Genotype-dependent N-glycosylation and newly exposed O-glycosylation affect plasmin-induced cleavage of histidine-rich glycoprotein (HRG).

Histidine-rich glycoprotein (HRG) is an abundant plasma protein harboring at least three N-glycosylation sites. HRG integrates many biological processes, such as coagulation, antiangiogenic activity, and pathogen clearance. Importantly, HRG is known to exhibit five genetic variants with minor allele frequencies of more than 10%. Among them, Pro204Ser can induce a fourth N-glycosylation site (Asn202). Considerable efforts have been made to reveal the biological function of HRG, whereas data on HRG glycosylation are scarcer. To close this knowledge gap, we used C18-based LC-MS/MS to study the glycosylation characteristics of six HRG samples from different sources. We used endogenous HRG purified from human plasma and compared its glycosylation to that of the recombinant HRG produced in Chinese hamster ovary cells or human embryonic kidney 293 cells, targeting distinct genotypic isoforms. In endogenous plasma HRG, every N-glycosylation site was occupied predominantly with a sialylated diantennary complex-type glycan. In contrast, in the recombinant HRGs, all glycans showed different antennarities, sialylation, and core fucosylation, as well as the presence of oligomannose glycans, LacdiNAcs, and antennary fucosylation. Furthermore, we observed two previously unreported O-glycosylation sites in HRG on residues Thr273 and Thr274. These sites together showed more than 90% glycan occupancy in all HRG samples studied. To investigate the potential relevance of HRG glycosylation, we assessed the plasmin-induced cleavage of HRG under various conditions. These analyses revealed that the sialylation of the N- and O-glycans as well as the genotype-dependent N-glycosylation significantly influenced the kinetics and specificity of plasmin-induced cleavage of HRG.

In-Depth Mass Spectrometry Analysis Reveals the Plasma Proteomic and N-Glycoproteomic Impact of an Amish-Enriched Cardioprotective Variant in B4GALT1.

B4GALT1 encodes ß-1,4-galactosyltransferase 1, an enzyme that plays a major role in glycan synthesis in the Golgi apparatus by catalyzing the addition of terminal galactose. Studies increasingly suggest that B4GALT1 may be involved in the regulation of lipid metabolism pathways. Recently, we discovered a single-site missense variant Asn352Ser (N352S) in the functional domain of B4GALT1 in an Amish population, which decreases the level of LDL-cholesterol (LDL-c) as well as the protein levels of ApoB, fibrinogen, and IgG in the blood. To systematically evaluate the effects of this missense variant on protein glycosylation, expression, and secretion, we developed a nano-LC-MS/MS-based platform combined with TMT-labeling for in-depth quantitative proteomic and glycoproteomic analyses in the plasma of individuals homozygous for the B4GALT1 missense variant N352S versus non-carriers (n = 5 per genotype). A total of 488 secreted proteins in the plasma were identified and quantified, 34 of which showed significant fold changes in protein levels between N352S homozygotes and non-carriers. We determined N-glycosylation profiles from 370 glycosylation sites in 151 glycoproteins and identified ten proteins most significantly associated with decreased galactosylation and sialyation in B4GALT1 N352S homozygotes. These results further support that B4GALT1 N352S alters the glycosylation profiles of a variety of critical target proteins, thus governing the functions of these proteins in multiple pathways, such as those involved in lipid metabolism, coagulation, and the immune response.

Effect of the 35 nm and 70 nm Size Exclusion Chromatography (SEC) Column and Plasma Storage Time on Separated Extracellular Vesicles.

The technical difficulty of separating extracellular vesicles (EVs) from plasma proteins in human blood presents a significant hurdle in EV research, particularly during nano ultra-high-performance liquid chromatography-tandem mass spectrometric (UHPLC-MS/MS) analysis, where detecting "vesicular" proteins among abundant plasma proteins is challenging. Standardisation is a pressing issue in EV research, prompting collaborative global efforts to address it. While the MISEV guidelines offer valuable recommendations, unanswered questions remain, particularly regarding sample storage. We compared size exclusion chromatography (SEC) columns with pore sizes of 35 nm and 70 nm to identify fractions with minimal contaminating proteins and the highest concentration of small EVs (sEVs). Following column selection, we explored potential differences in the quality and quantity of sEVs isolated from platelet-free plasma (PFP) after long-term storage at -80 °C (>2.5 years) compared to freshly drawn blood. Our methodologically rigorous study indicates that prolonged storage, under correct storage and processing conditions, does not compromise sEV quality. Both columns effectively isolated vesicles, with the 70 nm column exhibiting a higher abundance of "vesicular" proteins. We propose a relatively rapid and moderately efficient protocol for obtaining a comparatively pure sEV fraction from plasma, facilitating sEV processing in clinical trials.

