Total ion and selected ion current (m/z 195; C6F5CO+) traces were recorded and metabolites of LIDO were readily identified. Only one major metabolite, 3-hydroxy-N-(N-ethylglycyl)-2,6-xylidine, was excreted in urine. A new metabolite, 3-hydroxy-N-glycyl-2,6-xylidine was also present in significant amounts, as well as minor quantities of four oxygenated metabolites of N-(N-ethylglycyl)-2,6-xylidine. Other known metabolites of LIDO, including 3-hydroxylidocaine, were excreted in trace quantities. The results suggest that metabolism of LIDO in rats may be age- and/or dose-dependent.     

High-performance liquid chromatographic determination of nicotinamide and its metabolites in human and murine plasma and urine.

A high-performance liquid chromatographic method is described which enables the determination of nicotinamide and eight of its possible metabolites in human and murine plasma and urine, using ion-pairing on a base-deactivated reversed-phase column. Calibration curves were linear up to 2 mumol/ml for nicotinamide and 200 nmol/ml for the metabolites; both the intra- and inter-assay relative standard deviations ranged between 1 and 8%. In murine plasma, the N-oxide was the major nicotinamide metabolite, but in man, formation of 1-methylnicotinamide and the 2- and 4-pyridones was also significant. In urine, nicotinuric acid was seen in the mouse, but no nicotinic acid metabolites were seen in man.     

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.     

Application of overpressured layer chromatography combined with digital autoradiography and mass spectrometry in the study of deramciclane metabolism

Overpressured layer chromatography was combined with the highly sensitive and rapid digital autoradiography (DAR) and mass spectrometry to separate, detect, and identify 3H- and 14C-labeled deramciclane metabolites in different biological matrixes. Several minor and major metabolites were separated from plasma and urine samples. The radioactive metabolites localized by DAR were scraped from the thin-layer chromatographic plate and transferred to a mass spectrometer for structure identification. Several metabolites were isolated and characterized, including hydroxy-N-desmethyl deramciclane, which is described in detail. The combination of techniques is efficient and has good sensitivity: about 2 micrograms metabolite from a biological matrix was isolated and identified this way.     

Separation and identification of zaleplon metabolites in human urine using capillary electrophoresis with laser-induced fluorescence detection and liquid chromatography-mass spectrometry

A capillary electrophoresis (CE) method using laser-induced fluorescence (LIF) detection for the determination of the hypnotic drug zaleplon and its metabolites in human urine could be developed using carboxymethyl-beta-cyclodextrin as a charged carrier. By the help of a complementary HPLC method coupled to mass spectrometry, three metabolites present in human urine could be identified as 5-oxozaleplon, 5-oxo-N-deethylzaleplon and 5-oxozaleplon glucuronide. N-Deethylzaleplon, a previously described zaleplon metabolite, as well as zaleplon itself could not be detected in human urine by the CE-LIF assay. The results were confirmed by spiking with reference compounds of the phase I metabolites. The metabolites differed very much concerning their fluorescence intensities, thus the 5-oxo metabolites present as lactam tautomer fluoresced tenfold lower than the unchanged drug zaleplon and its N-deethylated metabolite. The glucuronide of the 5-oxozaleplon, however, showed high fluorescence due to its lactim structure. Limits of quantification yielded by the CE-LIF assay including a ten-fold preconcentration step by solid-phase extraction were 10 ng/ml for zaleplon and N-deethylzaleplon and 100 ng/ml for 5-oxozaleplon and 5-oxo-N-deethylzaleplon.     

Use of high-performance liquid chromatography-thermospray mass spectrometry and gas chromatography-electron-impact mass spectrometry in the identification of the metabolites of alpha-methylacetohydroxamic acids, potential anti-asthmatic agents

Two potential anti-asthmatic alpha-methylacetohydroxamic acids, compound 1 and compound II were metabolised to two major products (metabolite 1 and metabolite 2) after oral dosing to rabbits. Metabolite 1, extracted under acid conditions from the plasma and urine of dosed animals, was identified as a glucuronide by incubation with beta-glucuronidase and subsequent high-performance liquid chromatographic-mass spectrometric (HPLC-MS) analysis of the aglycone. HPLC-MS analysis of metabolite 2 suggested that it was the acetamide, however, unequivocal identification was obtained by further analysis using gas chromatography-mass spectrometry (GC-MS) of its trimethylsilyl derivative and by comparison with the mass spectra of the authentic acetamides. This study shows the advantages of combining HPLC-MS with other techniques such as GC-MS for the identification of metabolites.     

