Methanol Precipitation Research Articles

Subcutaneous Semaglutide during Breastfeeding: Infant Safety Regarding Drug Transfer into Human Milk.

Postpartum mothers and their healthcare providers often face the challenge of limited data regarding the safety of drug therapies during lactation. Pregnancy can lead to sustained weight gain, and obesity can negatively impact both physical and psychological well-being. The introduction of GLP-1 agonists to augment weight loss has become a topic of interest for many postpartum mothers. Our study aims to investigate the transmission of semaglutide into human milk in the first steps to ensure the safety and health of both lactating mothers and their breastfed infants. Semaglutide quantification was performed using high-resolution liquid chromatography-mass spectrometry. InfantRisk Center Human Milk biorepository released milk samples from eight women collected at 0, 12 and 24 h post-semaglutide administration. Semaglutide was extracted using protein precipitation in methanol, followed by chromatographic separation. Linear calibration curves for the method ranged between 2.5-30 ng/mL, with a limit of detection of 1.7 ng/mL and a limit of quantification of 5.7 ng/mL (LLOQ). Semaglutide was not detected in any of the collected human milk samples. A worst-case scenario of the relative infant dose (RID) was calculated using the LLOQ as the drug concentration in milk when considering semaglutide's bioavailability and long-acting dose profile. The maximum RID projected was 1.26%, far below the standard 10% safety threshold. While questions about long-term infant outcomes, the safety of maternal nutrient intake, and the nutrient content of breast milk remain, our findings suggest that semaglutide concentrations in human milk are unlikely to pose clinical concerns for breastfed infants. These results support healthcare providers in making informed decisions regarding postpartum therapeutic interventions.

Physicochemical Stimuli-Mediated Precipitation Approach for the Modulation of Rifampicin's Dissolution and Oral Bioavailability.

The intricate process of protein binding orchestrates crucial drug interactions within the bloodstream, facilitating the formation of soluble complexes. This research endeavours to improve the dissolution and oral bioavailability of Rifampicin (RMP) by strategically manipulating drug-protein binding dynamics and the hydrophobic characteristics of human serum albumin (HSA). Various precipitation techniques leveraging methanol, ammonium sulfate, and heat treatment were meticulously employed to tailor the properties of colloidal albumin (HSA NPs). The resultant complexes underwent comprehensive characterization encompassing evaluations of hydrophobicity, size distribution, surface charge, and structural analyses through FTIR, TG-DSC, XRD, and morphological examinations. The findings revealed a significant binding affinity of 78.07 ± 6.6% with native albumin, aligning with prior research. Notably, the complex RMP-HSA NPs-M13, synthesized via the methanolic precipitation method, exhibited the most substantial complexation, achieving a remarkable 3.5-fold increase, followed by the ammonium sulfate (twofold) and heat treatment (1.07-fold) methods in comparison to native albumin binding. The gastric simulated media exhibited accelerated drug release kinetics, with maximal dissolution achieved within two hours, contrasting with the prolonged release observed under intestinal pH conditions. These findings translated into significant improvements in drug permeation, as evidenced by pharmacokinetic profiles demonstrating elevated Cmax, AUC, t1/2, and MRT values for RMP-HSA NPs-M13 compared to free RMP. In summary, this innovative approach underscores the potential of precipitation methods in engineering stable colloidal carrier systems tailored to enhance the oral bioavailability of poorly soluble drugs, offering a pragmatic and scalable alternative to conventional surfactants, polymers, or high-energy methods for complex formation and production.

Ionic liquid pretreatment of lignocellulose for complete hemicellulose removal to produce high-purity cellulose mixed esters

