Analysis Of Biological Fluids Research Articles

Phytosterols in human serum as measured using a liquid chromatography tandem mass spectrometry

Phytosterols are lipophilic compounds found in plants with structural similarity to mammalian cholesterol. They cannot be endogenously produced by mammals and therefore always originate from diet. There has been increased interest in dietary phytosterols over the last few decades due to their association with a variety of beneficial health effects including low-density lipoprotein cholesterol lowering, anti-inflammatory and anti-cancerous effects. They are proposed as potential moderators for diseases associated with the central nervous system where cholesterol homeostasis is found to be imperative (multiple sclerosis, dementia, etc.) due to their ability to reach the brain. Here we utilised an enzyme-assisted derivatisation for sterol analysis (EADSA) in combination with a liquid chromatography tandem mass spectrometry (LC-MSn) to characterise phytosterol content in human serum. As little as 100 fg of plant sterol was injected on a reversed phase LC column. The method allows semi-quantitative measurements of phytosterols and their derivatives simultaneously with measurement of cholesterol metabolites. The identification of phytosterols in human serum was based on comparison of their LC retention times and MS2, MS3 spectra with a library of authentic standards. Free campesterol serum concentration was in the range from 0.30-4.10 µg/mL, β-sitosterol 0.16-3.37 µg/mL and fucosterol was at lowest concentration range from 0.05-0.38 µg/mL in ten individuals. This analytical methodology could be applied to the analysis of other biological fluids and tissues.

Development of D-π-A organic dyes for discriminating HSA from BSA and study on dye-HSA interaction

HSA (human serum albumin), a most abundant protein in blood serum, plays a key role in maintaining human health. Abnormal HSA level is correlated with many diseases, and thus has been used as an essential biomarker for therapeutic monitoring and biomedical diagnosis. Development of small-molecule fluorescent probes allowing the selective and sensitive recognition of HSA in in vitro and in vivo is of fundamental importance in basic biological research as well as medical diagnosis. Herein, we reported a series of new synthesized fluorescent dyes containing D-π-A constitution, which exhibited different optical properties in solution and solid state. Among them, dye M−H−SO3 with a hydrophilic sulfonate group at electron-acceptor part displayed selectivity for discrimination of HSA from BSA and other enzymes. Upon binding of dye M−H−SO3 with HSA, a significant fluorescence enhancement with a turn-on ratio about 96-fold was triggered. The detection limit was estimated to be ∼ 40 nM. Studies on the interaction mechanism revealed that dye M−H−SO3 could bind to site III of HSA with a 1:1 binding stoichiometry. Furthermore, dye M−H−SO3 has been applied to determine HSA in real urine samples with good recoveries, which provided a useful method for HSA analysis in biological fluids.

Characterization of the plasma proteome from healthy adult dogs.

Bloodwork is a widely used diagnostic tool in veterinary medicine, as diagnosis and therapeutic interventions often rely on blood biomarkers. However, biomarkers available in veterinary medicine often lack sensitivity or specificity. Mass spectrometry-based proteomics technology has been extensively used in the analysis of biological fluids. It offers excellent potential for a more comprehensive characterization of the plasma proteome in veterinary medicine. In this study, we aimed to identify and quantify plasma proteins in a cohort of healthy dogs and compare two techniques for depleting high-abundance plasma proteins to enable the detection of lower-abundance proteins via label-free quantification liquid chromatography-mass spectrometry. We utilized surplus lithium-heparin plasma from 30 healthy dogs, subdivided into five groups of pooled plasma from 6 randomly selected individuals each. Firstly, we used a commercial kit to deplete high-abundance plasma proteins. Secondly, we employed an in-house method to remove albumin using Blue-Sepharose. Among all the samples, some of the most abundant proteins identified were apolipoprotein A and B, albumin, alpha-2-macroglobulin, fibrinogen beta chain, fibronectin, complement C3, serotransferrin, and coagulation factor V. However, neither of the depletion techniques achieved significant depletion of highly abundant proteins. Despite this limitation, we could detect and quantify many clinically relevant proteins. Determining the healthy canine proteome is a crucial first step in establishing a reference proteome for canine plasma. After enrichment, this reference proteome can later be utilized to identify protein markers associated with different diseases, thereby contributing to the diagnosis and prognosis of various pathologies.

