Cross-column density functional theory-based quantitative structure-retention relationship model development powered by machine learning.

Quantitative structure-retention relationship (QSRR) modeling has emerged as an efficient alternative to predict analyte retention times using molecular descriptors. However, most reported QSRR models are column-specific, requiring separate models for each high-performance liquid chromatography (HPLC) system. This study evaluates the potential of machine learning (ML) algorithms and quantum mechanical (QM) descriptors to develop QSRR models that can predict retention times across three different reversed-phase HPLC columns under varying conditions. Four machine learning methods-partial least squares (PLS) regression, ridge regression (RR), random forest (RF), and gradient boosting (GB)-were compared on a dataset of 360 retention times for 15 aromatic analytes. Molecular descriptors were calculated using density functional theory (DFT). Column characteristics like particle size and pore size and experimental conditions like temperature and gradient time were additionally used as descriptors. Results showed that the GB-QSRR model demonstrated the best predictive performance, with Q2 of 0.989 and root mean square error of prediction (RMSEP) of 0.749 min on the test set. Feature analysis revealed that solvation energy (SE), HOMO-LUMO energy gap (∆E HOMO-LUMO), total dipole moment (Mtot), and global hardness (η) are among the most influential predictors for retention time prediction, indicating the significance of electrostatic interactions and hydrophobicity. Our findings underscore the efficiency of ensemble methods, GB and RF models employing non-linear learners, in capturing local variations in retention times across diverse experimental setups. This study emphasizes the potential of cross-column QSRR modeling and highlights the utility of ML models in optimizing chromatographic analysis.

Development of a 3D-Printable, Porous, and Chemically Active Material Filled with Silica Particles and its Application to the Fabrication of a Microextraction Device.

We report on the first successful attempt to produce a silica/polymer composite with retained C18 silica sorptive properties that can be reliably printed using three-dimensional (3D) FDM printing. A 3D printer provides an exceptional tool for producing complex objects in an easy and inexpensive manner and satisfying the current custom demand of research. Fused deposition modeling (FDM) is the most popular 3D-printing technique based on the extrusion of a thermoplastic material. The lack of appropriate materials limits the development of advanced applications involving directly 3D-printed devices with intrinsic chemical activity. Progress in sample preparation, especially for complex sample matrices and when mass spectrometry is favorable, remains a vital research field. Silica particles, for example, which are commonly used for extraction, cannot be directly extruded and are not readily workable in a powder form. The availability of composite materials containing a thermoplastic polymer matrix and dispersed silica particles would accelerate research in this area. This paper describes how to prepare a polypropylene (PP)/acrylonitrile-butadiene-styrene (ABS)/C18-functionalized silica composite that can be processed by FDM 3D printing. We present a method for producing the filament as well as a procedure to remove ABS by acetone rinsing (to activate the material). The result is an activated 3D-printed object with a porous structure that allows access to silica particles while maintaining macroscopic size and shape. The 3D-printed device is intended for use in a solid-phase microextraction (SPME) procedure. The proposed composite's effectiveness is demonstrated for the microextraction of glimepiride, imipramine, and carbamazepine. The complex honeycomb geometry of the sorbent has shown to be superior to the simple tubular sorbent, which proves the benefits of 3D printing. The 3D-printed sorbent's shape and microextraction parameters were fine-tuned to provide satisfactory recoveries (33-47%) and high precision (2-6%), especially for carbamazepine microextraction.

Monitoring of age- and gender-related alterations of endocannabinoid levels in selected brain regions with the use of SPME probes.