Strategic valorization of agricultural waste can serve as waste management and promote sustainable biorefineries. Sugarcane bagasse is a readily available lignocellulosic biomass and can be converted into valuable cellulose-based thermoplastics. However, the cellulose purity significantly influences the material properties of the resulting thermoplastics, potentially limiting their applications. Therefore, conventional production necessitates harsh pretreatment of lignocellulose to isolate high-purity cellulose, followed by chemical modification. Here, we demonstrate a facile and green pretreatment method for complete removal of hemicellulose by incubating bagasse in a mixed system of an ionic liquid, 1-ethyl-3-methylimidazolium acetate (EmimOAc), and dimethyl sulfoxide (DMSO) at 140 °C, followed by precipitation in water, achieving >99 % hemicellulose removal. The pretreated bagasse, containing 71 % cellulose and 28 % lignin, underwent homogeneous transesterification with vinyl decanoate and isopropenyl acetate, using EmimOAc as both the solvent and catalyst, assisted by a co-solvent of DMSO. The polysaccharide derivative in the resulting acylated bagasse was separated from the lignin derivative by precipitation in methanol. The obtained polysaccharide derivative with high cellulose purity displayed a high weight-average molar mass of 1.5×106 g mol−1 and a polydispersity of 4. It also exhibited superior thermal stability (thermal degradation temperature, Td−5 %: 359 °C; glass transition temperature, Tg: 144 °C) compared to pulp-derived cellulose acetate decanoate (Td−5 %: 349 °C; Tg: 129 °C).

A Quick and Sensitive LC-MS/MS Method for Simultaneous Quantification of Sofosbuvir Metabolite (GS-331007) in Human Plasma: Application to Hepatitis C Infected Patients with End-Stage Renal Disease.

Sofosbuvir (SOF) is a revolutionary treatment for patients with hepatitis C virus (HCV). However, its efficacy and safety among patients with end-stage renal disease (ESRD) remains controversial. In this study, we examined the levels of SOF metabolite (GS-331007) (SOF-007) in human plasma of patients infected with HCV having ESRD using an optimized liquid chromatography-mass spectrometry (LC-MS) analytical method. In this case-control study, 10 clinically confirmed cases and five controls were enrolled. SOF-007 was extracted from plasma using methanol precipitation. The limit of detection (LOD) for the drug and its metabolite were 0.85 and 2.3, respectively. Such a wide range of quantification in a period of separation time shorter than 3.0 minutes (run time) allowed monitoring of the plasma concentration of analytes up to 4 hours (pre-dialysis and post-dialysis) for 12 weeks in non-cirrhotic patients with HCV infection undergoing dialysis. SOF-007 in the plasma of HCV patients with healthy kidneys showed no cumulative effect. An analysis comparing patients with ESRD and healthy participants showed that their behaviour was similar, followed by dialysis with a relatively small cumulative effect. The plasma concentrations of SOF-007 decreased significantly after the 4-hour period of dialysis compared with the plasma concentrations hemodialysis of pre-dialysis in HCV patients with ESRD.

Simultaneous Determination of One-Carbon Folate Metabolites and One-Carbon-Related Amino Acids in Biological Samples Using a UHPLC-MS/MS Method.

One-carbon folate metabolites and one-carbon-related amino acids play an important role in human physiology, and their detection in biological samples is essential. However, poor stability as well as low concentrations and occurrence in different species in various biological samples make their quantification very challenging. The aim of this study was to develop a simple, fast, and sensitive ultra-high-performance liquid chromatography MS/MS (UHPLC-MS/MS) method for the simultaneous quantification of various one-carbon folate metabolites (folic acid (FA), tetrahydrofolic acid (THF), p-aminobenzoyl-L-glutamic acid (pABG), 5-formyltetrahydrofolic acid (5-CHOTHF), 5-methyltetrahydrofolic acid (5-CH3THF), 10-formylfolic acid (10-CHOFA), 5,10-methenyl-5,6,7,8-tetrahydrofolic acid (5,10-CH+-THF), and 4-α-hydroxy-5-methyltetrahydrofolate (hmTHF)) and one-carbon-related amino acids (homocysteine (Hcy), methionine (Met), S-ade-L-homocysteine (SAH), and S-ade-L-methionine (SAM)). The method was standardized and validated by determining the selectivity, carryover, limits of detection, limits of quantitation, linearity, precision, accuracy, recovery, and matrix effects. The extraction methods were optimized with respect to several factors: protease-amylase treatment on embryos, deconjugation time, methanol precipitation, and proteins' isoelectric point precipitation on the folate recovery. Ten one-carbon folate metabolites and four one-carbon-related amino acids were detected using the UHPLC-MS/MS technique in various biological samples. The measured values of folate in human plasma, serum, and whole blood (WB) lay within the concentration range for normal donors. The contents of each analyte in mouse plasma were as follows: pABG (864.0 nmol/L), 5-CH3THF (202.2 nmol/L), hmTHF (122.2 nmol/L), Met (8.63 μmol/L), and SAH (0.06 μmol/L). The concentration of each analyte in mouse embryos were as follows: SAM (1.09 μg/g), SAH (0.13 μg/g), Met (16.5 μg/g), 5,10-CH+THF (74.3 ng/g), pABG (20.6 ng/g), and 5-CH3THF (185.4 ng/g). A simple and rapid sample preparation and UHPLC-MS/MS method was developed and validated for the simultaneous determination of the one-carbon-related folate metabolites and one-carbon-related amino acids in different biological samples.

