LC-MS/DIA-based strategy for comprehensive flavonoid profiling: an Ocotea spp. applicability case.

We introduce a liquid chromatography - mass spectrometry with data-independent acquisition (LC-MS/DIA)-based strategy, specifically tailored to achieve comprehensive and reliable glycosylated flavonoid profiling. This approach facilitates in-depth and simultaneous exploration of all detected precursors and fragments during data processing, employing the widely-used open-source MZmine 3 software. It was applied to a dataset of six Ocotea plant species. This framework suggested 49 flavonoids potentially newly described for these plant species, alongside 45 known features within the genus. Flavonols kaempferol and quercetin, both exhibiting O-glycosylation patterns, were particularly prevalent. Gas-phase fragmentation reactions further supported these findings. For the first time, the apigenin flavone backbone was also annotated in most of the examined Ocotea species. Apigenin derivatives were found mainly in the C-glycoside form, with O. porosa displaying the highest flavone : flavonol ratio. The approach also allowed an unprecedented detection of kaempferol and quercetin in O. porosa species, and it has underscored the untapped potential of LC-MS/DIA data for broad and reliable flavonoid profiling. Our study annotated more than 50 flavonoid backbones in each species, surpassing the current literature.

Integrative open workflow for confident annotation and molecular networking of metabolomics MSE/DIA data.

Liquid chromatography coupled with high-resolution mass spectrometry data-independent acquisition (LC-HRMS/DIA), including MSE, enable comprehensive metabolomics analyses though they pose challenges for data processing with automatic annotation and molecular networking (MN) implementation. This motivated the present proposal, in which we introduce DIA-IntOpenStream, a new integrated workflow combining open-source software to streamline MSE data handling. It provides 'in-house' custom database construction, allows the conversion of raw MSE data to a universal format (.mzML) and leverages open software (MZmine 3 and MS-DIAL) all advantages for confident annotation and effective MN data interpretation. This pipeline significantly enhances the accessibility, reliability and reproducibility of complex MSE/DIA studies, overcoming previous limitations of proprietary software and non-universal MS data formats that restricted integrative analysis. We demonstrate the utility of DIA-IntOpenStream with two independent datasets: dataset 1 consists of new data from 60 plant extracts from the Ocotea genus; dataset 2 is a publicly available actinobacterial extract spiked with authentic standard for detailed comparative analysis with existing methods. This user-friendly pipeline enables broader adoption of cutting-edge MS tools and provides value to the scientific community. Overall, it holds promise for speeding up metabolite discoveries toward a more collaborative and open environment for research.

Bioprospecting-based untargeted metabolomics identifies alkaloids as potential anti-inflammatory bioactive markers of Ocotea species (Lauraceae).

BACKGROUND: Species within the Ocotea genus (Lauraceae), have demonstrated an interesting profile of bioactivities. Renowned for their diverse morphology and intricate specialized metabolite composition, Ocotea species have re-emerged as compelling candidates for bioprospecting in drug discovery research. However, it is a genus insufficiently studied, particularly regarding anti-inflammatory activity. PURPOSE: To investigate the anti-inflammatory activity of Ocotea spp. extracts and determine the major markers in this genus. METHODS: Extracts of 60 different Ocotea spp. were analysed by an ex vivo anti-inflammatory assay in human whole blood. The experiment estimates the prostaglandin E2 levels, which is one of the main mediators of the inflammatory cascade, responsible for the classical symptoms of fever, pain, and other common effects of the inflammatory process. Untargeted metabolomics analysis through liquid chromatography coupled with high-resolution mass spectrometry was performed, along with statistical analysis, to investigate which Ocotea metabolites are correlated with their anti-inflammatory activity. RESULTS: The anti-inflammatory screening indicated that 49 out of 60 Ocotea spp. extracts exhibited significant inhibition of PGE2 release compared to the vehicle (p < 0.05). Furthermore, 10 of these extracts showed statistical similarity to the reference drugs. The bioactive markers were accurately identified using multivariate statistics combined with a fold change (> 1.5) and adjusted false discovery rate analysis as unknown compounds and alkaloids, with a majority of aporphine and benzylisoquinolines. These alkaloids were annotated with an increased level of confidence since MSE spectra were compared with comprehensive databases. CONCLUSION: This study represents the first bioprospecting report revealing the anti-inflammatory potential of several Ocotea spp. The determination of their anti-inflammatory markers could contribute to drug discovery and the chemical knowledge of the Ocotea genus.