INTRODUCTION: The endocannabinoid system consists of different types of receptors, enzymes and endocannabinoids (ECs), which are involved in several physiological processes, but also play important role in the development and progression of central nervous system disorders. OBJECTIVES: The purpose of this study was to apply precise and sensitive methodology for monitoring of four ECs, namely anandamide (AEA), 2-arachidonoyl glycerol (2-AG), N-arachidonoyl dopamine (NADA), 2-arachidonyl glyceryl ether (2-AGe) in selected brain regions of female and male rats at different stages of development (young, adult and old). METHODS: Biocompatible solid-phase microextraction (SPME) probes were introduced into the intact (non-homogenized) brain structures for isolation of four ECs, and the extracts were subjected to LC-MS/MS analysis. Two chemometric approaches, namely hierarchical cluster analysis (HCA) and Principal Component Analysis (PCA) were applied to provide more information about the levels of 2-AG and AEA in different brain structures. RESULTS: 2-AG and AEA were extracted and could be quantified in each brain region; the level of 2-AG was significantly higher in comparison to the level of AEA. Two highly unstable ECs, NADA and 2-AGe, were captured by SPME probes from intact brain samples for the first time. CONCLUSION: SPME probes were able to isolate highly unstable endogenous compounds from intact tissue, and provided new tools for precise analysis of the level and distribution of ECs in different brain regions. Monitoring of ECs in brain samples is important not only in physiological conditions, but also may contribute to better understanding of the functioning of the endocannabinoid system in various disorders.

Novel 2-alkythio-4-chloro-N-[imino(heteroaryl)methyl]benzenesulfonamide Derivatives: Synthesis, Molecular Structure, Anticancer Activity and Metabolic Stability.

A series of novel 2-alkythio-4-chloro-N-[imino-(heteroaryl)methyl]benzenesulfonamide derivatives, 8-24, were synthesized in the reaction of the N-(benzenesulfonyl)cyanamide potassium salts 1-7 with the appropriate mercaptoheterocycles. All the synthesized compounds were evaluated for their anticancer activity in HeLa, HCT-116 and MCF-7 cell lines. The most promising compounds, 11-13, molecular hybrids containing benzenesulfonamide and imidazole moieties, selectively showed a high cytotoxic effect in HeLa cancer cells (IC50: 6-7 µM) and exhibited about three times less cytotoxicity against the non-tumor cell line HaCaT cells (IC50: 18-20 µM). It was found that the anti-proliferative effects of 11, 12 and 13 were associated with their ability to induce apoptosis in HeLa cells. The compounds increased the early apoptotic population of cells, elevated the percentage of cells in the sub-G1 phase of the cell cycle and induced apoptosis through caspase activation in HeLa cells. For the most active compounds, susceptibility to undergo first-phase oxidation reactions in human liver microsomes was assessed. The results of the in vitro metabolic stability experiments indicated values of the factor t½ for 11-13 in the range of 9.1-20.3 min and suggested the hypothetical oxidation of these compounds to sulfenic and subsequently sulfinic acids as metabolites.

Effects of Fe3O4 Magnetic Nanoparticle Functionalization with Ionic Liquids and a Double-Chained Surfactant on the Pretreatment of Plasma Samples during Drug Extraction.

Ionic liquids (ILs), also known as "designer solvents," comprise a large group of compounds that can improve overall sample preparation performance due to their unique physical and chemical properties. Some of them have a comparable structure to surfactants, which can be also considered as effective extraction solvents. In this study, nine different ILs and a double-chained surfactant were investigated as potential coating materials for iron oxide-based nanoparticles (NPs) used in the pretreatment of human plasma samples. Various methods of synthesizing and functionalizing NPs were employed in fabricating the magnetic sorbents, with the physicochemical properties of the resultant extraction phases (i.e., naked NPs, NPs coated with silica, and NPs coated with silica and selected IL or surfactant) being characterized via X-ray diffraction (XRD), Fourier transform infrared (FT-IR) spectroscopy, thermogravimetric analysis (TG), and transmission electron microscopy (TEM). The effectiveness of the developed NP-based extraction phases was tested by applying them for the extraction of epirubicin hydrochloride (EPI) from plasma samples, followed by analysis via liquid chromatography with fluorescence detection (LC-FL). The results showed that NPs coated with both silica and IL or silica and surfactant provided significantly higher extraction efficiency compared to naked NPs and NPs coated solely with silica. Additionally, the findings also revealed that the adsorption of analytes depends not only on the coating procedure but also on the type of coating material used to functionalize the NPs. Among the tested structures, didodecyldimethylammonium bromide provided the best performance for the functionalization of NP sorbents previously coated with silica.