Wide-scope targeted analysis of bioactive lipids in human plasma by LC/MS/MS

Quantitative information on blood metabolites can be used in developing advanced medical strategies such as early detection and prevention of disease. Monitoring bioactive lipids such as steroids, bile acids, and PUFA metabolites could be a valuable indicator of health status. However, a method for simultaneously measuring these bioactive lipids has not yet been developed. Here, we report a LC/MS/MS method that can simultaneously measure 144 bioactive lipids, including steroids, bile acids, and PUFA metabolites, from human plasma, and a sample preparation method for these targets. Protein removal by methanol precipitation and purification of bioactive lipids by solid-phase extraction improved the recovery of the targeted compounds in human plasma samples, demonstrating the importance of sample preparation methods for a wide range of bioactive lipid analyses. Using the developed method, we studied the plasma from healthy human volunteers and confirmed the presence of bioactive lipid molecules associated with sex differences and circadian rhythms. The developed method of bioactive lipid analysis can be applied to health monitoring and disease biomarker discovery in precision medicine.

Effects of dietary Andrographis paniculata extract on growth, haematological, immune responses, immune-related genes expression of ornamental goldfish (Carassius auratus) and its susceptibility to Aeromonas hydrophila infection

In the present study, goldfish (Carassius auratus) were fed diets supplemented with different levels of methanolic crude extracts of Andrographis paniculata to assess their efficacy against Aeromonas hydrophila. The A. paniculata extract showed antibacterial activity against A. hydrophila at different concentrations and was characterised by FTIR and GC-MS analysis. The fishes (C. auratus) were divided into three groups, the control fishes were fed with a basel diet, the DI and DII group fishes were fed with a basel diet mixed with 0.1% and 1% of methanolic crude extracts of A. paniculata, respectively for 4 weeks. Then, haematological and immunological parameters were examined in each experimental and control group after the 2nd and 4th week. Finally, all group fish were challenged with A. hydrophila at the end of the 4th week, and their cytokine genes expression were investigated. The results showed that even at low concentrations (50 μg) of A. paniculata leaf extracts showed a potent antibacterial effect against A. hydrophila. GC-MS analysis revealed that the methanol precipitation contains Benzylamine, 2-hydroxy-N,N-DI-[2-aminoethyl]-, 18-Nonadecen-1-OL, and andrographolide. Similarly, FTIR analysis revealed aliphatic primary amine, conjugated ketone and alcohols, which are known antimicrobials. The extracts also changed the growth, haematological and immunological parameters of treated fishes by significantly increased the total RBC counts and haemoglobin level, along with the phagocytic, total immunoglobulin and myeloperoxidase activities when compared to the BD group. Moreover, fish fed with 1% of A. paniculata extract showed a higher survival rate (80%) against A. hydrophila. Furthermore, 1% of A. paniculata extract dietary was significantly up-regulated pro-anti-inflammatory cytokine gene expression, compared with the BD group. In conclusion, dietary A. paniculata extract supplementation beneficial for C. auratus, as it assesses their efficacy against A. hydrophila.

Blood molecular profile to predict genotoxicity from exposure to antineoplastic drugs.

