Performance Liquid Chromatography-mass Spectrometry Research Articles

SIRT3-mediated deacetylation of NLRC4 promotes inflammasome activation.

Salmonella typhimurium (S. typhimurium) infection of macrophage induces NLRC4 inflammasome-mediated production of the pro-inflammatory cytokines IL-1β. Post-translational modifications on NLRC4 are critical for its activation. Sirtuin3 (SIRT3) is the most thoroughly studied mitochondrial nicotinamide adenine dinucleotide (NAD+) -dependent deacetylase. We wondered whether SIRT3 mediated-deacetylation could take part in NLRC4 inflammasome activation.Methods: We initially tested IL-1β production and pyroptosis after cytosolic transfection of flagellin or S. typhimurium infection in wild type and SIRT3-deficient primary peritoneal macrophages via immunoblotting and ELISA assay. These results were confirmed in SIRT3-deficient immortalized bone marrow derived macrophages (iBMDMs) which were generated by CRISPR-Cas9 technology. In addition, in vivo experiments were conducted to confirm the role of SIRT3 in S. typhimurium-induced cytokines production. Then NLRC4 assembly was analyzed by immune-fluorescence assay and ASC oligomerization assay. Immunoblotting, ELISA and flow cytometry were performed to clarify the role of SIRT3 in NLRP3 and AIM2 inflammasomes activation. To further investigate the mechanism of SIRT3 in NLRC4 activation, co-immunoprecipitation (Co-IP), we did immunoblot, cellular fractionation and in-vitro deacetylation assay. Finally, to clarify the acetylation sites of NLRC4, we performed liquid chromatography-mass spectrometry (LC-MS) and immunoblotting analysis.Results: SIRT3 deficiency led to significantly impaired NLRC4 inflammasome activation and pyroptosis both in vitro and in vivo. Furthermore, SIRT3 promotes NLRC4 inflammasome assembly by inducing more ASC speck formation and ASC oligomerization. However, SIRT3 is dispensable for NLRP3 and AIM2 inflammasome activation. Moreover, SIRT3 interacts with and deacetylates NLRC4 to promote its activation. Finally, we proved that deacetylation of NLRC4 at Lys71 or Lys272 could promote its activation.Conclusions: Our study reveals that SIRT3 mediated-deacetylation of NLRC4 is pivotal for NLRC4 activation and the acetylation switch of NLRC4 may aid the clearance of S. typhimurium infection.

Hplc-Ms/Ms Analysis of Key Components of the Amino Acid Metabolism in the Serum of Acute Myocardial Infarction Patients

Objective: The present study aimed to identify the key components of amino acids and potential mechanistic pathways of myocardial infarction. Methods: We performed case–control metabolite profiling via mass spectrometry in the serum obtained from 33 individuals with acute myocardial infarction, and compared the results to matched healthy volunteers (70 cases). Multi-reaction monitoring was used to evaluate the targeted metabolomics of amino acids by high performance liquid chromatography and tandem mass spectrometry; this method was validated using the standard solution. Moreover, we used the mixed effects models to compare the metabolite concentrations between the cases and controls by regulating relevant biological variables. Results: LC-MS/MS quantitatively determined 33 substances in amino acid metabolism and intestinal microbial metabolites, and provided good results in terms of accuracy, precision, and linearity. The method was validated according to Food and Drug Administration guidelines, and it provided good results in terms of precision, accuracy, and linearity. Thus, significant changes were observed in the aromatic amino acids (tyrosine, phenylalanine, and tryptophan), oxidative homeostasis (o-tyrosine), intestinal microbial metabolites (trimethylamine oxide, choline, acetylcholine), and methionine cycle (SAM, SAH, methionine, and homocysteine). Conclusions: A unique metabolite profile involving amino acid and intestinal microbial metabolites is observed in myocardial infarction disorders. The endogenous compounds evaluated in this study can be used as potential biomarkers. Funding Information: This work was supported by the National Natural Science Foundation of China (No. 81803689), the Natural Science Foundation of Guangdong Province (No. 2018A030310292), Project of Health Science and Technology of Guangzhou (No. 20191A011039), and the YIWEN Talent Project of The Third Affiliated Hospital of Guangzhou Medical University (No. 2021#9). Declaration of Interests: The authors declare no competing financial interest. Ethics Approval Statement: The study was approved by the Medical Ethics Committee of the Third Clinical School of Guangzhou Medical University. Acute myocardial infarction human serum samples and healthy human serum samples were obtained from The Third Clinical School of Guangzhou Medical University, and informed consent was obtained from all patients (batch numbers of ethical approval: 2021#026).

