Chromatography-electrospray Ionization-tandem Mass Spectrometry Method Research Articles

Targeted Metabolomics for the Analysis of p-Cresol in Mouse Brain: Impact of Biological Sex and Strain.

p-Cresol, an environmental contaminant and endogenous metabolite derived primarily from the conversion of l-tyrosine by intestinal microflora, is gaining increasing attention, due to its potential impact on human health. Recent studies have highlighted elevated levels of p-cresol and its metabolites, including p-cresyl sulfate and p-cresyl glucuronide, in various populations, suggesting a correlation with neurodevelopmental and neurodegenerative conditions. While the role of this compound as a uremic toxin is well established, its presence and concentration within the central nervous system (CNS) remain largely unexplored. To address this gap, an high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC-ESI-MS/MS) method was optimized and validated for the first time in this work for the targeted metabolomics of p-cresol in brain tissues. This method enabled the quantification of this compound in different brain areas of adult male and female C57BL/6J mice and in the cortex of various mouse strains, including CD-1 and the idiopathic autism model BTBR T+Itpr3tf/J. Additionally, preliminary analyses of human cortex samples confirmed the presence of p-cresol, suggesting its relevance in human brain health. Moreover, metabolomic analyses have further explored the correlations between p-cresol and neurotransmitters, with a particular focus on dopaminergic and noradrenergic pathways. These findings pave the way for understanding the potential impact of p-cresol on neurochemical networks and its implications for neurodevelopmental and neurodegenerative disorders.

Simultaneous determination of semi-essential nutrients taurine, l-carnitine and choline in infant formulas and adult/pediatric nutritional formulas by hydrophilic interaction liquid chromatography-electrospray ionization-tandem mass spectrometry.

The nutritional intake of formula-fed newborns is often limited to a single source, so it must be supplemented with essential nutrients for the growth and proper development of infants. Taurine, l-carnitine, and choline are considered conditionally essential nutrients especially in newborns and infants. In this work, a simple routine hydrophilic interaction liquid chromatography-electrospray ionization-tandem mass spectrometry (HILIC-ESI-MS/MS) method was developed and validated for the simultaneous determination of these semi-essential nutrients in infant and adult/pediatric milk formulas. The extraction recoveries were between 90% and 114%. Precision of the method offered relative standard deviation below 5% and 7% for intra-day and inter-day precision, respectively. The proposed method was successfully applied to quantification of taurine, l-carnitine, and choline in milk formula. The contents found were in good agreement with those provided on the product label for almost all samples. In view of these results, it can be concluded that the developed method can be a useful approach for the simultaneous determination of taurine, l-carnitine and choline in powdered milk samples, so it can be useful in the routine quality control of this kind of samples. © 2024 The Author(s). Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Unveiling Pharmacokinetics and Drug Interaction of Linagliptin and Pioglitazone HCl in Rat Plasma Using LC-MS/MS.

The linagliptin (LIN) and pioglitazone HCl (PIO) combination, currently undergoing phase III clinical trials for diabetes mellitus treatment, demonstrated significant improvements in glycemic control. However, the absence of an analytical method for simultaneous determination in biological fluids highlights a crucial gap. This underscores the pressing need for sensitive bioanalytical methods, emphasizing the paramount importance of developing such tools to advance diabetes management strategies and enhance patient care. Herein, a sensitive reverse-phase high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry method was developed for simultaneous determination of LIN and PIO in rat plasma using alogliptin as an internal standard. Chromatographic separation was performed on an Agilent Eclipse Plus C18 (4.6 × 100 mm, 3.5 μm) using an isocratic mobile phase system consisting of ammonium formate (pH 4.5) and methanol using an acetonitrile-induced protein precipitation technique for sample preparation. Multiple reaction monitoring in positive ion mode was used for quantitation of the precursor to production at m/z 473.2 → 419.9 for LIN, 357.1 → 134.2 for PIO, and 340.3 → 116.1 for ALO. The linearity range was 0.5 to 100 and 1 to 2000 ng/mL for LIN and PIO, respectively. The developed method was validated as per US-FDA guidelines and successfully applied to clinical pharmacokinetic and drug-drug interaction studies with a single oral administration of LIN and PIO in rat plasma. Pharmacokinetic parameters of LIN were significantly influenced by the concomitant administration of PIO and vice versa. Molecular modeling revealed the significant interaction of LIN and PIO with P-glycoprotein. Therefore, the drug-drug interaction between LIN and PIO deserves further study to improve drug therapy and prevent dangerous adverse effects.