A burst of fenoterol excretion during the recovery of a weight loss protocol.

Fenoterol is a sympathomimetic ß2 receptor agonist primarily used as a bronchodilator. Due to its sympathomimetic actions, the World Anti-Doping Agency (WADA) has banned it. Multiple acute weight loss protocols (WLP) are used by Olympic athletes for sports that segregate athletes by weight; these generally involve caloric and water deprivation combined with heat exposure. Athletes use WLP before weigh-in, then transition to different body acute weight regain protocols (WRP) before competitions. Here, we studied the pharmacokinetics of fenoterol under WLP conditions: energetic dietary restriction, decreased water intake, and exposure to a dry sauna (80 ± 2 °C), followed by a WRP. Five elite-level female judo athletes participated in the study. Four received fenoterol (200 µg; n = 2 or 400 µg; n = 2), while one was a control receiving placebo under identical conditions. We measured excretion of the fenoterol parent molecule and presented qualitative data of its sulfated metabolite using QqQ tandem quadrupole mass spectrometry for 118 h. The fenoterol parent appeared earlier in urine than did its conjugated metabolite; excretion profiles were similar among all subjects. The centers of mass for fenoterol parent curves were (time, fenoterol): athlete A (10.9, 7.3); athlete B (9.2, 27.3); athlete C (8.5, 6.9); athlete D (9.7, 5.0). After initiating WRP, we observed a burst in urinary fenoterol excretion once in complete decay. This trend was observed for all four athletes who received fenoterol. Our results suggest that during hypohydration, some of the unmetabolized fenoterol accumulates in tissues, then is released during rehydration. These findings can be important for detecting fenoterol use in athletes.

Anti-Inflammatory Markers of Hops Cultivars (Humulus lupulus L.) Evaluated by Untargeted Metabolomics Strategy.

Hops (Humulus lupulus L.) are edible flowers commonly used to add flavour and aroma to beer, besides they have rich chemical diversity and medicinal potential. In this work, an ex vivo anti-inflammatory assay via the LPS-induced signalling pathway and metabolomics approaches were performed to evaluate the ability of hops to inhibit the production of prostaglandin E2 (PGE2) inflammatory mediator and analyze which metabolites produced by the nine different hop cultivars are potential anti-inflammatory markers. Columbus, Chinook and Hallertau Mittelfrüh hop cultivars yielded extracts with PGE2 release inhibition rates of 86.7, 92.5 and 73.5 %, respectively. According to the multivariate statistical analysis, the majority of the metabolites correlated with the activity were prenylated phloroglucinol and phenolic homologs. These results suggest promissory anti-inflammatory hop metabolites.

In vivo anti-inflammatory activity of Fabaceae species extracts screened by a new ex vivo assay using human whole blood.

