Analysis Of Biological Samples Research Articles

Design-assisted HPLC-UV method for therapeutic drug monitoring of pholcodine, ephedrine, and guaifenesin in biological fluids.

Drug-drug interactions may amplify or diminish their intended effects, or even produce entirely new effects. Multicomponent mixture HPLC analysis offers a thorough and effective method for comprehending the makeup and behavior of complicated materials, advancing research and development across a range of scientific and industrial domains. A novel experimental design-assisted HPLC methodology for the concurrent investigation of the drug-drug interaction of pholcodine, ephedrine, and guaifenesin in biological fluids has been established. Rather than the routine methodology, the application of the factorial design-HPLC method offers a powerful and efficient tool for the analysis of these compounds. Both mixed and full factorial designs were employed to assess the impact of variable factors on chromatographic results. Utilizing an isocratic elution mode on a C18 column, the chromatographic separation was carried out. 15% Methanol, 5% acetonitrile, and 80% phosphate buffer with 0.1%(v/v) triethylamine set to pH 3 make up the mobile phase flowing at rate 1.0 mL/min. The calibration curves of the drugs show excellent linearity over a concentration ranges: 0.20-13.0µg/mL for PHO, 0.50-20.0µg/mL for EPH and 0.70-20.0µg/mL for GUA with LOQ values of 0.18, 0.38 and 0.50. The fast separation and quantitation in less than 6min is an advantage. Also, the method includes a robust sample preparation protocol for the analysis of complex biological samples, ensuring high selectivity and precision. The ease, speed and cost-effectiveness of the method are ideal for supporting in vitro studies, including drug-drug interaction investigations, especially in bioanalytical labs.

Smartphone microscopy: Design and implementation of a dual magnification system

This paper presents a dual magnification smartphone microscope designed for versatile imaging, enabling low magnification for wide-field viewing and high magnification for detailed biological sample analysis and automatic control of the working distance. It offers a cost-effective alternative to traditional lab microscopes, particularly in low-resource settings. Simulated, optimized, and designed using Ansys Zemax OpticStudio, the microscope was fabricated with 3D printing technology. Dual magnification (150x and 1000x) was achieved with two optical paths, improving resolution to 1 µm at higher and 1.5 µm at lower magnifications. The design provides clear and accurate images. The smartphone integration enhances usability, allowing easy magnification switching, image capture, and potential use of diagnostic applications.

Observation of biological and emulsion samples by newly developed three-dimensional impedance scanning electron microscopy

Imaging at nanometre-scale resolution is indispensable for many scientific fields such as biology, chemistry, material science and nanotechnology. Scanning electron microscopes (SEM) are widely used as important tools for the nanometre-scale analysis of various samples. However, because of the vacuum inside the SEM, a typical analysis requires fixation of samples, a drying process, and staining with heavy metals. Therefore, there is a need for convenient and minimally invasive methods of observing samples in solution. Recently, we have developed a new type of impedance microscopy, multi-frequency impedance SEM (IP-SEM), which allows nanoscale imaging of various specimens in water with minimal radiation damage. Here, we report a new IP-SEM system equipped with a linear-array terminal, which allows eight tilted images to be observed in a single capture by applying eight frequencies of input signals to each electrode. Furthermore, we developed a three-dimensional (3D) reconstruction method based on the Simulated Annealing (SA) algorithm, which enables us to construct a high-precision 3D model from the 8 tilted images. The method reported here can be easily used for 3D structural analysis of various biological samples, organic materials, and nanoparticles.

Innovative risk score to diagnose depression using data from the UAE Healthy Future Study