Determination of seventeen sugars and sugar alcohols in fruit juice samples using hydrophilic interaction liquid chromatography-tandem mass spectrometry combining response surface methodology design

Carbohydrates are the main source of energy for all organisms to sustain their life activities and probably the most common organic substances in nature, whose main source is grains, starchy vegetables and fruits. To clarify the content and distribution of sugars and sugar alcohols in different fruit species samples accurately, a rapid and sensitive hydrophilic interaction liquid chromatography-electrospray ionization-tandem mass spectrometry (HILIC-ESI-MS/MS) method combining ultrasonic-assisted extraction (UAE) and solid phase extraction (SPE) purification was developed for quantitative determination seventeen sugars and sugar alcohols in fruit juice. In this method, the targets were firstly extracted from 1 g of fruit juice sample using 40 mL double-distilled water by ultrasonic-assisted extraction (UAE) for 10 min according to the optimization of response surface methodology (RSM) design. Then, 1.0 mL of UAE extracted solution was cleaned using an amino (NH2) SPE cartridge in the following SPE procedure. Seventeen compounds were separated in 25 min using the HILIC column by a gradient with 0.1% of ammonium hydroxide and acetonitrile as mobile phase. The HILIC-MS/MS method was validated, with the following studies including limit of detection, linearity range, stability and recovery. The limits of detection (LOD) were between 1.2 and 228 ng/g and linear correlation coefficients (R2) were more than 0.994. The average recoveries of 17 sugars and sugar alcohols ranging from 81.8 to 114.9%. The results showed that inter-day and intra-day precision were found to be within acceptable range. The strategy was successfully used for profiling of sugars and sugar alcohols in various fruit juice matrices.

Development of a UPLC-ESI-MS/MS method for the determination of triamcinolone acetonide in human plasma and evaluation of its bioequivalence after a single intramuscular injection in healthy volunteers.

Introduction: Triamcinolone acetonide (TA) is commonly used in the treatment of various inflammatory conditions. To ensure its efficacy and safety, it is important to accurately determine its concentration in human plasma and evaluate its bioequivalence. In this study, an efficient ultra-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UPLC-ESI-MS/MS) method was developed for the quantification of TA in human plasma after a single intramuscular injection. The internal standard used in this method was cortisone acetate (CA). Methods: TA and CA were extracted from plasma using ethyl acetate and N-hexane (4:1, v/v), separated on a C18 reverse-phase column with a mobile phase of acetonitrile-water containing 1% formic acid (55:45, v/v), and analyzed by UPLC-ESI-MS/MS. Multiple-reaction monitoring was performed using the transitions m/z 435.4→397.3 for TA and m/z 403.4→163.1 for CA. Results: The developed UPLC-ESI-MS/MS method demonstrated linearity over a concentration range of 0.53-21.20ng/mL, with a lower limit of quantification of 0.53ng/mL. The intra- and inter-run precision values ranged from 3.007% to 9.960% and 3.528% to 11.26%, respectively. The intra- and inter-run accuracy ranges were -1.962% to -6.577% and -3.371% to 0.348%, respectively. The matrix effect, extraction recovery, and stability of TA all met the acceptance criteria recommended by the National Medical Products Administration (NMPA) for bioassays. In healthy volunteers who received a single intramuscular injection of 80mg of either the test or reference formulation of TA, various pharmacokinetic parameters were determined. C max was found to be 8.616 ± 1.232 and 8.285 ± 1.218ng/mL for the test and reference formulations, respectively. T max was approximately 1.833 ± 0.243 and 1.861 ± 0.230h. The t 1/2 was calculated to be 181.249 ± 78.585 and 201.782 ± 83.551h. AUC 0-720 was 835.642 ± 297.209 and 830.684 ± 331.168ngh/mL, AUC 0-∞ was 991.859 ± 355.939 and 1018.665 ± 420.769ngh/mL for the test and reference formulations, respectively. The average relative bioavailability of TA, determined using AUC 0-720, was 105.4 ± 26.9%. Bioequivalence was evaluated through variance analysis and a double unilateral test, and the 90% confidence intervals of AUC 0-720, C max, and AUC 0-∞ were 92.8%-113.4%, 99.1%-109.1%, and 89.7%-110.9%, respectively (all p > 0.05). Discussion: These results met the bioequivalence criteria set by the NMPA, indicating that the developed UPLC-ESI-MS/MS method accurately determined TA concentrations in the plasma of healthy Chinese volunteers and that the test and reference formulations exhibited bioequivalence in these individuals.