Quantifying carboxymethyl lysine and carboxyethyl lysine in human plasma: clinical insights into aging research using liquid chromatography-tandem mass spectrometry.

OBJECTIVE: The objective of this study was to establish a methodology for determining carboxymethyl lysine (CML) and carboxyethyl lysine (CEL) concentrations in human plasma using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The test results were also used for clinical aging research. METHODS: Human plasma samples were incubated with aqueous perfluorovaleric acid (NFPA), succeeded by precipitation utilizing trichloroacetic acid, hydrolysis facilitated by hydrochloric acid, nitrogen drying, and ultimate re-dissolution utilizing NFPA, followed by filtration. Cotinine-D3 was added as an internal standard. The separation was performed on an Agela Venusil ASB C18 column (50 mm × 4.6 mm, 5 µm) with a 5 mmol/L NFPA and acetonitrile/water of 60:40 (v/v) containing 0.15% formic acid. The multiple reaction monitoring mode was used for detecting CML, CEL, and cotinine-D3, with ion pairs m/z 205.2 > 84.1 (for quantitative) and m/z 205.2 > m/z 130.0 for CML, m/z 219.1 > 84.1 (for quantitative) and m/z 219.1 > m/z 130.1 for CEL, and m/z 180.1 > 80.1 for cotinine-D3, respectively. RESULTS: The separation of CML and CEL was accomplished within a total analysis time of 6 minutes. The retention times of CML, CEL, and cotinine-D3 were 3.43 minutes, 3.46 minutes, and 4.50 minutes, respectively. The assay exhibited linearity in the concentration range of 0.025-1.500 µmol/L, with a lower limit of quantification of 0.025 µmol/L for both compounds. The relative standard deviations of intra-day and inter-day were both below 9%, and the relative errors were both within the range of ±4%. The average recoveries were 94.24% for CML and 97.89% for CEL. CONCLUSION: The results indicate that the developed methodology is fast, highly sensitive, highly specific, reproducible, and suitable for the rapid detection of CML and CEL in clinical human plasma samples. The outcomes of the clinical research project on aging underscored the important indicative significance of these two indicators for research on human aging.

Exploring the potential of SrTi0.7Fe0.3O3 perovskite/Chitosan nanosheets for the development of a label-free electrochemical sensing assay for determination of naproxen in human plasma samples.

Naproxen is a nonsteroidal anti-inflammatory drug used to treat nonrheumatic inflammation, migraine, and gout. Therefore, the determination of naproxen in pharmaceutical and biological samples is of particular importance. In the present work, SrTi0.7Fe0.3O3 perovskite/Chitosan nanosheets were used to modify the surface of a glassy carbon electrode (GCE) for highly sensitive determination of naproxen. To ensure the successful synthesis of the perovskite nanosheets, morphological studies including scanning electron microscopy (SEM), Energy-dispersive X-ray (EDX), X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), and X-ray photoelectron spectroscopy (XPS) were carried out. The electrochemical investigations of naproxen on the modified surface of GCE were investigated and the limit of detection (LOD) and limit of quantification (LOQ) were acquired 0.50 and 1.67 µM, respectively. Additionally, the linear range (LR) of 1.99-130.84 µM was obtained for the oxidation of naproxen. The obtained results have been proved that the mentioned method is simple, sensitive, and specific with a short analysis time. The dominant analytical features of the designed sensor are possessing a low detection limit, excellent stability, repeatability, and high selectivity in the presence of naproxen. For investigation of the applicability of the designed assay in real sample analysis, human plasma samples have been examined and a recovery index was acquired 95%.

ω-(5-Phenyl-2H-tetrazol-2-yl)alkyl-substituted glycine amides and related compounds as inhibitors of the amine oxidase vascular adhesion protein-1 (VAP-1).

