International Council On Harmonization Research Articles

Novel spectrofluorometric approach for assessing vilazodone by blocking photoinduced electron transfer: analytical performance, and greenness-blueness evaluation.

In this paper, vilazodone (VLD), a serotonin modulator prescribed for major depressive disorder, was investigated using a rapid, highly sensitive, and eco-friendly spectrofluorometric approach. The native fluorescence of VLD, originating from its indole moiety, exhibited an emission peak at 486 nm upon excitation at 241 nm. However, the presence of a piperazinyl nitrogen atom in the VLD structure, acting as an electron donor, significantly diminished the fluorescence intensity through photoinduced electron transfer (PET) to the indole ring. However, by protonating this nitrogen atom using 0.02 M Teorell-Stenhagen buffer (pH 3.5), inhibition of the PET process effectively blocked electron transfer, restoring the fluorescent properties of the drug. Further, an enhancement in the fluorescence was achieved by employing methanol as the solvent, resulting in a 1.5-fold increase. The combined use of PET blockage and methanol enabled the detection of VLD at levels as low as 0.78 ng mL-1. Calibration analysis demonstrated linearity within the range 5-400 ng mL-1, exhibiting a correlation coefficient of 0.9998 and a limit of quantification of 2.37 ng mL-1. The method obeyed the requirements of International Council on Harmonization (ICH). The proposed approach was applied for the accurate measurement of VLD in pharmaceutical tablets, content uniformity testing based on USP requirements, and determining VLD concentration in spiked human plasma. Moreover, the environmental impact, in addition to practical effectiveness, of the proposed approach was evaluated using different metrics.

Exciting Advances in Sustainable Spectrophotometric Micro-Quantitation of an Innovative Painkiller "Tramadol and Celecoxib" Mixture in the Presence of a Toxic Impurity, Promoting Greenness and Whiteness Studies.

Tramadol (TRM) and celecoxib (CLX) form a novel mixture that helps relieve acute pain when other painkillers have no action. It is also reported that these drugs, TRM and CLX, are used to control COVID-19 symptoms. The current work highlights three important pillars of modern pharmaceutical analysis, which are as follows; impurity profiling, greenness/whiteness studies and simplicity accompanied by sensitivity. Since 4-methyl acetophenone inhibits the human carbonyl reductase enzyme (type I) and since this compound may pose a health risk, it is crucial to regulate its concentration in all dosage forms of CLX. Two simple and green spectrophotometric methods were developed, namely third derivative (D3) and Fourier self- deconvulation (FSD), for resolving severely overlapped spectra of TRM and CLX in the presence of 4-methyl acetophenone (4-MAP) as a process-related impurity in their novel tablet combination. The two approaches showed acceptable linearity with an excellent correlation coefficient. In both methods, TRM was measured when CLX and 4-methyl acetophenone were zero-crossing. The same procedure was applied for measuring CLX and its process-related impurity 4-MAP. The methodologies developed were thoroughly validated in compliance with ICH (International Council on Harmonisation) guidelines. Student t- and F-tests revealed no statistically significant variation among the current methods and the reported method. No spectrophotometric methods have been published previously for the simultaneous analysis of TRM and CLX along with 4-MAP. As a result, the newly developed spectrophotometric approaches have great relevance and originality in the field of pharmaceutical analysis.

Green electrochemical approach for simultaneous quantification of Dapoxetine hydrochloride and Tadalafil integrating design of experiments and functional principal component analysis

To ensure a successful therapy for impotence and erectile ejaculation issues, tablets containing Tadalafil (TD) and Dapoxetine hydrochloride (DP) must be verified for their ingredients. This study presents a green voltammetric technique for the simultaneous determination of DP and TD. The technique is based on the unique electrochemical properties of DP and TD, and it is simple, rapid, and cost-effective. To accomplish this goal, design of experiment (DoE) and functional principle component analysis (FPCA) were applied due to the difficulty of the optimization process. The International Council on Harmonization (ICH) guidelines were used to validate the proposed method, which displayed precision for DP and TD within the concentration range of 6.7–67.0 μg mL−1. The percentage recovery values were (96.83–100.67%) and (98.27–100.90%) for DP and TD respectively. To assess the ecological impact, four greenness evaluation tools were employed, including Eco-Scale Assessment (ESA), Environmental Methods Index (NEMI), Analytical Greenness Calculator (AGREE), and Complex Green Analytical Procedure Index (Complex GAPI). The proposed method was employed on the dosage form and compared statistically to a reported HPLC method, revealing no significant differences at a 95% confidence level. The proposed method is a promising green analytical tool for the simultaneous determination of DP and TD in pharmaceutical dosage forms with good precision and accuracy. Additionally, the method is environmentally friendly, as evidenced by the greenness evaluation results. In addition, this study aims to demonstrate how the latest nonparametric technique for analyzing functional data (curves) can produce reliable results in experimental settings.

