Spectrofluorimetric Assay Research Articles

A Rapid and Reliable Spectrofluorimetric Method to Measure the Urinary Lactulose/Mannitol Ratio for Dysbiosis Assessment.

Gut microbiota plays a crucial role in human health homeostasis, and the result of its alteration, known as dysbiosis, leads to several pathologies (e.g., inflammatory bowel disease, metabolic syndrome, and Crohn's disease). Traditional methods used to assess dysbiosis include the dual sugar absorption test and the urinary lactulose/mannitol ratio (LMR) measurement using mass spectrometry. Despite its precision, this approach is costly and requires specialized equipment. Hence, we developed a rapid and reliable spectrofluorimetric method for measuring LMR in urine, offering a more accessible alternative. This spectrofluorimetric assay quantifies the fluorescence of nicotinamide adenine dinucleotide (NADH) and nicotinamide adenine dinucleotide phosphate (NADPH) produced during the enzymatic oxidation of mannitol and lactulose, respectively. The assay requires 100 µL of urine samples and detects LMR values lower (eubiosis) and higher (dysbiosis) than 0.05, ultimately being amenable to high-throughput screening and automatization, making it practical for clinical and research settings. A validation of the method demonstrated its high precision, accuracy, and robustness. Additionally, this study confirmed analyte stability under various storage conditions, ensuring reliable results even with delayed analysis. Overall, this spectrofluorimetric technique reduces costs, time, and the environmental impact associated with traditional mass spectrometry methods, making it a viable option for widespread use in the assessment of dysbiosis.

Tuning fluorescence of fexofenadine by switching OFF intramolecular photoinduced electron transfer: Application to development of one-step green and high throughput microwell spectrofluorimetric assay for analysis of tablets and plasma.

Fexofenadine (FEX) is a non-sedating antihistamine commonly used for the treatment of allergic conditions such as seasonal rhinitis and chronic idiopathic urticaria. This study describes the tuning "ON" the intrinsic fluorescence of FEX by switching "OFF" its intramolecular photoinduced electron transfer (PET) through the protonation of the piperidinyl nitrogen atom using sulfuric acid. The resulting fluorescence was utilized as a basis for the development of a highly sensitive microwell spectrofluorimetric assay (MW-SFA) for the one-step determination of FEX in pharmaceutical tablets and plasma. The linear range of the assay was 10-500 ng ml-1, and its limit of quantitation was 25.9ng ml-1. The proposed MW-SFA was successfully applied to analyze FEX in pharmaceutical tablets and plasma samples, demonstrating good accuracy and precision. The greenness of the assay was confirmed using three metric assessment tools. In conclusion, the MW-SFA is a straightforward, single-step analysis that requires no experimental adjustments. It offers high sensitivity, efficient sample processing, and environmental sustainability. This assay is highly recommended for pharmaceutical quality control and clinical lab use, particularly for measuring FEX levels.

Dependence on relative humidity in the formation of reactive oxygen species in water droplets

Water microdroplets (7 to 11 µm average diameter, depending on flow rate) are sprayed in a closed chamber at ambient temperature, whose relative humidity (RH) is controlled. The resulting concentration of ROS (reactive oxygen species) formed in the microdroplets, measured by the amount of hydrogen peroxide (H2O2), is determined by nuclear magnetic resonance (NMR) and by spectrofluorimetric assays after the droplets are collected. The results are found to agree closely with one another. In addition, hydrated hydroxyl radical cations (•OH-H3O+) are recorded from the droplets using mass spectrometry and superoxide radical anions (•O2-) and hydroxyl radicals (•OH) by electron paramagnetic resonance spectroscopy. As the RH varies from 15 to 95%, the concentration of H2O2 shows a marked rise by a factor of about 3.5 in going from 15 to 50%, then levels off. By replacing the H2O of the sprayed water with deuterium oxide (D2O) but keeping the gas surrounding droplets with H2O, mass spectrometric analysis of the hydrated hydroxyl radical cations demonstrates that the water in the air plays a dominant role in producing H2O2 and other ROS, which accounts for the variation with RH. As RH increases, the droplet evaporation rate decreases. These two facts help us understand why viruses in droplets both survive better at low RH values, as found in indoor air in the wintertime, and are disinfected more effectively at higher RH values, as found in indoor air in the summertime, thus explaining the recognized seasonality of airborne viral infections.