Simultaneous determination of mitotane, its metabolite, and five steroid hormones in urine samples by capillary electrophoresis using ß-CD2 SDS1 complexes as hydrophobic compounds solubilizers.

Mitotane is a cytotoxic drug used in the treatment of inoperable adrenocortical carcinoma, it inhibits steroidogenesis as well, and therefore monitoring the level of steroid hormones in patients treated with mitotane is a crucial point of therapy. Hence, we have developed a simple, fast, and efficient electrophoretic method combined with reverse polarity sweeping as online preconcentration technique and dispersive liquid-liquid microextraction for the simultaneous determination of mitotane, its main metabolite DDA, and five steroid hormones (progesterone, testosterone, epitestosterone, cortisol, and corticosterone) in urine samples. In addition, a new sample matrix consisting of ß-CD2 SDS1 complexes for a high hydrophobic compounds solubilization was developed. Approach based on the application of ß-cyclodextrin and SDS complex of a ratio 2:1 allowed for hydrodynamic injection into the capillary of a solution containing both mitotane and other analytes. The detection limits of the analytes for the reverse polarity sweeping-dispersive liquid-liquid microextraction method were found to be in the range of 1.5-3 ng/mL, which were approximately 1000 times lower than in the conventional hydrodynamic injection (5 s, 0.5 psi) without any preconcentration procedure. All analytes were completely resolved in less than 13 min by uncoated silica capillary with an inner diameter of 75 µm (ID) × 60 cm. Electrophoretic separation was performed in reverse polarity with a voltage of -25 kV with a background electrolyte (BGE) consisting of 100 mM SDS, 25% ACN, 25 mM phosphate buffer (pH 2.5), and 7 mM ß-cyclodextrin.

Vitamin D and anti-Müllerian hormone concentration in human follicular fluid individually aspirated from all patient follicles.

OBJECTIVE: The main aim of this prospective study was to investigate the relationship between intrafollicular vitamin D and anti-Müllerian hormone (AMH) concentration and its impact on oocyte quality and developmental competence. METHODS: The analysis was performed on 208 follicular fluid (FF) samples obtained from 33 patients undergoing ovarian stimulation as part of in vitro fertilization (IVF) treatment that included intracytoplasmic sperm injection. RESULTS: Our study shows that vitamin D concentration in FF varies according to the developmental stage of the oocyte and corelates with embryo development status on day 3, while AMH concentration in FF is not correlated with the developmental potential of an oocyte. We demonstrated that the levels of vitamin D and AMH were higher in FF than in serum. Moreover we showed that AMH and vitamin D levels were positively correlated in FF but not in serum. CONCLUSION: FF-AMH levels do not appear to be a suitable as noninvasive test of the developmental potential of an oocyte, while FF-vitamin D level can be used to evaluate whether embryos obtained from particular oocytes have potential of reaching the third day of culture. However, our results encourage further research to be carried out on a larger number of patients and testing additional components found in FF such as androgens.

The Cooperative Anti-Neoplastic Activity of Polyphenolic Phytochemicals on Human T-Cell Acute Lymphoblastic Leukemia Cell Line MOLT-4 In Vitro.

Acute lymphoblastic leukemia (ALL) is the most common hematological malignancy affecting pediatric patients. ALL treatment regimens with cytostatics manifest substantial toxicity and have reached the maximum of well-tolerated doses. One potential approach for improving treatment efficiency could be supplementation of the current regimen with naturally occurring phytochemicals with anti-cancer properties. Nutraceuticals such as quercetin, curcumin, resveratrol, and genistein have been studied in anti-cancer therapy, but their application is limited by their low bioavailability. However, their cooperative activity could potentially increase their efficiency at low, bioavailable doses. We studied their cooperative effect on the viability of a human ALL MOLT-4 cell line in vitro at the concentration considered to be in the bioavailable range in vivo. To analyze their potential side effect on the viability of non-tumor cells, we evaluated their toxicity on a normal human foreskin fibroblast cell line (BJ). In both cell lines, we also measured specific indicators of cell death, changes in cell membrane permeability (CMP), and mitochondrial membrane potential (MMP). Even at a low bioavailable concentration, genistein and curcumin decreased MOLT-4 viability, and their combination had a significant interactive effect. While resveratrol and quercetin did not affect MOLT-4 viability, together they enhanced the effect of the genistein/curcumin mix, significantly inhibiting MOLT-4 population growth in vitro. Moreover, the analyzed phytochemicals and their combinations did not affect the BJ cell line. In both cell lines, they induced a decrease in MMP and correlating CMP changes, but in non-tumor cells, both metabolic activity and cell membrane continuity were restored in time. (4) Conclusions: The results indicate that the interactive activity of analyzed phytochemicals can induce an anti-cancer effect on ALL cells without a significant effect on non-tumor cells. It implies that the application of the combinations of phytochemicals an anti-cancer treatment supplement could be worth further investigation regardless of their low bioavailability.