Quantification of cefaclor in human plasma using SIL-IS LC-ESI-MS/MS for pharmacokinetics study in healthy Chinese volunteers.

A steady, high-efficiency, and precise liquid chromatography-electrospray ionization-tandem mass spectrometry method was established and validated using cefaclor-d5 as the stable isotope-labeled internal standard for quantification of cefaclor in human plasma. One-step protein precipitation was applied to extract human plasma samples using methanol as precipitant. An Ultimate XB C18 column (2.1 × 50.0mm, 5.0μm) was used to achieve chromatographic separation. Mobile phases of gradient elution were an aqueous solution containing 0.1% formic acid (mobile phase A) and an acetonitrile solution containing 0.1% formic acid (mobile phase B). Electrospray ionization in positive-ion mode was applied to detect under multiple reaction monitoring mode. Target fragment ion pairs of cefaclor and stable isotope-labeled internal standard, respectively, were m/z 368.2 → 191.1 and m/z 373.2 → 196.1. Linear range of this method was between 20.0 and 10,000.0ng/ml, with coefficient of determination (R2 ) >0.9900. Seven concentrations of quality control samples were used: 20.0ng/ml (lower limit of quantitation), 60.0ng/ml (low quality control), 650 ng/ml (middle quality control), 5000 ng/ml (arithmetic average middle quality control [AMQC]), 7500 ng/ml (high quality control), 10,000 ng/ml (upper limit of quantification), and 40,000 ng/ml (dilution quality control [DQC]). The method was validated for selectivity, lower limit of quantitation, linearity, accuracy, precision, recovery, matrix effect, dilution reliability, stability, carryover, and incurred sample reanalysis. This stable isotope-labeled internal standard liquid chromatography-electrospray ionization-tandem mass spectrometry approach has been successfully applied to study the pharmacokinetics of cefaclor dry suspension among healthy Chinese volunteers.

Simultaneous determination of six triterpenoid saponins in beagle dog plasma by UPLC-MS/MS and its application to a pharmacokinetic study after oral administration of the extract of the Eleutherococcus senticosus (Rupr. & Maxim.) Maxim. leaves

AbstractA rapid and simple ultra-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UPLC-ESI-MS/MS) method was developed and validated for simultaneous determination of six analytes from the Eleutherococcus senticosus (Rupr. & Maxim.) Maxim. leaves (ESL) in beagle dog plasma for the first time, including 3-O-α-l-rhamnopyranosyl-(1→2)-α-l-arabinopyranoside-29-hydroxy oleanolic acid, 3-O-β-d-glucopyranosyl-(1→2)-α-l-arabinopyranoside-29-hydroxy oleanolic acid, 3-O-β-d-glucopyranosyl-(1→2)-α-l-arabinopyranosyl-30-norlean-12,20 (29) –dien-28-olic acid, ciwujianoside E, guaianin N, and eleutheroside K. The chromatographic separation was performed using an ACQUITY UPLC BEH C18 column (2.1 × 100 mm, 1.7 μm) using a gradient elution way with a mobile phase of acetonitrile-water containing 0.1% formic acid. Analytes were detected on a triple-quadrupole mass spectrometer equipped with an electrospray ionization (ESI) source with multiple reaction monitoring (MRM) mode. Calibration curves were all linear (r ≥ 0.9933) over the concentration range. The mean extraction recoveries and matrix effect of analytes and I.S. were ranged from 80.26% to 98.32% and from 91.27% to 111.67%, respectively. The intra-day and inter-day precision were ranged from 2.20% to 14.81%, and the accuracy range was 1.60–14.60%. The analytical method was successfully applied for the pharmacokinetic characteristics of the six analytes in beagle plasma after oral administration of ESL extracts. The T1/2 of six analytes was more than 3.09 ± 0.78 h.

