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.

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.

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.

Synthesis, spectroscopic and computational characterization of charge transfer complex of remdesivir with chloranilic acid: Application to development of novel 96-microwell spectrophotometric assay.

Remdesivir (REM) is an adenosine triphosphate analog antiviral drug that has received authorization from European Commission and approval from the U.S. Food and Drug Administration for treatment of coronavirus disease 2019 (Covid-19). This study, describes, for the first time, the synthesis of a novel charge transfer complex (CTC) between REM, as electron donor, with chloranilic acid (CLA), as π electron acceptor. The CTC was characterized using different spectroscopic and thermogravimetric techniques. UV-visible spectroscopy 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 3.33 × 103 L mol-1 cm-1 and its band gap energy was 1.91 eV. The stoichiometric ratio of REM:CLA was found to be 1:1. The association constant of the complex was 1.11 × 109 L mol-1, and its standard free energy was 5.16 × 104 J mole-1. Computational calculation for atomic charges of energy minimized REM was conducted, the site of interaction on REM molecule was assigned and the mechanism of the reaction was postulated. The solid-state CTC was further characterized by FT-IR and 1H NMR spectroscopic techniques. Both FT-IR and 1H NMR confirmed the formation of the CTC and its structure. The reaction was adopted as a basis for developing a novel 96-microwell spectrophotometric method (MW-SPA) for REM. The assay limits of detection and quantitation were 3.57 and 10.83 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 REM in its bulk form and pharmaceutical formulation (injection). This assay is simple, economic, and more importantly, has high throughput property. Therefore, the assay can be valuable for routine in quality control laboratories for analysis of REM's bulk form and pharmaceutical injection.

Charge-transfer reaction of 2,3-dichloro-1,4-naphthoquinone with crizotinib: Spectrophotometric study, computational molecular modeling and use in development of microwell assay for crizotinib.

The reaction of 2,3-dichloro-1,4-naphthoquinone (DCNQ) with crizotinib (CZT; a novel drug used for treatment of non-small cell lung cancer) was investigated in different solvents of varying dielectric constants and polarity indexes. The reaction produced a red-colored product. Spectrophotometric investigations confirmed that the reaction proceeded through charge-transfer (CT) complex formation. The molar absorptivity of the complex was found to be linearly correlated with the dielectric constant and polarity index of the solvent; the correlation coefficients were 0.9567 and 0.9069, respectively. The stoichiometric ratio of DCNQ:CZT was found to be 2:1 and the association constant of the complex was found to be 1.07 × 10(2) l/mol. The kinetics of the reaction was studied; the order of the reaction, rate and rate constant were determined. Computational molecular modeling for the complex between DCNQ and CZT was conducted, the sites of interaction on CZT molecule were determined, and the mechanism of the reaction was postulated. The reaction was employed as a basis in the development of a novel 96-microwell assay for CZT in a linear range of 4-500 µg/ml. The assay limits of detection and quantitation were 2.06 and 6.23 µg/ml, respectively. The assay was validated as per the guidelines of the International Conference on Harmonization (ICH) and successfully applied to the analysis of CZT in its bulk and capsules with good accuracy and precision. The assay has high throughput and consumes a minimum volume of organic solvents thus it reduces the exposures of the analysts to the toxic effects of organic solvents, and significantly reduces the analysis cost.

Synthesis, spectroscopic and computational studies on hydrogen bonded charge transfer complex of duvelisib with chloranilic acid: Application to development of novel 96-microwell spectrophotometric assay.

Duvelisib (DUV) is a is a small-molecule with inhibitory action for phosphoinositide 3-kinase (PI3K). It has been recently approved for the effective treatment of chronic lymphocytic leukemia (CLL) and small lymphocytic lymphoma (SLL). Novel charge transfer complex (CTC) between DUV, as electron donor, with chloranilic acid (CLA), as π electron acceptor has been synthesized and characterized using different spectroscopic and thermogravimetric techniques. UV-visible spectroscopy ascertained the formation of the CTC in different solvents of varying polarity indexes and dielectric constants via formation of new broad absorption band with maximum absorption peak (λmax) in the range of 488-532 nm. The molar absorptivity of the CTC was dependent on the polarity index and dielectric constant of the solvent; the correlation coefficients were 0.9955 and 0.9749, respectively. The stoichiometric ratio of DUV:CLA was 1:1. Electronic spectral analysis was conducted for characterization of the complex in terms of its electronic constants. Computational calculation for atomic charges of energy minimized DUV was conducted and the site of interaction on DUV molecule was assigned. The solid-state CTC of DUV:CLA (1:1) was synthesized, and its structure was characterized by UV-visible, mass, FT-IR, and 1H NMR spectroscopic techniques. Both FT-IR and 1H NMR confirmed that both CT and hydrogen bonding contributed to the molecular composition of the complex. The reaction was adopted as a basis for developing a novel 96-microwell spectrophotometric assay (MW-SPA) for DUV. The assay limits of detection and quantitation were 0.57 and 1.72 µg/well, respectively. The assay was validated and all validation parameters were acceptable. The method was implemented successfully with great precision and accuracy to the analysis of the DUV in its bulk and capsules.

