Evaluation of Sample Preparation Methods for Non-Target Screening of Organic Micropollutants in Urban Waters Using High-Resolution Mass Spectrometry

Non-target screening (NTS) has gained interest in recent years for environmental monitoring purposes because it enables the analysis of a large number of pollutants without predefined lists of molecules. However, sample preparation methods are diverse, and few have been systematically compared in terms of the amount and relevance of the information obtained by subsequent NTS analysis. The goal of this work was to compare a large number of sample extraction methods for the unknown screening of urban waters. Various phases were tested for the solid-phase extraction of micropollutants from these waters. The evaluation of the different phases was assessed by statistical analysis based on the number of detected molecules, their range, and physicochemical properties (molecular weight, standard recoveries, polarity, and optical properties). Though each cartridge provided its own advantages, a multilayer cartridge combining several phases gathered more information in one single extraction by benefiting from the specificity of each one of its layers.

Epidemiological Characterization and Determination of TP53 and IGHV Mutational Status of a Large Series of Previously-Untreated Chronic Lymphocytic Leukemia (CLL) Patients in Spain: The Epicll Study

Introduction: Among the genetic lesions described in chronic lymphocytic leukemia (CLL), TP53 and IGHV mutational status are well-established prognostic biomarkers. While mutations resulting in dysregulation of TP53 are associated with chemo-resistance, mutated IGHV (IGHV-M) identifies a good prognosis and unmutated (IGHV-UM) is associated with an aggressive clinical outcome. Thus, molecular assessment of TP53 and IGHV mutational status is recommended to make treatment decisions. Moreover, 30% of CLL patients have a highly homologous complementarity-determining region 3 (CDR3), allowing their classification in subsets based on the stereotypical B-cell receptor immunoglobulins (BcR IG), which have been associated with different clinical features and outcomes. This study aimed to assess the mutational status of TP53 and IGHV and the frequency of stereotypical BcR IG subsets, including CLL#2 and CLL#4 associated with poor and good prognosis, respectively, in a large series of CLL patients in Spain. Methods: Observational, retrospective, cross-sectional, multicentric study of data from the RED53 project, a collaborative network between the Spanish Group of CLL (GELLC) and Janssen for the characterization of TP53 and IGVH mutational status in naïve CLL candidate patients to receive treatment. Blood samples from 225 institutions were collected between May 2016 and March 2021. Included patients had confirmed diagnosis of CLL and required first-line treatment. Basic demographic variables, leukocyte and lymphocyte counts, and the number of clonal CD5 +/CD19 + lymphocytes were recorded at sample extraction. Clonotypic IGHV-IGHD-IGHJ gene rearrangements and exons 4 to 10 of TP53 were amplified by PCR and sequenced (Sanger). Four analytical reference centers qualified by the European Initiative for CLL (ERIC) determined the mutational status following the ERIC guidelines. Results: A total of 1097 samples from patients with a median (range) age of 70.0 (27-97) years were analyzed. At sample extraction, patients had a median (range) of 54.5 (2-516) x10 9 leukocytes/mL and 46.1 (0-8810) x10 9 lymphocytes/mL, of which a median (range) of 80.0 (1-100) % (n=754) were clonal CD5 +/CD19 + lymphocytes. The most frequent indications for treatment initiation were progressive/tumoral adenopathy (n=525, 50.4%), progressive lymphocytosis (n=429, 41.2%), cytopenia (n=369, 35.4%), and systemic constitutional symptoms (n=252, 24.2%). Median (IQR) age was 63.0 (55.0, 71.0) years at diagnosis and 70.0 (62.0, 77.0) years at treatment onset. Median (range) time from diagnosis to treatment was 2.7 (0.6-6.1) years. Among 1097 patients, 100 (9.2%) had TP53 mutations with 103 variants, of which only 3 (3.0%) had 2 mutations. Of the 103 mutations, 91 (88.3%), 9 (8.7%), and 3 (2.9%) were pathogenic, likely pathogenic, and of uncertain significance, respectively. Fig. 1 shows mutation localization and type. IGHV was UM in 58% (471/812) and M in 33.1% (269/812) of patients, and unknown/undetermined in 1.8% (15/812), non-productive in 3.2% (26/812), and borderline in 3.8% (31/812) of patients. IGHV rearrangements were undetected in 25.6% (279/1091) of patients and 65.7% (717/1091), 8.5% (93/1091), and 0.2% (2/1091) had 1, 2, and 3 rearrangements, respectively . Of the 30 patients with IGHV3-21 rearrangements, 18 had available data, of which all had CLL#2 subset and, of the 15 patients with IGHV-M, 5 (33.3%) had CLL#2. Minor subsets were found in 7 (46.7%) and 17 (33.3%) of IGHV-M and UM, respectively. The most frequent stereotyped BcR IG subsets were CLL#2, CLL#1, and CLL#6, in 24.3% (18/74), 23% (17/74), and 10.8% (8/74) of patients, respectively. Among the 60 patients with mutated TP53 and IGHV mutational study available, 66.7% (40/60) had IGHV-UM. Conclusions: In our real-world experience, results regarding TP53 M and IGHV UM (9.2% and 58.0% of patients, respectively) are similar to those reported in previous series of patients requiring first-line treatment, with a slightly higher predominance of IGHV-UM over IGHV-M cases. Subset CLL#2 was the most frequently identified, whereas the frequency of CLL#6 was higher than that reported before. Considering the difficulties associated with the analysis of TP53 and IGHV mutational status of most laboratories diagnosing CLL, the RED53 network allows access to these determinations to naïve CLL patients with active disease by a simple, fast, and standardized method. Figure 1 Figure 1. Disclosures Terol: Roche: Consultancy; Gilead: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Travel, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Other: Travel, Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Other: Travel; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Travel; Roche: Membership on an entity's Board of Directors or advisory committees, Other: Travel; BMS: Consultancy; Hospital Clinico Valencia: Current Employment. Ferrer Lores: Janssen: Membership on an entity's Board of Directors or advisory committees. Bosch: Roche: Membership on an entity's Board of Directors or advisory committees, Other: Travel; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel, Research Funding; AbbVie: Membership on an entity's Board of Directors or advisory committees, Other: Travel; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel; TAKEDA: Membership on an entity's Board of Directors or advisory committees, Other: Travel. Gonzalez Diaz: Astra Zeneca: Membership on an entity's Board of Directors or advisory committees, Other: Lectures; Gilead: Membership on an entity's Board of Directors or advisory committees, Other: Lectures; Roche: Membership on an entity's Board of Directors or advisory committees, Other: Lectures; Janssen: Membership on an entity's Board of Directors or advisory committees, Other: Lectures; Abbvie: Membership on an entity's Board of Directors or advisory committees, Other: Lectures. Crespo: Janssen: Consultancy; Astra Zeneca: Research Funding; Roche/Genentech: Research Funding. Alcoceba: Janssen: Consultancy. Esteve: Janssen: Current Employment. Loriente: Janssen: Current Employment. Villanueva: Janssen: Current Employment.