Aqueous-phase oxidation of syringic acid emitted from biomass burning: Formation of light-absorbing compounds

Syringic acid is a methoxyphenol model compound derived from biomass burning, and its photooxidation processes have important effects on atmospheric chemistry. However, its aqueous-phase photochemistry remains unclear. In this study, we systematically report the photooxidation of syringic acid induced by OH radicals in the aqueous phase. Employing the relative rate technique, the bimolecular rate constant for syringic acid reaction with OH radicals was acquired to be (1.1 ± 0.3) × 1010 M−1 s−1. Notably, colored products were formed as the reaction progressed. Furthermore, the UV–vis and fluorescence spectra confirmed the formation of light-absorbing organic species, and the results agreed well with previous results on atmospheric and natural humic-like substances (HULIS). The photooxidation products were detected by high performance liquid chromatography mass spectrometry (HPLC/MS), and a possible reaction mechanism was proposed. The aqueous-phase reaction of syringic acid would undergo functionalization process forming a hydroxylation product that enhances the degree of oxidation of aqueous secondary organic aerosol (aqSOA), and goes through dimerization process by C-C or C-O coupling of phenoxy radicals which may conduce to the formation of HULIS. These findings suggest that the photooxidation of syringic acid is an important pathway for highly oxygenated phenolic aqSOA formation, providing a secondary source for HULIS in a liquid phase or in deliquescent particles surrounded by a layer of water.

In vitro enzyme inhibition, antibacterial, UHPLC-MS chemical profiling and in silico studies of Indigofera argentea Burm. f. for potential biopharmaceutical application

The study aimed to assess the phytochemical composition and enzyme inhibitory potential of methanol, n-hexane, chloroform, and n-butanol extracts/fractions of Indigofera argentea Berm. f. (whole plant), a medicinal plant from Pakistan. Chemical profiling of I. argentea crude methanol extract was done via ultra-high performance liquid chromatography mass spectrometry (UHPLC-MS) in both the positive and negative ionization modes. Standardin vitro bioassays were used to evaluate the enzyme inhibition potential against carbonic anhydrase, xanthine oxidase, and α-glucosidase. Additionally, nine dominant compounds as tentatively identified by UHPLC-MS profiling were docked against the tested enzymes to provide additional insights into the binding affinities against the target enzymes. UHPLC-MS chemical profiling showed the tentative presence of seventy-six secondary metabolites. The identified compounds include flavonoids and lithocholic acid derivatives of steroids in major amounts along with terpenes. Lithocholic acid derivatives were first reported from this plant and consider being potential candidates for treating diabetes. All extracts showed promising results against α-glucosidase with percentage inhibition of 82.8–21.2%, while for the xanthine oxidase the percentage inhibition values varying from 67.8–47.3%. Furthermore, moderate inhibition was observed against carbonic anhydrase. The methanolic extracts showed good inhibition against B. subtilis, K. pneumonia, P. vulgaris and P. aeruginosa as compared to Ciprofloxacin. Docking studies showed that atalanine showed higher inhibition affinity against the three studied enzymes as compared to morphine. Indigofera argentea showed promising enzyme inhibitory activities and can be considered as a potential source of bioactive secondary metabolites. Isolation of the bioactive compounds and further mechanistic studies could provide further insights into the biopharmaceutical potential of this plant as a nutraceutical and/or dietary supplement.

