Development of time-resolved fluoroimmunoassay with enhanced fluorescence reaction for the quantitation of atezolizumab in plasma.

Atezolizumab (ATZ) is a human monoclonal antibody, which has been granted multiple approvals from the US Food and Drug Administration (FDA) for the immunotherapy of different types of cancer. This study describes the prototype of a time-resolved fluoroimmunoassay (TRFIA) for the quantitation of ATZ in plasma. The assay involved the non-competitive binding of ATZ to its specific antigen [programmed death-ligand 1 (PD-L1) protein]. The immune complex formed on the inner surface of the assay plate wells was quantified by anti-human secondary antibody labeled with a chelate of europium-ethylenediaminetetraacetic acid. The enhanced fluorescence signal was generated by an enhanced fluorescence solution composed of thenoyltrifluoroacetone, trioctylphosphine oxide, and Triton X-100. The conditions of the TRFIA were refined, and its optimum procedures were established. The assay was validated in accordance with the immunoassay validation guidelines, and all the validation parameters were acceptable. The working range of the assay was 20-1000 pg mL-1, and its limit of quantitation was 20 pg mL-1. The assay was applied to the quantitation of ATZ in plasma samples with satisfactory accuracy and precision. The proposed TRFIA has significant benefits over the existing methodologies for the quantitation of ATZ in clinical settings.

Development of Eco-Friendly Scattering and Fluorimetric Methods for the Determination of Clemastine Through Its Interaction with Eosin Y: Assessment of Whiteness, Blueness, and Greenness Tools.

For the first time, clemastine was estimated in this work utilizing two validated resonance Rayleigh scattering (RRS) and fluorimetric methods. The methods relied on forming an association complex in an acidic medium between eosin Y reagent and clemastine. In the spectrofluorimetric approach, the investigated drug was quantified by quenching the fluorescence-emission intensity of eosin Y at 543.5 nm. The RRS method relied on enhancing the RRS spectrum at 331.8 nm, which is produced when eosin Y interacts with clemastine. Suitable conditions were established for the reaction to achieve maximum sensitivity. The linear values obtained from the spectrofluorimetric approach and the RRS method fall into the ranges of 0.2-1.5 µg mL- 1 and 0.25-2.0 µg mL- 1, respectively. It was established that the detection limits for these methods were 0.045 µg mL- 1 and 0.059 µg mL- 1, respectively. The developed methodologies yielded acceptable recoveries when used to estimate the quantity of clemastine in its pharmaceutical tablet dosage form. Regarding the use of greener solvents that were chosen, the suggested and reported methods were compared with the help of the Green Solvents Selecting (GSST) tool for assessing hazardous solvents to achieve sustainability. Furthermore, analytical Eco scale and comprehensive assessments of whiteness, blueness, and greenness were carried out utilizing Modified NEMI, ComplexGAPI, and AGREE evaluation tools. Additionally, recently developed tools such as BAGI and RGB 12 were applied to assess the blueness and the whiteness of the suggested methods.

A green approach for metoclopramide quantification in pharmaceutical products: new HPLC and spectrophotometric methods.

Green spectrophotometric and HPLC methods have been developed for the quantification of metoclopramide. In the spectrophotometric method, it was determined by direct absorbance measurement at 273 nm wavelength using ultrapure water as solvent. The Extend C18 column was used for the HPLC method. The mobile phase system consisted of a combination of ethanol and formic acid solution (pH 2.0; 30:70 v/v). Isocratic elution was applied and the flow rate was set at 1.0 mL min-1. Metoclopramide was detected at 273 nm. The methods performed were economical, rapid, environmentally friendly, and simple, providing metoclopramide analysis within 5 min. The methods have been successfully applied in pharmaceutical products without matrix interference. The results of the application of the developed methods to pharmaceutical products were statistically compared and no significant difference was observed between the methods. In addition, the greenness assessment of the developed methods was performed using AGREE software. Our developed methods, based on the use of solvents such as ethanol and water, are proposed as a more environmentally and analyst-friendly option for the quantification of metoclopramide in pharmaceutical products than other methods currently in use.