Nickel oxide electroplating and electrode surface modification for electrochemical detection of glutamate

The electrochemical behavior and catalytic properties of nickel oxide (NiOx) are enhanced through cathodic electroplating and subsequent electrochemical activation in an alkaline aqueous electrolyte. The electrochemical detection of ʟ-glutamate becomes feasible by introducing an amine group through immobilizing APTES ((3-aminopropyl)triethoxysilane) on the NiOx surface, resulting in a positive charge. The synergistic effect of electrochemically activated electroplated NiOx and the APTES-modified surface facilitates the effective electrochemical detection of trace ʟ-glutamate. The APTES-activated NiOx electrode exhibits a linear detection range of 0.2 nM to 2 mM for glutamate. This relatively wide concentration range is promising for the analysis of human biological fluids.

Quantification of Plasma Dopamine in Depressed Patients Using Silver-Enriched Silicon Nanowires as SERS-Active Substrates.

A decrease in the levels of dopamine (DA)─a key catecholamine biomarker for major depressive disorder─highlights the need for quantitative analysis of biological fluids to aid in the early diagnosis of diverse neuropsychiatric disorders. This study developed silicon nanowires enriched with silver nanoparticles to serve as a surface-enhanced Raman scattering (SERS) substrate to enable precise and sensitive quantification of blood plasma DA levels in humans. The silver-enriched silicon nanowires (SiNWs@Ag) yielded flower-like assemblies with densely populated SERS "hot spots," allowing sensitive DA detection. By correlating DA concentration with Raman intensity at 1156 cm-1, the plasma DA levels in treatment-naïve patients with major depression (n = 18) were 2 orders of magnitude lower than those in healthy controls (n = 18) (6.56 × 10-10 M vs 1.43 × 10-8 M). The plasma DA concentrations differed significantly between the two groups (two-tailed p = 5.77×10-7), highlighting a distinct demarcation between depression patients and healthy controls. Furthermore, the SiNWs@Ag substrate effectively differentiated between DA and norepinephrine (NE) in mixtures at nanomolar levels, demonstrating its selective detection capability. This study represents the first report on the quantitative detection of DA levels in human blood samples from individuals with major depression using an SERS technique, emphasizing its potential clinical utility in the evaluation and diagnosis of neuropsychiatric disorders.

Cancer screening through surface-enhanced Raman spectroscopy fingerprinting analysis of urinary metabolites using surface-carbonized silver nanowires on a filter membrane

BackgroundLabel-free surface-enhanced Raman spectroscopy (SERS)-based metabolic profiling has great potential for early cancer diagnosis, but further advancements in analytical methods and clinical evidence studies are required for clinical applications. To improve the cancer diagnostic accuracy of label-free SERS spectral analysis of complex biological fluids, it is necessary to obtain specifically enhanced SERS signals of cancer-related metabolites present at low concentrations. ResultsThis study presents a novel 3D SERS sensor, comprising a surface-carbonized silver nanowire (AgNW)-stacked filter membrane, alongside an optimized urine/methanol/chloroform extraction technique, which specifically changes the molecular adsorption and orientation of aromatic metabolites onto SERS substrates. By analyzing the pretreated urine samples on the surface-carbonized AgNW 3D SERS sensor, distinct and highly enhanced SERS peaks derived from semi-polar aromatic metabolites were observed for pancreatic cancer and prostate cancer samples compared with normal controls. Urine metabolite analysis using SERS fingerprinting successfully differentiated pancreatic cancer and prostate cancer groups from normal control group: normal control (n = 56), pancreatic cancer (n = 40), and prostate cancer (n = 39). Significance and noveltyWe confirmed the clinical feasibility of performing fingerprint analysis of urinary metabolites based on the surface-carbonized AgNW 3D SERS sensor and methanol/chloroform extraction for noninvasive cancer screening. This technology holds potential for large-scale screening owing to its high accuracy, and cost effective, simple and rapid detection method.

Synthesis of 15N-Pyridines and Higher Mass Isotopologs via Zincke Imine Intermediates.

