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When hospitalized, infants, particularly preterm, are often subjected to multiple painful needle procedures to collect sufficient blood for metabolic screening or diagnostic purposes using standard clinical tests. For example, at least 100 µL of whole blood is required to perform one creatinine plasma measurement with enzymatic colorimetric assays. As capillary electrophoresis-mass spectrometry (CE-MS) utilizing a sheathless porous tip interface only requires limited amounts of sample for in-depth metabolic profiling studies, the aim of this work was to assess the utility of this method for the determination of creatinine in low amounts of plasma using residual blood samples from adults and infants. By using a starting amount of 5 µL of plasma and an injection volume of only 6.7 nL, a detection limit (S/N = 3) of 30 nM could be obtained for creatinine, and intra- and interday precisions (for peak area ratios) were below 3.2%. To shorten the electrophoretic separation time, a multi-segment injection (MSI) strategy was employed to analyze up to seven samples in one electrophoretic run. The findings obtained by CE-MS for creatinine in pretreated plasma were compared with the values acquired by an enzymatic colorimetric assay typically used in clinical laboratories for this purpose. The comparison revealed that CE-MS could be used in a reliable way for the determination of creatinine in residual plasma samples from infants and adults. Nevertheless, to underscore the clinical efficacy of this method, a subsequent investigation employing an expanded pool of plasma samples is imperative. This will not only enhance the method's diagnostic utility but also contribute to minimizing both the amount and frequency of blood collection required for diagnostic purposes., (© 2024 The Authors. ELECTROPHORESIS published by Wiley‐VCH GmbH.)

The efficient profiling of highly polar and charged metabolites in biological samples remains a huge analytical challenge in metabolomics. Over the last decade, new analytical techniques have been developed for the selective and sensitive analysis of polar ionogenic compounds in various matrices. Still, the analysis of such compounds, notably for acidic ionogenic metabolites, remains a challenging endeavor, even more when the available sample size becomes an issue for the total analytical workflow. In this paper, we give an overview of the possibilities of capillary electrophoresis-mass spectrometry (CE-MS) for anionic metabolic profiling by focusing on main methodological developments. Attention is paid to the development of improved separation conditions and new interfacing designs in CE-MS for anionic metabolic profiling. A complete overview of all CE-MS-based methods developed for this purpose is provided in table format (Table 1) which includes information on sample type, separation conditions, mass analyzer and limits of detection (LODs). Selected applications are discussed to show the utility of CE-MS for anionic metabolic profiling, especially for small-volume biological samples. On the basis of the examination of the reported literature in this specific field, we conclude that there is still room for the design of a highly sensitive and reliable CE-MS method for anionic metabolic profiling. A rigorous validation and the availability of standard operating procedures would be highly favorable in order to make CE-MS an alternative, viable analytical technique for metabolomics., (© 2019 The Authors. Electrophoresis published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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). [ABSTRACT FROM AUTHOR]

Brain metabolomics is gaining interest because of the aging of the population, resulting in more central nervous system disorders such as Alzheimer's and Parkinson's disease. Most often these diseases are studied in vivo , such as for example by analysing cerebrospinal fluid or brain extracellular fluid. These sample types are often considered in pre-clinical studies using animal models. However, the scarce availability of both matrices results in some challenges related to sampling, sample preparation and normalization. Much effort has been made towards the development of alternative, less invasive sampling techniques for collecting small sample volumes (pL till mid μL range) over the past years. Despite recent advances, the analysis of low volumes is still a tremendous challenge. Therefore, proper preconcentration and sample pretreatment strategies are necessary together with sensitive analysis and detection techniques suitable for low-volume samples. In this review, an overview is given of the state-of-the-art mass spectrometry-based analytical workflows for probing (endogenous) metabolites in volume-restricted in-vivo brain samples. In this context, special attention is devoted to challenges related to sampling, sample preparation and preconcentration strategies. Finally, some general conclusions and perspectives are provided. • In vivo brain metabolomics is challenging because of low neuromodulator concentrations and volumes. • Novel trends in sampling, sample preparation and analysis of low volume samples are discussed. • Miniaturization of sampling probes largely reduces the invasiveness of brain fluid sampling. • Increased interest in solid-phase and liquid-phase microextraction. [ABSTRACT FROM AUTHOR]

• CE-MS method for direct metabolic profiling of rat brain microdialysis samples • On-line preconcentration employed to further improve CE-MS detection sensitivity • A range of endogenous metabolites reliably quantified in rat brain microdialysis samples Metabolic profiling of body fluids from small animal models is often used in (translational) biological studies in order to obtain insight into the underlying molecular mechanisms of (complex) diseases. An example is the use of brain microdialysis samples from rats to study neurological disorders by means of a metabolomics approach. From an analytical point of view, the profiling of (endogenous) metabolites in rat brain microdialysates is challenging because of the limited sample volume for both sample preparation and injection, notably in longitudinal studies. In this work, we have assessed the analytical performance of capillary electrophoresis-mass spectrometry (CE-MS) for the direct profiling of endogenous metabolites in rat brain microdialysates, i.e. without using any sample preparation or derivatization. An on-line preconcentration procedure with sample stacking, which was fully compatible with the high-salt concentration in microdialysates, was used to significantly improve the detection sensitivity of the CE-MS method for metabolic profiling. A response surface methodology, applying a Box-Behnken design, was considered to determine the optimal conditions for preconcentration. A linear response (R2>0.99) for selected metabolites in the concentration range from 0.05 to 10 µM was obtained in perfusate samples. Interday RSD values for peak area and migration time were 2.6-19% and below 3.8%, respectively. Limits of detection ranged from 11 to 284 nM when employing an injection volume of about 291 nL, corresponding to 17% of the total capillary volume. The utility of the CE-MS approach was demonstrated by the direct profiling of endogenous metabolites in rat brain microdialysates. At least 48 compounds could be analyzed of which 25 were provisionally identified and quantified. [ABSTRACT FROM AUTHOR]