Dual-plasmonic CuS@Au heterojunctions synergistic enhanced photothermal and colorimetric dual signal for sensitive multiplexed LFIA.

Multiplexed immunodetection, which achieves qualitative and quantitative outcomes for multiple targets in a single-run process, provides more sufficient results to guarantee food safety. Especially, lateral flow immunoassay (LFIA), with the ability to offer multiple test lines for analytes and one control line for verification, is a forceful candidate in multiplexed immunodetection. Nevertheless, given that single-signal mode is incredibly vulnerable to interference, further efforts should be engrossed on the combination of multiplexed immunodetection and multiple signals. Photothermal signal has sparked significant excitement in designing immunosensors. In this work, by optimizing and comparing the amount of gold, CuS@Au heterojunctions (CuS@Au HJ) were synthesized. The dual-plasmonic metal-semiconductor hybrid heterojunction exhibits a synergistic photothermal performance by increasing light absorption and encouraging interfacial electron transfer. Meanwhile, the colorimetric property is synergistic enhanced, which is conducive to reduce the consumption of antibodies and then improve assay sensitivity. Therefore, CuS@Au HJ are suitable to be constructed in a dual signal and multiplexed LFIA (DSM-LFIA). T-2 toxin and deoxynivalenol (DON) were used as model targets for the simulated multiplex immunoassay. In contrast to colloidal gold-based immunoassay, the built-in sensor has increased sensitivity by ≈ 4.42 times (colorimetric mode) and ≈17.79 times (photothermal mode) for DON detection and by ≈ 1.75 times (colorimetric mode) and ≈13.09 times (photothermal mode) for T-2 detection. As a proof-of-concept application, this work provides a reference to the design of DSM-LFIA for food safety detection.

Synthesis of dual models multivalent activatable aptamers based on HCR and RCA for ultrasensitive detection of Salmonella typhimurium.

Aptamers have superior structural properties and have been widely used in bacterial detection methods. However, the problem of low affinity still exists in complex sample detection. In contrast, hybridization chain reaction (HCR)-based model I and rolling circle amplification (RCA)-based model II multivalent activatable aptamers (multi-Apts) can fulfill the need for low-cost, rapid, highly sensitive and high affinity detection of S. typhimurium. In our research, two models of multi-Apts were designed. First, a monovalent activatable aptamer (mono-Apt) was constructed by fluorescence resonance energy transfer (FRET) with an S. typhimurium aptamer and its complementary chain of BHQ1. Next, the DNA scaffold was obtained by HCR and RCA, and the multi-Apts were obtained by self-assembly of the mono-Apt with a DNA scaffold. In model I, when target was presented, the complementary chain BHQ1 was released due to the binding of multi-Apts to the target and was subsequently adsorbed by UIO66. Finally, a FRET-based fluorescence detection signal was obtained. In mode II, the multi-Apts bound to the target, and the complementary chain BHQ1 was released to become the trigger chain for the next round of amplification of HCR with a fluorescence detection signal. HCR and RCA based multi-Apts were able to detect S. typhimurium as low as 2 CFU mL-1 and 1 CFU mL-1 respectively. Multi-Apts amplification strategy provides a new method for early diagnosis of pathogenic microorganisms in foods.

Development of Two Eco-Friendly and High-Throughput Microwell Spectrophotometric Methods for Analysis of an Antibacterial Drug Tulathromycin in Pharmaceutical Bulk Form.