Vascular adhesion protein-1 (VAP-1), also known as plasma amine oxidase or semicarbazide-sensitive amine oxidase, is an enzyme that degrades primary amines to aldehydes with the formation of hydrogen peroxide and ammonia. Among others, it plays a role in inflammatory processes as it can mediate the migration of leukocytes from the blood to the inflamed tissue. We prepared a series of ω-(5-phenyl-2H-tetrazol-2-yl)alkyl-substituted glycine amides and related compounds and tested them for inhibition of purified bovine plasma VAP-1. Compounds with submicromolar activity were obtained. Studies on the mechanism of action revealed that the glycine amides are substrate inhibitors, i.e., they are also converted to an aldehyde derivative. However, the reaction proceeds much more slowly than that of the substrate used in the assay, whose conversion is thus blocked. Examination of the selectivity of the synthesized glycine amides with respect to other amine oxidases showed that they inhibited diamine oxidase, which is structurally related to VAP-1, but only to a much lesser extent. In contrast, the activity of monoamine oxidase A and B was not affected. Selected compounds also inhibited VAP-1 in human plasma. The IC50 values measured were higher than those determined with the bovine enzyme. However, the structure-activity relationships obtained with the glycine amides were similar for both enzymes.

Fine-tuning Fluorescence of Amlodipine Adopting on Blocking of Photoinduced Electron Transfer (PET): Application in Human Plasma.

Assessing medication adherence through the determination of antihypertensive drugs in biological matrices holds significant importance. Amlodipine (AP), a potent antihypertensive medication extensively prescribed for hypertensive patients, is particularly noteworthy in this context. This article aims to introduce a rapid, simple, improved sensitivity, and reproducibility in detecting AP in its pure form, tablet formulation, and spiked human plasma than the other reported methods. The proposed method utilizes a fluorescence approach, relying on the inhibition of the intramolecular photoinduced electron transfer (PET) effect of the lone pair of the N-atom in the primary amino moiety of AP. This inhibition is achieved by acidifying the surrounding medium using 0.2 M acetic acid. By blocking PET, the target AP drug is sensitively detected, at [Formula: see text] 423 nm over a concentration range 25-500 ng mL- 1 showcasing an exceptionally low quantitation limit of 1.41 ng mL- 1. Notably, this innovative technique was successfully applied to detect AP in its solid dosage form and spiked human plasma. Remarkably, matrix interference was found to be insignificant, underscoring the robustness and applicability of the established approach. The combination of speed, sensitivity, and reproducibility makes this method particularly suitable for assessing medication adherence in patients prescribed AP for hypertension.

Design, Characterization and Biopharmaceutical Applications of Novel Green Boron-Carbon Quantum Dots for Quantification of Apalutamide; Greenness Evaluations.

The diagnosis of prostate cancer has been evolving in the current decade, with expected mortality rates of 499,000 death by the year 2030. Apalutamide (APL) has been approved in 2018 as the first drug for the controlling of prostate cancer. APL significant success warrantied its high global sales, which are expected to surpass 58% of segment market sales (together with another drug; enzalutamide). Therefore, new, fast and environmentally friendly analytical methods are required for its determination for the quality control and biological monitoring purposes. The proposed research designs and evaluates the first fluorimetric approach based on novel porous green boron-doped carbon quantum dots (B@CDs) for the determination of APL in biopharmaceutical matrices. The synthetic approach has high quantum yield (31.15%). B@CDs were characterized using several tools, including transmission electron microscopy (TEM), dynamic light scattering (DLS), FTIR and Energy dispersive X-ray spectroscopy (EDX) which proved their improved surface properties with an average nano-diameter of 3.0 nm. The interaction between B@CDs and APL led to enhancement their fluorescence at 441 nm (excitation at 372 nm). The approach was validated for the determination of APL within concentration range of 15.0-700.0 ng mL- 1 with quantification limit LOQ 4.37 ng mL- 1 and detection limit LOD 1.44 ng mL- 1. The approach was successfully applied for the determination of APL in human plasma and pharmaceutical monitoring of its marketed tablet form. Then, the approach was assessed for its environmental impact using different metrics and proved its ecological greenness.

Simultaneous determination of diquat, paraquat, glufosinate, and glyphosate in plasma by liquid chromatography/tandem mass spectrometry: from method development to clinical application.