Plasma Concentration of Cortisol Negatively Associates with Platelet Reactivity in Older Subjects.

The interaction of platelets with steroid hormones is poorly investigated. Age is one of the factors that increase the risk of pathological platelet reactivity and thrombosis. The aim of this study was to assess whether there were associations between platelet reactivity and plasma cortisol levels in volunteers aged 60-65 years. For this purpose, impedance aggregometry in whole blood measured after arachidonic acid, collagen, or ADP stimulation was used to estimate platelet reactivity and mass spectrometry was used to measure peripheral plasma cortisol concentration. Statistically significant negative correlations were observed between cortisol concentration and platelet reactivity in response to arachidonic acid and ADP, but not to collagen. The presented results suggest for the very first time that cortisol is a new endogenous modulator of platelet reactivity in the elderly population.

Serum metabolic fingerprinting of psoriasis and psoriatic arthritis patients using solid-phase microextraction-liquid chromatography-high-resolution mass spectrometry.

INTRODUCTION: Psoriatic arthritis (PsA), an inflammatory arthritis that develops in individuals with psoriasis, is associated with reduced quality of life. Identifying biomarkers associated with development of PsA as well as with PsA disease activity may help management of psoriatic disease. OBJECTIVES: To use metabolomic fingerprinting to determine potential candidate markers of disease conversion (psoriasis to PsA) and/or PsA activity. METHODS: A novel sample preparation protocol based on solid-phase microextraction (SPME) was used to prepare serum samples obtained from: (1) individuals with psoriasis, some of whom develop psoriatic arthritis (n = 20); (2) individuals with varying PsA activity (mild, moderate, severe; n = 10 each) and (3) healthy controls (n = 10). Metabolomic fingerprinting of the obtained extracts was performed using reversed-phase liquid chromatography coupled to high resolution mass spectrometry. RESULTS: Psoriasis patients who developed PsA had similar metabolomic profiles to patients with mild PsA and were also indistinguishable from patients with psoriasis who did not develop PsA. Elevated levels of selected long-chain fatty acids (e.g., 3-hydroxytetradecanedioic acid) that are associated with dysregulation of fatty acid metabolism, were observed in patients with severe PsA. In addition, 1,11-undecanedicarboxylic acid-an unusual fatty acid associated with peroxisomal disorders-was also identified as a classifier in PsA patients vs. healthy individuals. Furthermore, a number of different eicosanoids with either pro- or anti-inflammatory properties were detected solely in serum samples of patients with moderate and severe PsA. CONCLUSION: A global metabolomics approach was employed to analyze the serum metabolome of patients with psoriasis, PsA, and healthy controls in order to examine potential differences in the biochemical profiles at a metabolite level. A closer examination of circulating metabolites may potentially provide markers of PsA activity.

Metabolic stability studies of lead compounds supported by separation techniques and chemometrics analysis.

