Development of a rapid-fire drug screening method by probe electrospray ionization tandem mass spectrometry for human urine (RaDPi-U).

Drug screening tests are mandatory in the search for drugs in forensic biological samples, and immunological methods and mass spectrometry (e.g., gas chromatography-mass spectrometry and liquid chromatography-tandem mass spectrometry) are commonly used for that purpose. However, these methods have some drawbacks, and developing new screening methods is required. In this study, we develop a rapid-fire drug screening method by probe electrospray ionization tandem mass spectrometry (PESI-MS/MS), which is an ambient ionization mass spectrometry method, for human urine, named RaDPi-U. RaDPi-U is carried out in three steps: (1) mixing urine with internal standard (IS) solution and ethanol, followed by vortexing; (2) pipetting the mixture onto a sample plate for PESI; and (3) rapid-fire analysis by PESI-MS/MS. RaDPi-U targets 40 forensically important drugs, which include illegal drugs, hypnotics, and psychoactive substances. The analytical results were obtained within 3 min because of the above-mentioned simple workflow of RaDPi-U. The calibration curves of each analyte were constructed using the IS method, and they were quantitatively valid, resulting in good linearity (0.972-0.999) with a satisfactory lower limit of detection and lower limit of quantitation (0.01-7.1 ng/mL and 0.02-21 ng/mL, respectively). Further, both trueness and precisions were 28% or less, demonstrating the high reliability and repeatability of the method. Finally, we applied RaDPi-U to three postmortem urine specimens and successfully detected different drugs in each urine sample. The practicality of the method is proven, and RaDPi-U will be a strong tool as a rapid-fire drug screening method not only in forensic toxicology but also in clinical toxicology.

Development of two fully automated quick, easy, cheap, effective, rugged, and safe pretreatment methods for the extraction of psychotropic drugs from whole blood samples.

Quick, easy, cheap, effective, rugged, and safe extraction strategies are becoming increasingly adopted in various analytical fields to determine drugs in biological specimens. In the present study, we developed two fully automated quick, easy, cheap, effective, rugged, and safe extraction methods based on acetonitrile salting-out assisted liquid-liquid extraction (method 1) and acetonitrile salting-out assisted liquid-liquid extraction followed by dispersive solid-phase extraction (method 2) using a commercially available automated liquid-liquid extraction system. We applied these methods to the extraction of 14 psychotropic drugs (11 benzodiazepines and carbamazepine, quetiapine, and zolpidem) from whole blood samples. Both methods prior to liquid chromatography-tandem mass spectrometry analysis exhibited high linearity of calibration curves (correlation coefficients, > 0.9997), ppt level detection sensitivities, and satisfactory precisions (< 8.6% relative standard deviation), accuracies (within ± 16% relative error), and matrix effects (81-111%). Method 1 provided higher recovery rates (80-91%) than method 2 (72-86%), whereas method 2 provided higher detection sensitivities (limits of detection, 0.003-0.094 ng/mL) than method 1 (0.025-0.47 ng/mL) owing to the effectiveness of its dispersive solid-phase extraction cleanup step. These fully automated extraction methods realize reliable, labor-saving, user-friendly, and hygienic extraction of target analytes from whole blood samples.

Preliminary study to classify mechanisms of mitochondrial toxicity by in vitro metabolomics and bioinformatics.

