Eco-Friendly, Simple, Fast, and Sensitive UPLC-MS/MS Method for Determination of Pexidartinib in Plasma and Its Application to Metabolic Stability

Pexidartinib is the first drug approved by the U.S. Food and Drug Administration specifically to treat the rare joint tumor tenosynovial giant cell tumor. In the current study, a validated, selective, and sensitive UPLC-MS/MS assay was developed for the quantitative determination of pexidartinib in plasma samples using gifitinib as an internal standard (IS). Pexidartinib and IS were extracted by liquid-liquid extraction using methyl tert-butyl ether and separated on an acquity BEH C18 column kept at 40 °C using a mobile phase of 0.1% formic acid in acetonitrile: 0.1% formic acid in de-ionized water (70:30). The flow rate was 0.25 mL/min. Multiple reaction monitoring (MRM) was operated in electrospray (ESI)-positive mode at the ion transition of 418.06 > 165.0 for the analyte and 447.09 > 128.0 for the IS. FDA guidance for bioanalytical method validation was followed in method validation. The linearity of the established UPLC-MS/MS assay ranged from 0.5 to 1000 ng/mL with r > 0.999 with a limit of quantitation of 0.5 ng/mL. Moreover, the metabolic stability of pexidartinib in liver microsomes was estimated.

Electrochemical Degradation of Carbamazepine: A Case Study of Quantification of “10, 11 Dihydro-10 Hydroxy Carbamazepine And 10, 11-Epoxycarbamazepine” As The Main By-Products Using LC-TOF/MS.

Carbamazepine (CBZ) is one of the most widely used antiepileptic drugs in Malaysia, so, it was detected in wastewater frequently. The electrochemical treatment process has been applied for the degradation of CBZ using graphite-PVC as an anode. However, two main by-products, namely, 10,11-dihydro10-hydroxy carbamazepine (HDX-CBZ) and 10,11-epoxycarbamazepine (EPX-CBZ) have been analysed and quantified using liquid chromatography-time of flight/mass spectrometry (LC-TOF/MS). HDX-CBZ and EPX-CBZ were analysed in positive ionisation mode and were separated chromatographically using 5 mm, 2 mm´150 mm C18 column at a flow rate of 0.3 mL/min. To improve sensitivity and detectability, SPE was applied as a pre-concentration step for the treated carbamazepine samples to extract and pre-concentrate HDX-CBZ and EPX-CBZ. However, three different solvents, namely, methyl tertiary butyl ether, acetone and methanol, have been optimized to enhance the recovery. The recovery was 85% and 92% for HDX-CBZ and EPX-CBZ, respectively, in the presence of methanol. The limit of quantification (LOQ) was 0.588 and 0.109 µg/L for both by-products, respectively. The concentration of HDX-CBZ and EPX-CBZ was 343 and 144 μg/L, respectively, after 20 min of treatment, then, it was decreased to 17.2 and 9.8 μg/L at 40 min. Finally, both by-products were eliminated after 60 min.

Deep metabolic profiling assessment of tissue extraction protocols for three model organisms

Metabolic profiling harbors the potential to better understand various disease entities such as cancer, diabetes, Alzheimer's, Parkinson's disease or COVID-19. Deciphering these intricate pathways in human studies requires large sample sizes as a means of reducing variability. While such broad human studies have discovered new associations between a given disease and certain affected metabolites, i.e. biomarkers, they often provide limited functional insights. To design more standardized experiments, reduce variability in the measurements and better resolve the functional component of such dynamic metabolic profiles, model organisms are frequently used. Standardized rearing conditions and uniform sampling strategies are prerequisites towards a successful metabolomic study. However, further aspects such as the choice of extraction protocol and analytical technique can influence the outcome drastically. Here, we employed a highly standardized metabolic profiling assay analyzing 630 metabolites across three commonly used model organisms (Drosophila, mouse and Zebrafish) to find the optimal extraction protocols for various matrices. Focusing on parameters such as metabolite coverage, metabolite yield and variance between replicates we compared seven extraction protocols. We found that the application of a combination of 75% ethanol and methyl tertiary-butyl ether (MTBE), while not producing the broadest coverage and highest yields, was the most reproducible extraction protocol. We were able to determine up to 530 metabolites in mouse kidney samples, 509 in mouse liver, 422 in Zebrafish and 388 in Drosophila and discovered a core overlap of 261 metabolites in these four matrices. To enable other scientists to search for the most suitable extraction protocol in their experimental context and interact with this comprehensive data, we have integrated our data set in the open-source shiny app MetaboExtract. This will enable scientists to search for their metabolite or metabolite class of interest, compare it across the different tested extraction protocols and sample types as well as find reference concentrations.

EFFECT OF AN EXTRAGENT ON THE COMPOSITION OF THE LIPOPHILIC CONSTITUENTS OF THE EXTRACTS OF RHODIOLA ROSEA L. AND ON THE EXTRACTS ACTIVITY

The composition of the plant Rhodiola rosea L. lipophylic substances was studied. Acidic and neutral components were identified by gas-chromatography-mass-spectrometry. With methyl-tert-butyl ether (MTBE) as an extractant instead of the volatile solvent diethyl ether, lipophylic extract was obtained. Methyl-tert-butyl ether used as an extraction solvent for raw materials has all the advantages of diethyl ether, being free of its disadvantages. It does not form peroxides or produce elevated partial gas pressure due to its higher boiling point. As a result, comparison with databases identified some triterpene, phenolic and aliphatic acids with chain lengths 12 to 30 carbon atoms, including saturated, unsaturated, and dibasic acids. In addition to the components known from the literature, more than 50 triterpene and aliphatic compounds were detected in the unsaponifiable residue and acidic fractions for the first time. The hexane extract and the product obtained by the stepwise extraction of MTBE after the extraction of low-polarity compounds with hexane were investigated in a similar way. In the case of an extract obtained using MTBE after the extraction of low-polarity components with hexane, there was shown a more efficient extraction of benzoic and cinnamic acids compared to the exhaustive extraction of MTBE. These acids are absent in the hexane extract. Ethanol extraction was also carried out to test bioactivity: exhaustive and after hexane and MTBE extraction. Extracts obtained using MTBE and ethanol showed anti-virus activity against Ebola psevdovirus.

Novel Extraction Method for Combined Lipid and Metal Speciation From Caenorhabditis elegans With Focus on Iron Redox Status and Lipid Profiling

Parkinson´s disease progression is linked to iron redox status homeostasis via reactive oxygen species (ROS) formation, and lipids are the primary targets of ROS. The determination of iron redox status in vivo is challenging and requires specific extraction methods, which are so far tedious and very time-consuming. We demonstrated a novel, faster, and less laborious extraction method using the chelator ethylene glycol l-bis(β-aminoethyl ether)-N,N,N′,N′-tetra acetic acid (EGTA) as a stabilizing agent and synthetic quartz beads for homogenization under an argon atmosphere. Additionally, we combined the metal extraction with a well-established lipid extraction protocol using methyl-tert-butyl ether (MTBE) to avoid the problems of lipid precipitation in frozen samples and to determine lipid profiles and metal species from the same batch. The nonextractable matrix, such as the debris, is removed by centrifugation and digested to determine the total metal content of the sample as well. Lipid profiling using RP-LC–MS demonstrated high accordance of the modified extraction method to the reference method, and the organic solvent does not affect the iron redox status equilibrium. Furthermore, rigorous testing demonstrated the stability of the iron redox status equilibrium during the extraction process, secured by complexation, inert atmosphere, fast preparation, and immediately deep frozen extracts.