Stability of Wheat Floret Metabolites during Untargeted Metabolomics Studies

A typical metabolomic analysis consists of a multi-step procedure. Variation can be introduced in any analysis segment if proper care in quality assurance is not taken, thus compromising the final results. Sample stability is one of those factors. Although sophisticated studies addressing sample decay over time have been performed in the medical field, they are emerging in plant metabolomics. Here, we focus on the stability of wheat floret extracts on queue inside an auto-injector held at 25 °C. The objective was to locate an analytical time window from extraction to injection with no significant difference occurring in the sample. Total ion current chromatograms, principal component analysis, and volcano plots were used to measure changes in the samples. Results indicate a maximum work window time of 7:45 h for Steele-ND wheat methanolic extractions in an auto-sampler at 25 °C. Comparisons showed a significant gradual increase in the number and intensity of compounds observed that may be caused by the degradation of other molecules in the sample extract. The approach can be applied as preliminary work in a metabolite profiling study, helping to set the appropriate workload to produce confident results.

Isotope-Dilution Gas Chromatography-Mass Spectrometry Method for the Selective Detection of Nicotine and Menthol in E-Cigarette, or Vaping, Product Liquids and Aerosols

We developed a quantitative method for analyzing nicotine and menthol in e-cigarette, or vaping, products (EVPs). These products may adversely impact health through inhalational exposure to addictive and harmful chemicals. The presence of unknown substances in do-it-yourself e-liquids, counterfeits, or unregulated products may increase exposure to harmful chemicals, as underscored by the 2019 EVP use-associated lung injury (EVALI) outbreak. To minimize these risks, it is important to accurately quantify nicotine and menthol in e-liquids and aerosol emissions to evaluate EVP authenticity, verify product label accuracy, and identify potentially hazardous products. We developed a simple, versatile, high-throughput method using isotope-dilution gas chromatography-mass spectrometry for quantifying nicotine and menthol concentrations in both e-liquid contents and machine-generated aerosol emissions of EVPs. Rigorous validation has demonstrated that the method is specific, precise (CV<2.71%), accurate (percent error ≤7.0%), and robust. Linear calibration ranges from 0.01 to 1.00 mg/ml for both analytes was achieved, corresponding to expected analyte levels in e-liquids and machine-generated EVP aerosols. Limits of detection (LODs) in the final 10-ml sample extract were 0.4 μg/ml for nicotine and 0.2 μg/ml for menthol. The method was used to analyze aerosol emissions of 141 EVPs associated with the 2019 EVALI outbreak; detectable levels of nicotine (2.19–59.5 mg/g of aerosol) and menthol (1.09–10.69 mg/g of aerosol) were observed in 28 and 11%, respectively, of the samples analyzed. Nicotine was not detected in any of the tetrahydrocannabinol (THC), cannabidiol (CBD), or oil-based products, while menthol (2.95 mg/g of aerosol) was only detected in one of these products (THC-labeled). The analytical method can be used to quantify nicotine and menthol concentrations in the e-liquids and aerosols from a range of EVPs, and these findings highlight a difference between e-cigarette and other vaping products.

Method Validation and Determination of Leachable Metals from Infusion and Transfusion Medical Devices

This work deals with method validation for regulated metals (Cd, Pb, Ba, Sn, Cr and Cu) determination in infusion and transfusion medical devices. The investigated metals were extracted with water at (37 ± 1) °C followed by their determination in the extract by using inductively coupled plasma optical emission spectrometry (ICP-OES). The validated method was applied in the analysis of infusion and transfusion devices commercialized in Brazil to verify compliance with current legislation, which establishes that the sum of Pb, Ba, Sn, Cr and Cu in the extract must not exceed 1 mg L-1 and that of Cd must not 0.1 mg L-1. Samples from five manufacturers of infusion and transfusion devices, produced in Brazil or imported, were analysed. The results of the analysis showed that all devices complied with the legislation, whereas the sum of Pb, Ba, Sn, Cr and Cu concentrations and that of Cd in the extract were lower than the maximum permissible; Cd was not detected in any sample extract and the sum of the other elements was < 0.14 mg L-1 in all extracts of the analysed samples.

Analytical Evaluation of the Ideal Strategy for High-Throughput Flow Injection Analysis by Tandem Mass Spectrometry in Routine Newborn Screening

The introduction of tandem mass spectrometry (MS/MS) to clinical laboratories and the advent of expanded newborn screening (NBS) were crucial changes to public health programs worldwide. Speed, robustness, accuracy, selectivity, and specificity of analysis are all requirements of expanded NBS and are needed to minimize false positive results risks, to possibly eliminate false negatives, and to improve the positive predictive value of NBS. In this study, we firstly evaluated the analytical performances of the RenataDX Screening System, a fully integrated flow-injection MS/MS (FIA-MS/MS) IVD system for high-throughput dried blood spot (DBS) analysis in a routine NBS laboratory. Since a choice of several commercial NBS kits is available, we sought to compare NeoBaseTM 2 (PerkinElmer®) and MassChrom® (Chromsystems) non-derivatized kits on the RenataDX platform by evaluating their analytical performances. Moreover, we verified the degree of correlation between data obtained by the two different NBS MS/MS kits by FIA-MS/MS of over 500 samples. Our data suggest that both methods correlate well with clinically insignificant differences that do not impact the NBS result. Finally, while NeoBase™ 2 offers an easier and faster sample preparation, MassChrom® provides a cleaner sample extract which empirically should improve instrument reliability.