ICH Reflection paper on proposed international harmonization of real-world evidence terminology and convergence of general principles regarding planning and reporting of studies using real-world data, with a focus on effectiveness of drugs

Real-world data and real-world evidence play an increasingly important role in clinical research and healthcare decisionmaking. To use data and generate robust evidence effectively, they must be clearly defined and structured, semantically compatible and harmonized among stakeholders. On June 30, 2023, the International Council on Harmonization (ICH) presented for discussion a reflection paper on “International Harmonisation of Real-World Evidence Terminology and Convergence of General Principles Regarding Planning and Reporting of Studies Using Real-World Data, with a Focus on Effectiveness of Medicines”, which outlines their approach to achieve international integration in this area. This article provides an overview of the key aspects of the strategic plan proposed by the ICH.

Stability Indicating RP-HPLC Method Development for the Estimation of Molnupiravir in Capsule Dosage Form

In recent years much more attention has gained by antiviral drugs because of severe acute respiratory syndrome coronavirus-2 (SARS-CoV) infection. Molnupiravir is one of the favorable drugs for SARS-CoV-2 treatment. The present study aimed to develop and validate a simple, rapid, stable, selective, sensitive, accurate, robust, and economical RP-HPLC method for determining molnupiravir in its capsule dosage form. The chromatographic separation was achieved on inert sustain C18 column (250 mm x 4.6 mm x 5 μ) at 40°. Isocratic elution was performed with 25 mM KH2PO4 buffer (pH 3.0) and methanol (60:40 v/v) as mobile phase at flow rate of 1.0 mL/min with 50 μL injection volume. The detection was conceded out at 242 nm. The developed RP-HPLC method yielded a suitable retention time of 4.2 minutes for molnupiravir. The developed method was validated according to the International Council on Harmonization (ICH) guidelines and established to be linear in the range of 10 to 70 μg/mL with a linear regression coefficient of 0.9993. The %RSD for the method precision and system precision was found to be less than 2.0%. The %assay of the formulation is 101.52%. The LoD and LoQ were found to be 0.25 and 0.75 μg/mL, respectively. The specificity of the method established using forced degradation studies in which the drug is subjected to the stressed conditions such as thermal, acidic, basic oxidative and photolytic degradation. The developed and validated RP-HPLC method for molnupiravir takes short time and can be used for routine quality analysis of marketed Molnupiravir in capsule dosage form.

Quality by design-steered development and validation of analytical and bioanalytical methods for raloxifene: Application of Monte Carlo simulations and variance inflation factor.

A sensitive, rapid, reproducible, and economical HPLC method is reported for the quantification of raloxifene hydrochloride employing Quality by Design (QbD) principles. Factor screening studies, employing Taguchi design, indicated buffer volume percentage and isocratic flow rate as the critical method parameters (CMPs), which significantly influence the chosen critical analytical attributes, that is, tailing factor and theoretical plate number. Method conditions were subsequently optimized using face-centered cubic design with magnitude of variance inflation factor for assessing multicollinearity among CMPs. Method operable design region (MODR) was earmarked and liquid chromatographic separation optimized using 0.05 M citrate buffer, acetonitrile, and methanol (57:40:3 v/v/v) as ggmobile phase at 0.9 mL min-1 flow rate, λmax of 280 nm, and column temperature of 40°C. Validation of the developed analytical method was accomplished as per International Council on Harmonization (ICH) guidelines confirming high levels of linearity, precision, accuracy, robustness, and sensitivity. Application of Monte Carlo simulations enabled the attainment of best plausible chromatographic resolution and corroboration of the demarcated MODR. Establishment and validation of the bioanalytical method using rat plasma samples, along with forced degradation and stability studies, corroborated the aptness of developed HPLC methods for drug quantification in the biological fluids, as well as in bulk and marketed dosage forms.

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

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

Quantification of Luteolin, Apigenin and Chrysoeriol in Tecoma stans by RP-HPLC Method.

Tecoma stans (Fam. Bignoniaceae) is also popularly known as yellow bells and yellow trumpet bush in vernacular terminology. Limited and variable data are available from the literature regarding the quantification of luteolin, apigenin and chrysoeriol, which are considered as the most active pharmacological active constituents. High-performance liquid chromatography-photodiode array detection has been developed for the determination of the bioactive flavonoids, luteolin, apigenin and chrysoeriol, from the methanolic extract of the leaves of T. stans. A column packed with a pentafluorophenyl-based stationary phase was used for the separation of the analytes under gradient elution. The detection wavelength was 345nm. The validation of the method as per the International Council on Harmonisation (ICH) guidelines (ICH 2005) for linearity, accuracy and precision was investigated and found within limits specified by the ICH guidelines. The method was linear over with a good regression coefficient of more than 0.99. The limit of detection of the method was 0.68, 2.97 and 1.76μg/mL for luteolin, apigenin and chrysoeriol, respectively. In conclusion, a reliable and reproducible method was devised that can be used for the estimation of the said components from T. stans.

