Ultrafast photooxidation of protein-bound anionic flavin radicals.

The photophysical properties of anionic semireduced flavin radicals are largely unknown despite their importance in numerous biochemical reactions. Here, we studied the photoproducts of these intrinsically unstable species in five different flavoprotein oxidases where they can be stabilized, including the well-characterized glucose oxidase. Using ultrafast absorption and fluorescence spectroscopy, we unexpectedly found that photoexcitation systematically results in the oxidation of protein-bound anionic flavin radicals on a time scale of less than ∼100 fs. The thus generated photoproducts decay back in the remarkably narrow 10- to 20-ps time range. Based on molecular dynamics and quantum mechanics computations, positively charged active-site histidine and arginine residues are proposed to be the electron acceptor candidates. Altogether, we established that, in addition to the commonly known and extensively studied photoreduction of oxidized flavins in flavoproteins, the reverse process (i.e., the photooxidation of anionic flavin radicals) can also occur. We propose that this process may constitute an excited-state deactivation pathway for protein-bound anionic flavin radicals in general. This hitherto undocumented photochemical reaction in flavoproteins further extends the family of flavin photocycles.

Comparison of the effect of Sodium Chloride concentration on protein determination: Bradford and Biuret methods.

Protein quantification methods using spectrophotometry are widely used in laboratory routines for different purposes. Samples generally contain non-protein components that can interfere with the quality of the analysis. A simple and quick test with different concentrations of sodium chloride demonstrated that the Bradford method is significantly affected by the presence of salt, while Biuret remains stable. Therefore, the choice of method is an important factor in reducing errors and ensuring more reliable results.

Clofazimine pKa Determination by Potentiometry and Spectrophotometry: Reverse Cosolvent Dependence as an Indicator of the Presence of Dimers in Aqueous Solutions.

The weakly basic antibiotic and anti-inflammatory drug, clofazimine (CFZ), was first described in 1957. It has been used therapeutically, most notably in the treatment of leprosy. However, the compound is extremely insoluble in aqueous media, and, indeed, there is poor consensus about what its intrinsic solubility is since the reported values range from 0.04 to 11 ng/mL. To understand the speciation and solubilization of CFZ as a function of pH, it is of paramount importance to know the true aqueous pKa. However, there is also poor consensus about the value of the pKa (reported measured values range from 6.08 to 9.11). In the present study, we report the determination of the CFZ ionization constant using two independent techniques. A state-of-the-art potentiometric analysis was performed, drawing on titration data in methanol-water solutions (46-75 wt % MeOH) of CFZ, using the bias-reducing consensus of two different procedures of extrapolating the apparent psKa values to zero cosolvent to approximate the true aqueous pKa as 9.43 ± 0.12 (25 °C, I = 0.15 M reference ionic strength). In parallel, spectrophotometric UV/vis titration data were acquired (250-600 nm at different pH) in 10 mM HEPES buffer solutions containing up to 54 wt % MeOH. The alternating least squares (ALS) method was used in the analysis of the absorbance-pH spectra. Uncharacteristically, the cosolvent UV/vis data in our study showed reverse cosolvent dependence (apparent pKa values increased with increasing cosolvent) which could be explained by a dimerization of the free base. The analysis of UV/vis data obtained from 54 wt % MeOH-water solution containing 20 µM CFZ yielded the apparent pKa 9.51 ± 0.17 (I ≈ 0.005 M). To assess whether self-assembly of CFZ was energetically feasible, density functional theory (DFT) calculations were used to study the putative CFZ dimers in aqueous and methanol media. The DFT-optimized geometries and infrared spectra of CFZ dimers using water and methanol as solvents were calculated and analyzed. Based on the lack of negative frequencies in calculated infrared spectra, it was confirmed that optimized geometries correspond to the true energetic minima. Visual analysis of optimized structures indicates the presence of stacking interactions between two CFZ molecules. The protonation site (the imine nitrogen atom) was determined by 1H NMR spectroscopy.

Development of an azo functionalized oligomeric chitosan sensor for rapid visual, spectrophotometric and spectrofluorometric detection of KMnO4 up to micromolar concentrations.