BACKGROUND: Tulathromycin (TUL) is a triamilide antibacterial drug which has been approved for use in the European Union and the United States for the treatment and prevention of bovine respiratory diseases. The existing methods for determination of TUL in its pharmaceutical bulk form are very limited and suffer from major drawbacks. OBJECTIVES: The aim of this study was the development of two innovative microwell spectrophotometric methods (MW-SPMs) for determination of TUL in its pharmaceutical bulk form. METHODS: The formation of charge transfer complexes (CTCs) of TUL, as an electron donor, was investigated with 2,5-dihydroxy-3,6-dichlorocyclohexa-2,5-diene-1,4-dione (HCD) and 2,3-dichloro-5,6-dicyano-p-benzoquinone (CBQ), as π-electron acceptors. The CTCs were characterized by using UV-visible spectrophotometry and computational calculations. The reactions were employed for the development of two MW-SPMs with a one-step for the quantitative analysis of TUL. RESULTS: The formation of CTCs was confirmed via the formation of characteristic absorption bands with maximum absorption at 520 and 460 nm for CTCs with HCD and CBQ, respectively. The stoichiometry of both CTCs was found to be 1:1, and the values of different spectroscopic and electronic constants confirmed the stability of the CTCs. The mechanisms of the reactions were postulated. The linear range of both MW-SPMs was 10-500 µg/mL. The limits of quantitation were 13.5 and 26.4 µg/mL for methods involving reactions with HCD and CBQ, respectively. Both methods were successfully applied to the quantitation of TUL in pharmaceutical bulk form with acceptable accuracy and precision. The results of eco-friendliness/greenness assessment proved that both MW-SPMs fulfill the requirements of green analytical approaches. In addition, the one-step reactions and simultaneous handling of a large number of samples with micro-volumes in the proposed methods gave them the advantage of high throughput analysis. CONCLUSIONS: This study described two new MW-SPMs as valuable analytical tools for the determination of TUL.

A prototype of ultrasensitive time-resolved fluoroimmunoassay for the quantitation of lead in plasma using a fluorescence-enhanced europium chelate label for the detection system.

This study describes the prototype of a novel ultra-sensitive time-resolved fluoroimmunoassay (TRFIA) for the quantification of lead (Pb) in plasma. The assay procedures were conducted in 96-microwell plates and involved the competitive binding format. The assay used a mouse monoclonal antibody, designated as 2C33, that specifically recognized the diethylenetriamine pentaacetic acid chelate of Pb (Pb-DTPA) but did not recognize Pb-free DTPA chelator. The antigen used for coating onto the inner surfaces of assay plate microwells was Pb-DTPA conjugated with bovine serum albumin protein (Pb-DTPA-BSA). The competitive binding reaction occurred between Pb-DTPA chelates, formed in the sample solutions by treating the samples with an excess DTPA, and the coated Pb-DTPA-BSA for a limited quantity of 2C33 antibody binding sites. The antigen-antibody complex formed in the plate wells was quantified by a europium-DTPA-labeled secondary antibody and a fluorescence enhancement solution. The conditions of the assay were refined, and its optimum procedures were established. The TRFIA was validated following the immunoassay validation guidelines, and all of the validation criteria were acceptable. The working range of the assay was 20-300 pg mL-1 and its limit of quantitation was 20 pg mL-1. Metals that are commonly encountered in blood plasma did not interfere with Pb in the analysis by the proposed TRFIA. The assay was applied to the quantitation of Pb in plasma samples with satisfactory accuracy and precision. The results were compared favorably with those obtained by atomic emission spectroscopy. In conclusion, the present study represents the first TRFIA for the quantitation of Pb in plasma. The assay is superior to the existing atomic spectrometric methods and other immunoassays for Pb in terms of sensitivity, convenience, and analysis throughputs. The proposed TRFIA is anticipated to effectively contribute to assessing Pb concentrations and controlling the exposure of humans to its potential toxicity.

A novel ultrasensitive chemiluminescence enzyme immunoassay by employment of a signal enhancement of horseradish peroxidase-luminol-hydrogen peroxide reaction for the quantitation of atezolizumab, a monoclonal antibody used for cancer immunotherapy.