ATRT-17. TARGETING GLUTAMINE METABOLISM LOWERS METHYLATION POTENCIALS IN AT/RT AND SYNERGIZE WITH TAZEMETOSTAT

Atypical teratoid/rhabdoid tumors (AT/RT) have a single recurring genetic mutation in SMARCB1. This deletion leads to an abnormal SWI/SNF chromatin remodeling complex and the constitutive activation of EZH2. S-adenosyl-L-methionine (SAM) donates a methyl group to EZH2 which then methylates DNA and histones leading to the abnormal gene expression responsible for AT/RT’s aggressive phenotype. We have previously shown that glutamine metabolic inhibition with 6-diazo-5-oxo-L-norleucine (DON) confers a survival advantage in AT/RT. In this study, we identified with ultra-high performance liquid chromatography mass spectrometry that DON treatment lowered the methylation potential in AT/RT (Decreased SAM:SAH ratio, t-test in 5 AT/RT human-derived cell models comparing DON treatment to DMSO control, p<0.05). AT/RT cell lines grown in glutamine deplete media compared to normal growth conditions also had a reduced methylation potential (decreased SAM:SAH, t-test, p<0.05). DON treatment over 5 days decreased histone methylation (as determined by western blot for H3K27me3). Tazemetostat is a small molecule inhibitor that blocks the SAM methyl donor site on EZH2. We find that DON combines synergistically with Tazemetostat to slow AT/RT cell growth (MTS assay, p<0.01 t-test; MUSE viability assay, p<0.01 ANOVA) and enhances cytotoxicity (MUSE Annexin-V, p<0.01 by ANOVA). Synergies were especially pronounced at low concentrations of Tazemetostat which is significant given that Tazemetostat’s efficacy in AT/RT has been limited by poor CNS penetration. These studies identify a novel treatment strategy that has potential to improve survival in AT/RT.

Simultaneous quantitation of cytokinin bases and their glycoconjugates with stable isotope labelling ultrahigh performance liquid chromatography mass spectrometry

Cytokinins (CKs) are one class of important phytohormones widely investigated in most aspects of plant life. Similar to other phytohormones, CKs and their glycoconjugates are hydrophilic. Their ionization efficiencies for mass spectrometry (MS) detection are rather poor, whereas their retention and separation on reverse phase liquid chromatography (RPLC) are often unsatisfying. Chemical isotope labelling LC-MS analysis methods have been developed for most other phytohormones, enhancing their LC separations and quantitative sensitivity. However, there are currently no reports for chemical-labelled CKs. Here, we report a new chemical isotope labelling LC-MS analytical method for one-pot derivatization of CK bases and their glycoconjugates, based on differential benzylation labelling of the adenine skeleton of CKs with benzyl bromide and its deuterium isotope-labelled reagent. Benzylation alters the hydrophilicity of CKs and their glycoconjugates, improving their retention and separation on RPLC. The developed method demonstrated enhanced sensitivity, as the CKs and their glycoconjugates could be analysed with LODs within the range of 0.62–25.9 pg/mL. The method also demonstrated good intra- and inter-day precisions with standard deviations in the range of 1.9%–13.0%, and acceptable accuracy with recoveries in the range of 84.0%–119.9%. The developed method was employed on the quantitation of CKs in the fresh roots of Astragalus membranaceus collected from both fertilized and unfertilized fields. The significant impact that fertilizers had on endogenous CKs metabolism was observed. As such, monitoring endogenous CKs and their metabolites might be promising to control fertilizer abuse.

An analytical strategy for designer benzodiazepines and Z-hypnotics determination in plasma samples using ultra-high performance liquid chromatography/tandem mass spectrometry after microextraction by packed sorbent

The illicit market for new psychoactive substances (NPS) is continuously growing. Designer benzodiazepines (DBZD) and Z-hypnotics are increasingly being used for self-medication or recreational purposes. The limited regulation and little biological information available about NPS have raised the need for analytical methods capable of extracting and quantifying them in human biological fluids. In this work, a procedure based on microextraction by packed sorbent (MEPS) in combination with ultra-high performance liquid chromatography and tandem mass spectrometry (UHPLC–MS/MS) has been developed to determine the designer benzodiazepines (clonazolam, deschloroetizolam, nifoxipam, flubromazolam and meclonazepam), and the Z-hypnotics (zolpidem, zaleplon and zopiclone) in plasma. A 3342//16 asymmetric screening design was used to study extraction variables such as the type and volume of eluent, pH, number of extraction cycles, volume of washing solvent and type of sorbent. The ensuing analytical method was validated in terms of linearity by standard addition calibration curves at eight different analyte concentration levels from 0.5–500 ng mL–1. R2 values, limits of detection (LOD) and limits of quantification (LOQ) fell in the ranges 0.9900–0.9988, 0.5–5 ng mL–1 and 1–10 ng mL–1. Intra and interday precision expressed as relative standard deviations, were < 10.6 % and process efficiency ranged from 63 to 117 % for the quality control samples. The proposed method detected zolpidem and various other benzodiazepines in plasma samples from overdoses cases.