AIM: Mitochondrial toxicity is one of the causes for drug-induced liver injury, and the classification of phenotypes or mitochondrial toxicity are highly required though there are no molecular-profiling approaches for classifying mitochondrial toxicity. Therefore, the aim of this study was to classify the mechanisms of mitochondrial toxicity by metabolic profiling in vitro and bioinformatics. MAIN METHODS: We applied an established gas chromatography tandem mass spectrometry-based metabolomics to human hepatoma grade 2 (HepG2) cells that were exposed to mitochondrial toxicants, whose mechanisms are different, such as rotenone (0.1 µM), carbonyl cyanide-3-chlorophenylhydrazone (CCCP, 0.5 µM), nefazodone (20 µM), perhexiline (6.25 µM), or digitonin (positive cytotoxic substance, 4 µM). These concentrations were determined by the Mitochondrial ToxGlo Assay. Galactose medium was used for suppressing the Warburg effect in HepG2 cells, and the metabolome analysis successfully identified 125 metabolites in HepG2 cells. Multivariate, metabolic pathway and network analyses were performed by the R software. KEY FINDINGS: Metabolic profiling enabled the classifying the mitochondrial toxicity mechanisms of RCC inhibition and uncoupling. The metabolic profiles of respiratory chain complex (RCC) inhibitors (rotenone and nefazodone) and an uncoupler (CCCP) were fully differentiated from those of other compounds. The metabolic pathway analysis revealed that the RCC inhibitors and the uncoupler mainly disrupted TCA-cycle and related metabolic pathways. In addition, the correlation-based network analysis revealed that succinic acid, ß-alanine, and glutamic acid were potential metabolic indicators for RCC inhibition and uncoupling. SIGNIFICANCE: Our results provided new insights into classifying mechanisms of mitochondrial toxicity by in vitro metabolomics.

Easily Operable Quantification Method of 21 Plant-Derived Alkaloids in Human Serum by Automatic Sample Preparation and Liquid Chromatography-Tandem Mass Spectrometry.

In this study, we developed an easily operable quantification method for 21 plant-derived alkaloids in human serum by automatic sample preparation and liquid chromatography-tandem mass spectrometry. We designed to perform parallel sample preparation by a developed apparatus, which increased sample throughput. We conducted an automatic sample preparation through de-proteinization with 0.1% formic acid in methanol and achieved recovery rates of 89-107% (2.0-14% RSD) for all targeted analytes, demonstrating its high repeatability. The method validation results were satisfactory as follows: the linearity (r 2) of each calibration curve ranged from 0.978 to 1.000; the inter- and intra-day accuracies were 89.0-125% and 82.1-110%, respectively; the inter- and intra-day precisions were below 13% and 10%, respectively. Additionally, the lower limits of detection and quantification were 0.0044-0.047 and 0.013-0.14 ng/mL, respectively. Finally, the developed method was applied to pseudo-protoveratrine A poisoning serum and pseudo-colchicine poisoning serum, which were prepared by diluting acute-poisoning mice serum with human serum. Our method successfully quantitated protoveratrine A (0.15-0.25 ng/mL) and colchicine (4.8-6.0 ng/mL). Thus, our method is essential for prompt clinical treatment and critical care on patient in acute intoxication cases caused by plant-derived alkaloids. Supplementary Information: The online version contains supplementary material available at 10.1007/s10337-022-04212-5.

PiTMaP: A New Analytical Platform for High-Throughput Direct Metabolome Analysis by Probe Electrospray Ionization/Tandem Mass Spectrometry Using an R Software-Based Data Pipeline.

A new analytical platform called PiTMaP was developed for high-throughput direct metabolome analysis by probe electrospray ionization/tandem mass spectrometry (PESI/MS/MS) using an R software-based data pipeline. PESI/MS/MS was used as the data acquisition technique, applying a scheduled-selected reaction monitoring method to expand the targeted metabolites. Seventy-two metabolites mainly related to the central energy metabolism were selected; data acquisition time was optimized using mouse liver and brain samples, indicating that the 2.4 min data acquisition method had a higher repeatability than the 1.2 and 4.8 min methods. A data pipeline was constructed using the R software, and it was proven that it can (i) automatically generate box-and-whisker plots for all metabolites, (ii) perform multivariate analyses such as principal component analysis (PCA) and projection to latent structures-discriminant analysis (PLS-DA), (iii) generate score and loading plots of PCA and PLS-DA, (iv) calculate variable importance of projection (VIP) values, (v) determine a statistical family by VIP value criterion, (vi) perform tests of significance with the false discovery rate (FDR) correction method, and (vii) draw box-and-whisker plots only for significantly changed metabolites. These tasks could be completed within ca. 1 min. Finally, PiTMaP was applied to two cases: (1) an acetaminophen-induced acute liver injury model and control mice and (2) human meningioma samples with different grades (G1-G3), demonstrating the feasibility of PiTMaP. PiTMaP was found to perform data acquisition without tedious sample preparation and a posthoc data analysis within ca. 1 min. Thus, it would be a universal platform to perform rapid metabolic profiling of biological samples.