Abstract Introduction Depression has been recognized as a major mental health disorder. Diagnosing depression remains a challenge due to its complex multifactorial nature. The UAE Healthy Future Study is a long-term cohort study that focuses on understanding the factors that contribute to chronic disease among Emiratis. It involves answering a comprehensive questionnaire, undergoing physical measurements such as Body Mass Index (BMI), and biological samples for analysis. We analyized the eight-item depression screening instrument Patient Health Questionnaire (PHQ-8) for this project. Methods Out of 487 participants included in the PHQ-8, 205 participants (42.1%) were included in the statistical analysis after omitting missing values. A multivariate Least Absolute Selection Shrinkage Operator (LASSO) logistic regression model was performed using gender, age, BMI, waist circumference, hip circumference, body fat percentage, high-density lipoprotein (mg/dL), lowdensity lipoprotein (mg/dL), total cholesterol (mg/dL), diastolic blood pressure, systolic blood pressure, hemoglobin A1C, as predictors. The primary outcome was the binarized total PHQ-8 using a cutoff value of ten Tenfold cross-validation was applied. Results A cutoff value that yields an approximate sensitivity of 90% was selected to maximize the ability of the novel risk score to correctly identify subjects with depression symptoms. Sensitivity and specificity were estimated with a corresponding 95% confidence interval (95% CI). A novel depression risk score was computed as the linear predictor of the selected five variables in the LASSO regression analysis, as following: Risk score = -3.284 + 0.471*gender - 0.053*age + 0.049*BMI + 0.022*HDLmgdL - 0.072*HBA1c Conclusions We have shown that a novel depression risk score provides useful information for screening of depression. Further validation studies are needed to confirm the application of the risk score in daily practice. Key messages • A novel depression risk score provides useful information for screening of depression. • If the risk score can be verified in a different population, then it can improve the screening methods for depression.

A comprehensive study of AFM stiffness measurements on inclined surfaces: theoretical, numerical, and experimental evaluation using a Hertz approach

Atomic Force Microscopy (AFM) is a leading nanoscale technique known for its significant advantages in the analysis of soft materials and biological samples. Traditional AFM data analysis is often based on the Hertz model, which assumes perpendicular indentation of a planar sample. However, this assumption is not always valid due to the varying geometries of soft materials, whether natural, synthetic or biological. In this study, we present a new theoretical model that incorporates correction coefficients into Hertz’s model to account for cone-like and spherical probes, and to consider local tilt at the probe-sample interface. We validate our model using finite element analysis (FEA) simulations and experimental AFM measurements on tilted polyacrylamide gels. Our results highlight the need to include local tilt at the probe-sample contact to ensure accurate AFM measurements. This represents a step forward in our understanding of the elastic properties at the surface of soft materials in the broadest sense.

Extraction of synthetic cathinones from biological samples: A systematic review

Analysis of biological samples containing synthetic cathinones has become a significant area of interest for researchers in recent years, because synthetic cathinones can cause adverse reactions such as delusions and hallucinations. Generally, the intricate nature of the sample matrix and exceedingly low drug concentration in biological samples present significant challenges for the determination of target substances. Accordingly, scientists were dedicated to the advancement of effective sample preparation techniques to address these difficulties. However, the current reviews involved in synthetic cathinones all focused on detection methods. A systematic review focusing on sample preparation for the quantification of synthetic cathinones is still lacking. In light of that, this paper reviewed the most commonly used sample preparation techniques for synthetic cathinones. Recent advances in sample preparation techniques for synthetic cathinones in different biological samples such as blood, urine, oral fluid and hair samples were discussed. In addition, we prospected the challenges and the future perspectives of biological sample preparation techniques for synthetic cathinones determination.

Utility of Authentic 13C‐Labeled Disaccharide to Calibrate Hyaluronan Content Measurements by LC‐MS

ABSTRACTHyaluronan (hyaluronic acid, HA), a key glycosaminoglycan in the extracellular matrix, plays crucial roles in various physiological and pathological processes, including development, tissue hydration, inflammation, and tumor progression. Traditional methods for HA quantification, such as ELISA‐like assays, often have limitations in sensitivity and specificity, particularly for lower molecular weight HA. In this work, we introduce a coupled liquid chromatographic‐tandem mass spectrometric (LC‐MS/MS) method that employs a chemoenzymatically synthesized 13C‐labeled lyase‐derived authentic HA disaccharide calibrant for quantification of HA at the nanogram level. The method was validated against three HA polysaccharides with the sizes of ~33, 210, and 540 kDa. We applied this quantification technique to mouse tissues and plasma from both healthy and acetaminophen‐induced acute liver injury mice. Our data revealed a ~75‐fold increase in HA concentration in the liver of acetaminophen‐injured mice with a concomitant depletion from plasma. Overall, our method offers a robust, universal, and highly sensitive tool for HA analysis in diverse biological samples that will advance the investigation of the roles of this polysaccharide in human disease conditions.