A new toxicity mechanism of N-(1,3-Dimethylbutyl)-N′-phenyl-p-phenylenediamine quinone: Formation of DNA adducts in mammalian cells and aqueous organisms

N-(1,3-dimethylbutyl)-N′-phenyl-p-phenylenediamine-quinone (6PPDQ), one of the oxidation products of rubber antioxidant 6PPD, has been identified as a novel toxicant to many organisms. However, an understanding of its underlying toxicity mechanisms remained elusive. In this study, we reported that 6PPDQ could react with deoxyguanosine to form one isomer of 3-hydroxy-1, N2-6PPD-etheno-2′-deoxyguanosine (6PPDQ-dG). Next, by employing an ultra-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UPLC-ESI-MS/MS) method, we found that 6PPDQ-dG could be detected in genomic DNA from 6PPDQ-treated mammalian cells and Chlamydomonas reinhardtii. We observed positive correlations between concentrations of exogenous 6PPDQ and the amounts of 6PPDQ-dG, and a recovery period after removal of 6PPDQ also led to decreased levels of the adduct in both organisms, which suggested potential repair pathways for this adduct in mammalian cells and unicellular algae. Additionally, we extracted the genomic DNA from tissues of frozen capelin and observed substantial amounts of the adduct in roe and gills, as well as livers at a relatively lower level. These results provided insights into the target organs and tissues that 6PPDQ might accumulate or harm fish. Overall, our study provides a new understanding of the mechanisms of toxicity of 6PPDQ in mammalian cells and aqueous organisms.

Analysis of bioactive compounds using LC-ESI-MS/MS, cytotoxic, antimicrobial effects, and enzyme activities from Cyclotrichium origanifolium.

Cyclotrichium origanifolium is a medicinal plant belonging to the Lamiaceae family. In this study, phenolic content analysis, antimicrobial effects, and cytotoxic effects of extracts of C. origanifolium were investigated. In the extracts, phenolic compound analysis by the liquid chromatography-electrospray ionization-tandem mass spectrometry method, antimicrobial effect by the minimum inhibition concentration method, and cytotoxic effect on human dermal fibroblasts (HDF), glioblastoma cell (U87), ovarian adenocarcinoma cell (Skov-3), and human colorectal adenocarcinoma cell (CaCo-2) cancer cell lines were investigated. Cytotoxicity analyses were performed by the MTT method. In addition, the GST and AChE enzyme activities of the extracts were also measured. Around 18 compounds were detected in both the methanol and ethanol extract. It was found that the best antimicrobial effect on Gram-negative Pseudomonas aeruginosa was on methanol extract, while the ethanol extract was on Candida albicans fungus (respectively, 2.50 mg/ml, 5.0μg/ml). A 500 μg/ml of methanol extract has been shown to have cytotoxic activity high effect on HDF cells. GST and AChE activity were found to decrease in a concentration-dependent manner.

A UPLC- MS/MS Method to Quantify β-Sitosterol and Ferulic Acid of Pygeum Africanum Extract in Bulk and Pharmaceutical Preparation.

This study uses a liquid chromatography-electrospray ionization-tandem mass spectrometry method to determine β-Sitosterol and Ferulic acid in Pygeum africanum extract. Chromatographic separation of the two analytes was performed on an ACQUITY UPLC H-Class system coupled with Xevo TQD mass spectrometer and HSS T3 C18 column (2.1 X 50mm, 1.8μm). Mobile phase A consisted of an aqueous solution of 0.1% formic acid (v/v), and mobile phase B was 0.1% formic acid (v/v) in methanol pumped through a gradient elution mode. Mass spectrometer parameters were optimized using an electrospray ionization source in the positive and negative ionization modes. The quantification of the two analytes was performed using multiple reaction monitoring transitions. The method was fully validated per (FDA) guidelines regarding linearity, accuracy, precision, carryover and selectivity. The proposed method was applied successfully to determine the two investigated compounds in commercially available pharmaceutical products.

