HILIC‐MS‐based shotgun metabolomic profiling of maternal urine at 9–23 weeks of gestation – establishing the baseline changes in the maternal metabolome

In this data‐rich age it is no longer necessary to methodically isolate, characterize and measure specific molecules. What is important is to identify which of the hundreds or thousands of resolved and measured ‘unknown’ molecules are potentially associated with the pathophysiology of interest. We have taken LC‐MS data from pregnancy urine and applied SIMCA P+ data analysis software in shotgun metabolomics to search the large amount of data for significant metabolite changes that occur in the transition from the first to early second trimester of pregnancy. Seventy‐two individual urine samples were examined spanning 9–23 weeks of gestation. Three‐hundred and eighty‐three ions were identified and variations were mapped between profiles of different gestational age and the significance quantified. In urine collected during pregnancy, the transition from first to early second trimester revealed a relatively steady pattern of metabolites except for four that showed a dramatic fall in abundance as pregnancy progressed from the first to second trimester. The pattern of changes in urinary metabolites identified by Zwitterionic Hydrophilic Liquid Interaction Chromatography (ZIC‐HILIC) coupled to mass spectrometry was evaluated and we established a baseline of changes from which a search for metabolomic markers associated with clinical pathologies of pregnancy can be made as a part of wider ultraomics study. Copyright © 2014 John Wiley & Sons, Ltd.     

Metabolomic investigation of cholestasis in a rat model using ultra-performance liquid chromatography/tandem mass spectrometry

Metabolomics follows the changes in concentrations of endogenous metabolites, which may reflect various disease states as well as systemic responses to environmental, therapeutic, or genetic interventions. In this study, we applied metabolomic approaches to monitor dynamic changes in plasma and urine metabolites, and compared these metabolite profiles in Eisai hyperbilirubinemic rats (EHBR, an animal model of cholestasis) with those in the parent strain of EHBR - Sprague-Dawley (SD) rats - in order to characterize cholestasis pathophysiologically. Ultra-performance liquid chromatography/tandem mass spectrometry-based analytical methods were used to assay metabolite levels. More than 250 metabolites were detected in both plasma and urine, and metabolite profiles of EHBR differed from those of SD rats. The levels of antioxidative and cytoprotective metabolites, taurine and hypotaurine, were markedly increased in urine of EHBR. The levels of many bile acids were also elevated in plasma and urine of EHBR, but the extent of elevation depended on the particular bile acid. The levels of cytoprotective ursodeoxycholic acid and its conjugates were markedly elevated, while that of cytotoxic chenodeoxycholic acid remained unchanged, suggesting the balance of bile acids had shifted resulting in decreased toxicity. In EHBR, reduced biliary excretion leads to increased systemic exposure to harmful compounds including some endogenous metabolites. Our metabolomic data suggest that mechanisms exist in EHBR that compensate for cholestasis-related damage.     

General unknown screening procedure for the characterization of human drug metabolites in forensic toxicology: Applications and constraints

LC coupled to single (LC–MS) and tandem (LC–MS/MS) mass spectrometry is recognized as the most powerful analytical tools for metabolic studies in drug discovery. In this article, we describe five cases illustrating the utility of screening xenobiotic metabolites in routine analysis of forensic samples using LC–MS/MS. Analyses were performed using a previously published LC–MS/MS general unknown screening (GUS) procedure developed using a hybrid linear IT–tandem mass spectrometer. In each of the cases presented, the presence of metabolites of xenobiotics was suspected after analyzing urine samples. In two cases, the parent drug was also detected and the metabolites were merely useful to confirm drug intake, but in three other cases, metabolite detection was of actual forensic interest. The presented results indicate that: (i) the GUS procedure developed is useful to detect a large variety of drug metabolites, which would have been hardly detected using targeted methods in the context of clinical or forensic toxicology; (ii) metabolite structure can generally be inferred from their “enhanced” product ion scan spectra; and (iii) structure confirmation can be achieved through in vitro metabolic experiments or through the analysis of urine samples from individuals taking the parent drug.     