This study describes, for the first time, the development and validation of a novel ultrasensitive chemiluminescence enzyme immunoassay (CLEIA) for the quantification of atezolizumab (ATZ), a monoclonal antibody approved by the FDA for treatment of different types of cancer. The assay involved the non-competitive binding of ATZ to its specific antigen (PD-L1 protein). The immune complex of PD-L1/ATZ formed on the internal surface of the plate wells was quantified by a novel chemiluminescence (CL)-producing horseradish peroxidase (HRP) reaction. The reaction employed a highly efficient CL enhancer for the HRP-luminol-hydrogen peroxide reaction which was 4-(imidazol-1-yl)phenol. The conditions of the CLEIA and its detection system were refined, and the optimum procedures were established. The CLEIA was validated in accordance with the guidelines of immunoassay validation for bioanalysis, and all the validation criteria were acceptable. The assay's limit of detection and limit of quantitation were 12.5 and 37.5 pg mL-1, respectively, with a working dynamic range of 25-800 pg mL-1. The assay enables the accurate and precise quantitation of ATZ in human plasma samples without any interferences from endogenous substances and/or the plasma matrix. The results of the proposed CLEIA were favourably comparable with those of a pre-validated enzyme-linked immunosorbent assay using a colorimetric detection system. The CLEIA is characterized by simple and high throughput features. The CLEIA is superior to the existing analytical methodologies for ATZ. The proposed CLEIA has a great value in the quantitation of ATZ in clinical settings for assessment of its pharmacokinetics, therapeutic drug monitoring, and refining the safety profile.

Development of Green and High Throughput Microwell Spectrophotometric Methods for Determination of Galidesivir in Bulk Drug and Dosage Forms Based on Simple Oxidimetric Reactions with Inorganic Agents.

BACKGROUND: Galidesivir hydrochloride (GDV) is a new potent and safe antiviral drug used for the treatment of a broad spectrum of viral diseases, including COVID-19. In the literature, no analytical method exists for the determination of GDV in bulk and dosage form. OBJECTIVE: The objective of this study was the investigation of oxidation reactions of GDV with five inorganic oxidizing reagents and the employment of the reactions in the development of five green microwell spectrophotometric methods (MW-SPMs) with simple procedure and high throughputs for determination of GDV in its bulk and dosage forms (capsules). METHODS: The reactions were carried out in 96-well plates and the absorbances of reaction solutions were measured by an absorbance microplate reader. Variables influencing the reactions were carefully investigated and optimized. RESULTS: Under the refined optimum conditions, Beer's law with excellent correlation coefficients (0.9992-0.9997) was followed in GDV concentrations in a general range of 5-700 µg/mL, and the limits of detection were ≥1.8 µg/mL. All validation parameters of all methods were acceptable. The methods were successfully applied to the analysis of GDV in bulk drug and capsules with high accuracy and precision; the recovery percentages were 98.6-101.2 ± 0.58-1.14%. The greenness of MW-SPMs was evaluated by three comprehensive metric tools, which demonstrated the adherence of MW-SPMs to the principles of the green analytical chemistry approach. CONCLUSIONS: The proposed MW-SPMs combined the advantages of microwell-based practice and the use of common laboratory reagents for the analysis. The advantages of microwell analysis were the high throughput, readily available for semi-automation, reduced samples/reagents volume, precise measurements, and versatility. The advantages of using common laboratory reagents were the availability, consistency, compatibility, safety, and cost-effectiveness. HIGHLIGHTS: Overall, the proposed MW-SPMs are versatile valuable tools for the quantitation of GDV during its pharmaceutical manufacturing.

Duvelisib: A comprehensive profile.

Duvelisib (DUV) is chemically named as (S)-3-(1-((9H-Purin-6-yl)amino)ethyl)-8-chloro-2-phenylisoquinolin-1(2H)-one. It is a novel drug with a small molecular weight and characterized by dual phosphoinositide-3-kinase (PI3K)- and PI3K-inhibitory activity. The Food and Drug Administration (FDA) recently approved DUV for the management of small lymphocytic lymphoma (SLL) and relapsed or refractory chronic lymphocytic leukemia (CLL) in adult patients. DUV is marketed under the brand name of Copiktra® (Verastem, Inc., Needham, MA, USA). This chapter provides a critical extensive review of the literature, the description of DUV in terms of its names, formulae, elemental composition, appearance, and use in the treatment of CLL, SLL, and follicular lymphoma. The chapter also describes the methods for preparation of DUV, its physical-chemical properties, analytical methods for its determination, pharmacological properties, and dosing information.

Development and validation of synchronous spectrofluorimetric method for the simultaneous determination of duvelisib and moxifloxacin: greenness metric assessment and application to a pharmacokinetic study in rats.