A preliminary study of rapid-fire high-throughput metabolite analysis using nano-flow injection/Q-TOFMS.

In this study, we demonstrated nano-flow injection analysis (nano-FIA) with quadrupole time-of-flight mass spectrometry (Q-TOFMS) for 17 highly polar intermediates produced during glycolysis, the tricarboxylic acid (TCA) cycle, and the pentose phosphate pathway (PPP). We optimized the analytical conditions for nano-flow injection/Q-TOFMS, and set the flow rate and ion source temperature to 1000 nL/min and 150 °C, respectively. Under optimal conditions, a single run was finished within 3 min, and the RSD value of 50 sequential injections was 4.2%. The method also showed quantitativity of four stable-isotope-labeled compounds (r2 > 0.99), demonstrating its robustness, high repeatability, and specificity. In addition, we compared three sample-preparation methods for rodent blood samples and found that protein precipitation with threefold methanol was the most effective. Finally, we applied the method to plasma samples from the serotonin syndrome (SS) model and control rats, the results of which were evaluated by principal component analysis (PCA). The two groups showed clearly separated PCA score plots, suggesting that the method could successfully catch the differences in metabolic profiles between SS and control rats. The results obtained from our new method were further validated by using the established gas chromatography/tandem mass spectrometry method, which demonstrated that there were good correlations between the two methods (R = 0.902 and 0.958 for lactic acid and malic acid, respectively, each at p < 0.001), thus proving the validity of our method. The method described here enables high-throughput analysis of metabolites and will be of use for the rapid analysis of metabolic profiles. Graphical abstract.

Optimal inter-batch normalization method for GC/MS/MS-based targeted metabolomics with special attention to centrifugal concentration.

This study investigated the optimal inter-batch normalization method for gas chromatography/tandem mass spectrometry (GC/MS/MS)-based targeted metabolome analysis of rodent blood samples. The effect of centrifugal concentration on inter-batch variation was also investigated. Six serum samples prepared from a mouse and 2 quality control (QC) samples from pooled mouse serum were assigned to each batch, and the 3 batches were analyzed by GC/MS/MS at different days. The following inter-batch normalization methods were applied to metabolome data: QC-based methods with quadratic (QUAD)- or cubic spline (CS)-fitting, total signal intensity (TI)-based method, median signal intensity (MI)-based method, and isotope labeled internal standard (IS)-based method. We revealed that centrifugal concentration was a critical factor to cause inter-batch variation. Unexpectedly, neither the QC-based normalization methods nor the IS-based method was able to normalize inter-batch variation, though MI- or TI-based normalization methods were effective in normalizing inter-batch variation. For further validation, 6 disease model rat and 6 control rat plasma were evenly divided into 3 batches, and analyzed as different batches. Same as the results above, MI- or TI-based methods were able to normalize inter-batch variation. In particular, the data normalized by TI-based method showed similar metabolic profiles obtained from their intra-batch analysis. In conclusion, the TI-based normalization method is the most effective to normalize inter-batch variation for GC/MS/MS-based metabolome analysis. Graphical abstract.

In Vivo Real-Time Monitoring System Using Probe Electrospray Ionization/Tandem Mass Spectrometry for Metabolites in Mouse Brain.

Recent improvements in ambient ionization techniques combined with mass spectrometry has enabled to achieve real-time monitoring of analytes of interest, even for biogenic molecules in living animals. Here, we demonstrate a newly developed system for in vivo real-time monitoring of metabolites in a living mouse brain. It consists of a semiautomated manipulation system and a unique probe electrospray ionization unit, which uses an extremely thin solid needle (tip dia.: 700 nm) for direct sampling and ionization, coupled to a conventional tandem mass spectrometer. The system successfully monitored 8 cerebrum metabolites related to central energy metabolism in an isoflurane-anesthetized mouse in real time with a 20 s interval. Moreover, our system succeeded in capturing dynamics of energy metabolism in a mouse administered with cannabinoid type-1 receptor agonist, which is known to disrupt cerebrum energy metabolism. The present system now opens the door to the next stage of cutting-edge technique in achieving in vivo real-time monitoring.