Abstract PO-050: Solubilization of keratin 17 promotes the metastatic dissemination of the most lethal form of pancreatic cancer

Abstract CLINICAL NEED &amp; OBJECTIVES: There is an unmet need for the discovery and development of novel therapeutic approaches for PDAC. Molecular subtyping, however, has recently uncovered potential biomarkers that, subject to further investigation, may be druggable to extend PDAC patient survival. Expression of Keratin 17 (K17), an oncofetal intermediate filament (IF) protein and hallmark of the most aggressive subtype, associates negatively with survival and drives chemotherapy resistance in PDAC. This suggests a functional role of K17 in PDAC, though the underlying mechanisms are unknown. Nuclear localization, however, has been suggested as a part of the overarching processes through which K17 promotes tumor growth. In related keratins, post-translational modifications (PTM) such as phosphorylation drive IF dissociation (solubilization) and can contribute to migration and metastatic dissemination. Thus, we hypothesized that K17 may harbor PTM- and/or solubilization-dependent oncogenic functions that could reveal new PDAC therapeutic options. METHODS &amp; RESULTS: Using isogenic murine PDAC models, we found that K17 expression enhances tumor growth and metastatic potential. Furthermore, K17 solubility was associated with shorter survival, independent of total K17 expression or cell proliferation in both murine and human PDACs. To identify K17 PTMs potentially driving solubilization, we performed liquid chromatography mass spectrometry to sequence K17 isolated from patient PDACs and identified phosphorylation of a conserved N-terminal region (Ser 10-13) as the predominant PTMs. To determine if Ser 10-13 phosphorylation regulates K17 solubility, we generated mutants that mimic K17 Ser 10-13 phosphorylation and found that this was sufficient to solubilize K17 and promote nuclear localization. Building on these findings, we used kinase inhibitors to show that the PKC-MEK-RSK signaling pathway is involved in phosphorylation of K17 Ser 10-13. To assess the impact of K17 Ser 10-13 phosphorylation on tumors, nude mice were implanted orthotopically with isogenic human PDAC cell lines expressing either wild type (WT), loss of function (LOF) Ser 10-13 mutants, or Ser 10-13 phosphorylation gain of function (GOF) mutants. Compared to tumors expressing WT K17, GOF tumors resulted in increased metastases, and LOF mutants abrogated these characteristics. CONCLUSION: These results suggest that enhanced K17 solubility by phosphorylation at Ser 10-13 is sufficient to impact PDAC metastasis and survival and reveal novel druggable sites on K17. These results motivate future studies to uncover K17 phosphorylation targeting approaches for PDAC. If successful, they will be coupled with the identification of PDAC patients by tumor K17 expression, providing the basis of a potential precision medicine strategy. Citation Format: Ryan R. Kawalerski, Mariana T. Gonçalves, Lucia Roa-Peña, Cindy V. Leiton, Chun-Hao Pan, Luke A. Torre-Healy, Taryn Boyle, Natasha T. Snider, Kenneth R. Shroyer, Luisa F. Escobar-Hoyos. Solubilization of keratin 17 promotes the metastatic dissemination of the most lethal form of pancreatic cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2020 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2020;80(22 Suppl):Abstract nr PO-050.

Comparison of Diagnostic Value between Point of Care Testing (POCT) and Standardize HbA1c Testing in Primary Health Care

Diabetes Mellitus is one of the biggest health problems. HbA1c is used to diagnose, to monitor treatment and treatment adjustment. High Performance Liquid Chromatography (HPLC) and electron spray mass spectrometry are recommended to measure HbA1c. But both methods need expensive cost, facility, and potentially increase health cost burden. HbA1c measurement using Point of Care Testing (POCT) is developed in Indonesia. POCT is more comfortable, has lower cost and easily brought to primary health care. Aim: to assess the clinical efficacy of POCT compared with a standard examination by HPLC. Methods: It is a cross-sectional observational study conducted at a first-level health facility (FKTP) in Malang. The subject population was patients who went to an outpatient unit in FKTP with inclusion criteria aged 20-75 years, were participants in the chronic disease management program (PROLANIS), both diabetes and non-diabetes. Results: Three hundred and thirty eight subjects were participated in this study. Five subjects were excluded because the presence of anemia. We used Wilcoxon test to compare HbA1c level between two methods and Rank Spearman correlation test to find correlation between two methods. This study showed measurement HbA1c level using POCT method had good accuracy (&gt;80%). Other than diagnosis value, increased utilization of POCT HbA1c might also be caused by its portability and patient’s comfort. Conclusions: This study showed a strong correlation (R=0.016) between POCT HbA1c and standardized measurement. POCT HbA1c also showed good accuracy in all HbA1c groups.

