6 research outputs found
Quali–Quantitative Characterization of Volatile and Non-Volatile Compounds in Protium heptaphyllum (Aubl.) Marchand Resin by GC–MS Validated Method, GC–FID and HPLC–HRMS2
Protium heptaphyllum (Aubl.) Marchand (PH) trees are endemic to the tropical region of South America, mostly Brazil. Antibacterial, antinociceptive, anti-inflammatory, anxiolytic, antidepressant and anti-hyperlipidemic/anti-hypercholesterolemic effects were reported for its resinous exudate Protiumheptaphyllum resin (PHR). This work aims to provide a qualitative and quantitative consistent chemical profiling of the major constituents of this resin and two extracts enriched in acid (acidic triterpene concentrated extract, ATCE) and neutral triterpenes (α and β-amyrin concentrated extract, AMCE). GC–MS/GC–FID was used for volatile terpene fraction, a validated GC–MS method was developed for quantification of neutral α and β-amyrin and HPLC–APCI HRMS2 was used for acidic triterpenes analysis. The chemical investigation reported 29 molecules, including 14 volatile terpenes, 6 neutral triterpenes and 11 acid triterpenes. The most abundant compounds were α-amyrin (251.28 g kg−1, 123.98 g kg−1 and 556.82 g kg−1 in PHR, ATCE and AMCE, respectively), β-amyrin (172.66 g kg−1, 95.39 g kg−1 and 385.58 g kg−1 in PHR, ATCE and AMCE, respectively), 3-oxo-tirucalla-7,24-dien-21-oic acid (80.64 g kg−1, 157.10 g kg−1 and 15.31 g kg−1 in PHR, ATCE and AMCE, respectively) and 3α-hydroxy-tirucalla-8,24-dien-21-oic acid (77.71 g kg−1, 130.40 g kg−1 and 11.64 g kg−1 in PHR, ATCE and AMCE, respectively). Results showed specific enrichment of acidic and neutral triterpenoids in the two respective extracts
Development and application of high resolution mass spectrometry analytical method to study and identify the photoinduced transformation products of environmental pollutants
Release of Selected Non-Intentionally Added Substances (NIAS) from PET Food Contact Materials: A New Online SPE-UHPLC-MS/MS Multiresidue Method
Food contact materials (FCMs) are an underestimated source of food chemical contaminants and a potentially relevant route of human exposure to chemicals that are harmful to the endocrine system. Foods and water are the main sources of exposure due to contact with the packaging materials, often of polymeric nature. European Regulation 10/2011 requires migration tests on FCMs and foodstuffs to evaluate the presence of listed substances (authorized monomers and additives) and non-intentionally added substances (NIAS) not listed in the regulation and not subjected to restrictions. The tests are required to ensure the compliance of packaging materials for the contained foods. NIAS are a heterogeneous group of substances classified with a potential estrogenic or androgenic activity. Subsequently, the evaluation of the presence of these molecules in foods and water is significant. Here we present an online SPE/UHPLC-tandem MS method to quantify trace levels of NIAS in food simulants (A: aqueous 3% acetic acid; B: aqueous 20% ethanol) contained in PET preformed bottles. The use of online SPE reduces systemic errors thanks to the automation of the technique. For the developed analytical method, we evaluate the limit of detection (LOD), the limit of quantitation (LOQ), selectivity, RSD% and BIAS% for LLOQ for a total of twelve NIAS, including monomers, antioxidants, UV-filters and additives. LOD ranged between 0.002 µg/L for bisphenol S and 13.6 µg/L for 2,6-di-tert-butyl-4-methylphenol (BHT). LOQs are comprised between 0.01 µg/L for bisphenol S and 42.2 µg/L for BHT. The online-SPE/UHPLC-tandem MS method is applied to the food simulants contained in several types of PET packaging materials to evaluate the migration of the selected NIAS. The results show the presence (µg/L) of NIAS in the tested samples, underlining the need for a new regulation for these potentially toxic molecules
Quantitation of endogenous GnRH by validated nano-HPLC-HRMS method: a pilot study on ewe plasma