With metabolism being one of the main routes of drug elimination from the body (accounting for removal of around 75% of known drugs), it is crucial to understand and study metabolic stability of drug candidates. Metabolically unstable compounds are uncomfortable to administer (requiring repetitive dosage during therapy), while overly stable drugs increase risk of adverse drug reactions. Additionally, biotransformation reactions can lead to formation of toxic or pharmacologically active metabolites (either less-active than parent drug, or even with different action). There were numerous approaches in estimating metabolic stability, including in vitro, in vivo, in silico, and high-throughput screening to name a few. This review aims at describing separation techniques used in in vitro metabolic stability estimation, as well as chemometric techniques allowing for creation of predictive models which enable high-throughput screening approach for estimation of metabolic stability. With a very low rate of drug approval, it is important to understand in silico methods that aim at supporting classical in vitro approach. Predictive models that allow assessment of certain biological properties of drug candidates allow for cutting not only cost, but also time required to synthesize compounds predicted to be unstable or inactive by in silico models.

Synthesis of Novel Pyrido[1,2-c]pyrimidine Derivatives with 6-Fluoro-3-(4-piperidynyl)-1,2-benzisoxazole Moiety as Potential SSRI and 5-HT1A Receptor Ligands.

Two series of novel 4-aryl-2H-pyrido[1,2-c]pyrimidine (6a-i) and 4-aryl-5,6,7,8-tetrahydropyrido[1,2-c]pyrimidine (7a-i) derivatives were synthesized. The chemical structures of the new compounds were confirmed by 1H and 13C NMR spectroscopy and ESI-HRMS spectrometry. The affinities of all compounds for the 5-HT1A receptor and serotonin transporter protein (SERT) were determined by in vitro radioligand binding assays. The test compounds demonstrated very high binding affinities for the 5-HT1A receptor of all derivatives in the series (6a-i and 7a-i) and generally low binding affinities for the SERT protein, with the exception of compounds 6a and 7g. Extended affinity tests for the receptors D2, 5-HT2A, 5-HT6 and 5-HT7 were conducted with regard to selected compounds (6a, 7g, 6d and 7i). All four compounds demonstrated very high affinities for the D2 and 5-HT2A receptors. Compounds 6a and 7g also had high affinities for 5-HT7, while 6d and 7i held moderate affinities for this receptor. Compounds 6a and 7g were also tested in vivo to identify their functional activity profiles with regard to the 5-HT1A receptor, with 6a demonstrating the activity profile of a presynaptic agonist. Metabolic stability tests were also conducted for 6a and 6d.

Evaluation of Analytes Characterized with Potential Protective Action after Rat Exposure to Lead.

Lead (Pb) was revealed for its role as a neurodevelopmental toxin. The determination of neurotransmitters (NTs) in particular brain regions could ameliorate the precise description and optimization of therapeutic protocols able to restore the harmony of signaling pathways in nervous and immune systems. The determination of selected analytes from the group of NTs based on the liquid chromatography (LC)-based method was carried out to illustrate the changes of amino acid (AA) and biogenic amine (BA) profiles observed in chosen immune and nervous systems rat tissues after Pb intoxication. Also, a protective combination of AA was proposed to correct the changes caused by Pb intoxication. After the administration of Pb, changes were observed in all organs studied and were characterized by a fluctuation of NT concentrations in immune and nervous systems (hypothalamus samples). Using a protective mixture of bioactive compounds prevented numerous changes in the balance of NT. The combined analysis of the immune and nervous system while the normalizing effect of curative agents on the level of differentially secreted NTs and AA is studied could present a new approach to the harmonization of those two essential systems after Pb intoxication.

Prediction of Chromatographic Elution Order of Analytical Mixtures Based on Quantitative Structure-Retention Relationships and Multi-Objective Optimization.

Prediction of the retention time from the molecular structure using quantitative structure-retention relationships is a powerful tool for the development of methods in reversed-phase HPLC. However, its fundamental limitation lies in the fact that low error in the prediction of the retention time does not necessarily guarantee a prediction of the elution order. Here, we propose a new method for the prediction of the elution order from quantitative structure-retention relationships using multi-objective optimization. Two case studies were evaluated: (i) separation of organic molecules in a Supelcosil LC-18 column, and (ii) separation of peptides in seven columns under varying conditions. Results have shown that, when compared to predictions based on the conventional model, the relative root mean square error of the elution order decreases by 48.84%, while the relative root mean square error of the retention time increases by 4.22% on average across both case studies. The predictive ability in terms of both retention time and elution order and the corresponding applicability domains were defined. The models were deemed stable and robust with few to no structural outliers.