Metal cations recognition by bowl-shaped N-pyrrolic polycyclic aromatic hydrocarbons.

Bowl-shaped, nitrogen-doped polycyclic aromatic hydrocarbons were examined for the first time as molecular receptors for the recognition of metal cations. Potentiometric and spectrofluorimetric assays, supported with density functional theory computations, revealed that the title compounds recognise metal cations with a special focus on caesium (Cs+) cations.

Cytotoxic pathways activated by multifunctional thiosemicarbazones targeting sigma-2 receptors in breast and lung carcinoma cells

BackgroundMultifunctional thiosemicarbazones (TSCs) able to bind sigma receptors and chelate metals are considered as a promising avenue for the treatment of pancreatic cancer due to the encouraging results obtained on in vitro and in vivo models. Here, we assessed the biochemical mechanism of these TSCs also on lung (A549) and breast (MCF7) cancer cells.MethodsThe density of sigma-2 receptors in normal (BEAS-2B and MCF10A) and in lung and breast (A549 and MCF7) cancer cells was evaluated by flow cytometry. In these cells, cytotoxicity (MTT assay) and activation of ER- and mitochondria-dependent cell death pathways (by spectrofluorimetric assays to measure Caspases 3/7/9; qRT-PCR detection of GRP78, ATF6, IRE1, PERK; MitoSOX, DCFDA-AM and JC-1 staining), induced by the TSCs FA4, MLP44, PS3 and ACThio1, were evaluated.ResultsFA4 and PS3 exerted more potent cytotoxicity than MLP44 and ACThio1 in all cancer cell lines, where the density of sigma-2 receptors was higher than in normal cells. Remarkably, FA4 promoted ER- and mitochondria-dependent cell death pathways in both cell models, whereas the other TSCs had variable, cell-dependent effects on the activation of the two proapoptotic pathways.ConclusionsOur data suggest that FA4 is a promising compound that deserves to be further studied for lung and breast cancer treatment. However, the other multifunctional TSCs also hold promise for the development of therapies towards a personalized medicine approach. Indeed, the presence of the sigma-2 receptor-targeting moiety would lead to a more specific tumor delivery embracing the characteristics of individual tumor types.

Dual fluorescence enhancement of loratidine by photoinduced electron transfer blocking and micellization: Application to the development of novel highly sensitive microwell spectrofluorimetric assay for analysis of dosage forms and urine samples

This study describes the enhancement of the weak native fluorescence of loratadine (LOR) by dual strategy via photoinduced electron transfer (PET) blocking followed by micellization into sodium dodecyl sulfate micelles. The enhanced fluorescence was employed as a basis for the development of a novel microwell spectrofluorimetric assay (MW-SFA) for the determination of LOR in its pharmaceutical dosage forms and urine samples. The assay was conducted in 96-microwell assay plates, and the enhanced fluorescence signals were measured by a microplate reader at 290 and 435 nm for excitation and emission, respectively. The optimum conditions of the assay were established, calibration curve was generated, and the linear regression equation was computed. The relation between the fluorescence signals and LOR concentrations was linear with good determination coefficients (0.9992) in the range of 10 – 2000 ng mL−1. The assay limits of detection and quantitation were 4.1 and 12.5 ng mL−1, respectively. The precision was satisfactory, with values of relative standard deviation not exceeding 1.68%, and the assay’s accuracy was ≥ 99.1%. The proposed was successfully applied to the analysis of LOR in its pharmaceutical dosage forms with acceptable accuracy and precision. The label claims were 99.3 – 100.5% (±0.95 – 1.59%). Statistical analysis comparing the results of the proposed assay with those obtained by a reported pre-validated assay revealed no significant difference between both methods in terms of the accuracy and precision at the 95% confidence level. The assay was also applied to the analysis of urine samples containing LOR with accuracy ≥ 98.24%. The greenness of the proposed assay was confirmed by three efficient metric tools. In overall conclusion, the proposed assay is characterized by high sensitivity, procedure simplicity, and high throughput, enabling the simultaneous analysis of many samples in a short time. Therefore, it is a valuable tool for rapid routine application in pharmaceutical quality control units and clinical laboratories for the determination of LOR.