A water-soluble azo functionalized oligomeric chitosan reagent (ß-NAC) has been developed for the visual detection and quantification of KMnO4 at micromolar concentrations. The ß-NAC sensor was also explored as a detection probe for the spectrophotometric and spectrofluorometric detection of several metal ions and anions. The synthesized reagent was characterized by TGA-DTA-DTG analysis, DLS studies, BET analysis, and spectral analysis. The ß-NAC reagent produces conspicuous colours with different concentrations and different pH values of KMnO4 solution. This provides evidence for high selectivity in the visual detection of KMnO4 up to the micromolar level because of its interactions in the case of KMnO4 only. The colour of the ß-NAC reagent after interacting with KMnO4 (10-3 M) changes from brown to blood red. Furthermore, the ß-NAC sensor was employed for the spectrophotometric detection of KMnO4. The absorption spectrum of ß-NAC shows a peak at 327 nm and on interacting with KMnO4, it shows a bathochromic shift to 331 nm. The intensity of the peak at 331 nm increases as the concentration of KMnO4 was increased from 1 µM to 0.01 M. The detection and quantification limits in the spectrophotometric detection of KMnO4 were found to be 4.55 µM and 15.17 µM, respectively. The results of pH studies show that there is a pH effect of the KMnO4 solution on KMnO4 detection. The stability of the complex was determined by investigating the effect of time on the absorption intensity. In the spectrofluorometric detection, the fluorescence intensity of ß-NAC at the 427 nm emission maxima was decreased on adding KMnO4 solution. The fluorescence quenching increased on increasing the KMnO4 concentration from 1 µM to 0.008 M. The optimum pH for fluorescence quenching was found to be 8. The detection and quantification limits in the spectrofluorometric detection of KMnO4 were found to be 0.967 µM and 3.223 µM, respectively. The Stern-Volmer constant value was found to be 41 366.2 L mol-1, confirming the significant complexation between KMnO4 and the ß-NAC reagent. Interference studies were conducted to analyse the effect of various metal ions and anions on KMnO4 detection. Electrochemical studies were also performed to analyse the mechanism of complex formation.

Zippered G-quadruplex/hemin DNAzyme: exceptional catalyst for universal bioanalytical applications.

G-quadruplex (G4)/hemin DNAzyme is promising horseradish peroxidase (HRP)-mimic candidate in the biological field. However, its relatively unsatisfactory catalytic capacity limits the potential applications. Inspired by nature protease, we conducted a proximity-enhanced cofactor assembly strategy (PECA) to form an exceptional HRP mimic, namely zippered G4/hemin DNAzyme (Z-G4/H). The hybridization of short oligonucleotides induced proximity assembly of the DNA-grafted hemin (DGH) with the complementary G4 sequences (cG4s), mimicking the tight configuration of protease cofactor and apoenzyme. The detailed investigations of catalytic efficiency and mechanism verified the higher activity, more rapid catalytic rate and high environmental tolerance of the Z-G4/H than the classical G4/hemin DNAzymes (C-G4/H). Furthermore, a proximity recognition transducer has been developed based on the PECA for sensitive detection of gene rearrangement and imaging human epidermal growth factor receptor 2 protein (HER2) dimerization on cell surfaces. Our studies demonstrate the high efficiency of Z-G4/H and its universal application potential in clinical diagnostics and biomolecule interaction research. It also may offer significant opportunities and inspiration for the engineering of the protease-free mimic enzyme.

Transcriptome-wide in vivo mapping of cleavage sites for the compact cyanobacterial ribonuclease E reveals insights into its function and substrate recognition.

