Green and Versatile High Throughput Microwell Oxidation-Based Spectrophotometric Methods for Determination of Galidesivir in Capsules.

BACKGROUND: Galidesivir (GDV) is a promising new antiviral drug for the potent and safe 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 aim of this study was the development of versatile green and simple microwell spectrophotometric methods (MW-SPMs) for the determination of GDV in its bulk form and capsules. METHODS: Three MW-SPMs were developed involving the oxidation of GDV by ammonium metavanadate (AMV), chromium trioxide (CTO), and potassium iodate (PIO) in an acid medium. The reactions were carried out in 96-well plates at room temperature and the absorbances of chromogenic reaction products were measured by an absorbance microplate reader at 780, 595, and 475 nm for AMV, CTO, and PIO, respectively. Variables influencing the reactions were carefully investigated and optimized. RESULTS: Linear relations with excellent correlation coefficients (0.9991-0.9997) were found between the absorbances and GDV concentrations in a range of 25-500 µg/mL. The limits of detection were ≥8.3 µg/mL. The accuracy and precision of the three MW-SPMs were confirmed by recovery and replicate analysis, respectively. The recovery values were 98.6-101.2% and the relative standard deviations were ≤1.02%. The proposed MW-SPMs were successfully applied to the analysis of GDV in bulk drug and capsules with high accuracy and precisions. The greenness of MW-SPMs was confirmed by three comprehensive metric tools. CONCLUSIONS: The proposed MW-SPMs combined the inherent advantages of microwell-based analysis and the use of common laboratory reagents for the involved reaction. These advantages include high-throughput, readily automation, reduced samples/reagents volume, precise measurements, and versatility. The advantages of the use of common laboratory reagents include 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.

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 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.

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.

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.].

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 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.

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.

Tailoring two white chromatographic platforms for simultaneous estimation of ritonavir-boosted nirmatrelvir in their novel pills: degradation, validation, and environmental impact studies.

As the COVID-19 pandemic is not yet over, Pfizer has launched the novel pill Paxlovid® (Nirmatrelvir (NMV) co-packaged with ritonavir (RIT)) as an effective medication for hospitalized and non-hospitalized patients. Making pharmaceutical analysis greener and more sustainable has lately become the main direction of the research community. In this context, two fast, green, and stability-indicating chromatographic methods were designed for the neat quantitative determination of NMV and RIT in their bulk and dosage forms. Method I is deemed the first electro-driven attempt for the assay of Paxlovid®. Herein, the optimized conditions of the Micellar Electrokinetic Chromatographic (MEKC) method were 50 mM borate buffer at pH 9.2 with 25 mM sodium lauryl sulfate (SDS) being used as the background electrolyte (BGE) on a deactivated fused silica capillary (50 cm effective length × 50 µm id). Method II was an isocratic reversed-phase HPLC separation method using Zorbax-Eclipse C18 (4.6 × 250 mm, 5 µm particle size) column and 50 mM ammonium acetate buffer at pH 5 and acetonitrile as mobile phase constituents at a flow rate of 1 mL min-1. For the sake of simplicity and increasing sensitivity, a single wavelength of 210 nm was used for the two methods to assay both drugs. Linear correlations between peak areas and concentration were observed in the ranges of 10-200 µg mL-1 for NMV and 5-100 µg mL-1 RIT in both methods. The impact of versatile stress conditions such as hydrolysis, oxidation, and photolysis on the stability of NMV and RIT was studied. Fortunately, both methodologies were able to separate both drugs from their degradants. Thus, the stability indicating power of the methods was proved. The derived methods were statistically validated in agreement with the ICH guidelines. Furthermore, the environmental friendliness and sustainability of these methods were investigated and compared with the cited methods using the holistic multicriteria evaluation tools namely Hexagon, AGREE, and RGB12 metrics. Conclusively, the proposed methods offered reliable, feasible, economic, white, and stability-indicating alternatives to the cited chromatographic methods.

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.

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.