International audienceGonadotropin-releasing hormone isoform I (GnRH), a neuro-deca-peptide, plays a fundamental role in development and maintenance of the reproductive system in vertebrates. The anomalous release of GnRH is observed in reproductive disorder such as hypogonadotropic hypogonadism, polycystic ovary syndrome (PCOS), or following prenatal exposure to elevated androgen levels. Quantitation of GnRH plasma levels could help to diagnose and better understand these pathologies. Here, a validated nano-high-performance liquid chromatography–high-resolution mass spectrometry (HPLC-HRMS) method to quantify GnRH in ewe plasma samples is presented. Protein precipitation and solid-phase extraction (SPE) pre-treatment steps were required to purify and enrich GnRH and internal standard ( lamprey -luteinizing hormone-releasing hormone-III, l -LHRH-III). For the validation process, a surrogate matrix approach was chosen following the International Council for Harmonisation (ICH) and FDA guidelines. Before the validation study, the validation model using the surrogate matrix was compared with those using a real matrix such as human plasma. All the tested parameters were analogous confirming the use of the surrogate matrix as a standard calibration medium. From the validation study, limit of detection (LOD) and limit of quantitation (LOQ) values of 0.008 and 0.024 ng/mL were obtained, respectively. Selectivity, accuracy, precision, recovery, and matrix effect were assessed with quality control samples in human plasma and all values were acceptable. Sixteen samples belonging to healthy and prenatal androgen (PNA) exposed ewes were collected and analyzed, and the GnRH levels ranged between 0.05 and 3.26 ng/mL. The nano-HPLC-HRMS developed here was successful in measuring GnRH, representing therefore a suitable technique to quantify GnRH in ewe plasma and to detect it in other matrices and species
Sequencing intact membrane proteins using MALDI mass spectrometry
Membrane proteins are key players in many cellular events and represent crucial drug targets. Matrix-assisted laser desorption ionization mass spectrometry (MALDI MS) is a valuable approach to investigate them. To our knowledge, there are only a few reports of sequencing small membrane proteins using MALDI in-source decay (ISD). We report the successful fragmentation and sequencing of membrane proteins up to 46Â kDa by MALDI-ISD. We have 1) investigated key MALDI parameters that influence the sequencing of a soluble protein; 2) used atomic force microscopy to observe our samples and correlate their topological features with MALDI data, which allowed us to optimize fragmentation conditions; 3) sequenced N- and C-termini of three membrane proteins (SpoIIIAF, TIM23, and NOX), solubilized in three different ways. Our results indicate that detergent and buffer type are of key importance for successful MALDI-ISD sequencing. Our findings are significant because sequencing membrane proteins enables the unique characterization of challenging biomolecules. The resulting fragmentation patterns provide key insights into the identity of proteins, their sequences, modifications, and other crucial information, such as the position of unexpected truncation
DataSheet1_In vitro characterization of 3D culture-based differentiation of human liver stem cells.pdf
Introduction: The lack of functional hepatocytes poses a significant challenge for drug safety testing and therapeutic applications due to the inability of mature hepatocytes to expand and their tendency to lose functionality in vitro. Previous studies have demonstrated the potential of Human Liver Stem Cells (HLSCs) to differentiate into hepatocyte-like cells within an in vitro rotary cell culture system, guided by a combination of growth factors and molecules known to regulate hepatocyte maturation. In this study, we employed a matrix multi-assay approach to comprehensively characterize HLSC differentiation.Methods: We evaluated the expression of hepatic markers using qRT-PCR, immunofluorescence, and Western blot analysis. Additionally, we measured urea and FVIII secretion into the supernatant and developed an updated indocyanine green in vitro assay to assess hepatocyte functionality.Results: Molecular analyses of differentiated HLSC aggregates revealed significant upregulation of hepatic genes, including CYP450, urea cycle enzymes, and uptake transporters exclusively expressed on the sinusoidal side of mature hepatocytes, evident as early as 1Â day post-differentiation. Interestingly, HLSCs transiently upregulated stem cell markers during differentiation, followed by downregulation after 7Â days. Furthermore, differentiated aggregates demonstrated the ability to release urea and FVIII into the supernatant as early as the first 24Â h, with accumulation over time.Discussion: These findings suggest that a 3D rotation culture system may facilitate rapid hepatic differentiation of HLSCs. Despite the limitations of this rotary culture system, its unique advantages hold promise for characterizing HLSC GMP batches for clinical applications.</p