Simultaneous determination of three naphthoquinones from Arnebia euchroma (Royle) Johnst in beagle plasma by UPLC-MS/MS and application for pharmacokinetics study

Abstract A rapid, simple and efficient ultra-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UPLC-ESI-MS/MS) method was established to simultaneous determination of shikonin, isobutyryl shikonin, β, βʹ-dimethylacryl alkanin in beagle plasma and evaluated by using esculetin as internal standard. The electrospray ionization (ESI) source was operated in negative ionization mode. Multi-reaction monitoring (MRM) was used to quantitatively analyzed shikonin m/z 287.0 → 217.9, isobutyryl shikonin m/z 357.0 → 268.9, β, βʹ-dimethylacryl alkanin m/z 370.0 → 270.1 and esculetin m/z 177.0 → 89.0, respectively. The method was validated for selectivity, linearity, lower limit of quantification, precision, accuracy, recovery, matrix effect and stability. All validation parameters met the acceptance criteria according to regulatory guidelines. This method was successfully applied for the pharmacokinetic study of shikonin, isobutyryl shikonin, β, βʹ-dimethylacryl alkanin in beagle dogs plasma after oral administration of A. euchroma extract.

Determination and enantioselective separation of zilpaterol in human urine after mimicking consumption of contaminated meat using high-performance liquid chromatography with tandem mass spectrometry techniques.

The synthetic β-adrenoreceptor agonist zilpaterol is legitimately used as an animal feed supplement in selected countries due to its known effects on lipolysis and protein biosynthesis. These pharmacological characteristics of zilpaterol have contributed to its classification as doping agent in sport by the World Anti-Doping Agency. However, the use as a feed supplement can lead to residues of the drug in edible tissues and, possibly, also in the urine of consumers. To provide urinary elimination profiles of microdosed zilpaterol and to determine whether the ingestion of zilpaterol below or at the acceptable daily intake level of 0.04 μg/kg bodyweight can result in an adverse analytical finding (AAF) in doping controls, healthy volunteers were administered single or multiple oral doses of 0.5μg or 3μg zilpaterol to mimic ingestion of contaminated cattle meat. Urine samples were collected and analyzed using a validated high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC-ESI-MS/MS) method and a newly developed chiral high-performance liquid chromatography-atmospheric pressure chemical ionization-tandem mass spectrometry (HPLC-APCI-MS/MS) method. Urinary peak concentrations of zilpaterol were observed for all volunteers 1.5-12.5h after ingestion, and maximum levels >5ng/mL, which would constitute an AAF in doping controls, were found after the intake of 3μg of zilpaterol on five consecutive days in one out of five study participants. Noteworthy, the enantiomeric ratio of excreted zilpaterol remained constant over time. This study provides first insights into the urinary excretion of microdosed zilpaterol. Furthermore, a method was successfully developed and applied for the separation of the zilpaterol enantiomers with mass spectrometric detection.

Thermal Stability of Fructooligosaccharides Extracted from Defatted Rice Bran: A Kinetic Study Using Liquid Chromatography-Tandem Mass Spectrometry.

Thermal degradation kinetics of fructooligosaccharides (FOS) in defatted rice bran were studied at temperatures of 90, 100, and 110 °C. FOS extracted from rice bran and dissolved in buffers at pH values of 5.0, 6.0, and 7.0 were prepared for the thermal treatments. The residual FOS (including 1-kestose (GF2), nystose (GF3), and 1F-fructofuranosylnystose (GF4)) contents were determined using the ultra-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UPLC-ESI-MS/MS) method. The results showed that the thermal degradation kinetics of GF2, GF3, and GF4 followed a first-order kinetic model. Thermal degradation rate constants (k values) of GF2, GF3, and GF4 at different temperature and pH values were estimated using the first-order kinetic equation and SAS 9.1. As a result, these k values decreased gradually as the pH of the sample increased from 5.0 to 7.0. The Arrhenius model was applied to describe the heat dependence of the k-values. The activation energy (Ea) was calculated for each case of GF2, GF3, and GF4 degradation at pH values of 5.0, 6.0, and 7.0. The result showed that rice bran FOS is very thermostable at neutral pH while more labile at acidic pH.