Ribonucleases are crucial enzymes in RNA metabolism and post-transcriptional regulatory processes in bacteria. Cyanobacteria encode the two essential ribonucleases RNase E and RNase J. Cyanobacterial RNase E is shorter than homologues in other groups of bacteria and lacks both the chloroplast-specific N-terminal extension as well as the C-terminal domain typical for RNase E of enterobacteria. In order to investigate the function of RNase E in the model cyanobacterium Synechocystis sp. PCC 6803, we engineered a temperature-sensitive RNase E mutant by introducing two site-specific mutations, I65F and the spontaneously occurred V94A. This enabled us to perform RNA-seq after the transient inactivation of RNase E by a temperature shift (TIER-seq) and to map 1472 RNase-E-dependent cleavage sites. We inferred a dominating cleavage signature consisting of an adenine at the -3 and a uridine at the +2 position within a single-stranded segment of the RNA. The data identified mRNAs likely regulated jointly by RNase E and an sRNA and potential 3' end-derived sRNAs. Our findings substantiate the pivotal role of RNase E in post-transcriptional regulation and suggest the redundant or concerted action of RNase E and RNase J in cyanobacteria.

Utilization of Response Surface Methodology in Optimization of Proline Extraction from Castanea sativa Honey.

Proline constitutes approximately 85 % of the amino acid composition of honey. Therefore, the quantitative determination of this amino acid in honey samples is used by many national/international authorities to evaluate the quality of honey types. In this study, it was aimed to achieve maximum proline amino acid extraction from honey samples whose botanical origins were confirmed by melissopalynological analysis. For this reason, based on three different spectrophotometric methods used in the literature for proline analysis, proline extraction was optimized with the Response Surface Method (RSM) and Box-Behnken experimental design. Three independent variables were determined as treatment time (2, 6, and 10 min), treatment temperature (22, 46, and 70 °C), and cooling time (5, 25, and 45 min). As a result of the optimization, it was seen that only significantly effective independent variable on the proline content of honey was the processing temperature. The optimum conditions obtained as a result of the RSM were found to be 2 min for the treatment time, 70 °C for the treatment temperature and 45 min for the cooling time. The composite desirability of the optimum conditions (R2 ) was found to be 1.00. It was determined that the method proposed by International Honey Commission (IHC) is efficient for proline analysis, but it provides more proline extraction by reducing of time from 10 min to 2 min in hold time in boiling water bath only during the extraction step. As a result, the conditions to be used in order to achieve maximum proline extraction with different spectrophotometric methods were determined and optimum values were determined. In addition, since the botanical origin of honey samples significantly affects the proline content of honey, it can be suggested that this study be optimized for different monofloral honey samples as well.

Greenness-by-design approach for developing novel UV spectrophotometric methodologies resolving a quaternary overlapping mixture.

Greenness-by-design (GbD) is an approach that integrates green chemistry principles into the method development stage of analytical processes, aiming to reduce their environmental impact. In this work, we applied GbD to a novel univariate double divisor corrected amplitude (DDCA) method that can resolve a quaternary pharmaceutical mixture in a fixed-dose polypill product. We also used a genetic algorithm as a chemometric modeling technique to select the informative variables for the analysis of the overlapping mixture. This resulted in more accurate and efficient predictive models. We used a computational approach to study the effect of solvents on the spectral resolution of the mixture and to minimize the spectral interferences caused by the solvent, thus achieving spectral resolution with minimal analytical effort and ecological footprint. The validated methods showed wide linear concentration ranges for the four components (1-30 µg/mL for losartan, 2.5-30 µg/mL for atorvastatin and aspirin, and 2.5-35 µg/mL for atenolol) and achieved high scores on the hexagon and spider charts, demonstrating their eco-friendliness.

Development of Visible Spectrophotometric Methods for the Determination of Tricyclic Antidepressants Based on Formation of Molecular Complexes with p-Benzoquinones.

Tricyclic antidepressants are commonly employed in the management of major depressive disorders. The present work describes two visible (VIS) spectrophotometric techniques that utilize the formation of charge transfer complexes between four antidepressant compounds, namely, amitriptyline hydrochloride (AMI), imipramine hydrochloride (IMI), clomipramine hydrochloride (CLO), and trimipramine maleate (TRI) acting as electron donors and two p-benzoquinones, namely, p-chloranilic acid (pCA) and 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ), serving as electron acceptors. The stoichiometry of the compounds produced exhibited a consistent 1:1 ratio in all instances, as established by Job's method. Molar absorptivities, equilibrium association constants, and several other spectroscopic properties were determined for all complexes. The developed spectrophotometric techniques were validated intra-laboratory and successfully applied for quantitative assessment of the four antidepressant active ingredients in several commercial pharmaceutical formulations. The methods are relatively simple, fast, and use readily available laboratory instrumentation, making them easily applicable by most quality control laboratories worldwide.