Duvelisib (DUV) is a potent anticancer drug whereas Moxifloxacin (MOX) is an antimicrobial drug with anti-proliferative potency against cancerous cells, which is empirically administered in cancer treatment. DUV and MOX combination is commonly prescribed to combat infections in patients while they are under chemotherapy treatment. This study describes, for the first time, the development of a simple and green synchronous spectrofluorimetric (SSF) method for the simultaneous estimation of DUV and MOX in plasma. DUV and MOX were quantified at 273 and 362 nm, respectively without interference between each other at Δλof 120 nm. The experimental variables influencing fluorescence intensities were thoroughly investigated and the optimum conditions were established. At pH 3.5, the optimum synchronous fluorescence intensity (SFI) was achieved in water solvent by using sodium acetate buffer solution. Calibration curves for DUV and MOX, correlating the SFI with the corresponding drug concentration, were linear in the range of 50-1000 ng mL-1for both drugs, with good correlation coefficients. The method was extremely sensitive, with limits of detection of 24 and 22 ng mL-1, and limits of quantitation of 40 and 45 ngmL-1for DUV and MOX, respectively. The SSF method was validated according to the Food and Drug Administration (FDA) guidelines for validation of analytical procedures, and the validation parameters were acceptable. The proposed SSF method was applied to the pharmacokinetic and bioavailability studies in rats' plasma after single concurrent oral administration of both drugs. The results of the study revealed that caution should be taken with DUV dose when concurrently administered with MOX. The greenness of SSF method was assessed by three different metric tools namely Analytical Eco-scale, Green Analytical Procedure Index, and Analytical Greenness Calculator. The results confirmed that SSF method is an eco-friendly and green analytical approach. In conclusion, the proposed SSF method is a valuable tool for pharmacokinetic/bioavailability studies and therapeutic drug monitoring of simultaneously administered DUV and MOX.

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.

Retraction notice to "Development of a highly sensitive chemiluminescence immunoassay using a novel signal-enhanced detection system for quantitation of durvalumab, an immune-checkpoint inhibitor monoclonal antibody used for immunotherapy of lung cancer" [Heliyon 9 (2023) e15782].

[This retracts the article DOI: 10.1016/j.heliyon.2023.e15782.].

A novel mesna-based electrochemical sensor embellished with silver nanoparticles for ultrasensitive analysis of modafinil.

Modafinil (MOD) is a CNS stimulant used for the treatment of narcolepsy, shift work sleep disorder, excessive daytime sleepiness, and post-COVID 19 neurological symptoms. In the literature, there is no report of square wave voltammetric (SWV) methods being used for the determination of MOD. This study describes, for the first time, the construction and evaluation of the analytical performance of a novel sensor for ultrasensitive SWV detection of MOD. The sensor was constructed by integration of silver nanoparticles (AgNPs) on Mesna (MSN) layers over a pencil graphite electrode (PGE) surface. The interface and morphological characteristics of the fabricated AgNPs@MSN/PGE sensor were investigated via cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), and scanning electron microscopy (SEM). This sensor was found to enhance the electro-oxidation of MOD. The combination of AgNPs@MSN/PGE with SWV enabled the determination of MOD in its bulk form and in pharmaceutical and biological matrices at the nanomolar scale (LOD = 28.59 nM) with excellent recoveries. This study represents the first report describing an electrochemical procedure for MOD detection in human plasma. The established SWV method was also validated, and the results were consistent with ICH criteria. Finally, the presented SWV procedure provides a facile, sensitive, rapid, and cost-effective approach compared to other existing methods.

Development and Comparative Evaluation of Two Highly Sensitive Immunosensor Platforms for Trace Determination of Copper Ions in Drinking Water Using a Monoclonal Antibody Specific to Copper-EDTA Complex.