A target‐group‐change couple with mass defect filtering strategy to identify the metabolites of “Dogel ebs” in rats plasma,urine and bile

“Dogel ebs” was known as Sophora flavescens Ait., a classical traditional Chinese Mongolian herbal medicine, which had the effects on damp‐heat dysentery, scrofula, and syndrome of accumulated dampness toxicity. Although the chemical constituents have been clarified by our previous studies, the metabolic transformation of “Dogel ebs” in vivo was still unclear. To explore the mechanism of “Dogel ebs,” the metabolites in plasma, bile, and urine samples were investigated. A fast positive and negative ion switching technology was used for the simultaneous determination of flavonoids and alkaloids in “Dogel ebs” in a single run. And a target‐group‐change coupled with mass defect filtering strategy was utilized to analyze the collected data. 89 parent compounds and 82 metabolites were characterized by high‐performance liquid chromatography with quadrupole exactive Orbitrap mass spectrometry. Both phase I and phase II metabolites were observed and the metabolic pathways involved in oxidation, demethylation, acetylation, and glucuronidation. 69 metabolites of “Dogel ebs,” including three hydroxyls bonding xanthohumol, formononetin‐7‐O‐glucuronide, 2′‐hydroxyl‐isoxanthohumol decarboxylation metabolite, oxysophocarpine dehydrogen, 9α‐hydroxysophoramine‐O‐glucuronide, etc. were reported for the first time.     

Dimethocaine,a synthetic cocaine analogue: studies on its in-vivo metabolism and its detectability in urine by means of a rat model and liquid chromatography–linear ion-trap (high-resolution) mass spectrometry

Dimethocaine (DMC, larocaine), a synthetic derivative of cocaine, is a widely distributed “legal high” consumed as a “new psychoactive substance” (NPS) without any safety testing, for example studies of metabolism. Therefore, the purpose of this work was to study its in-vivo and in-vitro metabolism by use of liquid chromatography–(high resolution) mass spectrometry (LC–HRMS ⁿ ). DMC was administered to male Wistar rats (20 mg kg^?1) and their urine was extracted either by solid-phase extraction after enzymatic cleavage of conjugates or by use of protein precipitation (PP). The metabolites were separated and identified by LC–HRMS ⁿ . The main phase I reactions were ester hydrolysis, deethylation, hydroxylation of the aromatic system, and a combination of these. The main phase II reaction was N-acetylation of the p-aminobenzoic acid part of the unchanged parent compound and of several phase I metabolites. The metabolites identified were then used for identification of DMC in rat urine after application of a common user’s dose. By use of GC–MS and LC–MS ⁿ standard urine-screening approaches (SUSAs), DMC and its metabolites could be detected in the urine samples.     

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.     

Urinary exposure marker discovery for toxicants using ultra-high pressure liquid chromatography coupled with Orbitrap high resolution mass spectrometry and three untargeted metabolomics approaches

Human biomonitoring is the assessment of actual internal contamination of chemicals by measuring exposure markers, chemicals or their metabolites, in human urine, blood, serum, and other body fluids. However, the metabolism of chemicals within an organism is extremely complex. Therefore, the identification of metabolites is often difficult and laborious. Several untargeted metabolomics methods have been developed to perform objective searching/filtering of accurate-mass-based LC-MS data to facilitate metabolite identification. In this study, three metabolomics data processing approaches were used for chemical exposure marker discovery in urine with an LTQ-Orbitrap high-resolution mass spectrometry (HRMS) dataset; di-isononyl phthalate (DINP) was used as an example. The data processing techniques included the SMAIT, mass defect filtering (MDF), and XCMS Online. Sixteen, 83, and 139 probable DINP metabolite signals were obtained using the SMAIT, MDF, and XCMS procedures, respectively. Fourteen probable metabolite signals mined simultaneously by the three metabolomics approaches were confirmed as DINP metabolites by structural information provided by LC-MS/MS. Among them, 13 probable metabolite signals were validated as exposure-related markers in a rat model. Six (m/z 319.155, 361.127, 373.126, 389.157, 437.112 and 443.130) of the 13 exposure-related DINP metabolite signals have not previously been reported in the literature. Our data indicate that SMAIT provided an efficient method to discover effectively and systematically urinary exposure markers of toxicant. The DINP metabolism information can provide valuable information for further investigations of DINP toxicity, toxicokinetics, exposure assessment, and human health effects.     