The potential protective effects of malacca (Phyllanthus emblica L.) extract against doxorubicin-induced cardiotoxicity in male Wistar rats

Doxorubicin as a chemotherapy agent is most widely used in cancer treatment. Long-term use at a predetermined dose has a side effect, namely cardiotoxicity. Doxorubicin-induced cardiotoxicity is considered to be caused by reactive oxygen species (ROS), which is also characterized by increasing CK-MB enzyme levels in the blood. To reduce the impact of doxorubin-induced toxicity, a study was conducted on natural antioxidant sources with cardioprotective capabilities in vivo. Phyllanthus emblica L. fruit was used as a sample for a natural source of antioxidants extracted using ethanol. Then performed a phytochemical screening of secondary metabolites contained in it. This extract was administered orally in various doses to the experimental animal Wistar rats and the induced doxorubicin to these animals. The CK-MB enzyme levels were measured, and the heart organ histopathology test was performed. The results of this study indicate that P. emblica L. fruit extract contains alkaloids, tannins, flavonoids, terpenoids, phenolics, and triterpenoids compounds. Extract treatment at a 400 mg/kg BW dose showed the best reduction in CK-MB levels with great improvements in regular arrangement and shape of myocardial muscle cells of cardiac tissue. The sample extract at a 400 mg/kg BW dose showed remarkably decreasing of CK-MB great improvements of heart tissue on doxorubicin-induced cardiotoxicity. This study showed the potential protective effect of P. emblica L. against doxorubicin-induced cardiotoxicity.

Occurrence and Source Apportionment of Polycyclic Aromatic Hydrocarbons (PAHs) in Green Mussel (Perna viridis) from Cilincing Waters of Jakarta Bay, Indonesia

Green mussels (Perna viridis) as sedentary organisms have been adopted as bioindicators in the marine environment for many decades. They have been potentially affected by anthropogenic activity and organic contaminants such as polycyclic aromatic hydrocarbons (PAHs) in an aquatic environment. This study aims to determine concentration, distribution and potential sources of PAHs in green mussel biomass obtained from Cilincing waters, Jakarta Bay. Fresh green mussels were collected from aquaculture facilities at Cilincing waters of Jakarta Bay with various size categories of shell length and were extracted with solvent n-hexane-dichloromethane mixture using Soxhlet method. The concentrate of sample extract was purified using silica gel/sodium sulfate anhydrous with n-pentene-DCM effluent in fractionation. Finally, the purified sample was injected into the GC-MS instrument. The result showed that the highest concentration of total PAH (TPAH) was 126.47 ng.g-1 wet weight (ww), founded in the guts from the biggest group of green mussel (length of 6.00 to 7.99 cm), and 3-aromatic rings were predominantly detected in all group sizes of green mussel. The result of the LMW/ HMW and diagnostic binary ratio indicated that PAH contamination is caused by a mixed source of petrogenic and pyrogenic processes. It may be due to the presence of oil spilled and petroleum waste from shipyard and cargo activities and atmospheric deposition. Based on excess cancer risk (ECR) value and the concentration level of PAH carcinogenic according to National Agency of Drug and Food Control (BPOM) regulation, PAH level in green mussels are acceptable and minor negative impact due to typical human consumption. Nevertheless, it needs to be circumspect to exploiting green mussels as seafood resources for daily consumption due to the carcinogenic content.

Citrate Mediated Europium-Based Detection of Oxytetracycline in Citrus Tissues

Oxytetracycline (OTC) and streptomycin have been used for the control of several plant diseases and were recently permitted for the control of citrus greening disease, Huanglongbing. Consequently, sensitive and reliable methods are highly needed for the detection of OTC in citrus tissues. Herein, we studied the replacement of cetyltrimethylammonium chloride (CTAC) by citrate (Cit) as a sensitizing agent for the analysis of OTC in citrus tissues using the recently established europium (Eu) method. In addition, we determined the optimal conditions for the formation of the Eu-OTC-Cit ternary complex in tris buffer. Our results showed that the plant matrix significantly decreased the fluorescence intensity of the Eu-OTC-Cit complex even after the replacement of CTAC. Our investigations showed that phenols such as gallic acid degrade slowly at high pH and their degradation was enhanced in the presence of the (Eu+3) cation. To reduce the plant matrix interference, the sample extract was cleaned using solid-phase extraction (SPE). The OTC recoveries from spiked healthy and Candidatus Liberibacter asiaticus (CLas)-infected trees were 91.4 ± 7.8% and 82.4 ± 3.9%, respectively. We also used the citrate method to determine the level of OTC in trunk-injected trees. The level of OTC as measured using the Eu-OTC-Cit complex (117.5 ± 20.3 µg g−1 fresh weight “FWT”) was similar to that measured using Eu-OTC-CTAC complex (97.5 ± 14 µg g−1 FWT). In addition, we were able to visualize the OTC in citrus leaf extract, under ultraviolet light (400 nm), after it was cleaned with the SPE. Our study showed that the citrate can be successfully used to replace the harmful CTAC surfactant, which could also react with phenols.