Green Chemometric Determination of Cefotaxime Sodium in the Presence of Its Degradation Impurities Using Different Multivariate Data Processing Tools; GAPI and AGREE Greenness Evaluation.

Four eco-friendly, cost-effective, and fast stability-indicating UV-VIS spectrophotometric methods were validated for cefotaxime sodium (CFX) determination either in the presence of its acidic or alkaline degradation products. The applied methods used multivariate chemometry, namely, classical least square (CLS), principal component regression (PCR), partial least square (PLS), and genetic algorithm-partial least square (GA-PLS), to resolve the analytes' spectral overlap. The spectral zone for the studied mixtures was within the range from 220 to 320 nm at a 1 nm interval. The selected region showed severe overlap in the UV spectra of cefotaxime sodium and its acidic or alkaline degradation products. Seventeen mixtures were used for the models' construction, and eight were used as an external validation set. For the PLS and GA-PLS models, a number of latent factors were determined as a pre-step before the models' construction and found to be three for the (CFX/acidic degradants) mixture and two for the (CFX/alkaline degradants) mixture. For GA-PLS, spectral points were minimized to around 45% of the PLS models. The root mean square errors of prediction were found to be (0.19, 0.29, 0.47, and 0.20) for the (CFX/acidic degradants) mixture and (0.21, 0.21, 0.21, and 0.22) for the (CFX/alkaline degradants) mixture for CLS, PCR, PLS, and GA-PLS, respectively, indicating the excellent accuracy and precision of the developed models. The linear concentration range was studied within 12-20 µg mL-1 for CFX in both mixtures. The validity of the developed models was also judged using other different calculated tools such as root mean square error of cross validation, percentage recoveries, standard deviations, and correlation coefficients, which indicated excellent results. The developed methods were also applied to the determination of cefotaxime sodium in marketed vials, with satisfactory results. The results were statistically compared to the reported method, revealing no significant differences. Furthermore, the greenness profiles of the proposed methods were assessed using the GAPI and AGREE metrics.

Charge Transfer Complex of Lorlatinib with Chloranilic Acid: Characterization and Application to the Development of a Novel 96-Microwell Spectrophotometric Assay with High Throughput.

Lorlatinib (LRL) is the first drug of the third generation of anaplastic lymphoma kinase (ALK) inhibitors used a first-line treatment of non-small cell lung cancer (NSCLC). This study describes, for the first time, the investigations for the formation of a charge transfer complex (CTC) between LRL, as electron donor, with chloranilic acid (CLA), as a π-electron acceptor. The CTC was characterized by ultraviolet (UV)-visible spectrophotometry and computational calculations. The UV-visible spectrophotometry ascertained the formation of the CTC in methanol via formation of a new broad absorption band with maximum absorption peak (λmax) at 530 nm. The molar absorptivity (ε) of the complex was 0.55 × 103 L mol-1 cm-1 and its band gap energy was 2.3465 eV. The stoichiometric ratio of LRL/CLA was found to be 1:2. The association constant of the complex was 0.40 × 103 L mol-1, and its standard free energy was -0.15 × 102 J mole-1. The computational calculation for the atomic charges of an energy minimized LRL molecule was conducted, the sites of interaction on the LRL molecule were assigned, and the mechanism of the reaction was postulated. The reaction was adopted as a basis for developing a novel 96-microwell spectrophotometric method (MW-SPA) for LRL. The assay limits of detection and quantitation were 2.1 and 6.5 µg/well, respectively. The assay was validated, and all validation parameters were acceptable. The assay was implemented successfully with great precision and accuracy to the determination of LRL in its bulk form and pharmaceutical formulation (tablets). This assay is simple, economic, and more importantly has a high-throughput property. Therefore, the assay can be valuable for routine in quality control laboratories for analysis of LRL's bulk form and pharmaceutical tablets.