INTRODUCTION: Plants have been considered a promising source for discovering new compounds with pharmacological activities. The Fabaceae family comprises a large variety of species that produce substances with diverse therapeutic potential, including anti-inflammatory activity. The limitations of current anti-inflammatories generate the need to research new anti-inflammatory structures with higher efficacy as well as develop methods for screening multiple samples, reliably and ethically, to assess such therapeutic properties. OBJECTIVE: Validate and apply a quantification method for prostaglandin E2 (PGE2 ) production from an ex vivo assay in human blood in order to screen anti-inflammatory activity present in many Fabaceae species extracts. METHODS: Human blood was incubated with extracts from 47 Fabaceae species. After lipopolysaccharide (LPS)-induced inflammation, PGE2 was quantified in the plasma by liquid chromatography with tandem mass spectrometry (LC-MS/MS). The extracts that presented PGE2 production inhibition were further assessed through in vivo assay and then chemically characterised through an analysis of ultra-performance liquid chromatography electrospray ionisation quadrupole time-of-flight tandem mass spectrometry (UPLC-ESI-QTOF-MS2 ) data. RESULTS: The new ex vivo anti-inflammatory assay showed that five out of the 47 Fabaceae species inhibited PGE2 production. Results from an in vivo assay and the metabolic profile of the active extracts supported the anti-inflammatory potential of four species. CONCLUSION: The quantification method for PGE2 demonstrated fast, sensitive, precise, and accurate results. The new ex vivo anti-inflammatory assay comprised a great, reliable, and ethical approach for the screening of a large number of samples before an in vivo bioassay. Additionally, the four active extracts in both ex vivo and in vivo assays may be useful for the development of more efficient anti-inflammatory drugs.

Plasma Lipid Profile Reveals Plasmalogens as Potential Biomarkers for Colon Cancer Screening.

In this era of precision medicine, there is an increasingly urgent need for highly sensitive tests for detecting tumors such as colon cancer (CC), a silent disease where the first symptoms may take 10-15 years to appear. Mass spectrometry-based lipidomics is an emerging tool for such clinical diagnosis. We used ultra-performance liquid chromatography coupled to electrospray ionization quadrupole time-of-flight mass spectrometry operating in high energy collision spectral acquisition mode (MSE) mode (UPLC-QTOF-MSE) and gas chromatography (GC) to investigate differences between the plasmatic lipidic composition of CC patients and control (CTR) subjects. Key enzymes in lipidic metabolism were investigated using immuno-based detection assays. Our partial least squares discriminant analysis (PLS-DA) resulted in a suitable discrimination between CTR and CC plasma samples. Forty-two statistically significant discriminating lipids were putatively identified. Ether lipids showed a prominent presence and accordingly, a decrease in glyceronephosphate O-acyltransferase (GNPAT) enzyme activity was found. A receiver operating characteristic (ROC) curve built for three plasmalogens of phosphatidylserine (PS), named PS(P-36:1), PS(P-38:3) and PS(P-40:5), presented an area under the curve (AUC) of 0.998, and sensitivity and specificity of 100 and 85.7% respectively. These results show significant differences in CC patients' plasma lipid composition that may be useful in discriminating them from CTR individuals with a special role for plasmalogens.

Multiplatform Investigation of Plasma and Tissue Lipid Signatures of Breast Cancer Using Mass Spectrometry Tools.

Plasma and tissue from breast cancer patients are valuable for diagnostic/prognostic purposes and are accessible by multiple mass spectrometry (MS) tools. Liquid chromatography-mass spectrometry (LC-MS) and ambient mass spectrometry imaging (MSI) were shown to be robust and reproducible technologies for breast cancer diagnosis. Here, we investigated whether there is a correspondence between lipid cancer features observed by desorption electrospray ionization (DESI)-MSI in tissue and those detected by LC-MS in plasma samples. The study included 28 tissues and 20 plasma samples from 24 women with ductal breast carcinomas of both special and no special type (NST) along with 22 plasma samples from healthy women. The comparison of plasma and tissue lipid signatures revealed that each one of the studied matrices (i.e., blood or tumor) has its own specific molecular signature and the full interposition of their discriminant ions is not possible. This comparison also revealed that the molecular indicators of tissue injury, characteristic of the breast cancer tissue profile obtained by DESI-MSI, do not persist as cancer discriminators in peripheral blood even though some of them could be found in plasma samples.

Changes in the blood plasma lipidome associated with effective or poor response to atypical antipsychotic treatments in schizophrenia patients.