Intact Endogenous Metabolite Analysis of Mice Liver by Probe Electrospray Ionization/Triple Quadrupole Tandem Mass Spectrometry and Its Preliminary Application to in Vivo Real-Time Analysis.

Probe electrospray ionization (PESI) is a recently developed ionization technique that enables the direct detection of endogenous compounds like metabolites without sample preparation. In this study, we have demonstrated the first combination use of PESI with triple quadrupole tandem mass spectrometry (MS/MS), which was then applied to intact endogenous metabolite analysis of mice liver, achieving detection of 26 metabolites including amino acids, organic acids, and sugars. To investigate its practicality, metabolic profiles of control and CCl4-induced acute hepatic injury mouse model were measured by the developed method. Results showed clear separation of the two groups in score plots of principal component analysis and identified taurine as the primary contributor to group separation. The results were further validated by the established gas chromatography/MS/MS method, demonstrating the present method's usefulness. In addition, we preliminarily applied the method to real-time analysis of an intact liver of a living mouse. We successfully achieved monitoring of the real-time changes of two tricarboxylic acid cycle intermediates, α-ketoglutaric acid and fumaric acid, in the liver immediately after pyruvic acid injection via a cannulated tube to the portal vein. The present method achieved an intact analysis of metabolites in liver without sample preparation, and it also demonstrates future possibility to establish in vivo real-time metabolome analysis of living animals by PESI/MS/MS.

Time-course mass spectrometry imaging for depicting drug incorporation into hair.

In order to investigate the incorporation of drugs into hair, matrix-assisted laser desorption/ionization-time-of-flight tandem mass spectrometry (MS/MS) imaging was performed on the longitudinal sections of single scalp hair shafts sampled from volunteers after a single oral administration of methoxyphenamine (MOP), a noncontrolled analogue of methamphetamine. Hair specimens were collected by plucking out with the roots intact, and these specimens were prepped by an optimized procedure based on freeze-sectioning to detect the drug inside the hair shaft and hair root. Time-course changes in the imaging results, with confirmatory quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis for each 1-mm segment of single hair strands, revealed a substantial concentration of the drug first onto the hair bulbs after ingestion, while only a small portion appeared to be incorporated into the hair matrix, forming a 2-3 mm distinctive drug band with tailing. Comparable amount of the drug also appeared to be incorporated into the keratinized hair shaft in the upper dermis zone, forming another distinct drug band of about 2 mm, which both moved toward the distal side, following the strand's growth rate. These findings provide forensically crucial information: there are two major drug incorporation sites, at least for MOP, which cause overlap of the recordings and deteriorates its chronological resolution down to about 11 days or perhaps longer.

High-resolution mass spectrometric determination of the synthetic cannabinoids MAM-2201, AM-2201, AM-2232, and their metabolites in postmortem plasma and urine by LC/Q-TOFMS.

High-resolution mass spectrometry and accurate mass measurement by liquid chromatography/quadrupole-time of flight mass spectrometry (LC/Q-TOFMS) was applied to postmortem plasma and urine specimens from an autopsy of a fatal case involving synthetic cannabinoid use, resulting in the detection of three synthetic cannabinoids: MAM-2201, AM-1220, and AM-2232. We searched for their metabolites existing in postmortem plasma or urine by LC/Q-TOFMS and were able to detect N-dealkylated metabolites, defluorinated and further oxidized metabolites of MAM-2201, and some hydroxylated metabolites. Postmortem plasma concentrations of the parent drugs, N-dealkylated metabolites, and fluorinated and further oxidized metabolites of MAM-2201 were measured, and quantitation results revealed site differences between heart and femoral postmortem plasma concentrations of parent drugs and some metabolites, suggesting postmortem redistribution of the synthetic cannabinoids and their metabolites. Quantitation results suggest that defluorination is a major metabolic pathway for MAM-2201, and N-dealkylation is a common but minor pathway for the naphthoylindole-type synthetic cannabinoids in human.

A preliminary study on postmortem interval estimation of suffocated rats by GC-MS/MS-based plasma metabolic profiling.