Development of Green and High Throughput Microplate Reader-Assisted Universal Microwell Spectrophotometric Assay for Direct Determination of Tyrosine Kinase Inhibitors in Their Pharmaceutical Formulations Irrespective the Diversity of Their Chemical Structures.

This study discusses the development and validation of a universal microwell spectrophotometric assay for TKIs, regardless of the diversity in their chemical structures. The assay depends on directly measuring the native ultraviolet light (UV) absorption of TKIs. The assay was carried out using UV-transparent 96-microwell plates and the absorbance signals were measured by a microplate reader at 230 nm, at which all TKIs had light absorption. Beer's law correlating the absorbances of TKIs with their corresponding concentrations was obeyed in the range of 2-160 µg mL-1 with excellent correlation coefficients (0.9991-0.9997). The limits of detection and limits quantitation were in the ranges of 0.56-5.21 and 1.69-15.78 µg mL-1, respectively. The proposed assay showed high precision as the values of the relative standard deviations for the intra- and inter-assay precisions did not exceed 2.03 and 2.14%, respectively. The accuracy of the assay was proven as the recovery values were in the range of 97.8-102.9% (±0.8-2.4%). The proposed assay was successfully applied to the quantitation of all TKIs in their pharmaceutical formulations (tablets) with reliable results in terms of high accuracy and precision. The assay greenness was evaluated, and the results proved that the assay fulfils the requirements of green analytical approach. The proposed assay is the first assay that can analyse all TKIs on a single assay system without chemical derivatization or modifications in the detection wavelength. In addition, the simple and simultaneous handling of a large number of samples as a batch using micro-volumes of samples gave the assay the advantage of high throughput analysis, which is a serious demand in the pharmaceutical industry.

Development and Validation of Green and High-Throughput Microwell Spectrophotometric Assay for the Determination of Selective Serotonin Reuptake Inhibitors in Their Pharmaceutical Dosage Forms.

This study describes the development and validation of a new green and high-throughput microwell spectrophotometric assay (MW-SPA) for the determination of three selective serotonin reuptake inhibitors (SSRIs) in their pharmaceutical dosage forms. These SSRIs are fluoxetine, fluvoxamine, and paroxetine, the most prescribed drugs for the treatment of depression. The proposed assay was based on the formation of orange-colored N-substituted naphthoquinone derivatives upon the reaction of SSRIs with 1,2-naphthoquinone-4-sulphonate (NQS) in alkaline media. The assay was conducted in 96-microwell assay plates, and the absorbances of the reaction products were measured by a microplate reader at their maximum absorbance wavelengths. The optimum conditions of the reaction were refined and established. Under these conditions, calibration curves were generated, and linear regression equations were computed. The linear relations between the absorbances and drug concentrations were linear with good correlation coefficients (0.9992-0.9997) in the range of 2-80 µg/mL. The assay limits of detection were in the range of 1.5-4.2 µg/mL. The precision was satisfactory as the values of relative standard deviation did not exceed 1.70%. The accuracy of the assay was ≥98.2%. The proposed MW-SPA was successfully applied to the analysis of the SSRIs in their pharmaceutical dosage forms with acceptable accuracy and precision; the label claims were 99.2-100.5% (±0.96-1.35%). The results of the proposed MW-SPA were compared with those of the official/pre-validated assays by statistical analysis with respect to the accuracy (by t-test) and precision (by F-test). No significant differences were found between the calculated and theoretical values of the t- and F-tests at the 95% confidence level, proving similar accuracy and precision in the determination of SSRIs by both assays. The greenness of the proposed assay was confirmed by two metric tools. In addition, the assay is characterized with a high-throughput property which enables the simultaneous analysis of many samples in a short time. Therefore, the assay is a valuable tool for rapid routine application in pharmaceutical quality control units for the determination of the investigated SSRIs.