Genotoxicity is an important information that should be included in human biomonitoring programmes. However, the usually applied cytogenetic assays are laborious and time-consuming, reason why it is critical to develop rapid and economic new methods. The aim of this study was to evaluate if the molecular profile of frozen whole blood, acquired by Fourier Transform Infrared (FTIR) spectroscopy, allows to assess genotoxicity in occupational exposure to antineoplastic drugs, as obtained by the cytokinesis-block micronucleus assay. For that purpose, 92 samples of peripheral blood were studied: 46 samples from hospital professionals occupationally exposed to antineoplastic drugs and 46 samples from workers in academia without exposure (controls). It was first evaluated the metabolome from frozen whole blood by methanol precipitation of macromolecules as haemoglobin, followed by centrifugation. The metabolome molecular profile resulted in 3 ratios of spectral bands, significantly different between the exposed and non-exposed group (p<0.01) and a spectral principal component-linear discriminant analysis (PCA-LDA) model enabling to predict genotoxicity from exposure with 73 % accuracy. After optimization of the dilution degree and solution used, it was possible to obtain a higher number of significant ratios of spectral bands, i.e., 10 ratios significantly different (p<0.001), highlighting the high sensitivity and specificity of the method. Indeed, the PCA-LDA model, based on the molecular profile of whole blood, enabled to predict genotoxicity from the exposure with an accuracy, sensitivity, and specificity of 92 %, 93 % and 91 %, respectively. All these parameters were achieved based on 1μL of frozen whole blood, in a high-throughput mode, i.e., based on the simultaneous analysis of 92 samples, in a simple and economic mode. In summary, it can be conclude that this method presents a very promising potential for high-dimension screening of exposure to genotoxic substances.

Quantitative plasma profiling by 1H NMR-based metabolomics: impact of sample treatment.

Introduction: There is evidence that sample treatment of blood-based biosamples may affect integral signals in nuclear magnetic resonance-based metabolomics. The presence of macromolecules in plasma/serum samples makes investigating low-molecular-weight metabolites challenging. It is particularly relevant in the targeted approach, in which absolute concentrations of selected metabolites are often quantified based on the area of integral signals. Since there are a few treatments of plasma/serum samples for quantitative analysis without a universally accepted method, this topic remains of interest for future research. Methods: In this work, targeted metabolomic profiling of 43 metabolites was performed on pooled plasma to compare four methodologies consisting of Carr-Purcell-Meiboom-Gill (CPMG) editing, ultrafiltration, protein precipitation with methanol, and glycerophospholipid solid-phase extraction (g-SPE) for phospholipid removal; prior to NMR metabolomics analysis. The effect of the sample treatments on the metabolite concentrations was evaluated using a permutation test of multiclass and pairwise Fisher scores. Results: Results showed that methanol precipitation and ultrafiltration had a higher number of metabolites with coefficient of variation (CV) values above 20%. G-SPE and CPMG editing demonstrated better precision for most of the metabolites analyzed. However, differential quantification performance between procedures were metabolite-dependent. For example, pairwise comparisons showed that methanol precipitation and CPMG editing were suitable for quantifying citrate, while g-SPE showed better results for 2-hydroxybutyrate and tryptophan. Discussion: There are alterations in the absolute concentration of various metabolites that are dependent on the procedure. Considering these alterations is essential before proceeding with the quantification of treatment-sensitive metabolites in biological samples for improving biomarker discovery and biological interpretations. The study demonstrated that g-SPE and CPMG editing are effective methods for removing proteins and phospholipids from plasma samples for quantitative NMR analysis of metabolites. However, careful consideration should be given to the specific metabolites of interest and their susceptibility to the sample treatment procedures. These findings contribute to the development of optimized sample preparation protocols for metabolomics studies using NMR spectroscopy.

Evaluation of Two Simultaneous Metabolomic and Proteomic Extraction Protocols Assessed by Ultra-High-Performance Liquid Chromatography Tandem Mass Spectrometry.

Untargeted multi-omics analysis of plasma is an emerging tool for the identification of novel biomarkers for evaluating disease prognosis, and for developing a better understanding of molecular mechanisms underlying human disease. The successful application of metabolomic and proteomic approaches relies on reproducibly quantifying a wide range of metabolites and proteins. Herein, we report the results of untargeted metabolomic and proteomic analyses from blood plasma samples following analyte extraction by two frequently-used solvent systems: chloroform/methanol and methanol-only. Whole blood samples were collected from participants (n = 6) at University Hospital Sharjah (UHS) hospital, then plasma was separated and extracted by two methods: (i) methanol precipitation and (ii) 4:3 methanol:chloroform extraction. The coverage and reproducibility of the two methods were assessed by ultra-high-performance liquid chromatography-electrospray ionization quadrupole time-of-flight mass spectrometry (UHPLC-ESI-QTOF-MS). The study revealed that metabolite extraction by methanol-only showed greater reproducibility for both metabolomic and proteomic quantifications than did methanol/chloroform, while yielding similar peptide coverage. However, coverage of extracted metabolites was higher with the methanol/chloroform precipitation.