This study describes the development of two highly sensitive immunosensor platforms for the trace determination of copper ions, Cu(II), in drinking water. These platforms were a microwell-based enzyme-linked immunosorbent assay (ELISA) and a kinetic exclusion assay (KinExA) with a KinExATM 3200 immunosensor. Both ELISA and KinExA were developed utilizing the same antibody and coating reagent. The antibody was a mouse monoclonal antibody, designated as 8D66, that specifically recognized Cu(II)-ethylenediamine tetraacetic acid complex (Cu(II)-EDTA) but did not recognize Cu(II)-free EDTA. The 8D66 monoclonal antibody was generated by the fusion of spleen cells of an immunized BALB/c mouse with SP2/0-Ag14 myeloma cells. The immunogen was a protein conjugate of Cu(II)-EDTA with keyhole limpet hemocyanin protein. The coating reagent was Cu(II)-EDTA covalently linked to bovine serum albumin protein (Cu(II)-EDTA-BSA). Both assays involved the competitive binding reaction between Cu(II)-EDTA complexes, formed in the sample solution, and Cu(II)-EDTA-BSA conjugate which has been immobilized onto ELISA plates (in ELISA) or polymethylmethacrylate beads (in KinExA) for a limited quantity of binding sites of the 8D66 antibody. In ELISA, color signals were generated by a peroxidase-labeled secondary antibody and 3,3',5,5'-tetramethylbenzidine substrate. In KinExA, a fluorescein isothiocyanate-labeled secondary antibody was used to generate KinExAgram (trend-line fluorescence responses vs. time). The conditions of both ELISA and KinExA were investigated, and the optimum procedures were established. Both ELISA and KinExA were validated, and all validation parameters were acceptable. Many different metal ions that are commonly encountered in drinking water did not interfere with the Cu(II) analysis by both ELISA and KinExA. Both assays were applied to the determination of Cu(II) in drinking water with satisfactory accuracy and precision. Both assays were compared favorably with inductively coupled plasma atomic emission spectroscopy in terms of their abilities to accurately and precisely determine Cu(II) in drinking water samples. A comparative evaluation of ELISA and KinExA revealed that KinExA had a higher sensitivity and better precision than ELISA, whereas both assays had comparable accuracy. Both ELISA and KinExA were superior to the existing atomic spectrometric methods for Cu(II) in terms of sensitivity, convenience, and analysis throughputs. The proposed ELISA and KinExA are anticipated to effectively contribute to assessing Cu(II) concentrations and control the exposure of humans to its potential toxicities.

Three Innovative Green and High-Throughput Microwell Spectrophotometric Methods for the Quantitation of Ceritinib, a Potent Drug for the Treatment of ALK-Positive Non-Small Cell Lung Cancer: An Application to the Analysis of Capsules and Drug Uniformity Testing.

Ceritinib (CER) is a potent drug that has been recently approved by the Food and Drug Administration for the treatment of patients with non-small cell lung cancer harboring the anaplastic lymphoma kinase mutation gene. The existing methods for the quality control of CER are very limited and suffer from limited analytical throughput and do not meet the requirements of the green analytical principles. This study presented the first-ever development and validation of three innovative green and high-throughput microwell spectrophotometric methods (MW-SPMs) for the quality control of CER in its dosage form (Zykadia® capsules). These MW-SPMs were based on the formation of colored N-vinylamino-substituted haloquinone derivatives of CER upon its reactions with each of chloranil, bromanil, and 2,3-dichloro-1,4-naphthoquinone in the presence of acetaldehyde. The optimized procedures of the MW-SPMs were established, and their analytical performances were validated according to the ICH. The linear range of the MW-SPMs was 5-150 µg/mL, with limits of quantitation of 5.3-7.6 µg/mL. The accuracy and precision of the MW-SPMs were proved, as the average recovery values were 99.9-101.0%, and the relative standard deviations did not exceed 1.8%. The three methods were applied to the determination of CER content in Zykadia® capsules and drug content uniformity testing. The greenness of the MW-SPMs was proved using three different metric tools. In addition, these methods encompassed the advantage of high-throughput analysis. In conclusion, the three methods are valuable tools for convenient and reliable application in the pharmaceutical quality control units for CER-containing capsules.

Development of Two Novel One-Step and Green Microwell Spectrophotometric Methods for High-Throughput Determination of Ceritinib, a Potent Drug for Treatment of Anaplastic Lymphoma Kinase-Positive Non-Small-Cell Lung Cancer.