A Novel Spectrophotometric Method for Determination of Percarbonate by Using N, N-Diethyl-P-Phenylenediamine as an Indicator and Its Application in Activated Percarbonate Degradation of Ibuprofen.

Sodium percarbonate (SPC) concentration can be determined spectrophotometrically by using N, N-diethyl-p-phenylenediamine (DPD) as an indicator for the first time. The ultraviolet-visible spectrophotometry absorbance of DPD•+ measured at 551 nm was used to indicate SPC concentration. The method had good linearity (R2 = 0.9995) under the optimized experimental conditions (pH value = 3.50, DPD = 4 mM, Fe2+ = 0.5 mM, and t = 4 min) when the concentration of SPC was in the range of 0-50 µM. The blank spiked recovery of SPC was 95-105%. The detection limit and quantitative limit were 0.7-1.0 µM and 2.5-3.3 µM, respectively. The absorbance values of DPD•+ remained stable within 4-20 min. The method was tolerant to natural water matrix and low concentration of hydroxylamine (<0.8 mM). The reaction stoichiometric efficiency of SPC-based advanced oxidation processes in the degradation of ibuprofen was assessed by the utilization rate of SPC. The DPD and the wastewater from the reaction were non-toxic to Escherichia coli. Therefore, the novel Fe2+/SPC-DPD spectrophotometry proposed in this work can be used for accurate and safe measurement of SPC in water.

Spectrophotometric Study of Charge-Transfer Complexes of Ruxolitinib with Chloranilic Acid and 2,3-Dichloro-5,6-dicyano-1,4-benzoquinone: An Application to the Development of a Green and High-Throughput Microwell Method for Quantification of Ruxolitinib in Its Pharmaceutical Formulations.

Ruxolitinib (RUX) is a potent drug that has been approved by the Food and Drug Administration for the treatment of myelofibrosis, polycythemia vera, and graft-versus-host disease. This study describes the formation of colored charge-transfer complexes (CTCs) of RUX, an electron donor, with chloranilic acid (CLA) and 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ), the π-electron acceptors. The CTCs were characterized using UV-visible spectrophotometry. The formation of CTCs in methanol was confirmed via formation of new absorption bands with maximum absorption at 530 and 470 nm for CTCs with CLA and DDQ, respectively. The molar absorptivity and other physicochemical and electronic properties of CTCs were determined. The molar ratio was found to be 1:1 for both CTCs with CLA and CTCs with DDQ. The site of interaction on RUX molecules was assigned and the mechanisms of the reactions were postulated. The reactions were employed as basis for the development of a novel green and one-step microwell spectrophotometric method (MW-SPM) for high-throughput quantitation of RUX. Reactions of RUX with CLA and DDQ were carried out in 96-well transparent plates, and the absorbances of the colored CTCs were measured by an absorbance microplate reader. The MW-SPM was validated according to the ICH guidelines. The limits of quantitation were 7.5 and 12.6 µg/mL for the methods involving reactions with CLA and DDQ, respectively. The method was applied with great reliability to the quantitation of RUX content in Jakavi® tablets and Opzelura® cream. The greenness of the MW-SPM was assessed by three different metric tools, and the results proved that the method fulfills the requirements of green analytical approaches. In addition, the one-step reactions and simultaneous handling of a large number of samples with micro-volumes using the proposed method enables the high-throughput analysis. In conclusion, this study describes the first MW-SPM, a valuable analytical tool for the quality control of pharmaceutical formulations of RUX.

Evaluation of perfusion parameters of gingival inflammation using laser Doppler flowmetry and tissue spectrophotometry- a prospective comparative clinical study.