Efficient synthesis of Co2+–Co3+ hydrotalcite-like compounds via NaOH precipitation in methanol–water under mild alkaline conditions

Divalent/trivalent cobalt hydrotalcite-like compounds (Co2+–Co3+ HTlc) were synthesized via precipitation under mild alkaline conditions by using NaOH as precipitant and methanol–water as solvent. The influences of synthetic parameters including CH3OH:H2O volume ratio, precipitation pH, and hydrothermal treatment on the HTlc formation were investigated. The crystalline phase, morphology, and chemical composition of the synthesized samples were characterized by XRD, FTIR, SEM, TG–MS, and iodometric titration. The formation of Co2+–Co3+ HTlc was strongly affected by the methanol content and a single Co2+–Co3+ HTlc phase was obtained by raising the CH3OH:H2O ratio to 4:1–8:1. It was suggested that the presence of methanol increased the concentration of dissolved oxygen and promoted the oxidation of Co2+ into Co3+, leading to complete transformation of α-Co(OH)2 into Co2+–Co3+ HTlc. Moreover, single-phased Co2+–Co3+ HTlc was available at pH between 10 and 12, and they were hydrothermally stable by treatment at 60 °C for 24 h. Under the synthetic conditions, complete precipitation of cobalt ions was achieved. This highlights that the developed synthesis process is an efficiency way for quantitative synthesis of cobalt HTlc.

Metabolites Identification and Mechanism Prediction of Neobavaisoflavone In Vitro and In Vivo of Rats through UHPLC-Q-Exactive Plus Orbitrap MS Integrated Network Pharmacology

Neobavaisoflavone is an important isoflavone component isolated from Psoraleae Fructus. It is used extensively worldwide because of its antibacterial, antioxidant, anti-inflammatory, anticancer, and anti-osteoporotic activities. However, there is no systematic and comprehensive research on the metabolism of neobavaisoflavone in vivo and in vitro. The study aimed to analyze the metabolic characteristics and mechanism of neobavaisoflavone for the first time. Firstly, biological samples were pretreated by the solid-phase extraction (SPE) method, methanol precipitation, and acetonitrile precipitation. Secondly, the samples were analyzed on UHPLC-Q-Exactive Plus Orbitrap MS. Thirdly, metabolites were tentatively identified based on retention time, parallel reaction monitoring strategy, diagnostic product ions, and neutral loss fragments. A total of 72 metabolites of neobavaisoflavone were tentatively identified, including 28 in plasma, 43 in urine, 18 in feces, six in the liver, and four in the liver microsome. The results suggested that neobavaisoflavone mainly underwent glucuronidation, sulfation, hydroxylation, methylation, cyclization, hydration, and their composite reactions in vivo and in vitro. In addition, nine active components with high bioavailability and 191 corresponding targets were predicted by the Swiss Drug Design database. The 1806 items of GO and 183 KEGG signaling pathways were enriched. These results showed that metabolites expanded the potential effects of neobavaisoflavone. The present study would provide the scientific basis for the further exploitation and application of neobavaisoflavone.

Development of a UPLC-MS/MS method for the determination of active ingredients of Shuang Hu in rat blood and its application in pharmacokinetics.

A simple and sensitive method using in vivo microdialysis coupled with UPLC-MS/MS was established to evaluate the pharmacokinetics of Shuang Hu tincture (SHZTN). Xevo TQ-S was used to analyze the active ingredients of mesaconitine, hypaconitine, 4-hydroxycinnamic acid, ferulic acid and N-(2, 3-dimethyl phenyl)-2- aminobenzoic acid of SHZTN. Samples were prepared using a methanol precipitation method and the internal standards lannaconitine and p-hydroxybenzoic acid were added. The method validation was conducted according to the guidelines of the Pharmacopoeia of China. A good linear range was obtained in the range of 1-2,000 ng/ml. The intra-day and inter-day precisions were less than 14.7%, and the accuracy range of all the analytes was -10.5-9.3%. The recovery of each analyte was over 95.5%, and matrix effects can be neglected. After a single dose of 20 mg/kg SHZTN, the area under the curve and peak concentration of the five active ingredients were significantly increased by transdermal compared with oral administration, which indicated the high bioavailability of SHZTN. The time to peak concentration of all compounds was <3.4h, and the half-life was <15.4h, which indicated that the five compounds have the best absorption and rapid elimination. The method was successfully developed and applied to the pharmacokinetic study of SHZTN.

