Biomedical Evaluation of Cortisol, Cortisone, and Corticosterone along with Testosterone and Epitestosterone Applying Micellar Electrokinetic Chromatography

The validated micellar electrokinetic chromatography (MEKC) was proposed for the determination of five steroid hormones in human urine samples. That technique allowed for the separation and quantification of cortisol, cortisone, corticosterone, testosterone, and epitestosterone and was sensitive enough to detect low concentrations of these searched steroids in urine samples at the range of 2–300 ng/mL. The proposed MEKC technique with solid-phase extraction (SPE) procedure was simple, rapid, and has been successfully applied as a routine procedure to analyze steroids in human urine samples. The MEKC method offered a potential in clinical routine practice because of the short analysis time (8 min), low costs, and simultaneous analysis of five endogenous hormones. Due to its simplicity, speed, accuracy, and high recovery, the proposed method could offer a tool to determine steroid hormones as potential biomarkers in biomedical investigations, what was additionally revealed with healthy volunteers

Optimization of a Pre-MEKC Separation SPE Procedure for Steroid Molecules in Human Urine Samples

Many steroid hormones can be considered as potential biomarkers and their determination in body fluids can create opportunities for the rapid diagnosis of many diseases and disorders of the human body. Most existing methods for the determination of steroids are usually time- and labor-consuming and quite costly. Therefore, the aim of analytical laboratories is to develop a new, relatively low-cost and rapid implementation methodology for their determination in biological samples. Due to the fact that there is little literature data on concentrations of steroid hormones in urine samples, we have made attempts at the electrophoretic determination of these compounds. For this purpose, an extraction procedure for the optimized separation and simultaneous determination of seven steroid hormones in urine samples has been investigated. The isolation of analytes from biological samples was performed by liquid-liquid extraction (LLE) with dichloromethane and compared to solid phase extraction (SPE) with C18 and hydrophilic-lipophilic balance (HLB) columns. To separate all the analytes a micellar electrokinetic capillary chromatography (MECK) technique was employed. For full separation of all the analytes a running buffer (pH 9.2), composed of 10 mM sodium tetraborate decahydrate (borax), 50 mM sodium dodecyl sulfate (SDS), and 10% methanol was selected. The methodology developed in this work for the determination of steroid hormones meets all the requirements of analytical methods. The applicability of the method has been confirmed for the analysis of urine samples collected from volunteers—both men and women (students, amateur bodybuilders, using and not applying steroid doping). The data obtained during this work can be successfully used for further research on the determination of steroid hormones in urine samples

Investigation of Imidazolium-Based Ionic Liquids as Additives for the Separation of Urinary Biogenic Amines via Capillary Electrophoresis

Ionic liquids (ILs), such as imidazoles, can be used to prevent the sorption of analytes onto the walls of the capillary. Prior works have confirmed that coating the capillary wall with a cationic layer can increase its surface stability, thereby improving the repeatability of the separation process. In this study, micellar electrokinetic chromatography (MEKC) is employed to evaluate how two ILs with different anions—namely, 1-hexyl-3-methylimidazolium chloride [HMIM+Cl−] and 1-hexyl-3-methylimidazolium tetrafluoroborate [HMIM+BF4−]—affect the separation efficiency for biogenic amines (BAs) such as metanephrine (M), normetanephrine (NM), vanilmandelic acid (VMA), and homovanillic acid (HVA) in urine samples. To this end, solid-phase extraction (SPE) is employed using different sample pH values, with the results demonstrating that HVA and VMA is easily extracted at a sample pH of 5.5, while a sample pH of 9.0 facilitated the extraction of M and NM. In the applied SPE protocol, selected analytes were isolated from urine samples using hydrophilic–lipophilic-balanced (HLB) columns and eluted with methanol (MeOH). The validation data confirmed the method’s linearity (R2 > 0.996) for all analytes within the range of 0.25–10 µg/mL. The applicability of the optimized SPE-MEKC-UV method was confirmed by employing it to quantify clinically relevant BAs in real urine samples from pediatric neuroblastoma (NBL) patients

Comparative Study of Various Procedures for Extracting Doxorubicin from Animal Tissue Samples