BACKGROUND: The aim of this study was to determine the values of different perfusion parameters- such as oxygen saturation, the relative amount of hemoglobin, and blood flow- in healthy subjects compared to patients with gingivitis as a non-invasive measurement method. METHODS: A total of 114 subjects were enrolled in this study and separated into subjects with gingivitis (50) and without gingivitis (64) based on clinical examination. Gingival perfusion was measured at 22 points in the maxilla and mandible using laser Doppler flowmetry and tissue spectrophotometry (LDF-TS) with the "oxygen to see" device. All patients underwent measurement of gingival perfusion, followed by the clinical evaluation (measurement of probing depths, evaluation of bleeding on probing, plaque level, and biotype). Perfusion parameters were compared between the groups, associations between the non-invasive and clinical measurements were analyzed, and theoretical optimal cut-off values for predicting gingivitis were calculated with receiver operating characteristics. RESULTS: The mean oxygen saturation, mean relative amount of hemoglobin, and mean blood flow all significantly differed between the groups with and without gingivitis (p = 0.005, p < 0.001, and p < 0.001, respectively). The cut-off value for predicting gingivitis was > 40 AU (p < 0.001; sensitivity 0.90, specificity 0.67). CONCLUSIONS: As a non-invasive method, LDF-TS can help determine gingival hyperemia. Flow values above 40 AU indicate a higher risk of hyperemia, which can be associated with inflammation. The LDF-TS method can be used for the objective evaluation of perfusion parameters during routine examinations and can signal the progression of hyperperfusion before any change in clinical parameters is observed. TRIAL REGISTRATION: All procedures performed in this study involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. The study was approved by the institutional Clinical Research Ethics Committee (Ethik-Kommission der Medizinischen Fakultät der RWTH Aachen, Decision Number 286/20) and retrospectively registered by the German Clinical Trials Register (File Number DRKS00024048, registered on the 15th of October 2021).

The Role of Intraoral Scanners in the Shade Matching Process: A Systematic Review.

PURPOSE: The variation in findings with regards to the accuracy and precision of intraoral scanners for shade selection are no doubt confusing for clinicians who may find it difficult to make evidence-based decisions. The aim of this systematic review is to provide a comprehensive and in-depth assessment of available studies to determine the viability of using intraoral scanners for the purpose of shade matching. The PICO-guided research question is as follows: when shade matching, are intraoral scanners as valid as visual or other digital shade measuring devices in determining tooth colors. METHODS: Electronic databases including PubMed/MEDLINE, SCOPUS, EBSCO, Cochrane, and ProQuest were systematically searched for articles published between January 1, 2011 and December 30, 2021 using the main search terms: "intraoral scanners," "scanners," "TRIOS," "CEREC," "Planmeca," "Medit," "digital dentistry" in concurrence with one of the following keywords: "EasyShade" OR "shade selection" OR "shade matching" OR "shade" OR "tooth color" OR "tooth shade" OR "digital shade matching." Bibliographies of included articles and the following journals were searched for relevant articles: Journal of Prosthetic Dentistry, Journal of Prosthodontics, Journal of Esthetic and Restorative Dentistry, Journal of Advanced Prosthodontics, and Journal of Dentistry. A total of 15 articles were included in the review. RESULTS: Intraoral scanners are highly repeatable for shade matching, and outperformed visual shade matching. Accuracy varied significantly between studies, with the majority recommending the use of visual shade matching to confirm/verify the intraoral scanner results. Setting intraoral scanners to the Vita 3D Master shade guide improved both accuracy and precision. Shade matching with intraoral scanners may be influenced by external factors such as ambient light sources and incorrect use or manipulation. CONCLUSION: Intraoral scanners set to the Vita 3D Master shade guide may be used for shade matching, but shade should be verified with visual shade matching. Further studies are required to address limitations of current studies.

Novel High-Throughput Microwell Spectrophotometric Assay for One-Step Determination of Lorlatinib, a Novel Potent Drug for the Treatment of Anaplastic Lymphoma Kinase (ALK)-Positive Non-Small Cell Lung Cancer.