Synthesis of Thioglycolic Acid-Modified Molybdenum Disulfide Nanosheets for Electrochemical Sensing and Its Application in Molnupiravir Detection

In the modern world, population growth, industrialization, and lifestyle changes have led to a rise in existing and new diseases, increasing global drug consumption. Proper pharmaceutical dosage is vital since drugs are only effective within specific concentration ranges. Therefore, developing reliable analytical methods for drug analysis in pharmaceuticals and biological samples is essential. Electroanalytical methods are particularly advantageous due to their low cost, ease of use, and rapid response. This study introduces a highly sensitive electrochemical sensor based on thioglycolic acid (TA)-decorated metallic phase molybdenum disulfide (MP-MoS2) nanosheets for the selective detection of molnupiravir (MOL), an antiviral drug used in Covid-19 treatment. The TA@MP-MoS2 nanomaterial was characterized using FTIR, TEM, and EIS. Screen-printed carbon electrodes (SPCE) were modified with TA@MP-MoS2 nanosheets to evaluate their electro-chemical and catalytic behaviours towards MOL by cyclic voltammetry (CV) and square wave voltammetry (SWV). The sensor displayed a well-defined electro-oxidation signal for MOL at 0.534 V, with the linear responses in two concentration ranges: 0.50–3.40 μM and 3.40–9.55 μM, and a low detection limit of 22.6 nM. The proposed design that has promising results could be an alternative strategy to fabricate the sensitive sensor for the detection of antiviral agents in real samples.

Comparison of in-vacuum micro-PIXE and in-air micro-PIXE for unfixed plant sample

PIXE analysis is very useful for the simultaneous determination of various elements. Typically, when micro-PIXE analysis of biological samples with high water content is performed in a vacuum, freeze-drying methods are used to fix the structure and remove any ice crystals present in a controlled manner to prevent changes or redistribution of elemental content in the sample. This method is essential for cell size analysis. However, we considered that fixation is not always necessary for macroscopic analysis of plant tissue size. Therefore, we investigated the extent to which elemental density changes with vacuum extraction from elemental density ratios obtained from micro-PIXE analysis of Arabidopsis thaliana leaves in-air and in-vacuum. The deformation caused by beam irradiation and the quantitative changes of C, O, and H in the samples were also evaluated by STIM and RBS analyses of Arabidopsis thaliana petal.

Highly sensitive detection of RNase H via a novel DNA/RNA heteroduplex combining isothermal exponential amplification strategy

Ribonuclease H (RNase H) plays a crucial role in a variety of cellular processes and is emerging as an essential therapeutic target for many diseases. Various methods have been constructed to assay RNase H activity, but these methods are either time-consuming or have poor sensitivity. In this study, we developed a novel strategy that combined a specially designed DNA/RNA chimeric substrate with exponential amplification reaction (EXPAR) for rapid and sensitive detection of RNase H activity. In the presence of RNase H, the RNA strand of the DNA/RNA heteroduplex was specifically degraded, forming a 3′-hydroxyl group in the locking primer for recognition and extension by DNA polymerase. This ultimately triggered EXPAR and produced a large number of single-stranded DNA, which was monitored in real-time with fluorescent dye. Under optimized conditions, the proposed strategy can detect as low as 6 × 10−10 U/μL of RNase H, which was at least 1000 times more sensitive than several reported methods. Furthermore, we demonstrated the usage of this method for RNase H inhibitor analysis and practical application in complex biological samples, including serum and tumor cell extracts. Therefore, these results suggested that the developed method is a promising tool for highly sensitive detection of RNase H and RNase H-associated disease diagnosis.

High-loading ficin@AuNPs on polymer-UiO-66 surface with enhanced peroxidase-mimetic catalytic activity for colourimetric detection of dopamine.