Validated spectrofluorimetric assay of two co-administered drug mixtures containing hydroxychloroquine with either moxifloxacin or ofloxacin as a drug regimen for hospital-acquired pneumonia in covid-19 patients.

Moxifloxacin and ofloxacin are two broad-spectrum quinolone antibiotics. They are among the most widely used antibiotics nowadays in controlling the covid-19 pandemic. Hydroxychloroquine is an FDA-approved drug for the treatment of covid-19. This work describes a simple, green, selective, and sensitive spectrofluorimetric method for the assay of moxifloxacin and ofloxacin in presence of hydroxychloroquine as two co-administered mixtures used in the treatment of hospital-acquired pneumonia in the covid-19 patients. Simultaneous assay of hydroxychloroquine and moxifloxacin was carried out in methanol by direct spectrofluorimetric method (method I) at 375 nm and 550 nm respectively after excitation at 300 nm. The direct spectrofluorimetric assay was rectilinear over concentration range of (50.0-400.0) and (300.0-2500.0) ng/ml for hydroxychloroquine and moxifloxacin with LOD of 6.4 and 33.64 ng/ml and LOQ of 19.4 and 102.6 ng/ml for the two drugs respectively. While the assay of hydroxychloroquine and ofloxacin was carried out by measuring the first derivative synchronous amplitude for hydroxychloroquine at the zero crossing point of ofloxacin and vice versa at Δ λ = 140 nm (method II). Hydroxychloroquine was measured at 266 nm while ofloxacin at 340 nm over concentration range of (4.0-40.0 ng/ml) for hydroxychloroquine and (200.0-2000.0 ng/ml) for ofloxacin with LOD of 0.467 ng/ml and 25.3 ng/ml and LOQ of 1.42 ng/ml and 76.6 ng/ml for the two drugs respectively. the two methods were validated following ICH guidelines and were applied to the analysis of the two drugs in plasma with good percent recoveries (109.73-93.17 %).

Development of a Green Microwell Spectrofluorimetric Assay with High Analytical Throughput for the Determination of Selective Serotonin Reuptake Inhibitors in Pharmaceutical Dosage Forms and Plasma.

In this study, a new green microwell spectrofluorimetric assay (MW-SFA) with high throughput was developed and validated, for the first time, for the determination of three selective serotonin reuptake inhibitors (SSRIs) in pharmaceutical dosage forms and plasma. These SSRIs were fluoxetine (FLX), fluvoxamine (FXM), and paroxetine (PXT), which are commonly prescribed drugs for depression treatment. The MW-SFA is based on the condensation reaction of SSRIs with 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) in alkaline media to form highly fluorescent derivatives. The MW-SFA procedures were conducted in 96-microwell white opaque assay plates with a flat bottom and the fluorescence signals were measured using a microplate reader at their maximum excitation and emission wavelengths. The calibration curves were generated with good correlation coefficients (0.9992-0.9995) between the relative fluorescence intensity (RFI) and the SSRI concentrations in the range of 35-800 ng/mL. The limits of detection were in the range of 11-25 ng/mL, and the precision and accuracy were satisfactory. The proposed MW-SFA was successfully applied to the analysis of the SSRIs in their pharmaceutical dosage forms. The statistical analysis for the comparison between the MW-SFA assay results and those of pharmacopeial assays showed no significant differences between the assays in terms of their accuracy and precision. The application of the proposed MW-SFA was extended to successfully analyze SSRIs in plasma samples. The greenness of the assay was confirmed using three different metric tools. The assay was characterized with high throughput properties, enabling the sensitive simultaneous analysis of many samples in a short time. This assay is valuable for rapid routine applications in pharmaceutical quality control units and clinical laboratories for the determination of SSRIs.

Augmentation of Brexpiprazole fluorescence through photoinduced electron transfer inhibition for the sensitive spectrofluorimetric assay of pharmaceutical dosage forms and spiked human plasma: Application to content uniformity testing