Durvalumab (DUR) is a human monoclonal antibody used for the immunotherapy of lung cancer. It is a novel immune-checkpoint inhibitor, which blocks the programmed death 1 (PD-1) and programmed death-ligand 1 (PD-L1) proteins and works to promote the normal immune responses that attack the tumour cells. To support the pharmacokinetic (PK) studies, therapeutic drug monitoring (TDM) and refining the safety profile of DUR, an efficient assay is required, preferably immunoassay. This study describes, for the first time, the development of a highly sensitive chemiluminescence immunoassay (CLIA) for the quantitation of DUR in plasma samples with enhanced chemiluminescence detection system. The CLIA protocol was conducted in 96-microwell plates and involved the non-competitive binding reaction of DUR to its specific antigen (PD-L1 protein). The immune complex of DUR with PD-L1 formed onto the inner surface of the assay plate wells was quantified by a chemiluminescence (CL)-producing horseradish peroxidase (HRP) reaction. The reaction employed 4-(1,2,4-triazol-1-yl)phenol (TRP) as an efficient enhancer of the HRP-luminol-hydrogen peroxide (H2O2) CL reaction. The optimum protocol of the proposed CLIA was established, and its validation parameters were assessed as per the guidelines for the validation of immunoassays for bioanalysis. The working dynamic range of the assay was 10-800 pg mL-1 with a limit of detection (LOD) of 10.3 pg mL-1. The assay enables the accurate and precise quantitation of DUR in human plasma at a concentration as low as 30.8 pg mL-1. The CLIA protocol is simple and convenient; an analyst can analyse several hundreds of samples per working day. This high throughput property enables the processing of many samples in clinical settings. The proposed CLIA has a significant benefit in the quantitation of DUR in clinical settings for assessment of its PK, TDM and refining the safety profile.

Development and validation of a UPLC-MS/MS method for simultaneous detection of doxorubicin and sorafenib in plasma: Application to pharmacokinetic studies in rats.

An ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was developed for the simultaneous quantitation of doxorubicin (DOX) and sorafenib (SOR) in rat plasma. Chromatographic separation was performed using a reversed-phase column C18 (1.7 µm, 1.0 × 100 mm Acquity UPLC BEH™). The gradient mobile phase system consisted of water containing 0.1% acetic acid (mobile phase A) and methanol (mobile phase B) with a flow rate of 0.40 mL/min over 8 min. Erlotinib (ERL) was used as an internal standard (IS). The quantitation of conversion of [M + H]+, which was the protonated precursor ion, to the corresponding product ions was performed using multiple reaction monitoring (MRM) with a mass-to-charge ratio (m/z) of 544 > 397.005 for DOX, 465.05 > 252.03 for SOR, and 394 > 278 for the IS. Different parameters were used to validate the method including accuracy, precision, linearity, and stability. The developed UPLC-MS/MS method was linear over the concentration ranges of 9-2000 ng/mL and 7-2000 ng/mL with LLOQ of 9 and 7 ng/mL for DOX and SOR, respectively. The intra-day and inter-day accuracy, expressed as % relative standard deviation (RSD%), was below 10% for both DOX and SOR in all QC samples that have drug concentrations above the LLOQ. The intra-day and inter-day precision, expressed as percent relative error (Er %), was within the limit of 15.0% for all concentrations above LLOQ. Four groups of Wistar rats (250-280 g) were used to conduct the pharmacokinetic study. Group I received a single intraperitoneal (IP) injection of DOX (5 mg/kg); Group II received a single oral dose of SOR (40 mg/kg), Group III received a combination of both drugs; and Group IV received sterile water for injection IP and 0.9% w/v sodium chloride solution orally to serve as a control. Non-compartmental analysis was used to calculate the different pharmacokinetic parameters. Data revealed that coadministration of DOX and SOR altered some of the pharmacokinetic parameters of both agents and resulted in an increase in the Cmax and AUC and reduction in the apparent clearance (CL/F). In conclusion, our newly developed method is sensitive, specific, and can reliably be used to simultaneously determine DOX and SOR concentrations in rat plasma. Moreover, the results of the pharmacokinetic study suggest that coadministration of DOX and SOR might cause an increase in exposure of both drugs.

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.

Development and validation of an UPLC-ESI-MS/MS method for quantification of duvelisib in plasma: application to pharmacokinetic study in rats.