This article presents a comparative study of selected deproteinization-, liquid–liquid-extraction- (LLE), and solid-phase-extraction (SPE)-based procedures for the isolation of doxorubicin (DOX) and daunorubicin (DAU) as an internal standard (IS) from rat tissue samples. During the experiments, all samples were analyzed via liquid chromatography coupled with fluorescence detection (LC-FL), with analytes being monitored at excitation and emission wavelengths of 487 and 555 nm, respectively. The absolute recoveries of the sample-preparation procedure were then calculated and compared, and the advantages and disadvantages of each approach were considered in depth. Ultimately, SPE with hydrophilic–lipophilic balanced (HLB) sorbents was selected as the most effective extraction procedure as it enabled the absolute recovery of DOX from tissue samples at a level of 91.6 ± 5.1%. Next, the selected HLB-SPE protocol was coupled with LC-FL separation and the resultant method was validated according to FDA and ICH requirements. The validation data confirmed that the developed procedure met all required criteria for bioanalytical methods, with a limit of detection (LOD) and limit of quantification (LOQ) of 0.005 µg/g and 0.01 µg/g, respectively. Finally, the developed protocol was successfully tested on various rat tissues enriched with DOX, confirming its potential as an interesting alternative to previously reported protocols for pharmacokinetic studies and clinical investigations aimed at analysis of the level and biodistribution of DOX in tissue samples after systemic administration of this drug

Comparison of Three Extraction Approaches for the Isolation of Neurotransmitters from Rat Brain Samples

The determination of neurotransmitters (NTs) as relevant potential biomarkers in the study of various central nervous system (CNS) pathologies has been demonstrated. Knowing that NTs-related diseases mostly occupy individual regions of the nervous system, as observed, for instance, in neurodegenerative diseases (Alzheimer’s and Parkinson’s Diseases), the analysis of brain slices is preferred to whole-brain analysis. In this report, we present sample preparation approaches, such as solid-phase extraction, solid-phase microextraction, and dispersive liquid–liquid microextraction, and discuss the pitfalls and advantages of each extraction method. The ionic liquid (1-ethyl-3-methylimidazolium tetrafluoroborate)-assisted solid-phase microextraction (IL-SPME) is found to be, in our research, the relevant step towards the simultaneous determination of six NTs, namely, dopamine (DA), adrenaline (A), noradrenaline (NA), serotonin (5-HT), l-tryptophan (l-Trp), l-tyrosine (l-Tyr) in rat brain samples. The development of a novel bioanalytical technique for the evaluation of biomarkers in the context of green chemistry might be accelerated just with the use of IL, and this approach can be considered an advantageous strategy

Chemometric Evaluation of Urinary Steroid Hormone Levels as Potential Biomarkers of Neuroendocrine Tumors

Neuroendocrine tumors (NETs) are uncommon tumors which can secrete specific hormone products such as peptides, biogenic amines and hormones. So far, the diagnosis of NETs has been difficult because most NET markers are not specific for a given tumor and none of the NET markers can be used to fulfil the criteria of high specificity and high sensitivity for the screening procedure. However, by combining the measurements of different NET markers, they become highly sensitive and specific diagnostic tests. The aim of the work was to identify whether urinary steroid hormones can be identified as potential new biomarkers of NETs, which could be used as prognostic and clinical course monitoring factors. Thus, a rapid and sensitive reversed-phase high-performance liquid chromatographic method (RP-HPLC) with UV detection has been developed for the determination of cortisol, cortisone, corticosterone, testosterone, epitestosterone and progesterone in human urine. The method has been validated for accuracy, precision, selectivity, linearity, recovery and stability. The limits of detection and quantification were 0.5 and 1 ng mL−1 for each steroid hormone, respectively. Linearity was confirmed within a range of 1–300 ng mL−1 with a correlation coefficient greater than 0.9995 for all analytes. The described method was successfully applied for the quantification of six endogenous steroid levels in human urine. Studies were performed on 20 healthy volunteers and 19 patients with NETs. Next, for better understanding of tumor biology in NETs and for checking whether steroid hormones can be used as potential biomarkers of NETs, a chemometric analysis of urinary steroid hormone levels in both data sets was performed