Determination of estrogens in human serum using a novel chemical derivatization-assisted liquid chromatography-electrospray ionization-tandem mass spectrometry method.

Assessing estrogen concentrations in biological systems can provide valuable information on physiological processes, which is crucial for the early diagnosis of many diseases. Because estrogens are present in the human body in low concentrations and in a wide dynamic range, analytical methods with high sensitivity and specificity are required for their determination in complex biological matrices. To discover an appropriate derivatization reagent for estrogen mass spectrometry (MS) analysis, we compared five sulfonyl chloride derivatization reagents, namely 3-methyl-8-quinolinesulfonyl chloride (MQSCl) and 8-quinolinesulfonyl chloride (QSCl), 1-methyl-1H-pyrazole-4-sulfonyl chloride, 1,2-methyl-imidazole-5-sulfonyl chloride, and dansyl chloride. By selecting the derivatization reagent with the best performance, we developed and validated a novel chemical derivatization-assisted-liquid chromatography-electrospray ionization-tandem mass spectrometry (CD-LC-ESI-MS/MS) method to simultaneously determine the concentrations of estrone, estradiol, and estriol (E1, E2, and E3) in human serum. It was found that among the five investigated reagents, MQSCl-derivatized estrogens presented the highest sensitivity using LC-ESI-MS/MS. Based on this discovery, MQSCl was chosen to derivatize the analyzed estrogens to assist LC-ESI-MS/MS analysis. The limit of quantification of E1, E2, and E3 was measured as 2.7, 4.6, and 5.1pg/mL, respectively. Inter- and intra-day precision, expressed as the coefficient of variation, was shown to be lower than 13.2% for all concentrations. The mean recovery was 72.4% overall, with good reproducibility at low, medium, and high concentrations in the calibration range. The developed method was successfully applied to the quantitative determination of estrogens in clinical human serum from pediatric and adult women, demonstrating the suitability of estrogen analysis in the biological matrix at low concentration (pg/mL).

Mass spectrometry profiling analysis enables the identification of new modifications in ribosomal RNA

Ribosomal RNAs (rRNAs) provide the structural framework of ribosomes and play critical roles in protein translation. In ribosome biogenesis, rRNAs acquire various modifications that can influence the structure and catalytic activity of ribosomes. However, rRNA modifications in plants have yet to be fully defined. Herein, we proposed a method to purify rRNAs by a successive isolation with different strategies, including polyA-based mRNA depletion and agarose gel electrophoresis-based purification, with which highly pure rRNAs could be obtained. In addition, we developed a liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) method to systematically profile and characterize modifications from the isolated highly pure plant 18S rRNA and 25S rRNA. LC-ESI-MS/MS analysis showed that 10 and 12 kinds of modifications were present in plant 18S rRNA and 25S rRNA, respectively. Notably, among these identified modifications, 2 kinds of modifications of N2,N2-dimethylguanosine (m2,2G) and N6,N6-dimethyladenosine (m6,6A) in 18S rRNA, and 4 kinds of modifications of m2,2G, m6,6A, N7-methylguanosine (m7G) and 3-methyluridin (m3U) in 25S rRNA, were first reported to be present in plants. Moreover, exposure of Arabidopsis thaliana to cadmium (Cd) led to significant changes of modifications in both 18S rRNA and 25S rRNA of plants, indicating that rRNA modifications play important roles in response to environmental stress. The discovery of new modifications in plant rRNAs improves the spectra of plant rRNA modifications and may promote the investigation of the functional roles of plant ribosomes in regulating gene expression.

Metabolomic Workflow for the Accurate and High-Throughput Exploration of the Pathways of Tryptophan, Tyrosine, Phenylalanine, and Branched-Chain Amino Acids in Human Biofluids.