PATH-45. APOLLO: RAMAN-BASED PATHOLOGY OF MALIGNANT GLIOMA

BACKGROUND DNA methylation is an essential component for integrative diagnosis in glioma. Methylation subtype prediction of gliomas is currently done via sample extraction of high-quality of reasonable amount of DNA (~1ug), methylome profiling, followed by probe identification, curation and subsequent analysis via different random forest classifiers. However, the DNA methylation classification is not always available for all the samples. METHODS Raman Spectroscopy performed of the regions of interest using 1mm2 FFPE tissue spots from 45 patient samples with LGm1 to LGm6 methylation subtypes. Spectral information was then used to train a convolutional neural network (CNN) and develop a prediction algorithm. 70 % of dataset - model training while the remaining 30% for validation. Supervised wrapper methods and random forests were used to identify the top 109 most discriminatory Raman frequencies out of 1738. RESULTS We identified the most discriminatory features from these analyses and demonstrated that these frequencies show differential spectral intensities for these frequencies depending upon the glioma subtypes across the larger areas of the tissue. We compared the results of the Ward linkage clustering with the separation induced by the “frequency criterion”, an empirical observation that Raman spectra of tumor spots are characterized by intensities higher than 5000 on some of the frequencies from 1463 to 1473. For each of the 45 samples we ran Ward linkage clustering with a variable number of clusters (from 2 to 7), with the majority cluster corresponding to tumor spots and the others corresponding to (various types of) non-tumor spots. We found that the majority cluster matches very well the tumor spots characterized by the frequency criterion, The average accuracy over all samples was 90:3%, the average precision was 99:6% and the average recall was 90:2%. For most samples, two clusters were sufficient to distinguish between tumor and non-tumor spots with accuracy.

Review on in vivo profiling of drug metabolites with LC-MS/MS in the past decade

Metabolite profiling is an indispensable part of drug discovery and development, enabling a comprehensive understanding of the drug's metabolic behavior. Liquid chromatography-mass spectrometry facilitates metabolite profiling by reducing sample complexity and providing high sensitivity. This review discusses the in vivo metabolite profiling involving LC-MS/MS and the utilization of QTOF, QQQ mass analyzers with a particular emphasis on a mass filter. Further, a summary of sample extraction procedures in biological matrices such as plasma, urine, feces, serum and hair as in vivo samples are outlined. toward the end, we present 15 case studies in biological matrices and their LC-MS/MS conditions to understand the metabolic disposition.