The application of high-resolution mass spectrometry-based data-mining tools in tandem to metabolite profiling of a triple drug combination in humans

Patients are usually exposed to multiple drugs, and metabolite profiling of each drug in complex biological matrices is a big challenge. This study presented a new application of an improved high resolution mass spectrometry (HRMS)-based data-mining tools in tandem to fast and comprehensive metabolite identification of combination drugs in human. The model drug combination was metronidazole-pantoprazole-clarithromycin (MET-PAN-CLAR), which is widely used in clinic to treat ulcers caused by Helicobacter pylori. First, mass defect filter (MDF), as a targeted data processing tool, was able to recover all relevant metabolites of MET-PAN-CLAR in human plasma and urine from the full-scan MS dataset when appropriate MDF templates for each drug were defined. Second, the accurate mass-based background subtraction (BS), as an untargeted data-mining tool, worked effectively except for several trace metabolites, which were buried in the remaining background signals. Third, an integrated strategy, i.e., untargeted BS followed by improved MDF, was effective for metabolite identification of MET-PAN-CLAR. Most metabolites except for trace ones were found in the first step of BS-processed datasets, and the results led to the setup of appropriate metabolite MDF template for the subsequent MDF data processing. Trace metabolites were further recovered by MDF, which used both common MDF templates and the novel metabolite-based MDF templates. As a result, a total of 44 metabolites or related components were found for MET-PAN-CLAR in human plasma and urine using the integrated strategy. New metabolic pathways such as N-glucuronidation of PAN and dehydrogenation of CLAR were found. This study demonstrated that the combination of accurate mass-based multiple data-mining techniques in tandem, i.e., untargeted background subtraction followed by targeted mass defect filtering, can be a valuable tool for rapid metabolite profiling of combination drugs in vivo.     

Metabolic infrastructure of pregnant women with methylenetetrahydrofolate reductase polymorphisms: A metabolomic analysis

The aim of this study was to demonstrate the altered metabolic infrastructure of pregnant women with methylenetetrahydrofolate reductase (MTHFR) polymorphisms at first trimester and during delivery. Eight singleton pregnant women with MTHFR polymorphisms were compared with 10 normal pregnant women. Maternal blood samples were obtained twice during their pregnancy period (between the 11th and 14th gestational weeks and during delivery). Metabolomic analysis was performed using GC–MS. The GC–MS based metabolomic profile helped identify 95 metabolites in the plasma samples. In the MTHFR group, the levels of 1-monohexadecanoylglycerol, pyrophosphate, benzoin, and linoleic acid significantly decreased (P ˂ 0.05 for all), whereas the levels of glyceric acid, l -tryptophan, l -alanine, l -proline, norvaline, l -threonine, and myo-inositol significantly increased (P ˂ 0.01 for the first two metabolites, P ˂ 0.05 for the others) at 11–14 gestational weeks. Conversely, the levels of benzoin, 1-monohexadecanoylglycerol, pyruvic acid, l -proline, phosphoric acid, epsilon-caprolactam, and pipecolic acid significantly decreased in the MTHFR group, whereas metabolites such as hexadecanoic acid and 2-hydroxybutyric acid increased significantly in the study group during delivery. An impaired energy metabolism pathway, vitamin B complex disorders, tendency for metabolic acidosis (oxidative stress), and the need for cell/tissue support seem prevalent in pregnancies with MTHFR polymorphisms.     