Recently, MOFs@AuNPs composites-based catalysts via anchoring of AuNPs onto metal-organic-frameworks (MOFs) have attracted great attention. However, the influence of the AuNPs loading amounts on the catalytic activity of MOFs@AuNPs composites remains largely unexplored. Here, ficin (Fic) protected AuNPs (Fic@AuNPs) anchored onto the surface of UiO-66-NH2 (UiO) modified with poly(2-vinyl-4,4-dimethyl-2-oxazolidine) (PV) were designed and constructed. The UiOPVFic@AuNPs composites with longer PV chains leading to high-loading Fic@AuNPs exhibited intense peroxidase (POD)-mimetic activity in 3,3'5,5'-tetramethylbenzidine (TMB) oxidation. Further, following the colour-fading, dopamine (DA) was sensitively and selectively monitored in the composites-TMB-H2O2 system. The portable smartphone sensing platform-based colourimetric method had good linearity ranging from 3.34 to 36.7μM (R2 = 0.995), with a limit of detection of 0.3μM. This protocol explores high-loading AuNPs on polymer-MOFs composites, providing deep insights into understanding catalytic activity improvements of polymer-MOFs@AuNPs catalysts and revealing their application potential in real biological samples analysis.

A broad-spectrum LC-MS/MS method for screening and quantification of 100 analytes in clinical and autopsy blood samples

Liquid chromatography coupled with mass spectrometry (LC-MS) has been tremendously used for screening purposes in forensic toxicology, because of their great adaptability and reasonable time/resource consumption. Herein, a fully validated method based on liquid-liquid extraction (LLE) in human whole blood, by a multiple reaction monitoring (MRM) analysis through LC-MS/MS, is described. The proposed method simultaneously detects 100 analytes (plus three deuterated internal standard compounds) belonging to many different classes, including drugs of abuse, prescription and over-the-counter drugs commonly involved in poisoning and medical malpractice cases in our territory, as well as certain new psychoactive substances (NPS) and toxic substances potentially associated with adverse effects. The optimised LLE employs one extraction step of 200 μL blood using 0.1 M HCl methyl-tert-butyl-ether (MTBE) (acidified with concentrated HCl) proved to be suitable for the extraction of basic and neutral substances; as a reconstitution solvent a mixture of 88:12v/v, 0.1 % formic acid in 10 mM aqueous ammonium acetate, pH 3.5: 0.1 % formic acid in acetonitrile was used, yielding satisfactory recoveries for all analytes. The method was sensitive, showing low LOD/ LOQ for all substances ranging from 0.01 to 5/ 0.05–20 ng/mL, respectively. Linearity ranged between 0.05–500 ng/mL (R2 = 0.9811–0.9995), and the inter- and intra-day precisions ranged between 3–15 % and 7–18 %, respectively. Accuracy was evaluated in terms of percentage recovery, lying within acceptable range. The matrix effect expressed as ion suppression/enhancement of each analyte was in the range ±25 % for all analytes. Post-preparative stability of analytes was higher than 85 %, while no carryover between runs was observed. The developed method has been successfully applied in routine toxicological analyses for the analysis of biological samples from clinical and autopsy cases.

Comprehensive Microbiological and Metagenomic Analysis of the Guillain–Barré Syndrome Outbreak in Lima, 2019

In 2018/2019, two large Guillain–Barré Syndrome (GBS) outbreaks took place in Peru. Here, we report a comprehensive analysis of biological samples from GBS patients from the 2019 outbreak. We applied metagenomic, microbiologic, and serological analyses to different biological samples collected from GBS patients. Further phenotypic and genomic characterization was conducted on Campylobacter jejuni isolates from GBS samples. Microbiologic and metagenomic analyses revealed several patients with multiple co-infections, yet no common infectious agents were found other than C. jejuni. Four C. jejuni isolates were isolated from rectal swabs. Twenty-one patients had detectable IgG serum antibodies related to C. jejuni, of whom seven had IgM antibodies. Genomic analyses showed that these four strains were clonal (ST2993) and contained the class A lipooligosaccharide biosynthesis locus. These results further support the idea that that C. jejuni is the etiological agent that triggered the GBS outbreak in Peru in 2019 and that the strains are not restricted to Peru, hence could be regarded as a broad public health concern. Furthermore, though we cannot delineate the role played by co-infections in GBS development, results obtained herein highlight metagenomic analysis as a potential new tool for depicting a yet unknown area of research in GBS.