Methods to incorporate stable radioisotopes are integral to pharmaceutical and agrochemical development. However, despite the prevalence of pyridines in candidate compounds, methods to incorporate 15N atoms within their structures are limited. Here, we present a general approach to pyridine 15N-labeling that proceeds via ring-opening to NTf-Zincke imines and then ring-closure with commercially available 15NH4Cl salts. This process functions on a range of substituted pyridines, from simple building block-type compounds to late-stage labeling of complex pharmaceuticals, and 15N-incorporation is >95% in most cases. The reactivity of the Zincke imine intermediates also enables deuteration of the pyridine C3- and C5-positions, resulting in higher mass isotopologs required for LCMS analysis of biological fluids during drug development.

A greener RP-HPTLC-densitometry method for the quantification of apremilast in nanoformulations and commercial tablets: Greenness assessment by analytical eco-scale, ChlorTox, and AGREE methods

The greener “high-performance thin-layer chromatography (HPTLC)” methods for apremilast (APM) analysis in pharmaceutical products and biological fluids are not currently available in the literature. Accordingly, this study involves the development and validation of a rapid, simple, economical, and greener reversed-phase HPTLC methodology for the estimation of APM in prepared nanoparticles (NPs), nanoemulsion, and marketed tablets. The estimation of APM was conducted using “RP-18 silica gel 60 F254S HPTLC plates” as the stationary phase. The combination of ethanol/water (65:35, v/v) was used as the greener mobile phase for APM analysis. The greenness of the method was predicted using three different approaches, namely Analytical Eco-Score (AES), ChlorTox, and the Analytical GREENness (AGREE) approaches. The λmax = 238 nm was used for the APM detection. By contrasting its single band at Rf = 0.61 ± 0.01 with those of pure APM, the HPTLC peaks for APM in an prepared NP formulation, nanoemulsion, and marketed tablets were identified. In the concentration range of 100–700 ng/band, the proposed analytical methodology was linear. The values of AES, ChlorTox, and AGREE were determined to be 93, 0.66 g, and 0.89, respectively, demonstrated an outstanding greener profile for the existing method. The amount of APM in the tablet, NP formulation, and nanoemulsion was found to be 98.40, 101.60, and 99.37 %, respectively. According to the findings of validation tests and pharmaceutical analysis, the suggested analytical technique could be successfully applied for the routine examination of APM in marketed tablets and laboratory generated nanoformulations.

VALIDATION OF HEADSPACE SOLID-PHASE MICROEXTRACTION WITH GAS CHROMATOGRAPHY-FLAME IONISATION DETECTOR METHOD FOR ALCOHOL ANALYSIS ON GASTRIC FLUID

Identifying the concentration of alcohol compounds in postmortem analysis of biological fluids can help the investigation, just as postmortem analysis of gastric juices can reveal the concentrations of alcohol consumed. However, an efficient and effective combination of extraction and measurement methods is required when looking at complex postmortem samples. Therefore, a headspace-solid phase microextraction (HS-SPME) method was developed using gas chromatography with a flame ionization detector (GC-FID) to identify postmortem alcohol concentrations. This present study optimizes and validates an effective and efficient method for postmortem alcohol extraction and quantification in gastric fluid. The optimal conditions for HS-SPME extraction using 65μm Polydimethy lsiloxane Divinyl benzene (PDMS/DVB) fiber for analyte isolation were 15 minutes. at 60°C and an exposure time of 1 minute. The validation investigation shows that the suggested approach satisfies the criteria for linearity, precision, accuracy, LOD, and LOQ for postmortem measurement of alcohol in gastric fluid.

Strategies in sample preparation for proteome analysis of biological fluids from water buffaloes

Livestock species such as water buffalo have some unique characteristics like high fat percentage in milk and plasma, poor sample generation, peculiar behaviour, etc. making it difficult for proteomic analysis of such samples. Additionally, there is a limited database in terms of identified proteins, characterised antibodies and functional attributes of known molecules specific to buffalo. The body fluids like blood, milk, urine and foetal fluids offer a great opportunity in understanding physio-pathological processes and thus aid in developing reliable diagnostics. Standardised sample preparation protocols are key to advancement of proteomics science. Optimising sample preparation for 2-DE proteomics in buffalo serum, milk, and foetal fluids is crucial. Commercially available kits or laboratory methods for clean-up, enrichment, and condition optimising can be applied for getting optimum results. The 2-D clean up strategy is an important step in sample preparation procedures to get rid of inferring substances which hamper IEF. The sample preparation of the buffalo biological fluids (serum, foetal fluids, maternal uterine flushing and milk samples) reported in the present study shall provide valuable information for further research.

