A highly sensitive platform coupling capillary ion chromatography (Cap IC) with Q Exactive mass spectrometer has been developed for metabolic profiling of head and neck squamous cell carcinoma (HNSCC) cells. The Cap IC allowed an excellent separation of anionic polar metabolites, and the sensitivities increased by up to 100-fold compared to reversed-phase liquid chromatography and hydrophilic interaction chromatography performed at either high- or capillary-flow rates. The detection limits for a panel of standard metabolites were between 0.04 to 0.5 nmol/L (0.2 to 3.4 fmol) at a signal-to-noise ratio of 3. This platform was applied to an untargeted metabolomic analysis of head and neck cancer cells and stem-like cancer cells. Differential metabolomics analysis identified significant changes in energy metabolism pathways (e.g., glycolysis and tricarboxylic acid cycle). These experiments demonstrate Cap IC/MS as a powerful metabolomics tool by providing enhanced separation and sensitivity of polar metabolites combined with high resolution and accurate mass measurement (HR/AM) capabilities to differentiate isobaric metabolites.
In this study, we have demonstrated a targeted metabolomics method for analysis of cancer cells, based on high-performance ion chromatography (IC) separation, Q Exactive HF MS for high-resolution and accurate-mass (HR/AM) measurement and the use of stable isotope-labeled internal standards for absolute quantitation. Our method offers great technical advantages for metabolite analysis, including exquisite sensitivity, high speed and reproducibility, and wide dynamic range. The high-performance IC provided fast separation of cellular metabolites within 20 min and excellent resolving power for polar molecules including many isobaric metabolites. The IC/Q Exactive HF MS achieved wide dynamic ranges of 5 orders of magnitude for six targeted metabolites, pyruvate, succinic acid, malic acid, citric acid, fumaric acid, and alpha-ketoglutaric acid, with R(2) ≈ 0.99. Using this platform, metabolites can be simultaneously quantified from low fmol/μL to nmol/μL levels in cellular samples. The high flow rate IC at 380 μL/min has shown excellent reproducibility for a large set of samples (150 injections), with minimal variations of retention time (SD < ± 0.03 min). In addition, the IC-MS-based approach acquires targeted and global metabolomic data in a same analytical run, and the use of stable isotope-labeled standards facilitates accurate quantitation of targeted metabolites in large-scale metabolomics analysis. This metabolomics approach has been successfully applied to analysis of targeted metabolites in head and neck cancer cells as well as cancer stem-like cells (CSCs), and the findings indicate that the metabolic phenotypes may be distinct between high and low invasive head and neck cancer cells and between CSCs and non-SCCs.
Organic acids (OAs) serve as metabolites that play pivotal roles in a host of different metabolic and regulatory pathways. The polar nature of many OAs poses a challenge to their measurement using widely practiced analytical methods. In this study, a targeted metabolomics method was developed using ion chromatography/triple quadrupole mass spectrometry (IC/MS) to quantitate 28 polar OAs with limits of quantitation ranging from 0.25 to 50 μM. The interday assay precisions ranged from 1% to 19%, with accuracies ranging from 82% to 115%. The IC/MS assay was used to quantitate OAs in quadriceps muscle from sedentary mice compared to fatigued mice subjected to either a low intensity, long duration (LILD) or high intensity, short duration (HISD) forced treadmill regimen. Among the OAs examined, significant differences were detected for hippuric acid, malic acid, fumaric acid, and 2-ketoglutaric acid between the sedentary and fatigued mice. In conclusion, the IC/MS method enabled the separation and quantitative survey of a broad range of polar OAs that are difficult to analyze by chromatographic techniques.
Since July 2018 several drugs have been recalled due to contamination with
N
-nitrosodimethylamine (NDMA), a probable human carcinogen. Dimethylamine (DMA) and nitrite are precursors in the formation of NDMA. In this study, ion chromatography (IC) methods were developed for the determination of these two precursors in drug substances and drug products. Two methods were developed to determine DMA in two drug products using a cation exchange separation coupled to suppressed conductivity detection. The limit of detection of DMA is < 1 μg/g of active pharmaceutical ingredient (API) for both methods. Nitrite was determined using an anion exchange separation coupled with UV absorbance detection. The limit of detection of nitrite was 0.918 μg/g API. The developed methods were successfully applied to DMA and nitrite determinations in five drug products including metformin, losartan, ranitidine, Nytol, and Benadyrl, and two drug substances (APIs), losartan potassium and metformin hydrochloride. Some samples contained nitrite and DMA at detectable levels. Dimethylamine and nitrite recovery from pharmaceutical samples ranged from 96.0-104 %. The developed methods should be useful for the rapid screening and quantification of nitrite and DMA in pharmaceuticals and in-process samples to assess the likelihood of NDMA formation. The methods for DMA should be applicable to other amines to assess the likelihood of the formation of other nitrosamines in pharmaceutical products.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.