Brexpiprazole (BRX) is a new antipsychotic drug that recently was used in the treatment of schizophrenia and other psychosis. The presence of benzothiophene ring in its chemical structure makes BRX naturally fluoresces. However, the native fluorescence of the drug was low in neutral or alkaline medium owing to the occurrence of photoinduced electron transfer (PET) from the nitrogen of the piperazine ring to the benzothiophene ring. Protonation of this nitrogen atom using sulfuric acid could efficiently block PET process and thus retaining the strong fluorescence of the compound. Accordingly, a straightforward, highly sensitive, fast and green spectrofluorimetric approach was established for BRX determination. In 1.0 M sulfuric acid solution, BRX exhibited significant native fluorescence measured at emission wavelength of 390 nm after excitation at 333 nm. ICH requirements were used to evaluate the method. The fluorescence intensity and BRX concentration were found to be correlated linearly within the range of 5–220 ng mL−1 with a coefficient of correlation 0.9999. The limit of quantitation was 2.38 ng mL−1 while limit of detection was 0.78 ng mL−1. The developed approach was successfully used to analyze BRX in biological fluids and pharmaceutical dosage form. The suggested approach worked well when applied for testing the uniformity of content.

Development of green and high throughput microwell spectrophotometric and spectrofluorimetric assays for quantitation of roscovitine in its bulk form and capsules

Roscovitine (RCV) is a drug with potential for the treatment of different types of cancer, neurodegenerative disease, renal dysfunction, viral infections, and chronic inflammatory disorders. This study describes, for the first time, the development and validation of three novel microwell-based assays for quantitation of RCV in its bulk form and capsules. These assays are two spectrophotometric assays and one spectrofluorimetric assay. The first spectrophotometric assay (UV-SPA) is based on measuring the native ultraviolet (UV) radiation absorption of RCV. The second assay (CT-SPA) is based on the formation of colored charge transfer (CT) complex upon the reaction of RCV, as n-electron donors, with 2,5-dichloro-3,6-dihydroxybenzoquinone (CHBQ), as a π-electron acceptor. The spectrofluorimetric assay (NF-SFA) is based on measuring the native fluorescence of RCV. These three assays were carried out in 96-microwell assay plates, and the signals were measured by a microplate reader. The optimum conditions of the three assays were established and their analytical performances were validated according to the guidelines of the International Council for Harmonization (ICH) for validation of analytical procedures. The linear ranges for the three assays were 10–300 µg mL1, 25–800 µg mL1 and 25–500 ng mL1 for UV-SPA, CT-SPA and NF-SFA, respectively. The limits of detection (LOD) of the assays were 4.1 µg mL1, 8.8 µg mL1 and 9.2 ng mL1 for UV-SPA, CT-SPA and NF-SFA, respectively. The accuracy and precision of the three assays were proved as the recovery values were ≥98.8%, and the relative standard deviations (RSD) did not exceed 2.32%. The three assays were successfully applied to the quantitation of RCV in its bulk form and capsules; the mean recovery values were ≥99.5%. The greenness of the assays was evaluated by three different metric tools, and the results proved that the three assays fulfill the requirements of green analytical approaches. In addition, the simultaneous handling of large number of samples with micro-volumes in the proposed assays gave these assays the property of high throughput analysis. In conclusion, the three assays are valuable for the rapid routine application in the pharmaceutical quality control units for the quantitation of RCV.

One-Step Microwell Plate-Based Spectrofluorimetric Assay for Direct Determination of Statins in Bulk Forms and Pharmaceutical Formulations: A Green Eco-Friendly and High-Throughput Analytical Approach

This study describes the development of a one-step microwell spectrofluorimetric assay (MW-SFA) with high sensitivity and throughput for the determination of four statins in their pharmaceutical and formulations (tablets). These statins were pitavastatin (PIT), fluvastatin (FLU), rosuvastatin (ROS) and atorvastatin (ATO). The MW-SFA involves the measurement of the native fluorescence of the statin aqueous solutions. The assay was conducted in white opaque 96-microwell plates, and the fluorescence intensities of the solutions were measured by using a fluorescence microplate reader. The optimum conditions of the assay were established; under which, linear relationships with good correlation coefficients (0.9991-0.9996) were found between the fluorescence intensity and the concentration of the statin drug in a range of 0.2-200 µg mL-1 with limits of detection in a range of 0.1-4.1 µg mL-1. The proposed MW-SFA showed high precision, as the values of the relative standard deviations did not exceed 2.5%. The accuracy of the assay was proven by recovery studies, as the recovery values were 99.5-101.4% (±1.4-2.1%). The assay was applied to the determination of the investigated statins in their tablets. The results were statistically compared with those obtained by a reference method and the results proved to have comparable accuracy and precision of both methods, as evidenced by the t- and F-tests, respectively. The green and eco-friendly feature of the proposed assay was assessed by four different metric tools, and all the results proved that the assay meets the requirements of green and eco-friendly analytical approaches. In addition, ever-increasing miniaturization as handling of large numbers of micro-volume samples simultaneously in the proposed assay gave it a high-throughput feature. Therefore, the assay is a valuable tool for the rapid routine application in the pharmaceutical quality control units for the determination of statins.