Optimization of lipids extraction from Moringa oleifera seeds for biodiesel feedstock

This paper explore the mechanism of lipid extraction efficiency on Moringa oleifera seeds using Soxhlet extraction method. This present study essential to determine the effect of particle size of the sample, extraction time and type of solvent applied towards the efficiency in extracting the lipids from the material. Soxhlet extraction method utilizing Buchi B118 was use in this study and response surface method was applied to analysed the data and determine the optimum parameter condition to obtain the highest yield of Moringa oil extraction. Moringa oil derived from Moringa oleifera seeds was converted into biodiesel (FAME) via Transesterification process. Conversion of Moringa FAME was observed using three different alcohol oil to molar ratio by based-catalysed. This study shows significant strong correlations between particle size of the sample, extraction time and type of solvent use towards extraction yield. The Response surface analysis shows that 1.3611 mm particle size of sample, 3 hours of extraction time and hexane as extraction solvent was the optimum condition in order to get the highest yield of lipids extract from both Moringa oleifera samples. Authentication extraction based on RSM recommendation showed an average Moringa oil yield of 39.75 % by weight.

A Study of the Interaction of Ixabepilone As Anticancer Drug With Acetoxymercuric Fluorescein Reagent by Fluorescence Quenching Approach: A Validated Method

A spectrofluorimetric approach has been developed and validated for determination of sulfur-containing drug; ixabepilone in raw powder, vials and human plasma. This approach studies the quenching effect of IXA on the fluorescence intensity of acetoxymercuric fluorescein (AMF) reagent at λem of 530 nm and λex of 500 nm. All the parameters that can affect the reaction as pH, AMF solution concentration, temperature, time and solvents were studied and optimized. The linearity range of the studied approach was 20-100 ng mL-1 with correlation coefficient of (r = 0.9998). The proposed approach was validated and approved regarding to ICH guidelines in terms of accuracy, precision, linearity, LOD and LOQ, with mean percentage recovery of 99.79 and RSE% of 1.64. The previously obtained resultes were already statistically compared with that of established reported methods indicating no significant differences in accuracy and precision. Finally, the proposed approach is easy, sensitive, and inexpensive so it is suitable for routine determination of IXA in raw powder, vials and human plasma with no need for any prior separation or sample extraction.

Biological Significance of the Protein Changes Occurring in the Cerebrospinal Fluid of Alzheimer’s Disease Patients: Getting Clues from Proteomic Studies

The fact that cerebrospinal fluid (CSF) deeply irrigates the brain together with the relative simplicity of sample extraction from patients make this biological fluid the best target for biomarker discovery in neurodegenerative diseases. During the last decade, biomarker discovery has been especially fruitful for the identification new proteins that appear in the CSF of Alzheimer’s disease (AD) patients together with amyloid-β (Aβ42), total tau (T-tau), and phosphorylated tau (P-tau). Thus, several proteins have been already stablished as important biomarkers, due to an increase (i.e., CHI3L1) or a decrease (i.e., VGF) in AD patients’ CSF. Notwithstanding this, only a deep analysis of a database generated with all the changes observed in CSF across multiple proteomic studies, and especially those using state-of-the-art methodologies, may expose those components or metabolic pathways disrupted at different levels in AD. Deep comparative analysis of all the up- and down-regulated proteins across these studies revealed that 66% of the most consistent protein changes in CSF correspond to intracellular proteins. Interestingly, processes such as those associated to glucose metabolism or RXR signaling appeared inversely represented in CSF from AD patients in a significant manner. Herein, we discuss whether certain cellular processes constitute accurate indicators of AD progression by examining CSF. Furthermore, we uncover new CSF AD markers, such as ITAM, PTPRZ or CXL16, identified by this study.

Estimating Occupational Exposure to VOCs, SVOCs, Particles and Participant Survey Reported Symptoms in Central Thailand Rice Farmers Using Multiple Sampling Techniques

Thailand is known for its agricultural productivity and rice exportation. Most farms use small machines and manual labor, creating potential exposure to multiple health hazards. A cross-sectional study was conducted to measure pollutants liberated during preparation, pesticide application, and harvesting. Thirty rice farmers, mostly males from 41 to 50 years old, participated. The participant survey data showed that 53.3% of the respondents spent >2 h per crop on preparation, <1 h on pesticide application, and about 1–2 h harvesting; 86.7% of the respondents maintained and stored mechanical applicators at home, suggesting possible after-work exposures. Gloves, fabric masks, boots, and hats were worn during all activities, and >90% wore long sleeved shirts and pants. VOCs and SVOCs were collected using charcoal tubes and solid phase micro sample extraction (SPME). An analysis of the charcoal and SPME samplers found that 30 compounds were detected overall and that 10 were in both the charcoal tubes and SPME samplers. The chemicals most often detected were 1, 1, 1 Trichloro ethane and xylene. Additionally, farmers experienced the highest exposure to particulates during harvesting. These results demonstrated that farmers experience multiple exposures while farming and that risk communication with education or training programs may mitigate exposure.