Profiling acidic metabolites by capillary electrophoresis-mass spectrometry in low numbers of mammalian cells using a novel chemical derivatization approach.

The simultaneous analysis of a broad range of polar ionogenic metabolites using capillary electrophoresis-mass spectrometry (CE-MS) can be challenging, as two different analytical methods are often required, that is, one for cations and one for anions. Even though CE-MS has shown to be an effective method for cationic metabolite profiling, the analysis of small anionic metabolites often results in relatively low sensitivity and poor repeatability. In this work, a novel derivatization strategy based on trimethylmethaneaminophenacetyl bromide was developed to enable CE-MS analysis of carboxylic acid metabolites using normal CE polarity (i.e., cathode in the outlet) and detection by mass spectrometry in positive ionization mode. Optimization of derivatization conditions was performed using a response surface methodology after which the optimized method (incubation time 50 min, temperature 90°C, and pH 10) was used for the analysis of carboxylic acid metabolites in extracts from HepG2 cells. For selected metabolites, detection limits were down to 8.2 nM, and intraday relative standard deviation values for replicates (n = 3) for peak areas were below 21.5%. Metabolites related to glycolysis, tricarboxylic acid cycle, and anaerobic respiration pathways were quantified in 250,000 cell lysates, and could still be detected in extracts from only 25,000 HepG2 cell lysates (∼70 cell lysates injected).

LC-MS/MS analysis of the central energy and carbon metabolites in biological samples following derivatization by dimethylaminophenacyl bromide.

Recent advances in metabolomics have enabled larger proportions of the human metabolome to be analyzed quantitatively. However, this usually requires the use of several chromatographic methods coupled to mass spectrometry to cover the wide range of polarity, acidity/basicity and concentration of metabolites. Chemical derivatization allows in principle a wide coverage in a single method, as it affects both the separation and the detection of metabolites: it increases retention, stabilizes the analytes and improves the sensitivity of the analytes. The majority of quantitative derivatization techniques for LC-MS in metabolomics react with amines, phenols and thiols; however, there are unfortunately very few methods that can target carboxylic acids at the same time, which contribute to a large proportion of the human metabolome. Here, we describe a derivatization technique which simultaneously labels carboxylic acids, thiols and amines using the reagent dimethylaminophenacyl bromide (DmPABr). We further improve the quantitation by employing isotope-coded derivatization (ICD), which uses internal standards derivatized with an isotopically-labelled reagent (DmPABr-D6). We demonstrate the ability to measure and quantify 64 central carbon and energy-related metabolites including amino acids, N-acetylated amino acids, metabolites from the TCA cycle and pyruvate metabolism, acylcarnitines and medium-/long-chain fatty acids. To demonstrate the applicability of the analytical approach, we analyzed urine and SUIT-2 cells utilizing a 15-minute single UPLC-MS/MS method in positive ionization mode. SUIT-2 cells exposed to rotenone showed definitive changes in 28 out of the 64 metabolites, including metabolites from all 7 classes mentioned. By realizing the full potential of DmPABr to derivatize and quantify amines and thiols in addition to carboxylic acids, we extended the coverage of the metabolome, producing a strong platform that can be further applied to a variety of biological studies.