Background and Objectives: Lorlatinib (LOR) belongs to the third-generation anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitors. People who are diagnosed with ALK-positive metastatic and advanced non-small cell lung cancer (NSCLC) are eligible to get it as a first-line treatment option after it was given the approval by "the Food and Drug Administration (FDA)". However, no study has described constructing high-throughput analytical methodology for LOR quantitation in dosage form. For the first time, this work details the construction of a high-throughput, innovative microwell spectrophotometric assay (MW-SPA) for single-step assessment of LOR in its tablet form, for use in pharmaceutical quality control. Materials and Methods: Assay depended on charge transfer complex (CTC) formation between LOR, as electron donor, with 2,3-dichloro-3,5-dicyano-1,4-benzoquinone (DDQ), as π-electron acceptor. Reaction conditions were adjusted, the CTC was characterized by ultraviolet (UV)-visible spectrophotometry and computational molecular modeling, and its electronic constants were determined. Site of interaction on LOR molecule was allocated and reaction mechanism was suggested. Under refined optimum reaction conditions, the procedures of MW-SPA were performed in 96-well assay plates, and the responses were recorded by an absorbance plate reader. Validation of the current methodology was performed in accordance with guidelines of "the International Council on Harmonization (ICH)", and all validation parameters were acceptable. Results: Limits of detection and quantitation of MW-SPA were 1.8 and 5.5 µg/well, respectively. The assay was applied with great success for determining LOR in its tablets. Conclusions: This The assay is straightforward, economic and has high-throughput characteristics. Consequently, the assay is recommended as a valuable analytical approach in quality control laboratories for LOR's tablets' analysis.

Integrating-Sphere-Assisted Resonance Synchronous Spectroscopy for the Quantification of Material Double-Beam UV-Vis Absorption and Scattering Extinction.

Integrating spheres (IS) have been used extensively for the characterization of light absorption in turbid samples. However, converting the IS-based sample absorption coefficient to the UV-vis absorbance quantified with a double-beam UV-vis spectrophotometer is challenging. Herein, we report an integrating-sphere-assisted resonance synchronous (ISARS) spectroscopy method performed with conventional spectrofluorometers equipped with an integrating-sphere accessory. Mathematical models and experimental procedures for quantifying the sample, solvent, and instrument-baseline ISARS intensity spectra were provided. A three-parameter analytical model has been developed for correlating the ISARS-based UV-vis absorbance and the absorbance measured with double-beam instruments. This ISARS method enables the quantitative separation of light absorption and scattering contribution to the sample UV-vis extinction spectrum measured with double-beam UV-vis spectrophotometers. Example applications of this ISARS technique are demonstrated with a series of representative samples differing significantly in their optical complexities, from approximately pure absorbers, pure scatterers, to simultaneous light absorbers, scatterers, and emitters under resonance excitation and detection conditions.

Multiple spectrophotometric determinations of Chlorthalidone and Cilnidipine using propylene carbonate - As a step towards greenness.

Analyzing a drug based on its overlapping spectra requires the use of sophisticated equipment and more hazardous solvents, which is detrimental to ecological sustainability. There is a critical need to create a simple, unique, and cost-effective approach for detecting a mixture of compounds in a safer environment. The aim was to develop an eco-friendly, stability-indicating assay method to determine Chlorthalidone (CLD) and Cilnidipine (CIL) in bulk and tablet dosage form using four different Ultra-Violet (UV) spectrophotometric methods. The ratio difference method showed absorbance peaks at 256.01 nm, 220.87 nm, first ratio difference spectra at 267.21 nm, 288.03 nm, and second ratio difference spectra at 309.2 nm, 280.03 nm. The area under curve techniques showed an absorbance range of around 224-232 nm for CIL and 218-227 nm for CLD. Further, the spectral changes have been assessed at various conditions like acid, base, oxidation, and UV radiation, and it has been found that CLD tends to lose its spectral property by more than 45%. The method developed for two drugs has obeyed Beers law with the selected concentration range of 7-13 µg/mL for CLD and 8.75-16.25 µg/mL for CIL. The developed method was finally evaluated using four tools, and the results showed green pictographically representation in the GAPI and score near to 100 for AES and closer to 1 for AGREE indicated that the method was found to be most eco-friendly. The findings were easy to replicate, adoptive with major regulatory requirements, environmentally friendly, fast, and inexpensive to perform. This approach does not require any specific expensive equipment, and it might be inexpensive to use in the future to assess laboratory and commercial mixtures.