Case Study: Doping Substances in Equestrian Food Supplements

In the course of investigations on equestrian supplemental products for the presence of doping substances, two products were found to contain forbidden substances. As reported earlier a plant extract (Mexican cactus extract) named “Energy 5” contained the anabolic androgenic steroids (AAS) stanozolol, 17β-hydroxy-17α-methyl-5α-androstane-3β-ol (3β,5α-THMT) as well as mestanolone not declared on the label. In the present study, a product called “Super Kalm Paste” was tested. Analysis by gas chromatography - mass spectrometry (GC-MS) revealed that the preparation contained the class I anti-arrhythmics quinine (trade names Kinidin^TM, Durules) and cinchonine. The samples were prepared according to a sample preparation procedure established for anabolic steroids in nutritional supplements for humans. The sample treatment comprised the extraction and purification of the analytes as well as the chemical conversion with N-methyl-N-trimethylsilyl-trifluoracetamide (MSTFA) to yield the trimethylsilyl (TMS)-derivatives. To verify whether the administration of such products could lead to positive doping tests, a pilot excretion study on “Energy 5” was conducted with two geldings, and urine samples were collected. Gas chromatography - high resolution mass spectrometry (GC-HRMS) after solid phase extraction and mixed derivatisation has demonstrated the presence of the stanozolol metabolite 16β-hydroxy-stanozolol in urine samples after “Energy 5” application.     

SPE–NMR metabolite sub-profiling of urine

NMR-based metabolite profiling of urine is a fast and reproducible method for detection of numerous metabolites with diverse chemical properties. However, signal overlap in the (1)H NMR profiles of human urine may hamper quantification and identification of metabolites. Therefore, a new method has been developed using automated solid-phase extraction (SPE) combined with NMR metabolite profiling. SPE-NMR of urine resulted in three fractions with complementary and reproducible sub-profiles. The sub-profile from the wash fraction (100?% water) contained polar metabolites; that from the first eluted fraction (10?% methanol-90?% water) semi-polar metabolites; and that from the second eluted fraction (100?% methanol) aromatic metabolites. The method was validated by analysis of urine samples collected from a crossover human nutritional intervention trial in which healthy volunteers consumed capsules containing a polyphenol-rich mixture of red wine and grape juice extract (WGM), the same polyphenol mixture dissolved in a soy drink (WGM_Soy), or a placebo (PLA), over a period of five days. Consumption of WGM clearly increased urinary excretion of 4-hydroxyhippuric acid, hippuric acid, 3-hydroxyphenylacetic acid, homovanillic acid, and 3-(3-hydroxyphenyl)-3-hydroxypropionic acid. However, there was no difference between the excreted amounts of these metabolites after consumption of WGM or WGM_Soy, indicating that the soy drink is a suitable carrier for WGM polyphenols. Interestingly, WGM_Soy induced a significant increase in excretion of cis-aconitate compared with WGM and PLA, suggesting a higher demand on the tricarboxylic acid cycle. In conclusion, SPE-NMR metabolite sub-profiling is a reliable and improved method for quantification and identification of metabolites in urine to discover dietary effects and markers of phytochemical exposure.     

Simultaneous HPLC of twelve monoamines and metabolites shows neuroblastoma cell line releases HVA and HIAA

Neuroblastoma is a solid tumor occurring usually in children less than 5 years old. It has been difficult to distinguish neuroblastoma from other childhood tumors through morphological diagnosis. Urine homovanillic acid (HVA), which is a metabolite of dopamine, has been proposed as a diagnostic index. Although increased levels of a serotonin metabolite, 5-hydroxyindole-3-acetic acid (HIAA), have also been observed in urine samples of the patients, they were largely attributed to dietary amines. By using an HPLC system with electrochemical detection, which can simultaneously assay 12 monoamines and metabolites, we showed that HVA and HIAA are two of the most prominent monoamine metabolites in the medium after a neuroblastoma cell line (IMR-32) was cultured for 3 days. Moreover, we found that the levels of HVA and HIAA in the media are proportional to the cell densities. These results suggest that the levels of HVA and HIAA in tissue culture media, or in urine from patients whose dietary amines are well controlled, may provide a valuable diagnostic index for neuroblastoma.     

