ABSTRACT
A sensitive and accurate hydrophilic interaction liquid chromatography - tandem mass spectrometry method (HILIC-MS/MS) was developed and validated for the determination of phenylephrine concentration in Dried Blood Spot (DBS) samples from preterm infants, after ocular administration of an ophthalmic solution with phenylephrine. Sample preparation involved the extraction of the analyte from an 85 µL DBS sample with methanol - acetonitrile (50:50, v/v). Chromatographic separation was achieved on an ACQUITY UPLC BEH AMIDE column, under isocratic conditions within a 5 min run. Detection was achieved with a triple quadrupole MS applying electrospray ionization in positive mode. The method was fully validated and proved precise and accurate with in a linear range of 0.59-3.53 ng/ml in blood. The method was developed to provide insights on the level of exposure of infant population to phenylephrine after ocular administration.
Subject(s)
Chromatography, High Pressure Liquid/methods , Dried Blood Spot Testing/methods , Eye Diseases, Hereditary/diagnosis , Infant, Newborn, Diseases/diagnosis , Infant, Premature/blood , Mydriasis/diagnosis , Mydriatics/blood , Phenylephrine/blood , Tandem Mass Spectrometry/methods , Eye Diseases, Hereditary/blood , Female , Humans , Infant , Infant, Newborn , Infant, Newborn, Diseases/blood , Male , Mydriasis/blood , Mydriatics/administration & dosage , Ophthalmic Solutions , Phenylephrine/administration & dosageABSTRACT
A sensitive, accurate and precise method was developed for the quantification of a large number of organic acids in human urine by GC-MS/MS. The analytes were selected based on their role as key metabolic intermediates; intermediates of Krebs cycle, fatty acid oxidation, glycolysis, down-stream metabolites of neurotransmitter synthesis and degradation, metabolites indicative of nutritional deficiencies, byproducts of microbial activity in the gastrointestinal tract (GI) etc. The most efficient sample preparation protocol was selected based on tests for extraction with different solvents such as MTBE and ethyl acetate under acidic conditions, whereas finally a more general protocol was applied with methanol. Regarding derivatization, methoxyamine with MSTFA, 1% TMCS was applied. The method was extensively validated, including stability study, ensuring accurate determination of the studied organic acids in human urine. Proof of its utility was exhibited in a set of samples from human volunteers. The method can find wide applicability in the context of metabolomics for clinical or nutritional studies.
Subject(s)
Metabolomics , Tandem Mass Spectrometry , Gas Chromatography-Mass Spectrometry , HumansABSTRACT
Shikonin and its enantiomer alkannin, which are natural products, have been extensively studied in vitro and in vivo for, among others, their antitumor activity. The investigation of the molecular pathways involved in their action is of interest, since they are not yet clearly defined. Metabolic profiling in cells can provide a picture of a cell's phenotype upon intervention, assisting in the elucidation of the mechanism of action. In this study, the cytotoxic effect of shikonin on a human hepatocarcinoma cell line was studied. Huh7 cells were treated with shikonin at 5 µM, and it was found that shikonin markedly inhibited cell growth. Metabolic profiling indicated alterations in the metabolic content of the cells and the culture media upon treatment, detecting the metabolic response of the cells. This study demonstrates the potential of metabolomics to improve knowledge on the mechanisms involved in shikonin's antitumor action.
Subject(s)
Carcinoma, Hepatocellular/metabolism , Liver Neoplasms/metabolism , Metabolomics/methods , Naphthoquinones/pharmacology , Carcinoma, Hepatocellular/drug therapy , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , Chromatography, Liquid , Humans , Liver Neoplasms/drug therapy , Metabolome/drug effects , Signal Transduction/drug effects , Tandem Mass SpectrometryABSTRACT
AIM: Till now there is very limited knowledge on the molecular content of coelomic fluid and cells. This study presents the first attempt to elucidate the metabolic profile of such samples. METHODOLOGY: Samples were collected via coelocentesis from 41 women during the first trimester of gestation. Metabolic content was assessed using four different analytical platforms. For targeted analysis a hydrophilic interaction chromatography ultra high performance LC-MS/MS method was applied. Holistic analysis performed by GC-MS, NMR spectroscopy and ion cyclotron ultra-high resolution MS (FT-ICR-MS) instrumentation. RESULTS & CONCLUSIONS: Our observations suggest coelomic fluid and cells as promising biosamples, rich in metabolites with potential use in mammalian system biology studies.