Determination of pholcodine and its metabolites in urine by capillary gas chromatography

A sensitive and selective method for the determination of pholcodine and its metabolites in urine using capillary gas chromatography with nitrogen detection is described. The procedure includes enzymatic hydrolysis of urine by beta-glucuronidase and sample pretreatment on C2 solid-phase extraction columns. Validation of the method showed good sensitivity, precision and reproducibility. The method was useful for the study of pholcodine metabolism in man. Pholcodine was found to conjugate with glucuronic acid. Morphine was identified as a metabolite and another unidentified metabolite was also detected.     

Gas chromatography/flame ionisation detection mass spectrometry for the detection of endogenous urine metabolites for metabonomic studies and its use as a complementary tool to nuclear magnetic resonance spectroscopy

Metabonomics is a relatively new field of research in which the total pool of metabolites in body fluids or tissues from different patient groups is subjected to comparative analysis. Nuclear magnetic resonance (NMR) spectroscopy is the technology that is currently most widely used for the analysis of these highly complex metabolite mixtures, and hundreds of metabolites can be detected without any upfront separation. We have investigated in this study whether gas chromatography (GC) separation in combination with flame ionisation detection (FID) and mass spectrometry (MS) detection can be used for metabolite profiling from urine. We show that although GC sample preparation is much more involved than for NMR, hundreds of metabolites can reproducibly be detected and analysed by GC. We show that the data quality is sufficiently high--particularly if appropriate baseline correction and time-warping methods are applied--to allow for data comparison by chemometrics methods. A sample set of urines from eleven healthy human volunteers was analysed independently by GC and NMR, and subsequent chemometrics analysis of the two datasets showed some similar features. As judged by NIST database searches of the GC/MS data some of the major metabolites that are detected by NMR are also visible by GC/MS. Since in contrast to NMR every peak in GC corresponds to a single metabolite, the electron ionisation spectra can be used to quickly identify metabolites of interest if their reference spectra are present in a searchable database. In summary, we show that GC is a method that can be used as a complementary tool to NMR for metabolite profiling of urine samples.     

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.     

人参皂苷Rb1在大鼠体内的药物代谢研究

人参皂苷Rb1是人参中的达玛烷型三萜皂苷类化合物, 具有多种生物活性. 对人参皂苷Rb1代谢产物的分析已有报道, 在大鼠尿液、粪便、胃和大肠中共检出了5种代谢产物. 本文采用高效液相色谱-飞行时间串联质谱进行人参皂苷Rb1的体内代谢研究, 通过口服和静脉给予药物, 在大鼠尿液中共检出了人参皂苷Rb1的14种代谢产物, 并系统分析和推断了这些代谢物的转化规律和可能结构.     

Ultra-high performance liquid chromatography tandem mass spectrometry for comprehensive analysis of urinary acylcarnitines

We report an enabling mass spectrometric method for the analysis of lipid metabolites in order to define better the lipid metabolome in terms of chemical diversity and generate fragment ion spectra of these metabolites as a potential resource for unknown metabolite identification. This work focuses on the analysis of one important class of lipid metabolites, the acylcarnitines. Current analytical methods have only detected and identified a limited number of these metabolites. The method described herein provides the most comprehensive acylcarnitine profile in urine of healthy individuals up to date. It involves an optimized solid phase extraction technique for selective analyte extraction using cartridges containing both lipophilic and cation-exchange properties. The captured analytes are then subjected to ultra-high performance liquid chromatography (UPLC) separation, followed by tandem mass spectrometry (MS/MS) analysis using information-dependent acquisitions and selected reaction monitoring (SRM). The urine of six healthy individuals was analyzed using this method. A total of 355 acylcarnitines were detected; only 43 of them have been previously reported in the urine of healthy individuals. Detection of this large number of acylcarnitines illustrates the great diversity of the lipid metabolome as well as the usefulness of the method for profiling acylcarnitines. Furthermore, the MS/MS spectra of the 355 acylcarnitines will be uploaded to a public human metabolome database as a mass spectrometric resource for unknown metabolite identification.     