Spectrofluorimetric assay of amlodipine and perindopril in their raw materials, pharmaceutical formulations and spiked human plasma through the formation of complexes with Eosin Y

For quantitation of amlodipine (AML) and perindopril (PER) in their authentic, pharmaceutical formulations and spiked human plasma, a simple, sensitive, validated and inexpensive spectrofluorimetric method has been developed. The proposed method is developed to be based on quantitative quenching effect of two antihypertensive drugs on Eosin Y's native fluorescence which was achieved by developing binary complexes between each of the cited drugs in an acidic environment using acetate buffer (pH 4.4) with Eosin Y. Fluorescence quenching was recorded at 544 nm after excitation at 425 nm. For AML and PER, calibration curves were obtained over the range of 0.3–3.0 µg/mL and 0.2–2.0 µg/mL, respectively, with correlation coefficients of 0.9993 and 0.9995, respectively. The developed method was validated according to ICH guidelines. The proposed spectrofluorimetric method is regarded new and sensitive. As a result, the proposed method might be used to estimate the quality of the cited drugs in their pharmaceutical formulations and biological fluid.

Simultaneous Spectrophotometric & Spectrofluorimetric Assay of Silodosin & Solifenacin in their Co-formulated Binary Mixture

Simultaneous Spectrophotometric & Spectrofluorimetric Assay of Silodosin & Solifenacin in their Co-formulated Binary Mixture

Effects of Trimethylamine and Trimethylamine Oxide on Human Serum Albumin Observed by Tryptophan Fluorescence and Absorbance Spectroscopies

Trimethylamine (TMA) is an aliphatic tertiary amine produced by gut microbiota, starting from dietary precursors such as L-choline, L-carnitine and betaine. TMA and its metabolite trimethylamine-N-oxide (TMAO) are elevated in the plasma of cardiovascular disease (CVD) patients. Despite extensive literature on this topic, the scientific community is still divided on which of the two molecules is responsible for the harmful effects on human health. To assess whether the plasma levels of these molecules are also modulated by interactions with macromolecules present in the plasma, the weak bonds between TMA or/and TMAO with human serum albumin (HSA) were studied via molecular docking and spectrofluorimetric assay. The impact of TMA and TMAO on HSA and low-density lipoproteins (LDL) oxidation was also evaluated. Docking analysis shows three main binding sites for TMA and two for TMAO. Spectrofluorimetric results show interactions of HSA with TMA and TMAO; a significant (p = 0.010) decrease in Trp-214 intrinsic fluorescence of HSA was measured starting from the lowest concentrations of both TMA and TMAO (3.26 nM and 29.2 nM, respectively). Furthermore, at all concentrations tested, no significant effect on the formation of carbonyls in HSA was measured (p > 0.05) in the presence of TMA or TMAO. However, 28.6 mM TMAO significantly increased (p < 0.05) the degree of oxidation of LDL, suggesting that TMAO has a pro-oxidant role on LDL.

Protective Effect of Indomethacin-loaded Polymeric Nanoparticles Against Oxidative Stress-Induced Cytotoxicity in Human Breast Adenocarcinoma Cell Model

Introduction: Anti-inflammatory drugs are being utilized to treat cancer because of its inflammatory microenvironment. Objective: The objective of this study is to investigate the antioxidant potential of indomethacin and its genotoxicity, since free or loaded in polymeric nanocapsules using MCF-7 (human breast cancer) cells as an in vitro model. Method: Development of indomethacin-loaded polyepsiloncaprolactone (PCL) nanocapsules by interfacial deposition method. It is characterized by pH determination by potentiometer, mean diameter and polydispersity index by dynamic light scattering; zeta potential by electrophoretic mobility; encapsulation efficacy by high performance liquid chromatography method; corona effect formation; 2ʹ,7ʹ-dichlorofluorescin diacetate (DCFH-DA) method by spectrofluorimetric assay; nitric oxide (NO) determination by spectrophotometric and genotoxicity assay by plasmid DNA cleavage method. Results: The results showed a mild acidic pH (4.78 ± 0.10), sizes around 200 nm and PDI<0.2 with a zeta potential around -20 mV and encapsulation efficiency of 99% (1 mg mL-1), showing a dose-dependent corona formation profile in 24h incubation. Conclusion: DCFH-DA assay showed no production of reactive oxygen species (ROS) while NO determination showed that Ind-OH-NC from 26.7 to 100 μM increased reactive nitrogen species (RNS), demonstrating antioxidant potential against MCF-7 cells. No sample at the concentrations evaluated induced DNA cleavage, being considered a safe treatment.