Background and Objectives: Ceritinib (CER) is a potent drug of the third-generation tyrosine kinase inhibitor class. CER has been approved for the treatment of patients with non-small-cell lung cancer (NSCLC) harboring the anaplastic lymphoma kinase (ALK) mutation gene. In the literature, there is no green and high-throughput analytical method for the quantitation of CER in its dosage form (Zykadia® capsules). This study describes, for the first time, the development and validation of two novel one-step and green microwell spectrophotometric methods (MW-SPMs) for the high-throughput quantitation of CER in Zykadia® capsules. Materials and Methods: These two methods were based on an in microwell formation of colored derivatives upon the reaction of CER with two different benzoquinone reagents via two different mechanisms. These reagents were ortho-benzoquinone (OBQ) and 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ), and their reactions proceeded via condensation and charge transfer reactions, respectively. The reactions were carried out in 96-well transparent plates, and the absorbances of the colored reaction products were measured with an absorbance microplate reader at 540 and 460 nm for reactions with OBQ and DDQ, respectively. The optimum conditions of reactions were established, their molar ratios were determined, and reaction mechanisms were postulated. Under the refined optimum reaction conditions, procedures of MW-SPMs were established and validated according to the guidelines of the International Council on Harmonization. Results: The limits of quantitation were 6.5 and 10.2 µg/well for methods involving reactions with OBQ and DDQ, respectively. Both methods were applied with great reliability to the determination of CER content in Zykadia® capsules and their drug uniformity. Greenness of the MW-SPMs was evaluated using three different metric tools, and the results proved that the two methods fulfil the requirements of green analytical approaches. In addition, the simultaneous handling of a large number of samples with microvolumes in the proposed methods gave them the advantage of a high-throughput analysis. Conclusions: The two methods are valuable tools for rapid routine application in pharmaceutical quality control units for the quantitation of CER.

Development of two highly sensitive and selective sensor-assisted fluorescence immunoassays for trace determination of copper residues in food samples.

This study describes the development of two highly sensitive and selective sensor-assisted fluorescence immunoassays for the trace determination of copper ions, Cu(ii) residues, in food samples. These assays were the microwell-based fluoroimmuoassay (FIA) and the kinetic exclusion assay (KinExA). FIA and KinExA were assisted by a microplate reader and a KinExA™ 3200 immunosensor, respectively. Both FIA and KinExA were developed utilizing the same antibody, capturing reagent, and fluorescence signal-generating reagent. The antibody was a mouse monoclonal antibody, designated as 8D66, that specifically recognized the Cu(ii)-ethylenediaminetetraacetic acid complex (Cu(ii)-EDTA) but did not recognize Cu(ii)-free EDTA. The capturing reagent was Cu(ii)-EDTA covalently linked to bovine serum albumin protein (Cu(ii)-EDTA-BSA). The fluorescence-generating reagent was an anti-mouse IgG conjugated with fluorescein isothiocyanate (IgG-FITC). Both FIA and KinExA involved competitive binding reactions between Cu(ii)-EDTA complexes, formed in the sample solution, and Cu(ii)-EDTA-BSA conjugate which has been immobilized onto microwell fluorescence assay plates (in FIA) or polymethylmethacrylate beads (in KinExA) for a limited quantity of binding sites of 8D66 antibody. The conditions of both FIA and KinExA were investigated, and the optimum procedures were established. Both FIA and KinExA were validated, and all validation parameters were acceptable. Many different metal ions that are commonly encountered in food samples did not interfere with Cu(ii) analysis by both FIA and KinExA. Both assays were applied to the determination of Cu(ii) in food samples with satisfactory accuracy and precision. Both assays were compared favorably with inductively coupled plasma atomic emission spectroscopy. Comparative evaluation of FIA and KinExA revealed that KinExA had higher sensitivity and better precision than FIA, whereas, both assays had comparable accuracy. Both FIA and KinExA were superior to the existing atomic spectrometric methods for Cu(ii). The proposed FIA and KinExA are anticipated to effectively contribute to assessing Cu(ii) concentrations and controlling the exposure of humans to its potential toxicities.

Development and Comparative Evaluation of Two Different Label-Free and Sensitive Fluorescence Platforms for Analysis of Olaparib: A Recently FDA-Approved Drug for the Treatment of Ovarian and Breast Cancer.