Comprehensive Analysis of Pterostilbene Metabolites In Vivo and In Vitro Using a UHPLC-Q-Exactive Plus Mass Spectrometer with Multiple Data-Mining Methods

Pterostilbene, a stilbene phytoalexin, is mainly obtained from blueberries and grape vines; however, its metabolic mechanisms were unclear in vivo. In the present study, three different methods were used to prepare biological samples, and then, an efficient strategy based on ultrahigh-performance liquid chromatography coupled with mass spectrometry was developed to screen and identify pterostilbene metabolites in rat urine, plasma, liver, and feces. In order to elucidate pterostilbene or its metabolites involved in vitro, this study was assessed by the liver microsome system. As a result, a total of 88 pterostilbene metabolites were characterized. Among them, 77 metabolites in vivo and 14 metabolites in vitro were found; 50 and 38 metabolites were observed in rat plasma and urine, while only 4 and 12 metabolites were detected in rat feces and liver, inferring that plasma and urine possessed more diverse types of pterostilbene metabolites; 41 metabolic products were obtained by solid-phase extraction, and 9 and 10 metabolites were screened by methanol precipitation and acetonitrile precipitation, respectively, indicating that solid-phase extraction could be adopted as the most acceptable method for pterostilbene metabolism. The results also demonstrated that pterostilbene mainly underwent glucosylation, dehydrogenation, hydrogenation, demethoxylation, sulfation, NAC binding, methylene ketogenic, acetylation, and methylation. In summary, this research provides an idea for the further study of drug metabolism.

Drug metabolite cluster centers-based strategy for comprehensive profiling of Neomangiferin metabolites in vivo and in vitro and network pharmacology study on anti-inflammatory mechanism

Neomangiferin (NMF) is an extremely special xanthone that could be simultaneously attributed to C-glycoside and O-glycoside with a variety of biological activities, such as anti-inflammatory, antitumor, antipyretic, and so on. So far as we know, the metabolism profiling has been insufficient until now. Herein, Drug Metabolite Cluster Centers (DMCCs)-based Strategy has been developed to profile the NMF metabolites in vivo and in vitro. Firstly, the DMCCs was proposed depending on literature-related and preliminary analysis results. Secondly, the specific metabolic rule was implemented to screen the metabolites of candidate DMCCs from the acquired Ultra High Performance Liquid Chromatography Quadrupole Exactive Orbitrap Mass Spectrometry (UHPLC-Q-Exactive Orbitrap MS) data by extracted ion chromatography (EIC) method. Thirdly, candidate metabolites were accurately and tentatively identified according to the pyrolysis law of mass spectrometry, literature reports, comparison of reference substances, and especially the diagnostic product ions (DPIs) deduced preliminarily. Finally, network pharmacology was adopted to elucidate the anti-inflammatory action mechanism of NMF on the basis of DMCCs. As a result, 3 critical metabolites including NMF, Mangiferin (MF) and Norathyriol (NA) were proposed as DMCCs, and a total of 61 NMF metabolites (NMF included) were finally screened and characterized coupled with 3 different biological sample preparation methods including solid phase extraction (SPE), acetonitrile precipitation and methanol precipitation. Among them, 32 metabolites were discovered in rat urine, 30 in rat plasma, 12 in rat liver, 9 metabolites in liver microsomes and 8 in rat faeces, respectively. Our results also illustrated that NMF primarily underwent deglucosylation, glucuronidation, methylation, sulfation, dihydroxylation and their composite reactions in vivo and in vitro. Additionally, network pharmacology analysis based on DMCCs revealed 85 common targets of disease-metabolites, and the key targets were TNF, EGFR, ESR1, PTGS2, HIF1A, IL-2, PRKCA and PRKCB. They exerted anti-inflammatory effects mainly through the pathways of inflammatory response, calcium-dependent protein kinase C activity, nitrogen metabolism, pathways in cancer and so on. In general, our study constructed a novel strategy to comprehensive elucidate the biotransformation pathways of NMF in vivo and in vitro, and provided vital reference for further understanding its anti-inflammatory action mechanism. Moreover, the established strategy could be generalized to the metabolism and action mechanism study of other natural products.