The modulation of host and dietary metabolites by gut microbiota (GM) is important for maintaining correct host physiology and in the onset of various pathologies. An ultrahigh-performance liquid chromatography-electrospray ionization-tandem mass spectrometry method was developed for the targeted quantitation in human plasma, serum, and urine of 89 metabolites resulting from human-GM cometabolism of dietary essential amino acids tryptophan, tyrosine, and phenylalanine as well as branched-chain amino acids. Ninety-six-well plate hybrid-SPE enables fast clean-up of plasma and serum. Urine was diluted and filtered. A 15 min cycle enabled the acquisition of 96 samples per day, with most of the metabolites stable in aqueous solution for up to 72 h. Calibration curves were specifically optimized to cover expected concentrations in biological fluids, and limits of detection were at the order of ppb. Matrix effects were in acceptable ranges, and analytical recoveries were in general greater than 80%. Inter and intraday precision and accuracy were satisfactory. We demonstrated its application in plasma and urine samples obtained from the same individual in the frame of an interventional study, allowing the quantitation of 51 metabolites. The method could be considered the reference for deciphering changes in human-gut microbial cometabolism in health and disease. Data are available via Metabolights with the identifier MTBLS4399.

Simultaneous Determination of Fifteen Polyphenols in Fruit Juice Using Ultrahigh-Performance Liquid Chromatography-Tandem Mass Spectrometry Combining Dispersive Liquid-Liquid Microextraction.

Polyphenols are secondary metabolites of plants and used as effective antioxidants in dietary supplements, whose main sources are fruits, vegetables, and grains. To clarify the content and distribution of polyphenols in different fruit species samples accurately, a rapid and sensitive ultrahigh-pressure liquid chromatography-electrospray ionization-tandem mass spectrometry (UPLC-ESI-MS/MS) method combining dispersive liquid-liquid microextraction (DLLME) was developed for quantitative determination of fifteen polyphenol compounds in fruit juice. In this method, the targets were first extracted from 1 g of fruit juice sample using 10 mL of 80% ethanol solution by ultrasonic-assisted extraction (UAE). Then, 1.0 mL of UAE extracted solution, 60 μL of n-octanol and 2.0 mL of H2O were performed in the following DLLME procedure. A C18 reversed-phase column, ZORBAX SB (100 × 4.6 mm, 3.5 μm), was proposed under gradient elution with 0.1% formic acid aqueous solution and methanol mobile phases for the determination of 15 polyphenols, allowing us to obtain polyphenolic profiles in less than 23.0 min. Under the optimum conditions, the enrichment factors ranged from 162 to 194. The results showed that the 15 polyphenols had linear correlation coefficients (R2) more than 0.99. The limits of detection (LODs) were between 18.3 and 103.5 ng/g, and the average recoveries were between 96.9 and 116.3% with interday relative standard deviations (RSDs) ranging from 4.4 to 8.2% in all cases. The method was successfully applied to the analysis of real fruit juice samples and presented itself as a simple, rapid, practical, and environment-friendly technique.

Mass spectrometric determination of N7-HPTE-dG and N7-HPTE-Gua in mammalian cells and mice exposed to methoxychlor, an emergent persistent organic pollutant

Methoxychlor (MXC) is an organopesticide classified as a “Proposed Persistent Organic Pollutant” in the Stockholm Convention, and recent studies revealed that MXC could induce DNA strand breaks, whereas its underlying mechanisms were underinvestigated. Here, we first reported that hydroxymethoxychlor (HPTE), one of MXC’s active metabolites, could be oxidized in vivo to form quinone intermediate, which attacked N7 position of 2′-deoxyguanosine to form N7-HPTE-deoxyguanosine (N7-HPTE-dG), followed by depurination to produce N7-HPTE-guanine (N7-HPTE-Gua) in MXC-treated mammalian cells and tissues from mice fed with MXC, employing an ultra-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UPLC-ESI-MS/MS) method. We observed a positive correlation between the doses of MXC exposure and the levels of N7-HPTE-Gua and N7-HPTE-dG in cytoplasm and genomic DNA, respectively. Furthermore, after removal of exogenous MXC, the amount of genomic N7-HPTE-dG was significantly decreased during 24 h, while the level of cytoplasmic N7-HPTE-Gua was elevated during first 12 h, indicating the accumulation of the N7-HPTE-Gua in cells. Additionally, for animal experiment, genomic N7-HPTE-dG was observed in livers and cortexes from female C57BL/6 mice fed with MXC, suggesting a potential mechanism of its hepatoxicity and neurotoxicity. Overall, our study provides new understanding about the formation of MXC-induced DNA adducts in mammalian cells and animal models.

