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

Abstract

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).

Strategies for capillary electrophoresis: Method development and validation for pharmaceutical and biological applications—Updated and completely revised edition

“…Generally speaking, and among different protocols that can be applied to improve the preconcentration or separation of target analytes, perhaps the most common ones are off-line (also described as pre-column). These methods are generally manually performed by allowing researchers to control a wide range of conditions [523], including heating the sample and have been applied toward the analysis of acrylamide [524], mono-and oligosaccharides [525], sialylated N-glycan linkage isomers [526], 3-hydroxyaspartate [527], or acidic metabolites [528]. Off-line approaches have also allowed taking some advantage of advanced oxidation methods (combining H 2 O 2 and UV light) to follow the degradation of pharmaceuticals [529] or to convert alcohols into the corresponding acids, thus facilitating their separation by CE [350,530].…”

A Novel and Sensitive Method for the Analysis of Fatty Acid Biosignatures by Capillary Electrophoresis-Mass Spectrometry

“…The detection limits are a factor 5 to 90 lower compared to CE-MS using dicationic ion-pairing reagents 10 and 3 to 140 times lower compared to CE-MS with negative ionization mode. 9 Recently, the use of a derivatization strategy for small carboxylic acids showed a similar improvement with detection limits for positively charged carboxylic acids being a factor of 10−100 times lower when analyzed with CE-MS using positive ESI compared to direct analysis in negative mode CE-MS. 27 The derivatization of the carboxylic acids was obtained in 65 mM ammonium bicarbonate using an incubation temperature of 90 °C for 50 min with a stirring speed of 900 rpm. 27 These conditions are not well suited for implementation in flight hardware for future mission to ocean worlds.…”

“…9 Recently, the use of a derivatization strategy for small carboxylic acids showed a similar improvement with detection limits for positively charged carboxylic acids being a factor of 10−100 times lower when analyzed with CE-MS using positive ESI compared to direct analysis in negative mode CE-MS. 27 The derivatization of the carboxylic acids was obtained in 65 mM ammonium bicarbonate using an incubation temperature of 90 °C for 50 min with a stirring speed of 900 rpm. 27 These conditions are not well suited for implementation in flight hardware for future mission to ocean worlds. Our optimized derivatization procedure was mainly tested for the analysis of fatty acids, but could also be used for the analysis of small mono-, di-, and tricarboxylic acids.…”

Profiling of polar ionogenic metabolites in Polish wines by capillary electrophoresis‐mass spectrometry

“…For the CE‐MS methodology, we refer to our procedures described previously [ 14 ]. Briefly, CE‐MS experiments were carried out on a 7100 CE system hyphenated with a 6230 TOF, both from Agilent Technologies.…”