Lipids represent a large group of biomolecules that are responsible for various functions in organisms. Diseases such as diabetes, chronic inflammation, neurological disorders, or neurodegenerative and cardiovascular diseases can be caused by lipid imbalance. Due to the different stereochemical properties and composition of fatty acyl groups of molecules in most lipid classes, quantification of lipids and development of lipidomic analytical techniques are problematic. Identification of different lipid species from complex matrices is difficult, and therefore individual analytical steps, which include extraction, separation, and detection of lipids, must be chosen properly. This review critically documents recent strategies for lipid analysis from sample pretreatment to instrumental analysis and data interpretation published in the last five years (2019 to 2023). The advantages and disadvantages of various extraction methods are covered. The instrumental analysis step comprises methods for lipid identification and quantification. Mass spectrometry (MS) is the most used technique in lipid analysis, which can be performed by direct infusion MS approach or in combination with suitable separation techniques such as liquid chromatography or gas chromatography. Special attention is also given to the correct evaluation and interpretation of the data obtained from the lipid analyses. Only accurate, precise, robust and reliable analytical strategies are able to bring complex and useful lipidomic information, which may contribute to clarification of some diseases at the molecular level, and may be used as putative biomarkers and/or therapeutic targets.

Separation analytical methods, including liquid chromatography (LC) and capillary electrophoresis (CE), in combination with an appropriate detection technique, are dominant and powerful approaches preferred in the analysis of pharmaceutical and biomedical samples. Recent trends in analytical methods are focused on activities that push them to the field of greenness and sustainability. New approaches based on the implementation of greener solvents, non-hazardous chemicals, and reagents have grown exponentially. Similarly, recent trends are pushed in to the strategies based on miniaturization, reduction of wastes, avoiding derivatization procedures, or reduction of energy consumption. However, the real greenness of the analytical method can be evaluated only according to an objective and sufficient metric offering complex results taking into account all twelve rules of green analytical chemistry (SIGNIFICANCE mnemonic system). This review provides an extensive overview of papers published in the area of development of green LC and CE methods in the field of pharmaceutical and biomedical analysis over the last 5 years (2019-2023). The main focus is situated on the metrics used for greenness evaluation of the methods applied for the determination of bioactive agents. It critically evaluates and compares the demands of the real applicability of the methods in quality control and clinical environment with the requirements of the green analytical chemistry (GAC). Greenness and practicality of the summarized methods are re-evaluated or newly evaluated with the use of the dominant metrics tools, i.e., Analytical GREEnness (AGREE), Green Analytical Procedure Index (GAPI), Blue Applicability Grade Index (BAGI), and Sample Preparation Metric of Sustainability (SPMS). Moreover, general conclusions and future perspectives of the greening procedures and greenness evaluation metrics systems are presented. This paper should provide comprehensive information to analytical chemists, biochemists, and it can also represent a valuable source of information for clinicians, biomedical or quality control laboratories interested in development of analytical methods based on greenness, practicality, and sustainability.

Short-chain fatty acids (SCFAs) are the main metabolites produced by bacterial fermentation of non-digestible carbohydrates in the gastrointestinal tract. They can be seen as the major flow of carbon from the diet, through the microbiome to the host. SCFAs have been reported as important molecules responsible for the regulation of intestinal homeostasis. Moreover, these molecules have a significant impact on the immune system and are able to affect inflammation, cardiovascular diseases, diabetes type II, or oncological diseases. For this purpose, SCFAs could be used as putative biomarkers of various diseases, including cancer. A potential diagnostic value may be offered by analyzing SCFAs with the use of advanced analytical approaches such as gas chromatography (GC), liquid chromatography (LC), or capillary electrophoresis (CE) coupled with mass spectrometry (MS). The presented review summarizes the importance of analyzing SCFAs from clinical and analytical perspective. Current advances in the analysis of SCFAs focused on sample pretreatment, separation strategy, and detection methods are highlighted. Additionally, it also shows potential areas for the development of future diagnostic tools in oncology and other varieties of diseases based on targeted metabolite profiling.

Urine represents a convenient biofluid for metabolomic studies due to its noninvasive collection and richness in metabolites. Here, amino acids are valuable biomarkers for their ability to reflect imbalances of different biochemical pathways. An impact of amino acids on pathology, prognosis and therapy of various diseases, including inflammatory bowel disease (IBD), is therefore the subject of current clinical research. This work is aimed to develop a capillary electrophoresis-tandem mass spectrometry (CE-MS/MS) method for the quantification of the 20 proteinogenic amino acids in human urine samples obtained from patients suffering from IBD and treated with thiopurines. The optimized CE-MS/MS method, with minimum sample preparation (just “dilute and shoot”), exhibited excellent linearity for all the analytes (coefficients of determination were higher than 0.99), with inter-day and intra-day precision yielding relative standard deviations in the range of 0.91–15.12% and with accuracy yielding relative errors in the range of 85.47–112.46%. Total analysis time, an important parameter for the sample throughput demanded in routine practice, was shorter in ca. 17% when compared to established CE-MS methods. Favorable performance of the proposed CE-MS/MS method was also confirmed by the comparison with corresponding ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS) method. Consistent data for the investigated amino acid metabolome were obtained using both methods. For the first time, the amino acid profiling by CE-MS approach was applied on the clinical IBD samples. Here, significant differences observed in the concentration levels of some amino acids between IBD patients undergoing thiopurine treatment and healthy volunteers could result from the simultaneous action of the disease and the corresponding therapy. These findings indicate that amino acids analysis could be a valuable tool for the study of mechanism of the IBD treatment by thiopurines.