Estimation of postmortem interval (PMI) is an important goal in judicial autopsy. Although many approaches can estimate PMI through physical findings and biochemical tests, accurate PMI calculation by these conventional methods remains difficult because PMI is readily affected by surrounding conditions, such as ambient temperature and humidity. In this study, Sprague-Dawley (SD) rats (10 weeks) were sacrificed by suffocation, and blood was collected by dissection at various time intervals (0, 3, 6, 12, 24, and 48 h; n = 6) after death. A total of 70 endogenous metabolites were detected in plasma by gas chromatography-tandem mass spectrometry (GC-MS/MS). Each time group was separated from each other on the principal component analysis (PCA) score plot, suggesting that the various endogenous metabolites changed with time after death. To prepare a prediction model of a PMI, a partial least squares (or projection to latent structure, PLS) regression model was constructed using the levels of significantly different metabolites determined by variable importance in the projection (VIP) score and the Kruskal-Wallis test (P < 0.05). Because the constructed PLS regression model could successfully predict each PMI, this model was validated with another validation set (n = 3). In conclusion, plasma metabolic profiling demonstrated its ability to successfully estimate PMI under a certain condition. This result can be considered to be the first step for using the metabolomics method in future forensic casework.

Metabolic profiling of urine and blood plasma in rat models of drug addiction on the basis of morphine, methamphetamine, and cocaine-induced conditioned place preference.

The metabolic profiles of urine and blood plasma in drug-addicted rat models based on morphine (MOR), methamphetamine (MA), and cocaine (COC)-induced conditioned place preference (CPP) were investigated. Rewarding effects induced by each drug were assessed by use of the CPP model. A mass spectrometry (MS)-based metabolomics approach was applied to urine and plasma of MOR, MA, and COC-addicted rats. In total, 57 metabolites in plasma and 70 metabolites in urine were identified by gas chromatography-MS. The metabolomics approach revealed that amounts of some metabolites, including tricarboxylic acid cycle intermediates, significantly changed in the urine of MOR-addicted rats. This result indicated that disruption of energy metabolism is deeply relevant to MOR addiction. In addition, 3-hydroxybutyric acid, L-tryptophan, cystine, and n-propylamine levels were significantly changed in the plasma of MOR-addicted rats. Lactose, spermidine, and stearic acid levels were significantly changed in the urine of MA-addicted rats. Threonine, cystine, and spermidine levels were significantly increased in the plasma of COC-addicted rats. In conclusion, differences in the metabolic profiles were suggestive of different biological states of MOR, MA, and COC addiction; these may be attributed to the different actions of the drugs on the brain reward circuitry and the resulting adaptation. In addition, the results showed possibility of predict the extent of MOR addiction by metabolic profiling. This is the first study to apply metabolomics to CPP models of drug addiction, and we demonstrated that metabolomics can be a multilateral approach to investigating the mechanism of drug addiction.

A possible role of chenodeoxycholic acid and glycine-conjugated bile acids in fibrotic steatohepatitis in a dietary rat model.

BACKGROUND AND AIMS: Our previous study indicated that hepatic bile acids (BAs) may have deposited and stimulated the pathogenesis of a high fat-cholesterol (HFC) diet-induced fibrotic steatohepatitis in stroke-prone spontaneously hypertensive 5/Dmcr rats, based on dysregulated BA homeostasis pathways. We aimed to further characterize BA profiles in liver and evaluate their relationships to liver injury using this model. METHODS: Hepatic 21 BA levels were determined by ultra-performance liquid chromatography-tandem mass spectrometry, and their correlations with macrovesicular steatosis score, serum alanine aminotransferase (ALT) level and quantified fibrotic area were assessed using Spearman and Pearson correlations. RESULTS: Compared to control, BAs highly accumulated in HFC-fed rat liver at 2 weeks: cholic acid (CA), deoxycholic acid (DCA) and chenodeoxycholic acid (CDCA) were major species, thereafter, levels of CA and DCA declined, but CDCA species persistently increased, which induced a decrease in total CA/total CDCA ratio at 8 and 14 weeks. CDCA species positively, while total CA/total CDCA negatively, correlated with macrovesicular steatosis score, serum ALT and quantified fibrotic area. Unlike control, total ursodeoxycholic acid was minor in HFC-fed rat liver, and inversely correlated to aforementioned indicators of liver injury; total glyco-BAs, rather than tauro-BAs, were predominant in HFC-fed rat liver, and positively correlated with macrovesicular steatosis score. Moreover, its ratio to total tauro-BAs positively correlated with each parameter of liver injury, while inverse associations were detected for total tauro-BAs. CONCLUSIONS: Hepatic BA accumulation may potentiate liver disease. CDCA and glyco-BAs play a more important role in the pathogenesis of fibrotic steatohepatitis.