Untargeted metabolomic profiles of air pollution exposure during pregnancy and postpartum depression risk: a pilot study

Background. Postpartum depression (PPD) impacts 18% of mothers globally, adversely affecting the health of mothers and children. Evidence from the Programming Research on Obesity, Growth, Environment, and Social Stress (PROGRESS) cohort in Mexico City suggests that exposure to air pollution, namely PM2.5 (particulate matter &lt; 2.5 μm in diameter), in pregnancy is associated with increased PPD risk. Untargeted metabolomics, in which hundreds to thousands of metabolites are identified without specifying these metabolites a priori, offers a data-driven approach to identify mechanisms linking PM2.5 and PPD.Aim. To characterize metabolomic profiles associated with PM2.5 exposures and PPD in maternal serum collected during the second and third trimesters of pregnancy.Methods. We randomly selected 100 mothers from PROGRESS in a pilot study, estimated PM2.5 exposures during pregnancy using a satellite-based spatiotemporally-resolved prediction model, and measured maternal depression at 6 months postpartum using the Edinburgh Postnatal Depression Scale (EPDS). In maternal serum, we performed liquid chromatography-mass spectrometry (LC-MS)-based untargeted metabolomic profiling of polar, semi-polar, and non-polar metabolites.Results. After filtering, 202 putatively-identified metabolites were analyzed in linear models adjusted for maternal age, education, BMI, and laboratory batch. Of these, 20 and 29 metabolites in the second and third trimesters, respectively, were associated (p&lt;0.05) with average trimester PM2.5 levels in the respective trimester. Two third trimester metabolites (itaconate and nervonate) passed multiple testing correction (FDR&lt;0.05). Itaconate was also negatively associated with PM2.5 in second trimester (p=0.003) and marginally associated with lower PPD risk (EPDS ≥ 13; p=0.08).Conclusions. In maternal serum collected during pregnancy, we identified metabolites associated with PM2.5 exposure. Itaconate, which was negatively associated with both PM2.5 exposure and PPD, is a mitigator of inflammation and oxidative stress, consistent with processes known to be involved in both PM2.5 exposure and depression.

An untargeted LC-MS metabolomics approach to the metabolic profiles of bottom cultured scallops (Mizuhopecten yessoensis) subjected to mechanical shock in early post-harvest handling

From on-board handling to land-based depuration, scallops are subject to various stresses. Mechanical shock is one of the unavoidable factors of post-harvest handling. In this research, a metabolomics strategy based on ultra performance liquid chromatography–mass spectrometry (UPLC-MS) was used to investigate the metabolic characteristics of live scallops (Mizuhopecten yessoensis) subjected to mechanical shock. Phosphoarginine and ATP-related compound levels were also measured to understand the metabolic disturbances. Live scallops maintained in a circulating seawater system for 36 h formed the control group, while those individuals dropped 15 times onto a hard surface from a height of 1 m before being maintained in a circulating seawater system formed the shock group. Samples were collected from the adductor muscles, and multivariate statistical analyses were performed to identify the potential biomarkers of mechanical shock due to post-harvest handling. Our results suggested that obvious metabolic disturbances occurred when scallops suffered mechanical shock during post-harvest handling. A total of 18 biomarkers were identified, including 10 upregulated and 8 downregulated biomarkers. Pathway enrichment analysis showed that the biosynthesis of amino acids and cysteine and methionine metabolism were prone to interference when M. yessoensis was subjected to mechanical shock during early post-harvest handling. This study provides a new perspective into the metabolic profiles of scallops subjected to various stressors and can assist in the improvement of disposal methods of post-harvest scallops.

Determination of ultraviolet filters in human nails using an acid sample digestion followed by ultra-high performance liquid chromatography–mass spectrometry analysis

Ultraviolet filters (UV-filters) are specific chemicals that absorb and reflect UVA and UVB radiation from the sun. They are regularly used in sunscreens and in other personal care products (PCPs), and in products like plastics, adhesives, toys, or furniture finishes. This work develops and validates a new method to determine concentrations of UV-filters (BP-1, BP-2, BP-3, BP-6, BP-8, 4-OH-BP, THB, AVB) in human nail samples. Nails are easily available and are considered to be suitable indicators of cumulative and continued exposure to harmful chemicals. The treatment of nail samples includes microwave assisted digestion/extraction (MAE) in a methanolic solution of o-phosphoric acid (0.05 mol L−1) followed by analyte determination using ultra-high performance liquid chromatography–mass spectrometry (UHPLC–MS/MS) in multiple reaction monitoring mode. The analytes were separated in less than 10 min. The digestion procedure was optimized using multivariate techniques. Matrix-matched calibration with a pig hoof matrix was used for validating the method. A study of accuracy with spiked blank samples was also conducted. The calculated detection limits varied between 0.2 and 1.5 ng g−1, and quantification limits between 1.0 and 5.0 ng g−1. The trueness of the method was an estimation of the recovery, which was between 90.2% and 112.2%; with an estimated precision (relative standard deviation, % RSD) lower than 12.3% for all UV-filters. Nail samples were obtained from 22 volunteers (male and female). The results showed that BP-1 and BP-3 mainly bioaccumulate in human nails.