Metabolic profiling of valproic acid in patients using negative-ion chemical ionization gas chromatography-mass spectrometry

A negative-ion chemical ionization gas chromatographic-mass spectrometric method for the determination of valproic acid (VPA) and fourteen of its metabolites in a single chromatographic run is reported. The assay features the use of four internal standards and is applicable to the analysis of small serum and urine volumes. A combination of pentafluorobenzyl and trimethylsilyl derivatization resulted in the [M - 181]- ion as the base peak for all the metabolites measured. When these ions were monitored sensitivities in the low picogram levels were achieved. The VPA metabolite profile was determined in pediatric patients on VPA monotherapy and on combined VPA therapy with either carbamazepine or clobazam. The recently characterized diene metabolite, (E,E)-2,3'-diene-VPA, was found to be a major serum metabolite of VPA. In the patient groups taking VPA in combination with carbamazepine, the induction of omega and omega-1 pathways of VPA metabolism was apparent, while the levels of the beta-oxidation products were significantly decreased.     

Fast and simple method for the determination of urinary 1‐hydroxypyrene

A fast method for the measurement of metabolites of pyrene in urine was improved by HPLC with fluorescence detector using “heart‐cut” technique. This method can quantify the total amount of pyrene metabolites corresponding to glucuronic acid and sulfate conjugates as well as free 1‐OH‐Py. The hydrolyzed biological fluid was directly injected into the chromatographic system, via a column‐switching system. Pre‐treatment and analysis were performed within 0.5 and 9 min, respectively. Enzymatic hydrolysis has been optimized to not exceed 2 h. The best response function, in the 0.2–10 ng/mL range, is the linear regression, bringing simplicity, good accuracy, and low LOQ. The intra‐ and interday precision values were inferior to 1 and 2%, respectively. The proposed method provided a simple, convenient, and practical procedure to determine the level of the main urinary pyrene metabolites in biological samples.     

Analysis of Arecoline in Rat Urine, and Identification of its Metabolites, by Liquid Chromatography–Tandem Mass Spectrometry

A simple and rapid high-performance liquid chromatographic–electrospray ionization (ESI) tandem mass spectrometric method has been developed for elucidation of the structures of the metabolites of arecoline in rat urine after administration of a single dose (20 mg kg^?1). The urine samples were purified on a C₁₈ solid-phase extraction cartridge and analysis was then performed on a reversed-phase C₁₈ column with 60:40 (v/v) methanol–0.01% triethylamine solution (2 mmol L^?1, adjusted to pH 3.5 with formic acid) as mobile phase and detection by on-line MS–MS. Identification of the metabolites and elucidation of their structures were performed by comparing molecular masses (ΔM), retention-times, and product ion spectra with those of the parent drug. The parent drug arecoline, four phase-I metabolites, and one phase-II metabolite were identified in rat urine.     

2-Methiopropamine, a thiophene analogue of methamphetamine: studies on its metabolism and detectability in the rat and human using GC-MS and LC-(HR)-MS techniques

2-Methiopropamine [1-(thiophen-2-yl)-2-methylaminopropane, 2-MPA], a thiophene analogue of methamphetamine, is available from online vendors selling “research chemicals.” The first samples were seized by the German police in 2011. As it is a recreational stimulant, its inclusion in routine drug screening protocols should be required. The aims of this study were to identify the phase I and II metabolites of 2-MPA in rat and human urine and to identify the human cytochrome-P450 (CYP) isoenzymes involved in its phase I metabolism. In addition, the detectability of 2-MPA in urine samples using the authors’ well-established gas chromatography–mass spectrometry (GC-MS) and liquid chromatography-linear ion trap-mass spectrometry (LC-MSⁿ) screening protocols was also evaluated. The metabolites were isolated from rat and human urine samples by solid-phase extraction without or following enzymatic cleavage of conjugates. The phase I metabolites, following acetylation, were separated and identified by GC-MS and/or liquid chromatography–high-resolution linear ion trap mass spectrometry (LC-HR-MSⁿ) and the phase II metabolites by LC-HR-MSⁿ. The following major metabolic pathways were proposed: N-demethylation, hydroxylation at the side chain and at the thiophene ring, and combination of these transformations followed by glucuronidation and/or sulfation. CYP1A2, CYP2C19, CYP2D6, and CYP3A4 were identified as the major phase I metabolizing enzymes. They were also involved in the N-demethylation of the analogue methamphetamine and CYP2C19, CYP2D6, and CYP3A4 in its ring hydroxylation. Following the administration of a typical user’s dose, 2-MPA and its metabolites were identified in rat urine using the authors’ GC-MS and the LC-MSⁿ screening approaches. Ingestion of 2-MPA could also be detected by both protocols in an authentic human urine sample.     

