Reversed-phase high-performance liquid chromatography (RP-HPLC) is the most popular chromatographic mode, accounting for more than 90% of all separations. HPLC itself owes its immense popularity to it being relatively simple and inexpensive, with the equipment being reliable and easy to operate. Due to extensive automation, it can be run virtually unattended with multiple samples at various separation conditions, even by relatively low-skilled personnel. Currently, there are >600 RP-HPLC columns available to end users for purchase, some of which exhibit very large differences in selectivity and production quality. Often, two similar RP-HPLC columns are not equally suitable for the requisite separation, and to date, there is no universal RP-HPLC column covering a variety of analytes. This forces analytical laboratories to keep a multitude of diverse columns. Therefore, column selection is a crucial segment of RP-HPLC method development, especially since sample complexity is constantly increasing. Rationally choosing an appropriate column is complicated. In addition to the differences in the primary intermolecular interactions with analytes of the dispersive (London) type, individual columns can also exhibit a unique character owing to specific polar, hydrogen bond, and electron pair donor–acceptor interactions. They can also vary depending on the type of packing, amount and type of residual silanols, "end-capping", bonding density of ligands, and pore size, among others. Consequently, the chromatographic performance of RP-HPLC systems is often considerably altered depending on the selected column. Although a wide spectrum of knowledge is available on this important subject, there is still a lack of a comprehensive review for an objective comparison and/or selection of chromatographic columns. We aim for this review to be a comprehensive, authoritative, critical, and easily readable monograph of the most relevant publications regarding column selection and characterization in RP-HPLC covering the past four decades. Future perspectives, which involve the integration of state-of-the-art molecular simulations (molecular dynamics or Monte Carlo) with minimal experiments, aimed at nearly "experiment-free" column selection methodology, are proposed.
The approaches of combining analytical and pharmacokinetic tools can currently assist clinicians in routinely providing more effective and individualized treatment, including in complicated cases of pediatric acute myeloid leukemia. Anticancer drug dosing based on synchronized drug monitoring and pharmacokinetic analysis can provide a better estimation of the drug systemic exposure than that obtained with the stable dosing plan. Leukemia is difficult to treat and, in the case of children, has the most dramatic and tragic course. Therefore, it is important to search for new biomarkers of leukemia that will enable early diagnosis. Hence, a completed strategy with chromatographic methods as the core element of analytical procedures provides an efficient tool that is beneficial for clinicians involved in the treatment and diagnosis of leukemia.
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.
In this work, a target-based drug screening method is proposed exploiting the synergy effect of ligand-based and structure-based computer-assisted drug design. The new method provides great flexibility in drug design and drug candidates with considerably lower risk in an efficient manner. As a model system, 45 sulphonamides (33 training, 12 testing ligands) in complex with carbonic anhydrase IX were used for development of quantitative structure-activity-lipophilicity (property)-relationships (QSPRs). For each ligand, nearly 5,000 molecular descriptors were calculated, while lipophilicity (logkw) and inhibitory activity (logKi) were used as drug properties. Genetic algorithm-partial least squares (GA-PLS) provided a QSPR model with high prediction capability employing only seven molecular descriptors. As a proof-of-concept, optimal drug structure was obtained by inverting the model with respect to reference drug properties. 3509 ligands were ranked accordingly. Top 10 ligands were further validated through molecular docking. Large-scale MD simulations were performed to test the stability of structures of selected ligands obtained through docking complemented with biophysical experiments.
A rapid and effective method of nonionic micellar electrokinetic chromatography with laser-induced fluorescence detection and sweeping as an online preconcentration method was developed for the simultaneous determination of free essential amino acids in supplement formulations. The analytes were derivatized using fluorescein isothiocyanate isomer I dissolved in dimethyl sulfoxide. Optimal separation conditions were achieved using an uncoated fused silica capillary (40.0 cm effective length, 50.2 cm total length, 50 µm internal diameter), 20 mM Na2B4O7, (pH 9.3), 50 mM Brij-35, and 15% (v/v) methanol as running buffer. Complete separation of all analytes was achieved within 15 minutes. The optimized method exhibited good linearity (r2 > 0.997), acceptable precision (1.3 – 12.4%), accuracy (88 – 112%) in concentration range 20 – 1000 ng mL-1 The limit of detection was 60 pg mL-1. The method succesfully determined the quantity of free essential amino acids in a dietary supplement containing spirulina.
The goal of this study was to assess various analytical approaches for the simultaneous and efficient extraction of steroid hormones (cortisone, cortisol, prednisolone, corticosterone, testosterone, 17α‐methyltestosterone, epitestosterone, progesterone) from urine samples prior to separation based on field‐amplified sample stacking MEKC (FASS‐MEKC). FASS‐MEKC successfully allowed the compounds to be separated within 12 min using a BGE composed of 5 mM sodium tetraborate, 150 mM boric acid, 50 mM SDS, and 15% methanol. Therefore, many procedures such as solid‐phase microextraction, SPE, and dispersive liquid–liquid microextraction (DLLME) were tested and compared using a multivariate tool, namely, cluster analysis. Finally, DLLME‐FASS‐MEKC was validated and proved a good linearity of calibration curves ( R 2 above 0.9948) in a concentration range from 50 to 1000 ng/mL for all analytes. The LOD was established at 15 ng/mL, whereas the LOQ was 50 ng/mL. The intra‐ and interday precision, expressed as RSD%, did not exceed 9.97%. The DLLME‐FASS‐MEKC method was successfully applied to the analysis of urine samples from healthy volunteers and sportsmen. This methodology could prove to be useful in clinical studies and/or doping control depending on the steroid concentrations required in biomedical applications.
Abstract 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.
The influence of sample matrix on sample sweeping in MEKC was examined in the presented manuscript. Significant focusing effect was observed for relatively hydrophobic cationic compounds (emetine, strychnine and quinine) using high ionic strength sample matrix (900 mM H3 PO4 /720 mM Tris) which conductivity was about ninefold higher than utilized BGE. Moreover, the results were obtained using BGE composed of comparatively low surfactant concentration (10 mM SDS) and 40 mM H3 PO4 /32 mM Tris buffer solution. About 200 to 300-fold preconcentration of analytes was reached with the presented method. Basing on experimental results and computer simulation using Simul5 software, hypothetical mechanism of observed phenomenon was proposed.