ICP-MS and fluorescence dual-mode detection of ZIKV-RNA based on quantum dot labeling with hybridization chain reaction

The detection of Zika virus (ZIKV) is of great significance to human life and health. Herein, we presented an ICP-MS and fluorescent dual-mode sensor for quantitative analysis of Zika virus RNA fragments (ZIKV-RNA), which employed quantum dots (QDs) as signal tags and combined with hybridization chain reaction (HCR). The dual-mode sensor realized cross-checking of the analysis results and improved the assay accuracy. Firstly, the single-stranded DNA (ssDNA) was anchored on the surface of magnetic beads (MBs). Afterward, HCR was conducted with probe DNA-CdSe quantum dots conjugates (pDNA-QDs) and link DNA (lDNA), producing the MBs-ssDNA-[pDNA-QDs-lDNA]n conjugates. In the presence of target ZIKV-RNA, a strand displacement reaction occurred, leading to the dissociation of the [pDNA-QDs-lDNA]n labels from the conjugates into the solution. Finally, the signal intensity was detected using ICP-MS and fluorescence analysis, with achieved limits of detection of 131 pM and 152 pM, respectively. The inter-assay RSD values of fluorescence and ICP-MS were 3.94 % and 4.26 % at 10 nM level, respectively, showing that the method had good precision. This method showed high selectivity and was applied to the analysis of biological fluids. There was no significant difference between the results of ICP-MS modes and fluorescence mode. This method offers a new strategy for sensitivity analysis of ZIKV-RNA and exhibits promise in clinical applications for diagnosis.

Interpretation of results of automated analysis of pleural effusion (clinical cases)

Automated analysis of effusion fluids, providing a large amount of objective information about a biological sample, has significantly expanded the diagnostic capabilities of the cytological service. At the same time, there are currently no unified algorithms for interpreting the obtained data. The aim of the work was to develop, based on the results of automated analysis of pleural fluids, alternative predictions of the genesis of the effusion (benign / malignant). The study was carried out on the basis of the Clinical Regional Hospital No. 2 in Krasnodar (Russia). The results of automated analysis of biological fluids obtained using the Sysmex XN analyzer were compared with the data of cytological, laboratory and instrumental studies. The article presents some of our own developments that can serve as indicative criteria for interpreting the results of automated analysis of biological fluids. It has been established that in terms of differential diagnosis of the genesis of effusion, the indicators of total cytosis (TC-BF) and the absolute content of highly fluorescent mononuclear cells (HF-BF#) are the most informative. Accounting for clinical data, results of laboratory and instrumental studies increases the informative value of automated analysis of biological fluids.

Alterations in glutamate, arginine, and energy metabolism characterize cerebrospinal fluid and plasma metabolome of persons with HIV-associated dementia.

HIV-associated dementia (HAD) is the most severe clinical expression of HIV-mediated neuropathology, and the processes underlying its development remain poorly understood. We aimed to exploit high-dimensional metabolic profiling to gain insights into the pathological mechanisms associated to HAD. In this cross-sectional study, we utilized metabolomics to profile matched cerebrospinal fluid (CSF) and plasma samples of HAD individuals ( n = 20) compared with neurologically asymptomatic people with HIV (ASYM, n = 20) and healthy controls (NEG, n = 20). Identification of plasma and CSF metabolites was performed by liquid-chromatography or gas-chromatography following a validated experimental pipeline. The resulting metabolic profiles were analyzed by machine-learning algorithms, and altered pathways were identified by comparison with KEGG pathway database. In CSF, HAD patients displayed an imbalance in glutamine/glutamate ratio, decreased levels of isocitrate and arginine, and increased oxidative stress when compared with ASYM or NEG. These changes were confirmed in matched plasma samples, which in addition revealed an accumulation of eicosanoids and unsaturated fatty acids in HAD individuals. Pathway analysis in both biological fluids suggested that alterations in several metabolic processes, including protein biosynthesis, glutamate and arginine metabolism, and energy metabolism, in association to a perturbed eicosanoid metabolism in plasma, may represent the metabolic signature associated to HAD. These findings show that HAD may be associated with metabolic modifications in CSF and plasma. These preliminary data may be useful to identify novel metabolic biomarkers and therapeutic targets in HIV-associated neurological impairment.