Voltammetric and flow amperometric determination of drug guaifenesin in pharmaceutical and biological samples using screen-printed sensor with boron doped diamond electrode

New voltammetric and flow amperometric methods for the determination of guaifenesin (GFE) using a perspective screen-printed sensor (SPE) with boron-doped diamond electrode (BDDE) were developed. The electrochemical oxidation of GFE was studied on the surface of the oxygen-terminated BDDE of the sensor. The GFE provided two irreversible anodic signals at a potential of 1.0 and 1.1 V (vs. Ag|AgCl|KCl sat.) in Britton-Robinson buffer (pH 2), which was chosen as the supporting electrolyte for all measurements. First, a voltammetric method based on differential pulse voltammetry was developed and a low detection limit (LOD = 41 nmol L−1), a wide linear dynamic range (LDR = 0.1–155 μmol L−1), and a good recovery in the analysis of model and pharmaceutical samples (RSD <3.0 %) were obtained. In addition, this sensor demonstrated excellent sensitivity and reproducibility in the analysis of biological samples (RSD <3.2 %), where the analysis took place in a drop of serum (50 μL) without pretreatment and additional electrolyte. Subsequently, SP/BDDE was incorporated into a flow-through 3D printed electrochemical cell and a flow injection analysis method with electrochemical detection (FIA-ED) was developed, resulting in excellent analytical parameters (LOD = 86 nmol L−1, LDR = 0.1–50 μmol L−1). Moreover, the mechanism of electrochemical oxidation of GFE was proposed based on calculations of HOMO spatial distribution and spectroelectrochemical measurements focused on IR identification of intermediates and products.

Multi-class plant hormone HILIC-MS/MS analysis coupled with high-throughput phenotyping to investigate plant-environment interactions.

Plant hormones are chemical signals governing almost every aspect of a plant's life cycle and responses to environmental cues. They are enmeshed within complex signaling networks that can only be deciphered by using broad-scale analytical methods to capture information about several plant hormone classes simultaneously. Methods used for this purpose are all based on reversed-phase (RP) liquid chromatography and mass spectrometric detection. Hydrophilic interaction chromatography (HILIC) is an alternative chromatographic method that performs well in analyses of biological samples. We therefore developed and validated a HILIC method for broad-scale plant hormone analysis including a rapid sample preparation procedure; moreover, derivatization or fractionation is not required. The method enables plant hormone screening focused on polar and moderately polar analytes including cytokinins, auxins, jasmonates, abscisic acid and its metabolites, salicylates, indoleamines (melatonin), and 1-aminocyclopropane-1-carboxylic acid (ACC), for a total of 45 analytes. Importantly, the major pitfalls of ACC analysis have been addressed. Furthermore, HILIC provides orthogonal selectivity to conventional RP methods and displays greater sensitivity, resulting in lower limits of quantification. However, it is less robust, so procedures to increase its reproducibility were established. The method's potential is demonstrated in a case study by employing an approach combining hormonal analysis with phenomics to examine responses of three Arabidopsis ecotypes toward three abiotic stress treatments: salinity, low nutrient availability, and their combination. The case study showcases the value of the simultaneous determination of several plant hormone classes coupled with phenomics data when unraveling processes involving complex cross-talk under diverse plant-environment interactions.

Boric Acid Functionalized Hypercrosslinked Polymers for Selective Extraction of Trace Catecholamines and Their Metabolites in Rat Serum

ABSTRACTAbnormal levels of catecholamine (CA) neurotransmitters and their metabolites in biological fluids can lead to various neurological disorders. Herein, a boric acid‐functionalized hypercrosslinked polymer was prepared and utilized as a sorbent for the dispersive solid‐phase extraction of CAs and their metabolites in rat serum. By combination with a high‐performance liquid chromatography‐fluorescence detector, the extraction parameters for the seven target analytes were optimized. Under the optimal extraction condition, the methodology for the quantitative analysis of CAs and their metabolites in rat serum samples was established. The limits of detection and limits of quantification were found to be in the ranges of 0.010–0.015 and 0.033–0.050 ng/mL, respectively. The results demonstrated satisfactory recoveries, with values ranging from 88.02% to 113.27%, accompanied by relative standard deviations within the range of 2.69%–9.59%. In addition, the method showed good anti‐interference ability (matrix effect ranged from 2.64% to 18.07%). The developed method was validated for the determination of CAs and their metabolites in normal and Alzheimer's disease model rats’ serum, which proved the promising application of the method for CAs neurotransmitter analysis in biological samples.