Occurrence and Fate of Bisphenol A and its Congeners in Two Wastewater Treatment Plants and Receiving Surface Waters in Romania.

The present study investigated the distribution and environmental fate of Bisphenol A (BPA), the 4-hydroxyacetophenone (4-HAP) metabolite, and 5 other bisphenol congeners in 2 municipal wastewater treatment plants (WWTPs) and their receiving rivers in Romania. Accordingly, a new, highly sensitive and accurate solid-phase extraction-liquid chromatography-electrospray ionization-tandem mass spectrometry method was developed and validated. This technique generated low limit of quantitation values: below 2.3 ng/L for surface water and less than 2.4 and 2.7 ng/L for WWTP effluent and influent water. The sum of detected analytes in wastewater was between 1337 and 16 118 ng/L for influent samples and between 15 and 96 ng/L for effluent samples. In surface water, the total of all compounds was somewhere between 34 and 240 ng/L. The highest concentration observed was for BPA in all 3 types of analyzed water (up to 9140 ng/L for influent, as high as 75 ng/L for effluent, and a maximum of 135 ng/L in surface waters). All analyzed samples were free of bisphenols B, C, and F. For all analytes detected in surface water, the concentration values were higher than those determined in the effluent samples, which may be caused by intrinsic contamination of the 2 rivers (Danube and Jiu Rivers). Values of environmental risk coefficients, calculated for both effluents and surface waters, indicated a low ecological risk or no ecological risk for 3 types of organisms (algae, daphnia, and fish). Human risk assessment calculation suggests no risk to human health as a result of the presence of BPA in either of the 2 rivers. Environ Toxicol Chem 2021;40:435-446. © 2020 SETAC.

Pharmacokinetics and Tissue Distribution Study of Modified Xiaochaihu Granules Against Gastric Ulcer Induced by Ethanol in Rats by UPLC-MS/MS

Gastric ulcer (GU) is one of the major gastrointestinal disorder diseases, with increasing incidence and prevalence globally. Modified Xiaochaihu granules (MXCHG) have been used effectively for treating chronic gastritis and GU clinically. To investigate the pharmacokinetics and tissue distribution of MXCHG, an ultraperformance liquid chromatography-electrospray ionization-tandem mass spectrometry (UPLC-ESI-MS/MS) method was established for the simultaneous determination of 8 bioactive ingredients (baicalin, wogonoside, baicalein, liquiritin, glycyrrhizic acid, berberine hydrochloride, saikosaponin a, and saikosaponin d) in rat plasma and various tissues using puerarin as an internal standard (IS). The biological samples were pretreated by protein precipitation with acetonitrile. The chromatographic separation was carried out on a C18 column with a gradient mobile phase consisting of acetonitrile and 0.1% formic acid in water. All analytes and IS were quantitated through ESI in the positive/negative ion multiple reaction monitoring mode. The mass transitions were as follows: m/z 445.0 → 268.5 for baicalin, m/z 458.7 → 282.8 for wogonoside, m/z 269.2 → 222.6 for baicalein, m/z 417.0 → 254.8 for liquiritin, m/z 822.1 → 350.8 for glycyrrhizic acid, m/ z 336.0 → 319.9 for berberine hydrochloride, m/z 780.3 → 618.5 for saikosaponin, and m/z 415.0 → 294.6 for the IS. The validated method was successfully applied to the pharmacokinetics and tissue distribution study of 8 compounds in rat plasma and tissues after the intragastric administration of MXCHG. The results demonstrated that 8 components were distributed widely and rapidly in various rat tissues after intravenous administration. Tissue deposition of the compounds in the rats was mainly in the small intestine and stomach. The present study can provide more useful information to guide the clinical use of MXCHG and the developed analytical method can also be applied for further clinical pharmacokinetic studies.