A Biophysical Investigation of DNA-Binding Interactions of Push-Pull Dibenzodioxins and Implications for in Vitro anti-Cancer Activity

Dibenzodioxin-DNA interactions were studied using biophysical techniques. UV-visible spectral titrations were performed against calf thymus (CT)-DNA to determine the binding constants. Spectrofluorimetric dye-displacement assay and DNA viscometric assay were used to understand the nature of binding interactions. Molecules with intercalative binding preference increased DNA viscosity, similar to a standard intercalator. Dibenzodioxin cytotoxicity was evaluated against HeLa cells. The most active molecules also had the highest binding constants with CT-DNA. Select dibenzodioxins were docked against DNA in silico. The binding behavior was compared using a known DNA binding small molecule. Dibenzodioxins with good cytotoxicity also had high DNA-binding affinity, implying that DNA could be a likely target for these dibenzodioxins. One fluorescent dibenzodioxin was used to study intra-cellular localization in HeLa cells using fluorescence microscopy. This derivative with weaker cytotoxicity was located mainly in the cytoplasm than in the nuclei, again hinting toward dibenzodioxin-DNA-binding interaction as the main mechanism of cytotoxicity.

Green & Sensitive pH-dependent Spectrofluorimetric Assay of Tamsulosin Hydrochloride and Tadalafil in their New Combined Formulation for Benign Prostatic Hyperplasia: Application to Spiked Human Plasma

Sensitive and green spectrofluorimetric methods were utilized for Tamsulosin Hydrochloride (TAM) and Tadalafil (TDL) assessment in bulk and their newly available combined mixture for benign prostatic hyperplasia and erectile dysfunction. The technique relies on measuring native fluorescence of TAM in 0.1 N HCl at 324 nm and TDL in 0.1 N NaOH at 348 nm due to their different fluorimetric behavior in acidic and basic media where TAM has no fluorescence in basic medium and vice versa. To achieve better regression, the spectra were derivatized allowing determination of TAM at 314 nm and TDL at 320 and 380 nm (peak to peak) by applying third and first derivative, respectively. In addition, pH-dependent “constant-wavelength synchronous” spectrofluorimetry was applied where TAM and TDL were determined at 218 nm in acidic medium and at 268 nm in basic medium, respectively. Finally, derivatizing the latter emission spectra allowed determination of TAM and TDL at 232 nm and at 262 and 278 nm (peak to peak), respectively. Acidic and basic emission spectra where scanned at λexc = 225 nm (for TAM assay) and at λexc = 247 nm (for TDL assay), respectively. Fluorescence–concentration plots were linear and the proposed methods were used for analysis of TAM and TDL combined laboratory prepared formulation. These procedures are green, sensitive and of low cost which make them suitable for quality control analysis of the two drugs. In addition, the high selectivity of the proposed methods was tested by successfully applying them for TAM and TDL assay in plasma samples.

Spectrofluorimetric Approach for Quantification of Cyclizine in the Presence of its Toxic Impurities in Human Plasma; in silico Study and ADMET Calculations