Olaparib (OLA) is a PARP inhibitor drug which has been recently approved by the Food and Drug Administration (FDA) for the treatment of ovarian and breast cancer. A convenient analytical tool for the quantitation of OLA in its dosage form and plasma samples was urgently needed. This study describes, for the first time, the development of two different label-free and sensitive fluorescence-based platforms for the pharmaceutical and bioanalysis of OLA. These platforms were microwell-assisted with a fluorescence microplate reader (MW-FLR) and high-performance liquid chromatography with fluorescence detection (HPLC-FD). Both MW-FLR and HPLC-FD employed the native fluorescence of OLA as an analytical signal. The MW-FLR involved measuring the fluorescence signals in 96-well white-opaque plates. The HPLC-FD involved chromatographic separation of OLA and duvelisib (DUV), as an internal standard on a Nucleosil-CN HPLC column (250 mm length × 4.6 mm i.d., 5 µm particle diameter) with a mobile phase composed of acetonitrile: water (25:75, v/v) pumped at a flow rate of 1.7 mL/min. Elution of OLA and DUV was detected using a fluorescence detector. The optimal conditions of both MW-FLR and HPLC-FD were established, and they were validated according to the guidelines of the International Council for Harmonization for the validation of analytical procedures. The linear ranges of MW-FLR and HPLC-FD were 25-1000 and 5-200 ng/mL, respectively, with limits of detection of 15 and 1.7 ng/mL, respectively. The accuracy and precision of both platforms were confirmed as the recovery values were ≥98.2% and the relative standard deviations (RSD) were ≤2.89%. Both methodologies were satisfactorily applied to the quantitation of OLA in its commercial dosage form (Lynparza® tablets) and plasma samples with high accuracy and precision. The greenness of both MW-FLR and HPLC-FD was assessed using two different multiple parameter-based metric tools, and the results proved their greenness and adherence to the requirements of green analytical approaches. Both platforms have simple procedures and acceptable levels of analytical throughput. In conclusion, the proposed MW-FLR and HPLC-FD are valuable tools for routine use in quality control and clinical laboratories for the quantitation of OLA for the purposes of pharmaceutical quality control, pharmacokinetic studies, and bioequivalence testing.

Development of Novel Micellar-Enhanced High-Throughput Microwell Spectrofluorimetric Method for Quantification of Lorlatinib: Application to In Vitro Drug Release and Analysis of Urine Samples.

Lorlatinib (LOR) is a third-generation anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitor drug. The Food and Drug Administration (FDA) has granted an approval for the use of LOR as a first therapeutic intervention for individuals diagnosed with ALK-positive metastatic and advanced non-small-cell lung cancer (NSCLC). The present study outlines, for the first time, the development and validation of an innovative microwell-based spectrofluorimetric (MW-SFL) method for the quantification of LOR. The proposed method involved the enhancement of the weak native fluorescence of LOR by its micellization into the sodium lauryl sulfate (SLS) micelles. The procedures of the method were conducted in white opaque plates with 96 microwells, and the enhanced fluorescence signals were measured by a fluorescence plate reader at 405 nm after excitation at 310 nm. The measured relative fluorescence intensity (RFI) had a linear relationship with LOR concentrations in the range of 60-1600 ng mL-1. The limit of detection (LOD) and the limit of quantification (LOQ) were found to be 19 and 56 ng mL-1, respectively. The method's accuracy and precision were assessed using a recovery study; the recovery values ranged from 99.98% to 101.40%, accompanied by relative standard deviation (RSD) values of 0.42% to 1.59%. The proposed MW-SFL method combined the advantages of the intrinsically high sensitivity of the spectrofluorimetric measurement and the excellent throughput of the microwell-based approach. The results proved the method is effective in the determination of LOR in its pharmaceutical tablets, tablet dissolution testing, as well as in spiked urine with a high degree of precision and accuracy. The MW-SFL method is notable for its simple procedures and utilization of water as a solvent, as well as minimal quantities of sample solutions. These features align with its ecofriendly approach to green chemistry principles. These advantages gave the proposed MW-SFL method a high potential value for the determination of LOR in clinical and quality control laboratories.