The Influence of Ionic Liquids on the Effectiveness of Analytical Methods Used in the Monitoring of Human and Veterinary Pharmaceuticals in Biological and Environmental Samples-Trends and Perspectives.

Recent years have seen the increased utilization of ionic liquids (ILs) in the development and optimization of analytical methods. Their unique and eco-friendly properties and the ability to modify their structure allows them to be useful both at the sample preparation stage and at the separation stage of the analytes. The use of ILs for the analysis of pharmaceuticals seems particularly interesting because of their systematic delivery to the environment. Nowadays, they are commonly detected in many countries at very low concentration levels. However, due to their specific physiological activity, pharmaceuticals are responsible for bioaccumulation and toxic effects in aquatic and terrestrial ecosystems as well as possibly upsetting the body's equilibrium, leading to the dangerous phenomenon of drug resistance. This review will provide a comprehensive summary of the use of ILs in various sample preparation procedures and separation methods for the determination of pharmaceuticals in environmental and biological matrices based on liquid-based chromatography (LC, SFC, TLC), gas chromatography (GC) and electromigration techniques (e.g., capillary electrophoresis (CE)). Moreover, the advantages and disadvantages of ILs, which can appear during extraction and separation, will be presented and attention will be given to the criteria to be followed during the selection of ILs for specific applications.

Sensitive Analysis of Idarubicin in Human Urine and Plasma by Liquid Chromatography with Fluorescence Detection: An Application in Drug Monitoring.

A new approach for the sensitive, robust and rapid determination of idarubicin (IDA) in human plasma and urine samples based on liquid chromatography with fluorescence detection (LC-FL) was developed. Satisfactory chromatographic separation of the analyte after solid-phase extraction (SPE) was performed on a Discovery HS C18 analytical column using a mixture of acetonitrile and 0.1% formic acid in water as the mobile phase in isocratic mode. IDA and daunorubicin hydrochloride used as an internal standard (I.S.) were monitored at the excitation and emission wavelengths of 487 and 547 nm, respectively. The method was validated according to the FDA and ICH guidelines. The linearity was confirmed in the range of 0.1-50 ng/mL and 0.25-200 ng/mL, while the limit of detection (LOD) was 0.05 and 0.125 ng/mL in plasma and urine samples, respectively. The developed LC-FL method was successfully applied for drug determinations in human plasma and urine after oral administration of IDA at a dose of 10 mg to a patient with highly advanced alveolar rhabdomyosarcoma (RMA). Moreover, the potential exposure to IDA present in both fluids for healthcare workers and the caregivers of patients has been evaluated. The present LC-FL method can be a useful tool in pharmacokinetic and clinical investigations, in the monitoring of chemotherapy containing IDA, as well as for sensitive and reliable IDA quantitation in biological fluids.

Application of solid-phase microextraction in current biomedical research.

Extraction of endogenous compounds and drugs and their corresponding metabolites from complex matrices, such as biofluids and solid tissues, requires adequate analytical approach facilitating qualitative and quantitative analysis. To this end, solid-phase microextraction has been introduced as modern technology that is capable of efficient and high-throughput extraction of compounds due to its ability to amalgamate sampling, extraction, and pre-concentration steps, while requiring minimal use of organic solvents. The ability of solid-phase microextraction to enable analyses on small-volume biological samples and growing availability of biocompatible solid-phase microextraction coatings make it a highly useful technology for variety of applications. For example, solid-phase microextraction is particularly useful for identifying biomarkers in metabolomics studies, and it can be successfully applied in pharmaceutical and toxicological studies requiring the fast and sensitive determination of drug levels, especially those that are present at low levels in biological matrices such as plasma, urine, saliva, and hair. Moreover, solid-phase microextraction can be directly applied in in vivo studies because this extraction technique is non-exhaustive and its biocompatible probes offer minimal invasiveness to the analyzed system. In this article, we review recent progress in well-established solid-phase microextraction technique for in vitro and in vivo analyses of various metabolites and drugs in clinical, pharmaceutical, and toxicological applications.