Development and Validation of a Dispersive Liquid–Liquid Microextraction Method for Metoclopramide Analysis in Pharmaceuticals and Biological Samples

Development and Validation of a Dispersive Liquid–Liquid Microextraction Method for Metoclopramide Analysis in Pharmaceuticals and Biological Samples

A dual-recognition strategy based on pH-responsive molecularly imprinted membrane for highly selective capture of catecholamines: From construction to practical application

BackgroundCatecholamines (CAs) are involved in a wide range of physiological and pathological processes in the body and are progressively being used as important biomarkers for a variety of diseases. It is of great significance for accurate quantification of CAs to the diagnosis and treatment of diseases. However, the separation of CAs from complex biological matrices is still a great challenge due to the trace levels of CAs and the limited selectivity of existing pretreatment methods. ResultsIn this work, a dual-recognition imprinted membrane (BA-MIM) was developed to utilize the synergistic action of pH-responsive boron affinity and molecular imprinted cavities for highly selective capture and release of CAs. The prepared BA-MIM possessed remarkable adsorption capacity (maximum capacity, 43.3 mg g−1), desirable surface hydrophilicity (46.2°), superior selectivity (IF = 6.2, α = 14.3), as well as favorable reusability (number of cycles, 6 times). On this basis, an integrated analytical method based on BA-MIM extraction combined with ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) was innovatively developed to highly selective separation, enrichment, and detection of CAs in rat brain tissue. Under the optimum conditions, a low quantitation limits (0.05–0.10 ng mL−1), wide linear range (10–1000 ng mL−1), good linearity (r2 > 0.99), and satisfactory recoveries (88.5%–98.5 %) were obtained for CAs. The proven method was further applied to kidney-yang-deficiency-syndrome (KYDS) group rat model, revealed the intrinsic connection between kidney disease and catecholamine metabolism. SignificanceThis work provides an excellent reference paradigm for the effective construction of dual-recognition functional membrane material to the high-selective analysis of trace targets in complex matrices. Additionally, this integrated analytical strategy demonstrates its efficiency, sustainability, versatility, and convenience, showing remarkable prospect in a variety of applications for biological sample analysis.

Innovative voltammetric techniques for bumadizone analysis in pharmaceutical and biological samples: emphasizing green, white, and blue analytical approaches

There are no documented electroanalytical methods for quantifying the anti-inflammatory drug bumadizone (BUM) in pharmaceutical or biological matrices. So, a new voltammetric method was developed to determine BUM at nano concentrations in pharmaceutical forms, in the presence of its alkaline degradant, and in biological fluids. Five electrodes were tested, including three nano-reduced graphene oxide (nRGO) electrodes (5%, 15%, and 20%), a carbon paste electrode (CPE), and a 10% nRGO-modified CPE. The 10% nRGO-modified electrode showed the best performance, offering high selectivity and low detection limits, with good linearity in the concentration range of 0.9 × 102 to 15 × 102 ng mL−1. Differential pulse voltammetry successfully applied this electrode for BUM determination in various samples, achieving excellent recovery without preliminary separation. The method was validated according to ICH guidelines and compared favorably to the reference method. Its environmental impact was assessed using AGREE and Eco-scale metrics in addition to the RGB algorithm, showing superior greenness and whiteness profiles due to safer solvents and lower energy consumption, along with high practical effectiveness using the BAGI metric.

Analysis of human biological samples using porous graphitic carbon columns and liquid chromatography-mass spectrometry: a review.

Liquid chromatography-mass spectrometry (LC-MS) has emerged as a powerful analytical technique for analyzing complex biological samples. Among various chromatographic stationary phases, porous graphitic carbon (PGC) columns have attracted significant attention due to their unique properties-such as the ability to separate both polar and non-polar compounds and their stability through all pH ranges and to high temperatures-besides the compatibility with LC-MS. This review discusses the applicability of PGC for SPE and separation in LC-MS-based analyses of human biological samples, highlighting the diverse applications of PGC-LC-MS in analyzing endogenous metabolites, pharmaceuticals, and biomarkers, such as glycans, proteins, oligosaccharides, sugar phosphates, and nucleotides. Additionally, the fundamental principles underlying PGC column chemistry and its advantages, challenges, and advances in method development are explored. This comprehensive review aims to provide researchers and practitioners with a valuable resource for understanding the capabilities and limitations of PGC columns in LC-MS-based analysis of human biological samples, thereby facilitating advancements in analytical methodologies and biomedical research.