Analytical strategies to profile the internal chemical exposome and the metabolome of human placenta

As a non-invasive biological matrix, the placenta offers great and novel opportunities to monitor fetal exposure to exogenous chemicals and their biotransformation products (the internal chemical exposome), as well as the biological responses associated, in large-scale epidemiological studies. However, it is first crucial to ensure that analytical methods based on high-resolution mass spectrometry (HRMS) can detect the low abundant components of the internal chemical exposome present in these complex biological matrices. In this study, we aimed to develop a robust analytical method (extraction and sample preparation) sensitive enough to profile the internal chemical exposome and the metabolome of placenta using high-resolution mass spectrometry (HRMS) for future application in mother child cohorts. Several extraction solvents (methanol, methanol/H2O (50/50 v/v), methyl tert-butyl ether/methanol/H2O) were tested and their ability to extract components of the internal exposome and metabolome were compared. Then, sample preparation methods commonly used for metabolomics application (methanolic protein precipitation) were compared with solid phase extraction (SPE), protein and phospholipid removal plates (PPRP) and combination of SPE and PPRP. The methods were compared and validated using qualitative (i.e., numbers of features and chemical classes ID), quantitative parameters adopted from targeted multi-residue analysis (recovery experiments, repeatability and matrix effect) as well as the ability of these methods to be implemented for high-throughput applications. The analytical repeatability of the two most effective methods (methanolic extraction followed by either protein precipitation or PPRP) were tested at the batch level to determine the best concentration factors to be used for improving detection of components of the internal chemical exposome and metabolome without impacting on the analytical response. Finally, these two methods based on protein precipitation and PPRP were tested on 40 placenta samples from the French PELAGIE birth cohort, and annotation was performed on the related datasets to compare the respective impacts of PPT and PPRP. A wide range of exogenous (e.g., biocides, pharmaceuticals, personal care products) and endogenous chemicals (steroids, prostanoids, lipids, carnitins) could be detected and annotated (some of them for the first time in placenta). We show that both methods are complementary but that PPRP allows the injection of more concentrated extracts without impacting the LC repeatability and therefore improve the detection (presence and signal area fold change) of many exogenous and endogenous chemicals.

Diselenide as a Dual Functional Mechanophore Capable of Stress Self-Reporting and Self-Strengthening in Polyurethane Elastomers

Diselenide as a Dual Functional Mechanophore Capable of Stress Self-Reporting and Self-Strengthening in Polyurethane Elastomers

Abstract P5-13-20: Identifying a metabolite signature that correlates with tumor proliferation in early-stage breast cancer patients treated with CDK4/6 inhibitors from matched plasma and serum samples