A green sustainable ultrasensitive micelle-sensitized spectrofluorimetric approach for the estimation of tafamidis in different matrices at picogram levels

Tafamidis is a novel drug approved by the European Medicines Agency to treat certain forms of transthyretin amyloidosis. The current study introduces a new ultrasensitive validated spectrofluorimetric approach for tafamidis analysis in bulk, capsules, and biological fluids. The present study is considered the first spectrofluorimetry-based approach for tafamidis estimation. The proposed eco-friendly spectrofluorimetric method depended on the measurement of the enhanced native fluorescence of tafamidis exhibited at 310/370 nm without any derivatization reaction. It was found that about two-fold enhancement in the tafamidis fluorescence could be accomplished by the addition of Brij-35. Different experimental parameters affecting the fluorescence of tafamidis were examined to reach the optimal conditions and highest sensitivity. One milliliter of each of 0.02 M Britton-Robinson buffer at pH 2 and Brij-35 was added to give the maximum fluorescence intensity. The drug was successively assayed in the concentration range of (0.1–10.0 ng/mL) with a detection limit of 0.0107 ng/mL and a quantitation limit of 0.0324 ng/mL. Owing to the superior sensitivity and selectivity, the developed method was effectively applied to the quantitation of tafamidis in its capsules and spiked human plasma samples with high % recoveries and low % relative standard deviation values. The validation parameters were assessed by following the International Council for Harmonization (ICH) guidelines. Additionally, the eco-friendly property of the designed approach was considered, and the greenness was evaluated using the Green Analytical Procedure Index (GAPI) and Analytical GREENNESS metric approach (AGREE).

Spectroscopic and Chromatographic Estimation of Some Sartans and their Combinations with Thiazide Diuretics: A Review

Abstract: Sartans are often used as antihypertensives. They are also available in combination with thiazide diuretics for the management of hypertension. Analytical method development is a crucial part of successful drug development and characterization. Bioanalytical studies are of paramount importance while establishing pharmacokinetic and toxicokinetic data while forced degradation studies are important to elucidate degradation pathways and to establish stability of the drugs. : Different methods have been developed for the analysis of sartans and their combination with thiazide diuretics. We thought it imperative to summarize them so the data could be useful for analysis of newer sartans. The review describes various methods for analysis of some frequently employed sartans as well as the latest sartans and their combination with thiazide diuretics. The article also focuses on their analysis of biological fluids. Forced degradation studies have also been covered in the article. : Article is divided into three sections. First section covers introduction, second section focuses on different methods developed, including bioanalytical methods, while third section presents forced degradation studies carried out on the drugs. Important parameters of the analytical methods developed have been summarized in tabular form.

Voltammetric analysis of biological fluids: quantification of species in urine accounting for non-linear concentration effects

Voltammetric sensing of analytes in biological fluids is frequently accompanied by non-linear effects due to the analyte/matrix interactions. A theoretical approach is developed to describe such effects when standard addition methods are applied. Several asymptotic models are described considering equilibrium-like and kinetic-like interaction models and possible co-diffusion effects. Experimental data for non-linear features which appear in voltammetric sensing of serotonin and noradrenalin in urine are in satisfactory agreement with theoretical predictions.

Early diseases diagnosis in body fluids (serum and saliva) using infrared spectroscopy

Infrared spectroscopy it is becoming more and more useful in the field of biomedical research. Infrared spectroscopy has been used more and more to characterize biological matrixes, providing a simple way to obtain diagnostic and observational information from easily acquired samples. These tests are performed in order to monitor the changes, and in this way to characterize the biological matrix, with the aim of detecting the first signs that can diagnose a disease. Vibrational spectroscopy analysis of biological fluids has become more and more popular recently. Notably, the development of infrared spectroscopic screening of blood products, particularly blood serum and saliva, for illness diagnosis has attracted economic attention. This review examines some applications of the infrared spectroscopy method that was employed to examine human serum in order to detect disease at an early stage, published between 2017 and 2022.