Duvelisib (DUV) is a new oral phosphoinositide-3-kinase (PI3K)-δ and PI3K-γ inhibitor. It is used for the treatment of relapsed or refractory chronic lymphocytic leukemia (CLL) and small lymphocytic lymphoma (SLL). This study describes the development and validation of a new highly sensitive and efficient UPLC-ESI-MS/MS method for quantitation of DUV in plasma samples and its application to the pharmacokinetic study of DUV in rats. The method employed a very simple step for plasma sample pretreatment via precipitation of protein using methanol. DUV and ceritinib (CRB) as an internal standard (IS) were separated on a porous Hypersil BDS-C18 column (125 mm × 2 mm, 3 µm) using a mobile phase consisting of ammonium formate (10 mM, pH 4.2):acetonitrile (42 : 58, v/v), pumped isocratically at a flow rate of 0.3 mL min-1. DUV and CRB were eluted at 0.58 and 1.10 min, respectively. The mass spectrometric analysis was performed using an ESI in positive mode with multiple reaction monitoring (MRM). The technique was validated in accordance with the standards for validating bioanalytical methods established by the International Conference on Harmonization (ICH). The method's linear range was 5-500 ng mL-1, and its correlation coefficient was satisfactory as it is almost unity (0.9999). The limit of quantitation (LOQ) was 5 ng mL-1, while the limit of detection (LOD) was 1.7 ng mL-1. The recovery of the spiking DUV was between 94.95 and 102.21%, and the relative standard deviation (RSD) was less than 2.70%, confirming the method's accuracy and precision. The specificity/carryover of the method was proved. The robustness and ruggedness of the method was proved as the recovery values were 97.6-101.96% (±01.17-2.20%) and 98.74-102.00 (±1.18-4.02%) for robustness and ruggedness, respectively. The stability of DUV under the different analytical conditions were documented as the recovery values were in the range of 95.89-103.28% and the RSD values did not exceed 7.36%. The method was efficiently used to analyze DUV in human plasma samples that had been spiked with DUV and to conduct pharmacokinetic investigations of DUV in rats after giving them a single oral dosage of 25 mg kg-1 of the drug. The methodology is distinguished by excellent sensitivity, accuracy, and ease of sample pretreatment. Furthermore, it is efficient and has a short run time, which makes it high throughput and accordingly enables faster processing of many samples in clinical laboratories.

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.

Aspartames Alter Pharmacokinetics Parameters of Erlotinib and Gefitinib and Elevate Liver Enzymes in Wistar Rats.

Background: Erlotinib (ERL) and gefitinib (GEF) are extensively metabolized by CYP450 enzymes. Aspartame (ASP), an artificial sweetener, induces CYP2E1 and CYP3A2 enzymes in the brain and could increase liver enzymes. In this work, the influence of ASP on the pharmacokinetics (PK) of ERL and GEF in Wistar rats was evaluated. Methods: The PKs of ERL and GEF were evaluated after receiving 175 mg/kg or 1000 mg/kg of ASP for four weeks using UPLC-MS/MS. Levels of liver enzymes after four weeks of ASP consumption were also evaluated. Results: ASP 175 mg/kg was able to significantly alter levels of Cmax (36% increase for ERL, 38% decrease for GEF), AUC0-72 (205% increase for ERL, 41% increase for GEF), and AUC0-∞ (112% increase for ERL, 14% increase for GEF). Moreover, ASP 175 mg/kg decreased the apparent oral clearance ERL and GEF by 58% and 13%, respectively. ASP 1000 mg/kg increased Cmax of ERL by 159% and decreased GEF's Cmax by and 73%. Both AUC0-72 and AUC0-∞ were increased by ASP 1000 for ERL and decreased for GEF. CL/F decreased by 64% for ERL and increased by 38.8% for GEF. Moreover, data indicated that ASP significantly increased levels of liver enzymes within two weeks of administration. Conclusions: Although ASP 175 and 1000 mg/kg alter ERL and GEF PKs parameters, ASP 1000 mg/kg has the highest impact on most parameters. ASP 1000 mg/kg also can significantly increase activities of liver enzymes indicating the possibility of inducing liver injury. Therefore, it might be of clinical importance to avoid the administration of aspartame containing products while on ERL or GEF therapy.

Qualitative immunoassay for the determination of tetracycline antibiotic residues in milk samples followed by a quantitative improved HPLC-DAD method.

Environmental contaminant is one of several problems harming people and wildlife. An example of current emerging contaminants are antibiotics residues that can present in water and food. Although antibiotics are intended to treat or prevent human and animal infections, antibiotics have also been used as animal food supplements for their ability to promote growth and feed efficiency. This overuse of antibacterial has resulted in the accumulation of antibiotics residues in food products which are eventually consumed by human. The continuous unnecessary exposure of human to antibiotics through the direct animals meet or milk, or indirectly through plants or soil can increase the chance of the emergence of multi drug resistance bacteria and consequently adversely affecting human health. New regulations have been imposed regarding antibiotics utilization. Due to the scarce of data regarding antibiotics residue conditions in different types of food intended for human consumption in Saudi Arabia, this study proposed an optimized chromatographic method (HPLC-DAD) followed by an immunoassay approach for specifically detecting tetracyclines antibiotics in animal milk samples. The method was carried out using an RP-C18 column with a mobile phase consisting of 0.01 M KH2PO4: acetonitrile:methanol (70:20:10, v/v/v) adjusted to pH 4. Improvements were observed in the method in terms of resolution and sensitivity. The protein precipitation method used for extraction demonstrated high percent recoveries of 85-101%. The method was validated according to the guidelines of the International Conference for Harmonization (ICH). It is evidently clear from these findings that the presence of tetracycline and oxytetracycline antibiotics residues in milk products from the Saudi market are below the maximum residual limits (MRLs).

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.