Abstract Background: Cyclin-dependent kinase 4/6 inhibitors (CKD4/6i) have demonstrated clinical utility extending progression-free survival (PFS) and overall survival (OS) for advanced hormone receptor positive and HER2 negative (HR+/HER2-) breast cancer patients. The efficacy in early-stage breast cancer (eBC) is unclear, with conflicting results from adjuvant CDK4/6i trials on invasive disease-free survival. Thus, there is a critical need to identify biomarkers of response (BoR) to determine which, if any, eBC patients could benefit from this treatment. This BoR could also stratify advanced BC patients for likelihood to respond to CDK4/6i. Metabolism is influenced by both genome and environment, and changes in the metabolome can be correlated with drug responsiveness. Thus, metabolite BoRs may serve to identify eBC patients for which CDK4/6i would offer a therapeutic benefit.Methods: Plasma and serum samples from 50 early-stage ER+/HER2- breast cancer patients, treated with neoadjuvant CDK4/6 inhibitor palbociclib (palbo) and aromatase inhibitor (AI) anastrozole on NeoPalAna trial (ClinicalTrials.gov identifier NCT01723774), were collected from treatment-naïve patients (BL) and 3 consecutive time points: anastrozole,1 mg daily for 4 weeks (C1D1), anastrozole plus palbo,125 mg daily, for 15 days (C1D15), and for 4-5 months before surgery (SURG). Metabolites were extracted from all samples via methanol and chloroform precipitation and quantified using an unbiased, non-destructive, nuclear magnetic resonance (NMR)-based profiling platform (Olaris®, Inc., Waltham, MA). Statistical analysis and machine learning was used to identify differential metabolites and generate predictive models. A separate validation set of samples was collected from a subset of patients (N=6) who received an additional cycle of palbo treatment prior to surgery to assess model accuracy. Results: Non-parametric differential expression analysis of BL/C1D1, BL/C1D15, and C1D1/C1D15 identified 53 ,97, and 90 differential NMR resonances in plasma (p&amp;lt;0.05) and 36, 34, and 25 differential NMR resonances in serum (p&amp;lt;0.05), respectively. Based on the proliferative marker Ki67 levels at C1D15, 37 patients were classified as responders (Ki67≤2.7%) and 6 patients as non-responders (Ki67&amp;gt;2.7%). Analysis of the responder (R) and non-responder (NR) groups identified that 13 plasma and 14 serum resonances (21 unique resonances and 6 overlapping) were differentially expressed (p&amp;lt;0.05) at C1D1. Many of the differential resonances could be mapped back to amino acid metabolites including several branched chain amino acids such as leucine, valine, and isoleucine, and positively charged amino acids such as lysine. A Olaris® BoR score was generated using 5 differential resonances that had an AUC of 0.931 (training set) and 100% accuracy when predicting palbo-response in a blinded test set (N=6).Conclusion: The differential metabolites identified from matching plasma and serum samples suggest that, compared to serum, plasma has a better representation of the metabolic changes associated with palbo treatment-response. While comparing samples from R and NR patients, amino acids were found to be consistently altered in both serum and plasma before palbo treatment. In addition, a BoR model based on select metabolites could precisely stratify palbo-response in a blinded dataset. A larger independent validation cohort is ongoing. Citation Format: Chen Dong, Shana Thomas, Chandrashekhar Honrao, Leonardo O. Rodrigues, Nathalie Tessier, Bo Zhang, Souzan Sanati, Kiran Vij, Brenda J. Ernst, Karen S. Anderson, Mateusz Opyrchal, Foluso Ademuyiwa, Lindsay L. Peterson, Matthew P. Goetz, Donald Northfelt, Elizabeth O'Day, Cynthia Ma. Identifying a metabolite signature that correlates with tumor proliferation in early-stage breast cancer patients treated with CDK4/6 inhibitors from matched plasma and serum samples [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-13-20.

Quantification of Free and Total Carnitine in Serum Using Liquid Chromatography Tandem Mass Spectrometry.

L-carnitine is a crucial component for transporting long-chained fatty acids from the cytosol into the mitochondrial matrix for fatty acid oxidation. During this process, carnitine forms numerous acylcarnitines before being recycled into the cytosol. Abnormal levels of free carnitine, total carnitine, and acylcarnitines in serum can be indicative of a metabolic disorder before symptoms are present. A liquid chromatography tandem mass spectrometry (LC-MS/MS) method is described for the determination of free and total carnitine in serum. To measure total carnitine, samples are spiked with deuterated carnitine (internal standard) and hydrolyzed with potassium hydroxide to convert acylcarnitines to carnitine. The reaction is quenched by the addition of hydrochloric acid. Carnitine is extracted via a methanolic protein precipitation. The solution is then injected on LC-MS/MS for analysis to determine the carnitine concentration using multiple-reaction monitoring.

Proteomic and Metabolomic Analyses of Maggots in Porcine Corpses for Post-Mortem Interval Estimation

Post-mortem interval (PMI) estimation is a critical task in forensic science. In this study, we used maggots collected from pig carcasses and applied an integrated proteomics and metabolomics approach to determine potential candidate substances for the estimation of PMI. After methanol precipitation, the supernatant containing metabolites and the protein pellet were separated and subjected to metabolomic and proteomic analyses using liquid chromatography-tandem mass spectrometry (LC-MS/MS). MS/MS data were analyzed for identification and quantification using Proteome Discoverer and Compound Discoverer software. A total of 573 metabolites and more than 800 porcine proteins were identified in maggots. This is the first dataset of proteins and metabolites in maggots collected from porcine carcasses. In this study, guanosine monophosphate, xanthine, inosine, adenosine, and guanine were detected with a similar tendency to increase during early days of maggot development and then decreased gradually. We broadly profiled various biomolecules through analysis in the spot of incident. Especially, we confirmed that proteome and metabolome profiling could be performed directly and indirectly.