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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.

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Nowadays, lipidomics plays a crucial role in the investigation of novel biomarkers of various diseases. Its implementation into the field of clinical analysis led to the identification of specific lipids and/or significant changes in their plasma levels in patients suffering from cancer, Alzheimer's disease, sepsis, and many other diseases and pathological conditions. Profiling of lipids and determination of their plasma concentrations could also be helpful in the case of drug therapy management, especially in combination with therapeutic drug monitoring (TDM). Here, for the first time, a combined approach based on the TDM of colistin, a last-resort antibiotic, and lipidomic profiling is presented in a case study of a critically ill male patient suffering from Pseudomonas aeruginosa-induced pneumonia. Implementation of innovative analytical approaches for TDM (online combination of capillary electrophoresis with tandem mass spectrometry, CZE-MS/MS) and lipidomics (liquid chromatography-tandem mass spectrometry, LC-MS/MS) was demonstrated. The CZE-MS/MS strategy confirmed the chosen colistin drug dosing regimen, leading to stable colistin concentrations in plasma samples. The determined colistin concentrations in plasma samples reached the required minimal inhibitory concentration of 1 µg/mL. The complex lipidomics approach led to monitoring 545 lipids in collected patient plasma samples during and after the therapy. Some changes in specific individual lipids were in good agreement with previous lipidomics studies dealing with sepsis. The presented case study represents a good starting point for identifying particular individual lipids that could correlate with antimicrobial and inflammation therapeutic management.

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Many biologically active metabolites of the essential amino acid L-tryptophan (Trp) are associated with different neurodegenerative diseases and neurological disorders. Precise and reliable methods for their determination are needed. Variability in their physicochemical properties makes the analytical process challenging. In this case, chemical modification of analyte derivatization could come into play. Here, we introduce a novel fast reversed-phase ultra-high-performance liquid chromatography (RP-UHPLC) coupled with tandem mass spectrometry (MS/MS) method for the determination of Trp and its ten metabolites in human plasma samples after derivatization with 2-bromo-4'-nitroacetophenone (BNAP). The derivatization procedure was optimized in terms of incubation time, temperature, concentration, and volume of the derivatization reagent. Method development comprises a choice of a suitable stationary phase, mobile phase composition, and gradient elution optimization. The developed method was validated according to the ICH guidelines. Results of all validation parameters were within the acceptance criteria of the guideline, i.e., intra- and inter-day precision (expressed as relative standard deviation; RSD) were in the range of 0.5-8.2% and 2.3-7.4%, accuracy was in the range of 93.3-109.7% and 94.7-110.1%, limits of detection (LODs) were in the range of 0.15-9.43 ng/mL, coefficients of determination (R2) were higher than 0.9906, and carryovers were, in all cases, less than 8.8%. The practicability of the method was evaluated using the blue applicability grade index (BAGI) with a score of 65. Finally, the developed method was used for the analysis of Alzheimer's disease and healthy control plasma to prove its applicability. Statistical analysis revealed significant changes in picolinic acid (PA), anthranilic acid (AA), 5 hydroxyindole-3-acetic acid (5-OH IAA), and quinolinic acid (QA) concentration levels. This could serve as the basis for future studies that will be conducted with a large cohort of patients.

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Carboxylic acids (CAs) represent a large group of important molecules participating in various biologically significant processes. Analytical study of these compounds is typically performed by liquid chromatography (LC) combined with various types of detection. However, their analysis is often accompanied by a wide variety of problems depending on used separation system or detection method. The dominant ones are: i) poor chromatographic behavior of the CAs in reversed-phase LC; ii) absence of a chromophore (or fluorophore); iii) weak ionization in mass spectrometry (MS). To overcome these problems, targeted chemical modification, and derivatization, come into play. Therefore, derivatization still plays an important and, in many cases, irreplaceable role in sample preparation, and new derivatization methods of CAs are constantly being developed. The most commonly used type of reaction for CAs derivatization is amidation. In recent years, an increased interest in the isotopic labeling derivatization method has been observed. In this review, we comprehensively summarize the possibilities and actual trends in the derivatization of CAs that have been published over the past decade.

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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.

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POM analysis and related approaches are significant tools based on calculating various physico-chemical properties and predicting biological activity, ADME parameters, and toxicity of a molecule. These methods are used to evaluate a molecule's potential to become a drug candidate. Avenanthramides (AVNs) are promising secondary metabolites specific to Avena spp. (oat). They comprise the amides of anthranilic acid linked to various polyphenolic acids with or without post-condensation molecule transformation. These natural compounds have been reported to exert numerous biological effects, including antioxidant, anti-inflammatory, hepatoprotective, antiatherogenic, and antiproliferative properties. To date, almost 50 various AVNs have been identified. We performed a modified POM analysis of 42 AVNs using MOLINSPIRATION, SWISSADME, and OSIRIS software. The evaluation of primary in silico parameters revealed significant differences among individual AVNs, highlighting the most promising candidates. These preliminary results may help coordinate and initiate other research projects focused on particular AVNs, especially those with predicted bioactivity, low toxicity, optimal ADME parameters, and promising perspectives.