Study on the mechanism of degradation of tetracycline hydrochloride by microwave-activated sodium persulfate.

Among the different antibiotics, tetracycline hydrochloride (TCH) is one of the most commonly used. In this study, the activated sodium persulfate (SPS) process induced by microwave (MW) energy was used to treat TCH. The effect of different operational parameters of MW/SPS-treated TCH, such as SPS concentration, TCH concentration, initial pH, and MW power, was investigated. The concentration changes of TCH were determined using a spectrophotometer. The results of radical scavenger experiments indicated that the sulfate radical (SO4 ·-) was stronger than the hydroxyl radical (·OH). On the basis of high performance liquid chromatography-mass spectrometry (HPLC-MS) analysis, a possible degradation pathway of TCH was proposed. This research indicates that the MW/SPS system is a promising prospect for the treatment of TCH.

Liquid chromatography-tandem mass spectrometric assay for TPN171 in human plasma

A simple, rapid and accurate method for quantitative analysis of a highly selective phosphodiesterase-5 inhibitor (PDE5), TPN171 by high performance liquid chromatography and tandem mass spectrometry in human plasma was proposed and validated successfully using D3-TPN171 as internal standards (ISTD). An aliquot of 100 μL of plasma was mixed with internal standard and was precipitated with acetonitrile. Gradient elution was performed on a ACQUITY HSS T3 column (50 × 2.1 mm, 1.8 μm) coupled with a ACQUITY column in-line filter at 40℃, by 5 mM ammonium acetate in water containing 0.1 % formic acid and 0.1 % formic acid in acetonitrile as the mobile phase. The total analytical run time was 3.5 min. The analyte was monitored using multiple reaction monitoring (MRM) scan in positive polarity mode. The ion transition was m/z 442.2→113.2 and 445.2→116.2 for TPN171 and D3-TPN171 respectively. The method was validated for specificity, sensitivity, precision, accuracy, and other analytical parameters. The results found were satisfactory over the linear calibration range of 1−500 ng/mL. Within-day precisions ranged from 1.8 to 7.3 %, and between-day precisions from 2.3 to 4.9 %, accuracies were 95.5–99.8 %.The validated method was successfully applied to determine the plasma concentration after oral administration of 10 mg TPN171 in six healthy volunteers.

Actinomycetes strain Streptomyces sp. Je 1-42: phylogenetic analysis, biological activity and secondary metabolite profile

Aim. Screening of new natural bioactive molecules is one of the effective approach in combating multidrug-resistant pathogens and tumor cells. The aim of this work was to study the taxonomic characteristic, biological activity and secondary metabolites production of isolate Je 1-42 from the rhizosphere of Juniperus excelsa Bieb. Methods. Microbiological, molecular genetic, high performance liquid chromatography mass spectrometry and scanning electron microscopy methods were used in this work. Results. Based on a phylogenetic analysis of the 16S rRNA and gyrB gene sequences, microbiological and physiological characterization the isolate Je 1-42 has been affiliated to the genus Streptomyces. The isolate Je 1-42 showed a wide range of antibacterial activity. The antibiotics desertomycin A, kanchanamycin A, butylcycloheptylprodigiosine and spectinabilin were annotated in the Je 1-42 extract using dereplication analysis in the database DNP. Conclusions. Actinomycetes strain Streptomyces sp. Je 1-42 is an antagonist of gram-positive, gram-negative bacteria, fungi and produces antibiotics desertomycin A, canchanamycin A, butylcycloheptylprodigiozin, spectinabilin, which has a broad spectrum of antimicrobial activity. The production of these compounds obviously determines the ability of Je 1-42 to inhibit the growth of a wide range of bacteria and fungi. The strain of Streptomyces sp. Je 1-42 was deposited in the Microbial Culture Collection of Antibiotics Producers at Ivan Franko National University of Lviv (collection number Lv 1-042).&#x0D; Keywords: Streptomyces, secondary metabolites, antibacterial activity, phylogenetic analysis.