Atypical antipsychotics are widely used to manage schizophrenia symptoms. However, these drugs can induce deleterious side effects, such as MetS, which are associated with an increased cardiovascular risk to patients. Lipids play a central role in this context, and changes in lipid metabolism have been implicated in schizophrenia's pathobiology. Furthermore, recent evidence suggests that lipidome changes may be related to antipsychotic treatment response. The aim of this study was to evaluate the lipidome changes in blood plasma samples of schizophrenia patients before and after 6 weeks of treatment with either risperidone, olanzapine, or quetiapine. Liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis showed changes in the levels of ceramides (Cer), glycerophosphatidic acids (PA), glycerophosphocholines (PC), phosphatidylethanolamines (PE), phosphatidylinositols (PI), glycerophosphoglycerols (PG), and phosphatidylserines (PS) for all treatments. However, the treatment with risperidone also affected diacylglycerides (DG), ceramide 1-phosphates (CerP), triglycerides (TG), sphingomyelins (SM), and ceramide phosphoinositols (PI-Cer). Moreover, specific lipid profiles were observed that could be used to distinguish poor and good responders to the different antipsychotics. As such, further work in this area may lead to lipid-based biomarkers that could be used to improve the clinical management of schizophrenia patients.

Neuroprotective potential of Ayahuasca and untargeted metabolomics analyses: applicability to Parkinson's disease.

ETHNOPHARMACOLOGY RELEVANCE: Ayahuasca is a tea produced through decoction of Amazonian plants. It has been used for centuries by indigenous people of South America. The beverage is considered to be an ethnomedicine, and it is traditionally used for the treatment of a wide range of diseases, including neurological illness. Besides, some scientific evidence suggests it may be applicable to Parkinson's disease (PD) treatment. Thus, Ayahuasca deserves in depth studies to clarify its potential role in this disease. AIM OF THE STUDY: This study aimed to use an untargeted metabolomics approach to evaluate the neuroprotective potential of the Ayahuasca beverage, the extracts from its matrix plants (Banisteriopsis caapi and Psychotria viridis), its fractions and its main alkaloids on the viability of SH-SY5Y neuroblastoma cells in an in vitro PD model. MATERIAL AND METHODS: The cytotoxicity of Ayahuasca, crude extracts, and fractions of B. caapi and P. viridis, as well as neuroprotection promoted by these samples in a 6-hydroxydopamine (6-OHDA)-induced neurodegeneration model, were evaluated by the MTT assay at two time-points: 48 h (T1) and 72 h (T2). The main alkaloids from Ayahuasca matrix plants, harmine (HRE) and N,N-dimethyltryptamine (DMT), were also isolated and evaluated. An untargeted metabolomics approach was developed to explore the chemical composition of samples with neuroprotective activity. Ultra-Performance Liquid Chromatography coupled to Electrospray Ionisation and Time-of-Flight (UPLC-ESI-TOF) metabolome data was treated and further analysed using multivariate statistical analyses (MSA): principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA). The metabolites were dereplicated using the Dictionary of Natural Products and an in house database. The main alkaloids were also quantified by UPLC-MS/MS. RESULTS: The samples did not cause cytotoxicity in vitro and three of samples intensely increased cell viability at T1. The crude extracts, alkaloid fractions and HRE demonstrated remarkable neuroprotective effect at T2 while the hydroalcoholic fractions demonstrated this neuroprotective effect at T1 and T2. Several compounds from different classes, such as ß-carbolines and monoterpene indole alkaloids (MIAs) were revealed correlated with this property by MSA. Additionally, a total of 2419 compounds were detected in both ionisation modes. HRE showed potent neuroprotective action at 72 h, but it was not among the metabolites positively correlated with the most efficacious neuroprotective profile at either time (T1 and T2). Furthermore, DMT was statistically important to differentiate the dataset (VIP value > 1), although it did not exhibit sufficient neuroprotective activity by in vitro assay, neither a positive correlation with T1 and T2 neuroprotective profile, which corroborated the MSA results. CONCLUSION: The lower doses of the active samples stimulated neuronal cell proliferation and/or displayed the most efficacious neuroprotection profile, namely by preventing neuronal damage and improving cell viability against 6-OHDA-induced toxicity. Intriguingly, the hydroalcoholic fractions exhibited enhanced neuroprotective effects when compared to other samples and isolated alkaloids. This finding corroborates the significance of a holistic approach. The results demonstrate that Ayahuasca and its base plants have potential applicability for PD treatment and to prevent its progression differently from current drugs to treat PD.