Quantitative Structure-Retention Relationships with Non-Linear Programming for Prediction of Chromatographic Elution Order.

In this work, we employed a non-linear programming (NLP) approach via quantitative structure-retention relationships (QSRRs) modelling for prediction of elution order in reversed phase-liquid chromatography. With our rapid and efficient approach, error in prediction of retention time is sacrificed in favor of decreasing the error in elution order. Two case studies were evaluated: (i) analysis of 62 organic molecules on the Supelcosil LC-18 column; and (ii) analysis of 98 synthetic peptides on seven reversed phase-liquid chromatography (RP-LC) columns with varied gradients and column temperatures. On average across all the columns, all the chromatographic conditions and all the case studies, percentage root mean square error (%RMSE) of retention time exhibited a relative increase of 29.13%, while the %RMSE of elution order a relative decrease of 37.29%. Therefore, sacrificing %RMSE(tR) led to a considerable increase in the elution order predictive ability of the QSRR models across all the case studies. Results of our preliminary study show that the real value of the developed NLP-based method lies in its ability to easily obtain better-performing QSRR models that can accurately predict both retention time and elution order, even for complex mixtures, such as proteomics and metabolomics mixtures.

Lipophilicity Determination of Antifungal Isoxazolo[3,4-b]pyridin-3(1H)-ones and Their N1-Substituted Derivatives with Chromatographic and Computational Methods.

The lipophilicity of a molecule is a well-recognized as a crucial physicochemical factor that conditions the biological activity of a drug candidate. This study was aimed to evaluate the lipophilicity of isoxazolo[3,4-b]pyridine-3(1H)-ones and their N1-substituted derivatives, which demonstrated pronounced antifungal activities. Several methods, including reversed-phase thin layer chromatography (RP-TLC), reversed phase high-performance liquid chromatography (RP-HPLC), and micellar electrokinetic chromatography (MEKC), were employed. Furthermore, the calculated logP values were estimated using various freely and commercially available software packages and online platforms, as well as density functional theory computations (DFT). Similarities and dissimilarities between the determined lipophilicity indices were assessed using several chemometric approaches. Principal component analysis (PCA) indicated that other features beside lipophilicity affect antifungal activities of the investigated derivatives. Quantitative-structure-retention-relationship (QSRR) analysis by means of genetic algorithm-partial least squares (GA-PLS)-was implemented to rationalize the link between the physicochemical descriptors and lipophilicity. Among the studied compounds, structure 16 should be considered as the best starting structure for further studies, since it demonstrated the lowest lipophilic character within the series while retaining biological activity. Sum of ranking differences (SRD) analysis indicated that the chromatographic approach, regardless of the technique employed, should be considered as the best approach for lipophilicity assessment of isoxazolones.

Simultaneous electrokinetic and hydrodynamic injection and sequential stacking featuring sweeping for signal amplification following MEKC during the analysis of rapamycin (sirolimus) in serum samples.

Simultaneous electrokinetic and hydrodynamic injection of rapamycin (sirolimus) with off-line and online sample preconcentration techniques and using MEKC has been studied. Compared to conventional hydrodynamic injection, a 168-fold improvement in the signal was obtained with a combination of simultaneous electrokinetic and hydrodynamic injectionand field enhanced sample injection in conjunction with a sweeping technique called sequential stacking featuring sweeping. However, the coupling of the developed electrophoretic method and solid-phase microextraction allowed the signal intensity to increase more than 231 times. In this approach, the injection of the sample at negative polarity (anode at the detector end) into the capillary and the MEKC separation was achieved within 5 min using an electrolyte (composed of 10 mM sodium tetraborate and 40 mM SDS) when ultraviolet (UV) detection was performed at 280 nm. Thus, by combining the application of the sequential stacking featuring sweeping supported by the solid-phase microextraction clean-up procedure, the detection limit (LOD) for rapamycin in a serum sample was significantly decreased, and was set at 25 ng/mL. The proposed combined simultaneous electrokinetic and hydrodynamic injection with field enhanced sample injection -sweeping technique following MEKC separation of sirolimus in human serum could be an effective tool in biomedical and clinical applications.