Hydrophilic Phase Extraction Research Articles

Hydrophilic-Phase Extraction: A New Avenue of Solid-Phase Extraction for Oligonucleotide Bioanalysis

A novel mode of bioanalytical solid-phase extraction (SPE) has been developed and used for a quantitative application involving an RNA oligonucleotide therapeutic candidate. Previously established protocols and media showed very little recovery, and so this new approach, based on hydrophilic interaction liquid chromatography (HILIC) with an aminopropyl-bonded phase on a silica support and providing an essentially uncharted avenue of selectivity, was explored. The procedure featured only one step prior to sample load, high-organic sorbent conditioning, two washes following the load that switched between low and high pH, followed by elution in an a highly eluotropic HILIC composition. The moniker of “hydrophilic-phase extraction” (HPE) was given to the procedure. The HPE was proven to be a critical part of a fully quantitative HILIC–high-resolution mass spectrometry (HRMS) method for the RNA oligonucleotide and has potential for general application.

Novel hydrophilic-phase extraction, HILIC and high-resolution MS quantification of an RNA oligonucleotide in plasma.

Aim: In the theme of quantitative LC-MS bioanalysis of oligonucleotides free of ion-pairing, a 22-mer RNA oligonucleotide took center stage. The focus was on a unique polar-based retention scheme to produce a high-recovery extraction presenting a high-performance alternative extraction means, also there was the opportunity to involve hydrophilic-interaction liquid chromatography and contemporary high-resolution MS as the end point. Results: Original LC-MS methodology was developed for the oligonucleotide and the performance was robust for both nominal and accurate mass detection, the latter affording 10× improvement in sensitivity and 4000-fold linear dynamic range, 500pM to 2000nM. Conclusion: A novel means of solid-phase extraction is exhibited within a robust pair-free methodology, reaching pM sensitivity with the demonstrably beneficial accurate mass platform.

Effects of titanium dioxide nanoparticles on fecal metabolome in rats after oral administration for 90 days

To explore the effects and related mechanisms of oral exposure titanium dioxide nanoparticles (TiO2 NPs) for 90 days on the intestinal and the gut microbiota of rats, through fecal metabolomics. Twelve 4-week-old clean-grade Sprague Dawley (SD) rats were randomly de-vided into 2 groups by body weight, treated with TiO2 NPs at dose of 0 or 50 mg/kg body weight everyday respectively for 90 days. The solution of each infection was freshly prepared and shocked fully by ultrasonic. Characterization of the particle size, crystal form, purity, and specific surface area of TiO2 NPs was conducted. And the fresh feces of the rats were collected on the 90th day. After lyophilized and hydrophilic phase extraction, ultra performance liquid chromatography-Q-exactive orbitrap-high-resolution mass spectrometry system (UPLC-QEMS) was utilized for non-targeted determination of fecal meta-bolites. The metabolites were identified and labeled through Compound Discoverer 3.0 software, and used for subsequent metabolomics analysis. Bioinformatics analysis was carried out including unsupervised principal component analysis and supervised orthogonal projection to latent structure discriminant analysis for the differential metabolites between the two groups. The differential metabolites were followed-up for Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. Compared with the control group, the body weight of the rats was significantly reduced (P<0.05) in the treatment group. A total of 22 metabolites in fecal metabolomics showed significant changes. Among them, xanthine, 1-methyladenine, 3-hydroxypyridine, methionine sulfoxide, pyridoxine, 1,5-isoquinolinediol, N-acetylornithine, N-acetyl-D-galactosamine, L-citrulline, L-methionine, leucine, DL-tryptophan, L-ornithine, 4-methyl-5-thiazoleethanol, and L-glutamic acid totaled 15 metabolites increased significantly. N-acetylhistamine, D-pipecolinic acid, imidazolelactic acid, L-valine, 2,3,4,6-tetramethylpyrazine, caprolactam, and histamine totaled 7 metabolites decreased significantly. N-acetylhistamine, L-valine and methionine sulfoxide were changed more than 16 times. Analysis of KEGG pathway revealed that the two metabolic pathways arginine biosynthesis and aminoacyl-tRNA biosynthesis were significantly changed (false discover rate < 0.05, pathway impact > 0.1). Oral exposure to TiO2 NPs for 90 days could disrupt the metabolism of the intestine and gut microbiota, causing significant changes in metabolites and metabolic pathways which were related to inflammatory response, oxidative stress, glucose homeostasis, blood system and amino acid homeostasis in rat feces. It is suggested that the toxic effect of TiO2 NPs on rats may be closely related to intestinal and gut microbiota metabolism.