Diquat (DQ), paraquat (PQ), glufosinate (GLU), and glyphosate (GLYP) are commonly used herbicides that have been confirmed to be toxic to humans. Rapid and accurate measurements of these toxicants in clinical practice are beneficial for the correct diagnosis and timely treatment of herbicide-poisoned patients. The present study aimed to establish an efficient, convenient, and reliable method to achieve the simultaneous quantification of DQ, PQ, GLU, and GLYP in human plasma using liquid chromatography-tandem mass spectrometry (LC-MS/MS) without using derivatization or ion-pairing reagents. DQ, PQ, GLU, and GLYP were extracted by the rapid protein precipitation and liquid-liquid extraction method and then separated and detected by LC-MS/MS. Subsequently, linearity, limit of detection (LOD), limit of quantification (LOQ), precision, accuracy, extraction recovery, matrix effect, dilution integrity, and stability were evaluated to validate the method based on the FDA criteria. Finally, the validated method was applied to real plasma samples collected from 166 Chinese patients with herbicide poisoning. The results showed satisfactory linearity with low LOD (1 ng/mL for DQ and PQ, 5 ng/mL for GLU, and 10 ng/mL for GLYP, respectively) and low LOQ (5 ng/mL for DQ and PQ, 25 ng/mL for GLU and GLYP, respectively). In addition, the precision, accuracy, extraction recovery, and stability of the method were acceptable. The matrix effect was not observed in the analyzed samples. Moreover, the developed method was successfully applied to determine the target compounds in real plasma samples. These data provided reliable evidence for the application of this LC-MS/MS method for clinical poisoning detection.

Mass Spectrometry-Based Characterization of Protein Aggregates in Tissues and Biofluids.

Protein aggregation is a common mechanism in multiple neurodegenerative and heart diseases and the accumulation of proteins in aggregates is toxic to cells, causing injury and death. The degree of protein aggregation directly correlates with the severity of the disease. Misfolded proteins present thermodynamic barriers that culminate in the loss of structure and function and the exposure of hydrophobic residues. The exposure of hydrophobic residues is the driving force behind protein aggregation, as it reduces surface free energy and increases the propensity for the formation of large insoluble aggregates. Exploring the protein content of aggregates is fundamental to understanding their formation mechanism and pathophysiological effects. We demonstrate here a method for isolating aggregated protein content in human plasma and mouse brain samples. The samples were characterized by mass spectrometry analysis, transmission electron microscopy, and western blotting. We report the identification of proteins associated with neurodegenerative diseases in the isolated pellets. The western blotting analyses of the isolated pellet showed the positivity for CD89 and CD63, consolidated markers of exosomes, confirming the presence of exosomes within the pellet but not in the supernatant in human plasma. Notably, the concomitant isolation of exosomes together with the protein aggregates was feasible starting from 200 µL of human plasma. Moreover, the presented methodology separated albumin from the aggregated pellet, allowing identification of larger diversity of proteins through mass spectrometry analysis.

Characterization of a new SARS-CoV-2 variant that emerged in Brazil.

The spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) plays a key role in viral infectivity. It is also the major antigen stimulating the host's protective immune response, specifically, the production of neutralizing antibodies. Recently, a new variant of SARS-CoV-2 possessing multiple mutations in the S protein, designated P.1, emerged in Brazil. Here, we characterized a P.1 variant isolated in Japan by using Syrian hamsters, a well-established small animal model for the study of SARS-CoV-2 disease (COVID-19). In hamsters, the variant showed replicative abilities and pathogenicity similar to those of early and contemporary strains (i.e., SARS-CoV-2 bearing aspartic acid [D] or glycine [G] at position 614 of the S protein). Sera and/or plasma from convalescent patients and BNT162b2 messenger RNA vaccinees showed comparable neutralization titers across the P.1 variant, S-614D, and S-614G strains. In contrast, the S-614D and S-614G strains were less well recognized than the P.1 variant by serum from a P.1-infected patient. Prior infection with S-614D or S-614G strains efficiently prevented the replication of the P.1 variant in the lower respiratory tract of hamsters upon reinfection. In addition, passive transfer of neutralizing antibodies to hamsters infected with the P.1 variant or the S-614G strain led to reduced virus replication in the lower respiratory tract. However, the effect was less pronounced against the P.1 variant than the S-614G strain. These findings suggest that the P.1 variant may be somewhat antigenically different from the early and contemporary strains of SARS-CoV-2.