Urinary metabolites of cannabidiol in dog, rat and man and their identification by gas chromatography-mass spectrometry

Urinary metabolites of cannabidiol (CBD), a non-psychoactive cannabinoid of potential therapeutic interest, were extracted from dog, rat and human urine, concentrated by chromatography on Sephadex LH-20 and examined by gas chromatography-mass spectrometry as trimethylsilyl (TMS), [2H9]TMS, methyl ester-TMS and methyloxime-TMS derivatives. Fragmentation of the metabolites under electron-impact gave structurally informative fragment ions; computer-generated single-ion plots of these diagnostic ions were used extensively to aid metabolite identification. Over fifty metabolites were identified with considerable species variation. CBD was excreted in substantial concentration in human urine, both in the free state and as its glucuronide. In dog, unusual glucoside conjugates of three metabolites (4"- and 5"-hydroxy- and 6-oxo-CBD), not excreted in the unconjugated state, were found as the major metabolites at early times after drug administration. Other metabolites in all three species were mainly acids. Side-chain hydroxylated derivatives of CBD-7-oic acid were particularly abundant in human urine but much less so in dog. In the latter species the major oxidized metabolites were the products of beta-oxidation with further hydroxylation at C-6. A related, but undefined pathway resulted in loss of three carbon atoms from the side-chain of CBD in man with production of 2"-hydroxy-tris,nor-CBD-7-oic acid. Metabolism by the epoxide-diol pathway, resulting in dihydro-diol formation from the delta-8 double bond, gave metabolites in both dog and human urine. It was concluded that CBD could be used as a probe of the mechanism of several types of biotransformation; particularly those related to carboxylic acid metabolism as intermediates of the type not usually seen with endogenous compounds were excreted in substantial concentration.     

In vitro metabolism studies of desoxy‐methyltestosterone (DMT) and its five analogues,and in vivo metabolism of desoxy‐vinyltestosterone (DVT) in horses

The positive findings of norbolethone in 2002 and tetrahydrogestrinone in 2003 in human athlete samples confirmed that designer steroids were indeed being abused in human sports. In 2005, an addition to the family of designer steroids called ‘Madol’ [also known as desoxy‐methyltestosterone ( DMT )] was seized by government officials at the US–Canadian border. Two years later, a positive finding of DMT was reported in a mixed martial arts athlete's sample. It is not uncommon that doping agents used in human sports would likewise be abused in equine sports. Designer steroids would, therefore, pose a similar threat to the horseracing and equestrian communities. This paper describes the in vitro metabolism studies of DMT and five of its structural analogues with different substituents at the 17α position (R ? H, ethyl, vinyl, ethynyl and ²H₃‐methyl). In addition, the in vivo metabolism of desoxy‐vinyltestosterone ( DVT ) in horses will be presented. The in vitro studies revealed that the metabolic pathways of DMT and its analogues occurred predominantly in the A‐ring by way of a combination of enone formation, hydroxylation and reduction. Additional biotransformation involving hydroxylation of the 17α‐alkyl group was also observed for DMT and some of its analogues. The oral administration experiment revealed that DVT was extensively metabolised and the parent drug was not detected in urine. Two in vivo metabolites, derived respectively from (1) hydroxylation of the A‐ring and (2) di‐hydroxylation together with A‐ring double‐bond reduction, could be detected in urine up to a maximum of 46 h after administration. Another in vivo metabolite, derived from hydroxylation of the A‐ring with additional double‐bond reduction and di‐hydroxylation of the 17α‐vinyl group, could be detected in urine up to a maximum of 70 h post‐administration. All in vivo metabolites were excreted mainly as glucuronides and were also detected in the in vitro studies. Copyright © 2015 John Wiley & Sons, Ltd.     