Corrigendum: Effects of extracellular adhesion molecules on immune cell mediated solid tumor cell killing.

[This corrects the article DOI: 10.3389/fimmu.2022.1004171.].

Effects of extracellular adhesion molecules on immune cell mediated solid tumor cell killing.

Adoptive cell therapy (ACT) using ex vivo engineered/expanded immune cells demonstrated poor efficacy against solid tumors, despite its great success in treating various hematopoietic malignancies. To improve ACT for solid tumors, it is crucial to comprehend how the numerous components of the tumor microenvironment (TME) surrounding solid tumor cells influence killing ability of immune cells. In this study, we sought to determine the effects of extracellular adhesion provided by extracellular matrix (ECM) of TME on immune cell cytotoxicity by devising microwell arrays coated with proteins either preventing or promoting cell adhesion. Solid tumor cells in bovine serum albumin (BSA)-coated microwells did not attach to the surfaces and exhibited a round morphology, but solid tumor cells in fibronectin (FN)-coated microwells adhered firmed to the substrates with a flat shape. The seeding densities of solid tumor cells and immune cells were tuned to maximize one-to-one pairing within a single microwell, and live cell imaging was performed to examine dynamic cell-cell interactions and immune cell cytotoxicity at a single cell level. Both natural killer (NK) cells and T cells showed higher cytotoxicity against round tumor cells in BSA-coated microwells compared to flat tumor cells in FN-coated microwells, suggesting that extracellular adhesion-mediated firm adhesion of tumor cells made them more resistant to immune cell-mediated killing. Additionally, NK cells and T cells in FN-coated microwells exhibited divergent dynamic behaviors, indicating that two distinct subsets of cytotoxic lymphocytes respond differentially to extracellular adhesion cues during target cell recognition.

Innovative use of σ and π electron acceptors in the development of three high throughput 96-microwell spectrophotometric assays for crizotinib.

Crizotinib (CZT) is a potent and selective tyrosine kinase inhibitor used for treatment of non-small cell lung cancer (NSCLC). The development of high-throughput assays for its quality control (QC) is very essential to assure its therapeutic benefits. CZT molecule has multiple electron-donating atoms that can contribute to the formation of colored charge-transfer (CT) complex with iodine as σ-electron acceptor and with 2,5-dichloro-3,6-dihydroxy-1,4-benzoquinone (CHBQ) and 7,7,8,8-tetracyanoquinodimethane (TCNQ) as π-electron acceptors. These reactions were prospective basis for development of three innovative 96-microwell-based spectrophotometric assays for CZT. The reactions of CZT with iodine, CHBQ and TCNQ were performed in 96-microwell assay plates and absorbances of the CT complexes were measured by microwell absorbance reader at their corresponding maximum absorption peaks. The measured absorbances were correlated with the CZT concentrations in its sample solutions. Beer's law was obeyed with excellent correlation coefficients in the range of 0.5-30, 2-500, and 5-500 µg mL-1 for assays using iodine, CHBQ and TCNQ, respectively. The limits of detection were 2.17, 0.85 and 6.23 µg mL-1 for assays using iodine, CHBQ and TCNQ, respectively. The validation studies confirmed the accuracy and precision of all the proposed assays. The assays were successfully applied in the determination of CZT in Xalkori capsules. The proposed assays have very simple procedures to run in QC laboratories. Also, both assays enable analyst to process large number of samples and use of very small volumes of the organic solvent (ecofriendly and inexpensive).

Charge-transfer reaction of 1,4-benzoquinone with crizotinib: spectrophotometric study, computational molecular modeling and use in development of microwell assay for crizotinib.

The reaction of 1,4-benzoquinone (BQ) with crizotinib (CZT); a novel drug used for treatment of non-small cell lung cancer) was investigated in different solvents of varying dielectric constants and polarity indexes. The reaction resulted in the formation of a red-colored product. Spectrophotometric investigations confirmed that the reaction proceeded through charge-transfer (CT) complex formation. The molar absorptivity of the complex was found to be linearly correlated with the dielectric constant and polarity index of the solvent; the correlation coefficients were 0.9425 and 0.8340, respectively. The stoichiometric ratio of BQ:CZT was found to be 2:1 and the association constant of the complex was found to be 0.26×10(3)lmol(-1). The kinetics of the reaction was studied; the order of the reaction, rate and rate constant were determined. Computational molecular modeling for the complex between BQ and CZT was conducted, the sites of interaction on CZT molecule were determined, and the mechanism of the reaction was postulated. The reaction was employed as a basis in the development of a novel 96-microwell assay for CZT. The assay limits of detection and quantitation were 5.2 and 15.6µgml(-1), respectively. The assay was validated as per the guidelines of the International Conference on Harmonization (ICH) and successfully applied to the analysis of CZT in its bulk and capsules with good accuracy and precision. The assay has high throughput and consumes minimum volume of organic solvent thus it reduces the exposures of the analysts to the toxic effects of organic solvents, and significantly reduces the analysis cost.