LC-MS/MS method for quick detection of vonoprazan fumarate in human plasma: Development, validation and its application to a bioequivalence study.

A liquid chromatography-tandem mass spectrometry method with vonoprazan fumarate-d4 as a stable isotope-labeled internal standard was developed and validated aiming at quantification of vonoprazan fumarate in human plasma for a bioequivalence study. Chromatographic separation was achieved by acetonitrile one-step protein precipitation using a gradient elution of 0.1% formic acid aqueous solution and acetonitrile with a run time of 3.65 min. Detection was carried out on a tandem mass spectrometer in multiple reaction monitoring mode via a positive electrospray ionization interface. The multiple reaction monitoring mode of precursor-product ion transitions for vonoprazan fumarate and vonoprazan fumarate-d4 were m/z 346.0 → 315.1 and 350.0 → 316.0, respectively. The linear range was 0.150-60.000 ng/ml. This method was fully validated with acceptable results in terms of selectivity, carryover, lower limit of quantification, calibration curve, accuracy, precision, dilution effect, matrix effect, stability, recovery and incurred sample reanalysis. A successful application of this method was realized in the bioequivalence study of vonoprazan fumarate tablet (20 mg) among healthy Chinese volunteers.

Quantitation of tazemetostat in human plasma using liquid chromatography-tandem mass spectrometry.

To support a phase 1 trial in patients with lymphomas, we developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for tazemetostat quantitation in 20 µL of human plasma. After protein precipitation, chromatographic separation employed a Kinetex C18 column and a gradient of 0.1% formic acid in both water and acetonitrile, during a 3-min run time. Detection was achieved using a SCIEX 6500+ tandem mass spectrometer with electrospray positive-mode ionization. Validation was based on the latest Food and Drug Administration guidance. With a stable isotopic internal standard, the assay was linear within the range of 10-5000 ng/mL and proved to be accurate (91.9%-103.7%) and precise (<4.4% imprecision). Recovery varied between 93.3% and 121.1%, and matrix effect ranged from -25.5% to -4.9%. Hemolysis, lipemia, and dilution did not impact quantitation. Plasma stability was confirmed after three freeze-thaw cycles, 24 h at room temperature, and 4 months at -80°C. Incurred sample reanalysis yielded 94.4% samples within 20% difference (n = 36). External validation showed a mean bias of -11.1%. Pharmacokinetic (PK) data obtained from three patients suggested variable concentration time profiles, warranting collection of further data. The assay proved to be suitable for tazemetostat quantitation in human plasma and will support clinical studies by defining tazemetostat PKs.

Assessment of remdesivir and its nucleoside metabolite in beagle dogs and healthy humans by liquid chromatography coupled with triple quadrupole mass spectrometry.

The aim of this study was to assess the pharmacokinetics of the existing remdesivir intravenous formulation (100 mg dose) against the newly developed oral formulation (20 mg dose) for remdesivir and its active nucleoside metabolite (GS-441524) in beagle dogs followed by healthy human volunteers. A quantification method for remdesivir and its active nucleoside metabolite (GS-441524) in beagle dog and human plasma has been developed and validated using liquid chromatography coupled to triple quadrupole mass spectrometry detection. The analytical methods for beagle dogs and human differ in the calibration curve range, plasma matrix, processing volume, reconstitution volume and injection volume; however all other parameters were same in both methods. A simple protein precipitation extraction was carried out using acetonitrile containing the internal standard remdesivir D5. Remdesivir and GS-441524 were separated on an Endurus C-18P, 100 × 4.6 mm, 3 µm column and detected using a mass spectrometer with electrospray ionization in positive ion mode. The ion transitions used were m/z 603.1 → m/z 200.0 for remdesivir, m/z 292.0 → m/z 202.2 for GS-441524 and m/z 608.2 → m/z 205.1 for remdesivir D5. The calibration curve results were linear in beagle dog plasma (2.0-2,000.8 ng/ml range for remdesivir and 2.0-1,500.4 ng/ml for GS-441524) and human plasma (30.0-4,503.9 ng/ml range for remdesivir and 2.0-200.4 ng/ml for GS-441524). The recovery was >90% in beagle dog and human plasma. These methods were successfully used to determine the pharmacokinetic parameters of the intravenous injection and subcutaneous tablets dosage forms in beagle dogs and healthy humans.