A mass spectrometric study on meloxicam metabolism in horses and the fungus Cunninghamella elegans,and the relevance of this microbial system as a model of drug metabolism in the horse

This paper describes a study where the metabolism of the non‐steroidal anti‐inflammatory drug meloxicam was investigated in six horses and in the filamentous fungus Cunninghamella elegans. The metabolites identified were compared between the species, and then the fungus was used to produce larger amounts of the metabolites for future use as reference material. C. elegans proved to be a good model of phase I meloxicam metabolism in horses since all four metabolites found were the same in both species. Apart from the two main metabolites, 5′‐hydroxymethylmeloxicam and 5′‐carboxymeloxicam, a second isomer of hydroxymeloxicam and dihydroxylated meloxicam were detected for the first time in horse urine and the microbial incubations. Phase II metabolites were not discovered in the C. elegans samples but hydroxymeloxicam glucuronide was detected intact in horse urine for the first time in this study. Urine from six horses was further analyzed in a semi‐quantitative sense and 5′‐hydroxymethylmeloxicam gave peaks with much higher intensity compared to the parent drug and the other metabolites, and was detected for at least 14 days after the last given dose in some of the horses. From the results presented in this article, we suggest that analytical methods developed for the detection of meloxicam in horse urine after prohibited use should focus on the 5′‐hydroxymethyl metabolite and that C. elegans can be used to produce large amounts of this metabolite for potential future use as a reference compound. Copyright © 2009 John Wiley & Sons, Ltd.     

Concentration profiles of cocaine, pyrolytic methyl ecgonidine and thirteen metabolites in human blood and urine: determination by gas chromatography-mass spectrometry

When cocaine is smoked, a pyrolytic product, methyl ecgonidine (anhydroecgonine methyl ester), is also consumed with the cocaine. The amount of methyl ecgonidine formed depends on the pyrolytic conditions and composition of the illicit cocaine. This procedure describes detection of cocaine and 10 metabolites--cocaethylene, nor-cocaine, nor-cocaethylene, methyl ecgonine, ethyl ecgonine, benzoylecgonine, nor-benzoylecgonine, m-hydroxybenzoylecgonine, p-hydroxybenzoylecgonine and ecgonine--in blood and urine. In addition, the detection of pyrolytic methyl ecgonidine and three metabolites--ecgonidine (anhydroecgonine), ethyl ecgonidine (anhydroecgonine ethyl ester) and nor-ecgonidine (nor-anhydroecgonine)--are included. The newly described metabolites, ethyl ecgonidine and nor-ecgonidine, were synthesized and characterized by gas chromatography-mass spectrometry (GC-MS). All 15 compounds were extracted from 3 mL of blood or urine by solid-phase extraction and identified by a GC-MS method. The overall recoveries were 49% for methyl ecgonine, 35% for ethyl ecgonine, 29% for ecgonine and more than 83% for all other drugs. The limits of detection were between 0.5 and 4.0 ng/mL except for ecgonine, which was 16 ng/mL. Linearity for each analyte was established and in all cases correlation coefficients were 0.9985-1.0000. The procedure was applied to examine the concentration profiles of analytes of interest in post-mortem (PM) blood and urine, and in urine collected from living individuals (LV). These specimens previously were shown to be positive for the cocaine metabolite, benzoylecgonine. Ecgonidine, the major metabolite of methyl ecgonidine, was present in 77% of PM and 88% of the LV specimens, indicating smoking as the major route of cocaine administration. The new pyrolytic metabolites, ethyl ecgonidine and nor-ecgonidine, were present in smaller amounts. The urine concentrations of nor-ecgonidine were 0-163 ng/mL in LV and 0-75 ng/mL in PM specimens. Ethyl ecgonidine was found only in PM urine at concentrations 0-39 ng/mL. Ethanol-related cocaine metabolites, ethyl ecgonine or cocaethylene, were present in 69% of PM and 53% of cocaine-positive LV specimens, implying alcohol consumption with cocaine use. The four major metabolites of cocaine--benzoylecgonine, ecgonine, nor-benzoylecgonine and methyl ecgonine--constituted approximately 88 and 97% of all metabolites in PM and LV specimens, respectively. The concentrations of nor-cocaine and nor-cocaethylene were consistently the lowest of all cocaine metabolites. At benzoylecgonine concentrations below 100 ng/mL, ecgonine was present at the highest concentrations. In 20 urine specimens, benzoylecgonine and ecgonine median concentrations (range) were 54 (0-47) and 418 ng/mL (95-684), respectively. Therefore, detection of ecgonine is advantageous when benzoylecgonine concentrations are below 100 ng/mL.