Single-polyp metabolomics for coral health assessment.

Coral reef ecosystems supported by environmentally sensitive reef-building corals face serious threats from human activities. Our understanding of these reef threats is hampered by the lack of sufficiently sensitive coral environmental impact assessment systems. In this study, we established a platform for metabolomic analysis at the single-coral-polyp level using state-of-the-art mass spectrometry (probe electrospray ionization/tandem mass spectrometry; PESI/MS/MS) capable of fine-scale analysis. We analyzed the impact of the organic UV filter, benzophenone (BP), which has a negative impact on corals. We also analyzed ammonium and nitrate samples, which affect the environmental sensitivity of coral-zooxanthella (Symbiodiniaceae) holobionts, to provide new insights into coral biology with a focus on metabolites. The method established in this study breaks new ground by combining PESI/MS/MS with a technique for coral polyps that can control the presence or absence of zooxanthellae in corals, enabling functions of zooxanthellae to be assessed on a polyp-by-polyp basis for the first time. This system will clarify biological mechanisms of corals and will become an important model system for environmental impact assessment using marine organisms.

Simple and rapid assay method for simultaneous quantification of urinary nicotine and cotinine using micro-extraction by packed sorbent and gas chromatography-mass spectrometry.

A simple and rapid method for determination of nicotine and cotinine levels in urine was developed using samples prepared by micro-extraction by packed sorbent (MEPS) and subjected to gas chromatography-mass spectrometry (GC-MS) analysis. This method provided good reproducibility, as well as good linearity of calibration curves in the range of 1-100 and 50-1000 ng/mL for quality control samples spiked with nicotine and cotinine, respectively. The detection limit of nicotine and cotinine was as low as 0.25 and 20 ng/mL, respectively. An evaporation procedure is not suitable for nicotine determination, thus an advantage of the present MEPS assay method is direct testing with GC-MS without the need for evaporation to a dry solvent. Our findings show that it may be useful for determining nicotine levels in various types of research studies.

High-throughput analysis of anthocyanins in horticultural crops using probe electrospray ionization tandem mass spectrometry (PESI/MS/MS).

Plant secondary metabolites exhibit various horticultural traits. Simple and rapid analysis methods for evaluating these metabolites are in demand in breeding and consumer markets dealing with horticultural crops. We applied probe electrospray ionization (PESI) to evaluate secondary metabolite levels in horticultural crops. PESI does not require pre-treatment and separation of samples, which makes it suitable for high-throughput analysis. In this study, we targeted anthocyanins, one of the primary pigments in horticultural crops. Eighty-one anthocyanins were detected in approximately 3 minutes in the selected reaction-monitoring mode. Tandem mass spectrometry (MS/MS) could adequately distinguish between the fragments of anthocyanins and flavonols. Probe sampling, an intuitive method of sticking a probe directly to the sample, could detect anthocyanins qualitatively on a micro-area scale, such as achenes and receptacles in strawberry fruit. Our results suggest that PESI/MS/MS can be a powerful tool to characterize the profile of anthocyanins and compare their content among cultivars.

Impact of elobixibat on liver tumors, microbiome, and bile acid levels in a mouse model of nonalcoholic steatohepatitis.