LC-MS/MS를 이용한 우유 중 농약 성분 동시 분석법 개발 및 유효성 검증

해마다 연간 우유 소비량이 증가하는 우리나라의 경우, 유해물질 관리와 안전은 더욱 중요하다. 한국작물보호협회에서 자료에 의하면 2014~2018년 농약의 약제별 생산 및 출하 현황 중 살충제가 가장 많은 것으로 보고되어 있다. 카바메이트계 농약은 생물학적 효과가 좋아 살충제, 살균제 및 구충제 등 다양하게 사용되고 있다. 카바메이트계 농약은 일부는 높은 급성 독성을 가지고 있기 때문에 식품에 사용되는 것을 조심해야 하고, 일부 물질의 경우 발암물질을 발생시키고 돌연변이를 야기할 수 있다고 보고되었다. 본 연구에서는 다양한 카바메이트계 농약 중 국내에서 많이 사용되고 있는 살충제의 3가지 농약을 선정하여 우유 원료 중 bendiocarb, carbaryl과 carbofuran 등에 대한 효율적이고 효과적인 분석법을 개발하였고 유효성 검증을 실시하였다. 시료를 무수황산나트륨과 디클로로메탄을 넣고 진탕추출 후 탈수한 뒤 농축하였다. 이를 헥산과 헥산포화아세토니트릴을 이용하여 지방을 제거하고 C18 카트리지를 이용하여 정제 후 LCMS/MS에 주입하여 분석한 결과, bendiocarb, carbaryl 및 carbofuran의 표준원액을 blank를 이용하여 5~100 ng/mL 범위에서 결정계수(r2) 0.999 이상의 직선성을 확인하였다. Bendiocarb의 일내 정밀성은 4.0% 이하, 정확성은 96.2~103.9%, carbaryl의 정밀성은 4.3% 이하, 정확성은 96.9~104.5%, carbofuran의 정밀성은 5.3% 이하, 정확성은 90.9~109.1%로 확인되었고, bendiocarb의 일간 정밀성은 6.5% 이하, 정확성은 90.1~103.7%, carbaryl의 정밀성은 10.5% 이하, 정확성은 95.3~103.3%, carbofuran의 정밀성은 14.8% 이하, 정확성은 94.2~108.5%로 확인되었다. Bendiocarb, carbaryl 및 carbofuran의 평균 회수율은 103.6~110.7%였다. 이때 상대표준편차는 모두 4.9% 이하로 확인되었다. 3가지 성분 각각의 검출한계는 0.8~1.5 ng/mL, 정량한계는 3.0~6.0 ng/mL로 확인되었다. 측정불확도를 산출한 결과 bendiocarb는 14.17±0.82 ㎎/㎏, carbaryl은 14.34±0.91 ㎎/㎏, carbofuran은 14.28±0.79 ㎎/㎏(신뢰수준 95%, K=2)으로 나타났다. 따라서 본 연구에서는 우유 중 bendiocarb, carbaryl 및 carbofuran의 정성 및 정량분석을 위해 유효성이 검증된 동시 분석법을 확립함으로써 향후 우유 중 잔류물질 기준규격 설정 및 관리에 참고 자료가 될 수 있다고 판단된다.

Maternal Transfer of Cetirizine Into Human Milk.

Cetirizine hydrochloride is a second-generation H1 histamine antagonist with Food and Drug Administration approval for treatment of allergic rhinitis and urticaria. Currently, the Food and Drug Administration does not recommend use of cetirizine during breastfeeding, as there are insufficient studies on both the transference of cetirizine into human milk and the effects of cetirizine in infants. To determine the concentration of cetirizine in human milk, samples were analyzed using high performance liquid chromatography mass spectrometry. Based on calculations, relative infant dose was found to be 1.77% at 24 hr. In addition, there were no reported adverse effects seen in the infants. We suggest that transfer of cetirizine into human milk is minimal and unlikely to pose a significant risk to the breastfeeding infant. This is the first report presenting the transfer of cetirizine in human milk.