Human Cerebral Organoids and Fetal Brain Tissue Share Proteomic Similarities.

The limited access to functional human brain tissue has led to the development of stem cell-based alternative models. The differentiation of human pluripotent stem cells into cerebral organoids with self-organized architecture has created novel opportunities to study the early stages of the human cerebral formation. Here we applied state-of-the-art label-free shotgun proteomics to compare the proteome of stem cell-derived cerebral organoids to the human fetal brain. We identified 3,073 proteins associated with different developmental stages, from neural progenitors to neurons, astrocytes, or oligodendrocytes. The major protein groups are associated with neurogenesis, axon guidance, synaptogenesis, and cortical brain development. Glial cell proteins related to cell growth and maintenance, energy metabolism, cell communication, and signaling were also described. Our data support the variety of cells and neural network functional pathways observed within cell-derived cerebral organoids, confirming their usefulness as an alternative model. The characterization of brain organoid proteome is key to explore, in a dish, atypical and disrupted processes during brain development or neurodevelopmental, neurodegenerative, and neuropsychiatric diseases.

Lipid Metabolism Alterations in a Rat Model of Chronic and Intergenerational Exposure to Arsenic.

Chronic exposure to arsenic (As), whether directly through the consumption of contaminated drinking water or indirectly through the daily intake of As-contaminated food, is a health threat for more than 150 million people worldwide. Epidemiological studies found an association between chronic consumption of As and several pathologies, the most common being cancer-related disorders. However, As consumption has also been associated with metabolic disorders that could lead to diverse pathologies, such as type 2 diabetes mellitus, nonalcoholic fatty liver disease, and obesity. Here, we used ultra-performance liquid chromatography (UPLC) coupled to electrospray ionization/quadrupole time-of-flight mass spectrometry (ESI-QToF) to assess the effect of chronic intergenerational As exposure on the lipid metabolism profiles of serum from 4-month-old Wistar rats exposed to As prenatally and also during early life in drinking water (3 ppm). Significant differences in the levels of certain identified lysophospholipids, phosphatidylcholines, and triglycerides were found between the exposed rats and the control groups, as well as between the sexes. Significantly increased lipid oxidation determined by the malondialdehyde (MDA) method was found in exposed rats compared with controls. Chronic intergenerational As exposure alters the rat lipidome, increases lipid oxidation, and dysregulates metabolic pathways, the factors associated with the chronic inflammation present in different diseases associated with chronic exposure to As (i.e., keratosis, Bowen's disease, and kidney, liver, bladder, and lung cancer).

Glycerophospholipid Metabolism Alterations in Patients with Type 2 Diabetes Mellitus and Tuberculosis Comorbidity.

Type-2 Diabetes (T2D) is a predisposing cause for developing tuberculosis (TB) in low- and middle-income countries. TB-T2D comorbidity worsens clinical control and prognosis of the affected individuals. The underlying metabolic alterations for this infectious-metabolic disease are still largely unknown. Possible mediators of the increased susceptibility to TB in diabetic patients are lipids levels, which are altered in individuals with T2D. To evaluate the modulation of glycerophospholipids in patients with TB-T2D, an untargeted lipidomic approach was developed by means of ultra-performance liquid chromatography (UPLC) coupled to electrospray ionization/quadrupole time-of-flight mass spectrometry (ESI-QToF). In addition, tandem mass spectrometry was performed to determine the identity of the differentially expressed metabolites. We found that TB infected individuals with or without T2D share a common glycerophospholipid profile characterized by a decrease in phosphatidylcholines. A total of 14 glycerophospholipids were differentially deregulated in TB and TB-T2D patients and could potentially be considered biomarkers. It is necessary to further validate these identified lipids as biomarkers, focusing on the anticipate diagnosis for TB development in T2D predisposed individuals.