A Modified Folin-Ciocalteu Assay for the Determination of Total Phenolics Content in Honey

The Folin-Ciocalteu assay is a widely used method for measuring the total phenolic content (TPC) in honey, but it can be affected by the presence of reducing sugars in honey, which can lead to interference and an over-estimation of its TPC. To optimize the Folin-Ciocalteu assay for honey analysis, the effect of pH on the assay was investigated. A number of pH scenarios were tested using different concentrations of Na2CO3 (0.00%, 0.75%, 0.94%, and 7.50%) in order to minimize reducing sugar interference and maximize the reaction of phenolics in the assay. The modified TPC method was then validated in accordance with current International Council on Harmonisation (ICH) guidelines. The findings of this study demonstrate that the traditional Folin-Ciocalteu assay (using 7.50% aqueous Na2CO3 solution, pH 10.8) leads to a significant overestimation of the TPC of honey due to the interference of reducing sugars. However, a pH of 7.9, achieved by using a 0.75% aqueous Na2CO3 solution, provides suitable conditions to account for most of the phenolic compounds without interference from reducing sugars. This finding was further confirmed by testing various sugar solutions and artificial honey which yielded TPC values below the established limit of detection and quantification of the assay. However, a slight increase in pH, even by a moderate deviation (pH 8.9), leads to significant discrepancies in absorbance readings, indicating that pH control is crucial for the accurate analysis of TPC in honey.

In Vitro Permeation Studies on Carvedilol Containing Dissolving Microarray Patches Quantified Using a Rapid and Simple HPLC-UV Analytical Method

Analytical method validation is a vital element of drug formulation and delivery studies. Here, high-performance liquid chromatography in conjunction with UV detection (HPLC-UV) has been used to produce a straightforward, quick, yet sensitive analytical approach to quantify carvedilol (CAR). A C18 column was used to isolate the analyte from the mixture by isocratic elution with a mobile phase comprising a mixture of 0.1% v/v trifluoroacetic acid in water and acetonitrile in a ratio of 65:35 v/v at a flow rate of 0.6 mL min−1. Linearity was observed for CAR concentrations within the range of 1.5–50 μg mL−1 (R2 = 0.999) in phosphate buffer saline and within the range of 0.2–6.2 μg mL−1 (R2 = 0.9999) in methanol. The International Council on Harmonization (ICH) requirements were followed throughout the validation of the isocratic approach, rendering it specific, accurate, and precise. Moreover, robustness tests indicated that the method remained selective and specific despite small deliberate changes to environmental and operational factors. An efficient extraction procedure was also developed to extract and quantify CAR from excised neonatal porcine skin, resulting in recovery rates ranging from 95 to 97%. The methods reported here have been successfully utilised to evaluate CAR permeation, both transdermally and intradermally following application of a dissolving microarray patch (MAP) to excised neonatal porcine skin.Graphical

Spectrofluorimetric determination of selected genotoxic impurities in pharmaceutical raw materials and final products

A green spectrofluorimetric method was introduced for the determination of selected genotoxic impurities; 2-aminopyridine and 3-aminopyridine in different pharmaceutical raw materials and dosage forms. The method relied on the native fluorescence of these impurities in acidic medium. The experimental conditions were carefully studied and optimized, and the method was validated according to International Council on Harmonisation (ICH) guidelines. The linear range for both analytes was 2.50–100 ng/mL with good determination coefficients of 0.9995 and 0.9992 and detection limits of 0.62 ng/mL and 0.74 ng/mL for 2-aminopyridine and 3-aminopyridine, respectively. The method was successfully applied for determination of 2-aminopyridine and 3-aminopyridine in four active pharmaceutical ingredients and nine dosage forms with satisfactory percentage recoveries and without interference from co-formulated excipients. Analytical performance of the proposed method was comparable to that of the reported methods; hence, the proposed method can be used as a simple and low-cost alternative in quality control laboratories.