Ceramides and ceramide synthases in cancer: Focus on apoptosis and autophagy

Different studies corroborate a role for ceramide synthases and their downstream products, ceramides, in modulation of apoptosis and autophagy in the context of cancer. These mechanisms of regulation, however, appear to be context dependent in terms of ceramides’ fatty acid chain length, subcellular localization, and the presence or absence of their downstream targets. Our current understanding of the role of ceramide synthases and ceramides in regulation of apoptosis and autophagy could be harnessed to pioneer the development of new treatments to activate or inhibit a single type of ceramide synthase, thereby regulating the apoptosis induction or cross talk of apoptosis and autophagy in cancer cells. Moreover, the apoptotic function of ceramide suggests that ceramide analogues can pave the way for the development of novel cancer treatments. Therefore, in the current review paper we discuss the impact of ceramide synthases and ceramides in regulation of apoptosis and autophagy in context of different types of cancers. We also briefly introduce the latest information on ceramide synthase inhibitors, their application in diseases including cancer therapy, and discuss approaches for drug discovery in the field of ceramide synthase inhibitors. We finally discussed strategies for developing strategies to use lipids and ceramides analysis in biological fluids for developing early biomarkers for cancer.

High-Throughput Nanoliter Sampling and Direct Analysis of Biological Fluids Using Droplet Imbibition Mass Spectrometry.

A high-throughput droplet imbibition mass spectrometry (MS) experiment is reported for the first time that allows direct analysis of ultra-small volumes of complex mixtures. In this experiment, an array of optimized tips of glass capillaries containing the analyte solution is sampled by rapidly moving charged microdroplets, which picks up (i.e., imbibes) the analyte and transfers it to a proximal mass spectrometer. The advantages associated with this droplet imbibition experiment include (1) ultra-small sample consumption (1.3 nL/min), which reduces the matrix effect in complex mixture analysis, and (2) high surface activity, which eliminates ion suppression effects caused by competition for the space charge on the droplet surface. Collectively, the enhanced surface effect and small flow rates dramatically increase the sensitivity of the droplet imbibition MS approach. This was experimentally shown by constructing calibration curves for cocaine analysis in human raw urine and whole blood, achieving 2 and 7 pg/mL limits of detection, respectively. The high-throughput feature was demonstrated by analyzing five structurally different compounds in 20 s intervals. With the measured flow rate of 1.3 nL/min on a 5 μm glass tip size, the results described in the current study showcase droplet imbibition MS to be a powerful and high-throughput alternative for conventional nano-electrospray ionization (flow rate <100 nL/min), which is the most efficient method for transferring small sample volumes to mass spectrometers.

SERS Determination of Oxidative Stress Markers in Saliva Using Substrates with Silver Nanoparticle-Decorated Silicon Nanowires.

Glutathione and malondialdehyde are two compounds commonly used to evaluate the oxidative stress status of an organism. Although their determination is usually performed in blood serum, saliva is gaining ground as the biological fluid of choice for oxidative stress determination at the point of need. For this purpose, surface-enhanced Raman spectroscopy (SERS), which is a highly sensitive method for the detection of biomolecules, could offer additional advantages regarding the analysis of biological fluids at the point of need. In this work, silicon nanowires decorated with silver nanoparticles made by metal-assisted chemical etching were evaluated as substrates for the SERS determination of glutathione and malondialdehyde in water and saliva. In particular, glutathione was determined by monitoring the reduction in the Raman signal obtained from substrates modified with crystal violet upon incubation with aqueous glutathione solutions. On the other hand, malondialdehyde was detected after a reaction with thiobarbituric acid to produce a derivative with a strong Raman signal. The detection limits achieved after optimization of several assay parameters were 50 and 3.2 nM for aqueous solutions of glutathione and malondialdehyde, respectively. In artificial saliva, however, the detection limits were 2.0 and 0.32 μM for glutathione and malondialdehyde, respectively, which are, nonetheless, adequate for the determination of these two markers in saliva.