Urinary Metabolites Altered during the Third Trimester in Pregnancies Complicated by Gestational Diabetes Mellitus: Relationship with Potential Upcoming Metabolic Disorders.

Gestational diabetes mellitus (GDM) is a disorder in pregnancy with highest impact in the future life of both mother and newborn. Increasing incidence, economic impact, and potential for severe GDM-related pregnancy complications are some factors that have motivated the deep study of physiopathology, risk factors for developing GDM, and potential biomarkers for its diagnosis. In the present pilot study, we analyzed the urinary metabolome profile of GDM patients in the 3rd trimester of pregnancy, when GDM is already established and the patients are under dietary and pharmacological control. An untargeted metabolomics method based on liquid chromatography⁻mass spectrometry analysis was developed to identify differentially expressed metabolites in the GDM group. We identified 14 metabolites that are significantly upregulated in the urine of GDM patients, and, more importantly, we identified those related with the steroid hormone biosynthesis and tryptophan (TRP) metabolism pathways, which are associated with GDM pathophysiology. Thus, these metabolites could be screened as potential prognostic biomarkers of type two diabetes mellitus, coronary artery disease and chronic renal failure in future follow-up studies with GDM patients.

Blood-Based Lipidomics Approach to Evaluate Biomarkers Associated With Response to Olanzapine, Risperidone, and Quetiapine Treatment in Schizophrenia Patients.

This is the first study to identify lipidomic markers in plasma associated with response of acutely ill schizophrenia patients in response to specific antipsychotic treatments. The study population included 54 schizophrenia patients treated with antipsychotics for 6 weeks. Treatment led to significant improvement in positive and negative symptoms for 34 patients with little or no improvement for 20 patients. In addition, 37 patients showed an increase in body mass index after the 6 week treatment period, consistent with effects on metabolism and the association of such effects with symptom improvement. Profiling of plasma samples taken prior to therapy using liquid chromatography tandem mass spectrometry (LC-MS/MS) resulted in identification of 38, 10, and 52 compounds associated with the olanzapine, risperidone, and quetiapine treatment groups, which could be used to distinguish responders from non-responders. Limitations include the retroactive active nature of the study and the small sample size. Further investigations with larger sample sets could lead to the development of a molecular test that could be used to help psychiatrists determine the best treatment options for each patient.

Application of Atmospheric Solids Analysis Probe Mass Spectrometry (ASAP-MS) in Petroleomics: Analysis of Condensed Aromatics Standards, Crude Oil, and Paraffinic Fraction.

Atmospheric solids analysis probe mass spectrometry (ASAP-MS) is a powerful tool for analysis of solid and liquid samples. It is an excellent alternative for crude oil analysis without any sample preparation step. Here, ASAP-MS in positive ion mode, ASAP(+)-MS, has been optimized for analysis of condensed aromatics (CA) standards, crude oil, and paraffinic fraction samples using a Synapt G2-S HDMS. Initially, two methodologies were used to access the chemical composition of samples: (1) using a temperature gradient varying from 150 to 600 °C at a heating rate of 150 °C min-1, and (2) with constant temperature of 300 and 400 °C. ASAP(+)-MS ionized many compounds with a typical petroleum profile, showing a greater signals range of m/z 250-1300 and 200-1400 for crude oil and paraffin samples, respectively. Such performance, mainly related to the detection of high molecular weight compounds (>1000 Da), is superior to that of other traditional ionization sources, such as ESI, APCI, DART, and DESI. Additionally, the CA standards were identified in both forms: radicals, [M]+•, and protonated cations, [M + H]+, with minimum fragmentation. Therefore, ASAP was more efficient in accessing the chemical composition of nonpolar and polar compounds. It is promising in its application with ultrahigh resolution MS instruments, such as FT-ICR MS and Orbitrap, since molecular formulas with greater resolution and mass accuracy (<1 ppm) would be assigned. Graphical Abstract ᅟ.