FTIR-based fingerprinting combined with chemometrics for discrimination of Sonchus arvensis leaves extracts of various extracting solvents and the correlation with its antioxidant activity

Sonchus arvensis, the local name for tempuyung, is acknowledged to have many biological activities, including the antioxidant activity. This study aimed to cluster the leaves extracts based on the extracting solvent and to determine the functional groups significantly contributing to the antioxidant activity. From the water, 10%, 30%, 50%, 70% ethanol, and absolute ethanol extracts, we analyzed the total phenolics content (Folin-Ciocalteu method), antioxidant activity (DPPH method), and the FTIR spectra. The 70% ethanol extract exhibits the highest total phenolics and the highest antioxidant activity. The extracts were grouped based on the extracting solvent using the principal component analysis (PCA) with 95% total variance from its principal component 1 and 2. The partial least square (PLS) regression was employed for finding a functional group from the antioxidant constituents present in the sample extract. We predicted by PLS regression that the –OH and the C-O groups are attributed to the phenolics that give a significant contribution to the antioxidant activity of the S. arvensis leaves.

A Novel and Reliable Rat Model of Autism

Background: Autism spectrum disorders (ASD) is a complex neurodevelopmental disorder that lacks an ideal animal model to recapitulate the disease state of ASD. Previous studies have reported that transplanting gut microbiota of ASD patients into pregnant mice is sufficient to promote the changes of autism-like behavior in offspring. This study aims to explore whether fecal microbiota transplantation (FMT) can be used as a new method to establish the ASD animal model.Methods: We transplanted the fecal sample extract of ASD children into pregnant rats (rFMT) repeatedly to establish an ASD rat model (oFMT) and compare it with the classical valproic acid (VPA) model (oVPA).Results: First, we reveal that oFMT shows hypoevolutism and typical behavioral characteristics of ASD, consistent with the previous study. Second, the gut microbiota of oFMT mainly consists of Firmicutes and Bacteroidetes, recapitulating the abnormal gut microbiota of ASD. In oFMT, the abundance of Lactobacillus and Collinsella increased (Lactobacillus: oFMT 60.16%, oVPA 64.13%, oCON 40.11%; Collinsella: oFMT 3.73%, oVPA 1.39%, oCON 1.28%), compared with oVPA, gut microbiota also showed high consistency. Third, the expression of 5-hydroxytryptamine (5-HT) in oFMT serum increased, γ-aminobutyric acid (GABA) and norepinephrine (NE) in oFMT serum decreased. Fourth, the gut microbiota of oFMT also has some ASD characteristic gut microbiota not found in oVPA. Fifth, pregnant rat with VPA showed significant immune activation, while those with FMT showed relatively minor immune activation.Limitations: Although the mechanism of establishing FMT autism rat model (oFMT) has not clearly defined, the data show that the model has high structural validity, and FMT model is likely to be a new and reliable potential animal model of ASD, and will have potential value in studying gut microbiota of ASD.Conclusions: The FMT autism rat model has high structural validity, and the FMT model is likely to be a new and reliable potential animal model of ASD.

Validation of a simple HPLC method to quantify mycophenolic acid concentrations in human plasma

Introduction: Mycophenolic acid (MPA) is an active metabolite of mycophenolate mofetil and mycophenolate sodium which are widely prescribed to prevent organ rejection after solid organ transplantations. However, MPA induced many side effects on gastrointestinal tract and haematological system. Objectives: The purpose of this study is to establish a high-performance liquid chromatography (HPLC) method to determine the MPA concentration in plasma in order to optimize the treatment efficacy of MPA or apply to bioequivalence studies. MPA and visnadine (as an internal standard) were extracted from plasma samples with methanol by solid phase extraction using Osis HLB 1cc cartridge. 10 µL of sample extract was injected onto LiChroCART®125-4 (C18 reversed-phase column) at 43 °C on a Waters 2695 XE system. The signals were detected by PDA detector (photodiodes array) at 254 nm. The mobile phase was a mixture of acetonitrile and phosphate buffer (pH 3) with a flow rate of 1 mL/min. The validation criteria included: selectivity, linearity, accuracy, precision, recovery, lower limit of quantification. Results: Total chromatographic runtime was 15 min. MPA and visnadine were found at 6.45 and 10.79 min, respectively. MPA concentrations were in the linear range from 0.25 to 50 µg/mL. The coefficient of variation (CV) of mean intra-day and inter-day precision levels for MPA was less than 7.5%. The lower limit of quantification was 0.25 µg/mL. No interference was found in the assay. Conclusion: A simple and reliable HPLC method was developed to quantify the MPA concentration in plasma.