A LC-MS/MS method for the simultaneous determination of 6-cyanodopamine, 6-nitrodopamine, 6-nitrodopa, 6-nitroadrenaline and 6-bromodopamine in human plasma and its clinical application in patients with chronic kidney disease.

The aim of this study was to develop a high-performance liquid chromatography-tandem mass spectrometry method for the determination of 6-cyanodopamine, 6-nitrodopamine, 6-nitrodopa, 6-nitroadrenaline and 6-bromodopamine in human plasma samples. Strata-X 33 µm solid-phase extraction cartridges were used for the extraction of the catecholamines from human plasma samples. The catecholamines were separated in a 150 × 3 mm Shim-pack GIST C18-AQ column with 3 µm particle size, placed in an oven at 40°C and perfused with 82% mobile phase A (acetonitrile-H2O; 90:10, v/v) + 0.4% acetic acid and 18% mobile phase B (deionized H2O) + 0.2% formic acid at a flow rate of 340 µl/min in isocratic mode. The injected volume was 4 µl and the run lasted 4 min. The method was linear from 0.1 to 20 ng/ml and the lower limit of quantification was 0.1 ng/ml for all analytes. The method was applied to evaluate the plasma levels of catecholamines in plasma of patients with chronic kidney disease and allowed the detection for the first time of circulating levels of the novel catecholamines 6-bromodopamine and 6-cyanodopamine.

Cell-free hemoglobin and hemin catalyzing triclosan oxidative coupling in plasma: A novel exogenous phenolic pollutants coupling pathway.

Previous studies reported that hemoprotein CYP450 catalyzed triclosan coupling is an "uncommon" metabolic pathway that may enhance toxicity, raising concerns about its environmental and health impacts. Hemoglobin, a notable hemoprotein, can catalyze endogenous phenolic amino acid tyrosine coupling reactions. Our study explored the feasibility of these coupling reactions for exogenous phenolic pollutants in plasma. Both hemoglobin and hemin were found to catalyze triclosan coupling in the presence of H2O2. This resulted in the formation of five diTCS-2 H, two diTCS-Cl-3 H, and twelve triTCS-4 H in phosphate buffer, with a total of nineteen triclosan coupling products monitored using LC-QTOF. In plasma, five diTCS-2 H, two diTCS-Cl-3 H, and two triTCS-4 H were detected in hemoglobin-catalyzed reactions. Hemin showed a weaker catalytic effect on triclosan transformation compared to hemoglobin, likely due to hemin dimerization and oxidative degradation by H2O2, which limits its catalytic efficiency. Triclosan transformation in the human plasma-like medium still occurs with high H2O2, despite the presence of antioxidant proteins that typically inhibit such transformations. In plasma, free H2O2 was depleted within 40 minutes when 800 µM H2O2 was added, suggesting a rapid consumption of H2O2 in these reactions. Antioxidative species, or hemoglobin/hemin scavengers such as bovine serum albumin, may inhibit but not completely terminate the triclosan coupling reactions. Previous studies reported that diTCS-2 H showed higher hydrophobicity and greater endocrine-disrupting effects compared to triclosan, which further underscores the potential health risks. This study indicates that hemoglobin and heme in human plasma might significantly contribute to phenolic coupling reactions, potentially increasing health risks.

A highly sensitive spectrofluorimetric method for the determination of bilastine in human plasma: Application of content uniformity testing.

Bilastine, a new second generation antihistaminic drug, has been widely used for relieving symptoms of allergic rhinitis and urticaria without a sedative effect. A simple, cost-effective, and highly sensitive fluorimetric method was developed for the estimation of bilastine in human plasma, in addition to its pure state and tablets. The suggested method depended on binary complex formation of eosin with bilastine in a buffered medium at pH 4.2. The formed complex resulted in quantitative quenching of eosin emission at 538 nm after excitation at 335 nm. This method demonstrates a broad range of linearity, spanning from 200 to 1000 ng/mL, and exhibits exceptional sensitivity, with a limit of detection and quantitation of 30.85 and 93.48 ng/mL, respectively. In addition, this spectrofluorimetric method may be employed to determine the amount of bilastine in human plasma and tablets with satisfactory accuracy and excellent precision. Furthermore, the content uniformity of bilastine in commercially available tablets was successfully tested by this approach. Compared with the reference method, there were no significant variations in terms of precision or accuracy. In conclusion, the proposed protocol is highly recommended to quantitatively estimate bilastine in different quality control settings.