The screening of organic matter in mineral and tap water by UHPLC-HRMS.

It is highly desirable to screen for a large variety of organic compounds in water. Ultra-high performance liquid chromatography coupled to high-resolution mass spectrometry (UHPLC-HRMS) can analyse semi-polar to polar organic compounds in tap and mineral water. The use of UHPLC-HRMS is well consolidated for lipidomic, metabolomic and proteomic studies; based on the detection of a very large number of compounds of a variety of organic functions. Water analysis is usually performed by gas chromatography -mass spectrometry (GC-MS) techniques that are efficient for volatile organic compounds. Therefore the use of UHPLC-HRMS as a screening method based on an untargeted omic approach for the analysis of organic compounds with polar functional groups is welcome. This UHPLC-HRMS method was developed and tested with tap water and mineral water. We analysed six different brands of mineral water from France, Spain, Norway and Brazil and tap water from Jandaia do Sul, PR, Brazil to test the approach and demonstrate how UHPLC-HRMS may be used as a screening method for water quality. Forty-seven different ions were observed in tap and mineral water and their intensities were submitted to principal component analysis (PCA) evaluation. Fifteen ions were identified comparing the mass spectrometric results to metabolomic and lipidomic libraries.

The development of a specific and sensitive LC-MS-based method for the detection and quantification of hydroperoxy- and hydroxydocosahexaenoic acids as a tool for lipidomic analysis.

Docosahexaenoic acid (DHA) is an n-3 polyunsaturated fatty acid that is highly enriched in the brain, and the oxidation products of DHA are present or increased during neurodegenerative disease progression. The characterization of the oxidation products of DHA is critical to understanding the roles that these products play in the development of such diseases. In this study, we developed a sensitive and specific analytical tool for the detection and quantification of twelve major DHA hydroperoxide (HpDoHE) and hydroxide (HDoHE) isomers (isomers at positions 4, 5, 7, 8, 10, 11, 13, 14, 16, 17, 19 and 20) in biological systems. In this study, HpDoHE were synthesized by photooxidation, and the corresponding hydroxides were obtained by reduction with NaBH4. The isolated isomers were characterized by LC-MS/MS, and unique and specific fragment ions were chosen to construct a selected reaction monitoring (SRM) method for the targeted quantitative analysis of each HpDoHE and HDoHE isomer. The detection limits for the LC-MS/MS-SRM assay were 1-670 pg for HpDoHE and 0.5-8.5 pg for HDoHE injected onto a column. Using this method, it was possible to detect the basal levels of HDoHE isomers in both rat plasma and brain samples. Therefore, the developed LC-MS/MS-SRM can be used as an important tool to identify and quantify the hydro(pero)xy derivatives of DHA in biological system and may be helpful for the oxidative lipidomic studies.

New gadolinium complex with efficient hydrolase-like activity: a 100-million-fold rate enhancement in diester hydrolysis.

The synthesis, structure, and hydrolase-like catalytic activity of a new mononuclear gadolinium complex [Gd(L)(NO3)(H2O)3](NO3)2 (1) are reported. A clean two-stage kinetic reaction for hydrolysis of the diester 2,4-bis(dinitrophenyl)phosphate by 1 was followed, and the rate constants were determined. A high DNA cleavage activity was also demonstrated. The active species in the hydrolytic process is proposed based on the X-ray structure, electrospray ionization mass spectrometry analysis, and kinetic and potentiometric equilibrium studies of 1.