Suspect screening of natural toxins in surface and drinking water by high performance liquid chromatography and high-resolution mass spectrometry

Besides anthropogenic contamination, freshwater environments can also be affected by the presence of natural toxins. Mycotoxins, plant toxins and cyanotoxins are the most relevant groups that can be found in the aquatic system. However, until now, only cyanotoxins have been more carefully studied. In the present work, single workflow for the assessment of natural toxins in waters, based on suspect screening and target screening of a selected group of toxins is presented. The approach is based on a triple-stage solid-phase extraction (SPE) able to isolate a wide range of natural toxins of different polarities, followed by liquid chromatography coupled to high-resolution mass spectrometry (HPLC-ddHRMS2) using a Q-Exactive Orbitrap analyser. The acquisition was performed in full-scan (FS) and data-dependant acquisition (ddMS2) mode, working under positive and negative mode. For the tentative identification, different on-line databases such as ChemSpider and MzCloud and an in-house natural toxins list with 2384 structures, that includes cyanotoxins, plant toxins and mycotoxins, were used. Also, thanks to the MS2 data, it was possible to achieve a high level of tentative identification confidence, but confirmation was only possible comparing the standards of the suspected compounds. For those, the analytical parameters of the developed method were also validated, and the quantification was possible by external calibration. Validation showed recoveries in the range between 53 and 95%, and method limits of detection (MDL) between 0.02 and 1.22 μg/L. This approach was applied to study natural toxins in 4 sampling sites along the Ter River in Catalonia (NE Spain). In this preliminary study 23 natural toxins were tentatively identified, and 9 of them confirmed (aflatoxin B1, anatoxin-a, nodularin, microcystin-LR, baicalein, kojic acid, cinchonine, B-asarone and atropine). The results of the quantification of these compounds showed concentrations below 1 μg/L in all cases, that is considered safe according to the actual legislation. This suspect screening approach allows a more comprehensive assessment of natural toxins in natural waters.

Development and Validation of Montelukast with Grape/Licorice Juices and its Application to Pharmacokinetic Studies by LC/MS

A simple, rapid and sensitive simultaneous method for validation and determination of Montelukast in rat plasma in the presence of grape and licorice juices has been done by using High Performance Liquid Chromatography–Mass Spectrometry (HPLC/MS). A mixture of (77.5 % methanol, 22.5 % of 0.2% FA in water) was used as a mobile phase, ACE 5 C8 column (50 X 2.1 mm, 5μ), and a flow rate of 0.1 ml/min were used, the auto-sampler injection volume was 5 microliters, and candesartan was used as an internal standard. According to the result obtained, the precision of predicted measurements for Montelukast was good (mean CV % 0.05) of grape on Montelukast Cmax (163.492 ng/ml ±113.27). Neither licorice gives significant decrease in the Montelukast Cmax (143.861ng/ml ±54.52), its only delay the average Montelukast Cmax to half an hour. Even for the AUC, there were no significant difference between Montelukast alone and Montelukast with grape, and Montelukast with licorice (P>0.05). The kinetic data shows that Montelukast plasma level was the same with both combination of Montelukast and grape, and Montelukast and licorice.

Delineating a novel metabolic high triglycerides-low waxes syndrome that affects lipid homeostasis in meibomian and sebaceous glands

Meibomian glands that are embedded in tarsal plates of human eyelids, and sebaceous glands found in the skin, including that of eyelids, are two related types of holocrine glands that produce lipid-rich secretions called meibum and sebum. Pervasive ocular disorders, such as Meibomian gland dysfunction and dry eye, have been linked to changes in the lipid composition of meibum. However, in most described cases the changes were either small, or random, or insufficiently characterized on the molecular level. Here, we present results of comprehensive lipidomic analyses of meibum, tears and sebum of a patient whose secretions were highly abnormal (abnormal meibum, tears and sebum, or AMTS, patient). The lipidomes were characterized on the level of individual lipid species using ultra-high performance liquid chromatography and high resolution mass spectrometry. The major differences between the AMTS patient and normal age- and gender-matched subjects included, among others, severely diminished pools of normal meibomian lipids such as wax esters and cholesteryl esters in meibum and tears, a 2x increase in total cholesteryl esters to wax esters ratio, their skewed molecular profiles, a ~3x increase in free cholesterol to cholesteryl esters ratio, and, most importantly, a 20x to 30x increase in the triglicerides fraction over the norm. Sebum of the AMTS patient was also highly abnormal lacking major wax esters. Notably, the routine blood lipid panel test of the AMTS patient showed no abnormalities. The data imply that the AMTS patient had a severe, previously unreported, metabolic disorder that affected meibogenesis in Meibomian glands and sebogenesis in sebaceous glands. This is, to the best of our knowledge, a first observation of the condition that we have termed High Triglycerides/Low Waxes (HTLW) syndrome.