Environmentally benign first derivative synchronous spectrofluorimetry for the analysis of two binary mixtures containing duloxetine with avanafil or tadalafil in spiked plasma samples.

Antidepressants can cause sexual dysfunction side effects, necessitating the co-administration of phosphodiesterase type 5 inhibitors. The simultaneous determination of these drugs in biological fluids is critical for therapeutic drug monitoring. For the first time, two binary mixtures containing duloxetine with either avanafil or tadalafil were estimated utilizing simple green spectrofluorimetric methods without the need for a previous separation step. The study was based on first derivative synchronous spectrofluorimetry in ethanol using a change in wavelength difference (∆λ) of 20 and 25 nm for the first and second combinations, respectively. Duloxetine and avanafil were estimated at 297.7 and 331 nm in their binary mixture, while duloxetine and tadalafil were determined at 290.3 and 297.7 nm, respectively. The linearity was achieved over the ranges of 0.1-1.5 µg mL-1 for both duloxetine and avanafil and 0.01-0.40 µg mL-1 for tadalafil, with limits of detection of 0.013, 0.022, and 0.004 µg mL-1 for duloxetine, avanafil, and tadalafil, respectively. Successful application of the developed approaches was accomplished for the estimation of the two mixtures in dosage forms as well as human plasma with excellent percentage recoveries (96-103.75% in plasma), which supports their suitability for use in quality control laboratories and pharmacokinetic studies. Moreover, the adopted approaches' greenness was evidenced by applying three tools.

Fast concurrent determination of guaifenesin and pholcodine in formulations and spiked plasma using first derivative synchronous spectrofluorimetric approach.

Guaifenesin and pholcodine are frequently co-formulated in certain dosage forms. A new fast first derivative synchronous spectrofluorometric method has been used for their simultaneous analysis in mixtures. Here, first derivative synchronous spectrofluorometry enabled the successful simultaneous estimation of guaifenesin at 283 nm and pholcodine at 275 nm using a wavelength difference (Δλ) of 40 nm. The method was fully validated following International Council of Harmonization guidelines. For guaifenesin and pholcodine, linearity was determined within the corresponding ranges of 0.05-0.30 and 0.10-6.0 µg/ml. The two drugs were effectively analyzed using the developed approach in their respective formulations, and the results showed good agreement with those attained using reference methods. The method demonstrated excellent sensitivity, with detection limits down to 0.007 and 0.030 µg/ml and quantitation limits of 0.020 and 0.010 µg/ml for guaifenesin and pholcodine, respectively. Therefore, the procedure was successful in determining these drugs simultaneously in vitro in spiked plasma samples and syrup dosage form. The developed methodology also offered an environmentally friendly advantage by utilizing water as the optimal diluting solvent throughout the whole work. Different greenness approaches were investigated to ensure the method's ecofriendly properties.

First derivative synchronous spectrofluorimetric method for the simultaneous determination of tramadol and celecoxib in their dosage forms and human plasma.

One of the most common features of many different clinical conditions is pain; hence, there is a crucial need for eliminating or reducing it to a tolerable level to retrieve physical, psychological and social functioning. A first derivative synchronous spectrofluorimetry technique is proposed for the simultaneous determination of celecoxib and tramadol HCl, a recent coformulation authorized for treating acute pain in adults. The method includes using synchronous spectrofluorimetry at ∆λ = 80 nm where tramadol HCl was determined using first derivative technique at λ = 230.2 nm, while celecoxib was determined at λ = 288.24 nm. The proposed method was successfully applied to their co-formulated dosage forms in addition to spiked human plasma and validated in agreement with the guidelines of the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH). The linear ranges were found to be 0.50-5.0 and 0.15-0.50, the limits of detection to be 0.088 and 0.011 and the limits of quantification to be 0.266 and 0.032 µg/ml for celecoxib and tramadol, respectively. Statistical analysis revealed no significant difference when compared with previously reported methods as evidenced by the values of the variance ratio F-test and Student t-test. The proposed method was successfully applied to commercial dosage forms and spiked human samples. Moreover, the greenness of the proposed method was investigated based on the analytical eco-scale approach, with the results showing an excellent green scale with a score of 95.