Cyclizine (CYZ); an antiemetic compound; is widely misused for its euphoric or hallucinatory effects, either by oral or intravenous routes. The concomitant abuse of CYZ among addicted adolescents contributes to neuromuscular disorders that are life-threatening. Consequently, with the company of 1-Methylpiperazine (MPZ) and diphenylmethanol (DPM, Benzhydrol) as pharmacopoeia-reported CYZ impurities, a novel spectrofluorimetric assay for the detection of CYZ, has been established either in human plasma samples or in its parenteral formulation. The native fluorescence of CYZ has been investigated under various conditions. Different parameters affecting relative fluorescence intensity of CYZ including diluting solvent, surfactant, plasma protein solvent, and pH were studied and optimized. The linearity obtained between the fluorescence intensity at emission wavelength 350 nm after excitation at 244 nm and the corresponding CYZ concentrations was in the range 10–1000 ng/mL for measurement of CYZ either in pure form or in human plasma samples, with a appropriate correlation coefficient (r = 0.9999) and 3.10 ng/mL as the limit of detection and 9.41 ng/mL as the limit of quantitation. The suggested procedure was created and validated in accordance with ICH guidelines for quantification of CYZ either in its pure form or its dosage form, and FDA guidelines for the assay of CYZ in human plasma. Finally, in silico study and ADMET predictions were conducted for the studied drug impurities to estimate their pharmacokinetic behaviors. The results showed that both CYZ impurities have higher cellular permeability and maximum tolerated doses, DPM has higher BBB and CNS permeability than MPZ, while MPZ exceeds DPM in total clearance and volume of distribution.Graphic

Innovative localized surface plasmon resonance sensing technique for a green spectrofluorimetric assay of ketoprofen, paracetamol and chlorzoxazone in pharmaceutical preparations and biological fluids.

A green, quick and sensitive spectrofluorimetric technique was investigated and validated for the assay of three different drugs namely, ketoprofen (KPN), paracetamol (PAR), and chlorzoxazone (CLX). The method is based on fluorescence quenching of the fluorescence probe, silver nanoparticles (SNPs). The fluorescence quenching of SNPs may be attributed to the complexation between each of the studied drugs with SNPs. The fluorescence of SNPs alone or after complexation with the studied drugs were measured at 485 nm (λ ex 242 nm) without the need to extract the formed complex. Chemical reduction was employed for preparing SNPs, where silver nitrate was reduced by sodium borohydride in deionized water without adding organic stabilizer. SNPs were found soluble in water, had high stability and had a narrow emission band. The studied drugs were found to decrease the fluorescence of SNPs significantly through static quenching according to Stern-Volmer equation. Factors affecting the reaction between the drugs and NPs were carefully examined and optimized. Using the optimum conditions, the difference in the fluorescence intensity of SNPs before and after complexation with the studied drugs was in a good linear relationship with the concentration of the studied drugs, where (R 2 = 0.9998, 0.9998 and 0.9991) in the ranges of 0.5-5.0, 0.15-3.0 and 0.5-9.0 μg mL-1 for KPN, PAR and CLX, respectively. Validity of the proposed method was investigated according to ICH recommendations. The proposed technique was also employed for the analysis of each of the three drugs in commercial or laboratory prepared tablets and in spiked human plasma with very good recoveries as well as high level of accuracy and precision. This method was intended to the analysis of the proposed drugs in their single formulation and single drug administration. The suggested technique is considered an eco-friendly method, as it uses water as the safest and least expensive solvent. Moreover, the recommended technique does not involve solvent extraction of the formed complexes. Greenness assessment of the suggested procedure was accomplished by applying the four standard assessment tools. Consequently, the recommended method can be used in the routine quality control analysis of the cited drugs with minimum harmful effect on the environment as well as the individuals.

New validated spectrofluorimetric protocol for colistin assay through condensation with 2,2-dihydroxyindan-1,3-dione: application to content uniformity testing.

A new, cost-effective and sensitive spectroscopic assay for the quantification of Colistin Sulfate (CS) and its prodrug colistimethate sodium (CMS) has been developed and validated. The validated technique depends on the condensation of the studied drug with 2,2-dihydroxyindan-1,3-dione (ninhydrin) and phenylacetaldehyde using Teorell and Stenhagen buffer (pH = 6) to yield a fluorescent product that is estimated at emission wavelength (λ em = 474 nm) after excitation wavelength (λ ex = 390 nm). The reaction's affecting factors were carefully studied and adjusted accurately. Over the following range (0.4-2.4 μg mL-1), the produced calibration plot looked rectilinear, and the estimated limits of detection and quantification (LOD and LOQ) were 0.051 & 0.154 μg mL-1 respectively. The recommended approach was utilized to evaluate market products containing the investigated drug. Moreover, content uniformity testing was employed as a new procedure not found in the previously reported fluorimetric technique.