BACKGROUND: Elevated bile acid levels have been associated with liver tumors in fatty liver. Ileal bile acid transporter inhibitors may inhibit bile acid absorption in the distal ileum and increase bile acid levels in the colon, potentially decreasing the serum and hepatic bile acid levels. This study aimed to investigate the impact of these factors on liver tumor. METHODS: C57BL/6J mice received a one-time intraperitoneal injection of 25-mg/kg diethylnitrosamine. They were fed a choline-deficient high-fat diet for 20 weeks starting from 8 weeks of age, with or without elobixibat (EA Pharma, Tokyo, Japan). RESULTS: Both groups showed liver fat accumulation and fibrosis, with no significant differences between the two groups. However, mice with elobixibat showed fewer liver tumors. The total serum bile acid levels, including free, tauro-conjugated, glyco-conjugated, and tauro-α/ß-muricholic acids in the liver, were noticeably reduced following elobixibat treatment. The proportion of gram-positive bacteria in feces was significantly lower in the group treated with elobixibat (5.4%) than in the group without elobixibat (33.7%). CONCLUSION: Elobixibat suppressed tumor growth by inhibiting bile acid reabsorption, and decreasing total bile acid and primary bile acid levels in the serum and liver. Additionally, the presence of bile acids in the colon may have led to a significant reduction in the proportion of gram-positive bacteria, potentially resulting in decreased secondary bile acid synthesis.

Simultaneous enantiomeric determination of MDMA and its phase I and phase II metabolites in urine by liquid chromatography-tandem mass spectrometry with chiral derivatization.

A liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) procedure was developed for the simultaneous determination of enantiomers of the prevalent designer drug 3,4-methylenedioxymethamphetamine (MDMA) and its phase I and phase II metabolites in urine with chiral derivatization. The analytes in urine were directly derivatized with chiral Marfey's reagent, N(α)-(5-fluoro-2,4-dinitrophenyl)-D-leucinamide, without extraction. The diastereomers of the N(α)-(2,4-dinitrophenyl)-D-leucinamide derivatives generated were determined by LC-MS/MS. Satisfactory chromatographic separation was achieved for the enantiomers of MDMA and its metabolites 3,4-methylenedioxyamphetamine, 4-hydroxy-3-methoxymethamphetamine (HMMA), HMMA glucuronide, and HMMA sulfate on a semimicro octadecylsilane column using linear gradient elution. With use of multiple reaction monitoring mode, the limits of detection of these analytes ranged from 0.01 to 0.03 µg/mL. Linear calibration curves were obtained for all enantiomers from 0.1 to 20 µg/mL in urine. The method showed sufficient reproducibility and quantitative ability. This is the first report of a simple LC-MS/MS-based analytical procedure with direct chiral derivatization in aqueous media that allows simultaneous enantiomeric determination of drugs and their metabolites, including glucuronide and sulfate derivatives.

Metabolomics and Data-Driven Bioinformatics Revealed Key Maternal Metabolites Related to Fetal Lethality via Di(2-ethylhexyl)phthalate Exposure in Pregnant Mice.

We performed serum metabolome analysis of di(2-ethylhexyl)phthalate (DEHP)-exposed and control pregnant mice. Pregnant mice (n = 5) were fed a DEHP-containing diet (0.1% or 0.2% DEHP) or a normal diet (control) from gestational days 0-18. After maternal exposure to 0.2% DEHP there were no surviving fetuses, indicating its strong fetal lethality. There were no significant differences in the numbers of fetuses and placentas between the 0.1% DEHP and control groups, although fetal viability differed significantly between them, suggesting that maternal exposure to 0.1% DEHP could inhibit fetal growth. Metabolomics successfully detected 169 metabolites in serum. Principal component analysis (PCA) demonstrated that the three groups were clearly separated on PCA score plots. The biological interpretation of PC1 was fetal lethality, whereas PC2 meant metabolic alteration of pregnant mice via DEHP exposure without fetal lethality. In particular, the first component was significantly correlated with fetal viability, demonstrating that maternal metabolome changes via DEHP exposure were strongly related to fetal lethality. Levels of some amino acids were significantly increased in the DEHP-exposed groups, whereas those of some fatty acids, nicotinic acid, and 1,5-anhydroglucitol were significantly decreased in the DEHP groups. DEHP-induced increases in glycine levels could cause fetal neurological disorders, and decreases in nicotinic acid could inhibit fetal growth. In addition, a machine-learning Random forest could determine 16 potential biomarkers of DEHP exposure, and data-driven network analysis revealed that nicotinic acid was the most influential hub metabolite in the metabolic network. These findings will be useful for understanding the effects of DEHP on the maternal metabolome in pregnancy and their relationship to fetal lethality.