Exploring the In Vivo Existence Forms (23 Original Constituents and 147 Metabolites) of Astragali Radix Total Flavonoids and Their Distributions in Rats Using HPLC-DAD-ESI-IT-TOF-MSn

Astragali Radix total flavonoids (ARTF) is one of the main bioactive components of Astragali Radix (AR), and has many pharmacological effects. However, its metabolism and effective forms remains unclear. The HPLC-DAD-ESI-IT-TOF-MSⁿ technique was used to screen and tentatively identify the in vivo original constituents and metabolites of ARTF and to clarify their distribution in rats after oral administration. In addition, modern chromatographic methods were used to isolate the main metabolites from rat urine and NMR spectroscopy was used to elucidate their structures. As a result, 170 compounds (23 original constituents and 147 metabolites) were tentatively identified as forms existing in vivo, 13 of which have the same pharmacological effect with ARTF. Among 170 compounds, three were newly detected original constituents in vivo and 89 were new metabolites of ARTF, from which 12 metabolites were regarded as new compounds. Nineteen original constituents and 65 metabolites were detected in 10 organs. Four metabolites were isolated and identified from rat urine, including a new compound (calycoisn-3’-O-glucuronide methyl ester), a firstly-isolated metabolite (astraisoflavan-7-O-glucoside-2’-O-glucuronide), and two known metabolites (daidzein-7-O-sulfate and calycosin-3’-O-glucuronide). The original constituents and metabolites existing in vivo may be material basis for ARTF efficacy, and these findings are helpful for further clarifying the effective forms of ARTF.     

Elimination profiles of flurbiprofen and its metabolites in equine urine for doping analysis

Flurbiprofen and its main acidic metabolites were detected in equine urine after a single-dose administration of 500 mg flurbiprofen to two 2.5–3.5-years-old mares, in order to be used in equine doping control routine analysis. The urine levels of the parent drug were determined using GC/MS. Five acidic metabolites were found in the urine. The structure of the proposed metabolites was confirmed by HRMS accurate mass measurements. The highest flurbiprofen concentration was 204 μg ml⁻¹ at 1–3 h post administration. Flurbiprofen could be detected for 24–37 h in urine using the standard screening procedure. All metabolites were present 25 h post administration, while 4′-hydroxyflurbiprofen could be traced for more than 48 h and it is regarded as the long-term metabolite of flurbiprofen in horse.     

Pharmacokinetic Characterization of (Poly)phenolic Metabolites in Human Plasma and Urine after Acute and Short-Term Daily Consumption of Mango Pulp

Pharmacokinetic (PK) evaluation of polyphenolic metabolites over 24 h was conducted in human subjects (n = 13, BMI = 22.7 ± 0.4 kg/m²) after acute mango pulp (MP), vitamin C (VC) or MP + VC test beverage intake and after 14 days of MP beverage intake. Plasma and urine samples were collected at different time intervals and analyzed using targeted and non-targeted mass spectrometry. The maximum concentrations (C_max) of gallotannin metabolites were significantly increased (p < 0.05) after acute MP beverage intake compared to VC beverage alone. MP + VC beverage non-significantly enhanced the C_max of gallic acid metabolites compared to MP beverage alone. Pyrogallol (microbial-derived metabolite) derivatives increased (3.6%) after the 14 days of MP beverage intake compared to 24 h acute MP beverage intake (p < 0.05). These results indicate extensive absorption and breakdown of gallotannins to galloyl and other (poly)phenolic metabolites after MP consumption, suggesting modulation and/or acclimation of gut microbiota to daily MP intake.