Development and Validation of an Analytical Method for the Simultaneous Estimation of Azadirachtin and Camphor in a New Herbal Formulation

Background: High-performance liquid chromatography (HPLC) is an analytical tool for determining the quality of a drug product. Separating, identifying, and quantifying the various drugs should be able to HPLC methods. To quantify azadirachtin and camphor, a novel HPLC technique was created that is sensitive and selective. Objective: The analysis of azadirachtin and camphor has been designed and verified using a simple, efficient, selective, and precise HPLC technique. Material and Method: With the help of an HPLC C18 column (4.6 x 250 mm, 5 m), a mobile phase made up of buffer (pH adjusted to 3.0 with orthophosphoric acid) and acetonitrile in the ratio 15:85, flow rate of 1.0 mL/min, and column temperature kept at 35°C, good chromatographic separation was achieved in the current study. The resulting effluents were seen at 294 nm using a UV-visible detector. Result and Discussion: Sharp peaks for azadirachtin and camphor were found at retention times of 3.5 and 4.8 minutes, respectively, during the detection process at 294 nm. Over the concentration ranges of 0.1–0.9 g/mL for azadirachtin and 20–180 g/mL for camphor, the calibration curve was linear. According to International Council on Harmonisation (ICH) guidelines, the method was verified for accuracy, precision, repeatability, specificity, robustness, and detection and quantification limits. Conclusion: The proposed method has specificity, accuracy, and sensitivity, with an excellent linear.

Simultaneous Estimation of Terbutaline sulphate, Ambroxol hydrochloride, and Guaifenesin in Combined Dosage Form by HPTLC Method

A simple, step by step, cost-effective, accurate, sensitive, and selective validated High Performance Thin Layer Chromatographic method for simultaneous determination of Terbutaline sulphate, Ambroxol hydrochloride, and Guaifenesin in syrup formulation has been developed and validated. HPTLC separation was achieved on Merck aluminum HPTLC plates precoated with silica gel 60 F254. The solvent system comprised of Chloroform: Methanol: Ethyl acetate: Acetic acid: Formic acid (7.0:1.4: 0.8:1.0:0.5 v/v). Densitometric detection wavelength at 200 nm was used in reflectance-absorbance mode. The retention factors were found to be 0.32±0.02, 0.55± 0.02, and 0.72±0.02, for Terbutaline sulphate, Ambroxol hydrochloride, and Guaifenesin respectively. Results were found to be linear over a range of 200 - 700ng band-1, 1000 - 7000ng band-1, and 400-1200ng band-1 for Terbutaline sulphate, Ambroxol hydrochloride, and Guaifenesin, respectively. The percent assay was found to be 98.47%, 98.47%, and 99.23%, for Terbutaline sulphate, Ambroxol hydrochloride, and Guaifenesin, respectively in marketed formulation. The developed densitometric method was validated by following the International Council on Harmonisation (ICH) guideline. The developed and validated chromatographic method can be applied for routine quality control of Terbutaline sulphate, Ambroxol hydrochloride, and Guaifenesin in the combined syrup dosage form used in study.

A Qbd Driven Approach for Development and Validation of an RP-UHPLC Method for Simultaneous Estimation of Ofloxacin and Fluconazole: An Application to Pharmaceutical Analysis

A simple reverse-phase UHPLC (RP-UHPLC) analytical method was developed and validated according to International Council on Harmonization (ICH) guidelines for simultaneous estimation of ofloxacin (OFX) and fluconazole (FCZ) using a Response surface aided central composite design (CCD, Design-Expert®, Version 11.1.2.0). The method was validated for linearity, selectivity, sensitivity, accuracy, precision, limits of detection and quantification, solution stability and robustness. OFX and FCZ separation were achieved using Agilent 1290 Infinity II UHPLC system encompassed with Waters Atlantis® T3 (150 × 4.6 mm2 i.d., 3 µm particle size) column, water containing 0.1% v/v formic acid and methanol as isocratic mobile phase. OFX and FCZ eluted at 4.1 and 10.5 min with no interference of the excipients used in formulation at λmax 260 nm. The developed method was envisaged to determine total drug content, in vitro drug release study and ex-vivo corneal permeation study of conventional as well as novel drug delivery formulations. The proposed method is simple, cost-effective, dependable and reproducible and could be used for the regular analysis of OFX and FCZ in bulk and quality controls in the research and pharmaceutical industries.

Determination of Oxaliplatin and Curcumin in Combination via Micellar HPLC and Its Method Validation.

A micellar-HPLC method was developed for the determination of oxaliplatin (OHP) and curcumin (CUR) employing a C18 column [4.6 × 250 mm, particle size (dp) = 5 µm] and diode array detector. A rapid, cost-effective, environmentally friendly, time-efficient, easy-to-handle, and safe method was developed. The conditions were optimized for the estimation of OHP and CUR: 0.15 M sodium dodecyl sulfate (SDS) in 6% (v/v) pentanol buffered to pH 5.0 with a flow rate of 1.0 mL/min, injection volume of 20 µL, and detection at 325 nm. Different analytical parameters, including linearity, accuracy, precision, robustness, specificity, LOD, and LOQ, were determined in compliance with the International Council on Harmonisation (ICH) guidelines. The LOD (S/N = 3) of OHP was 0.004 µg/mL and for CUR it was 0.005 µg/mL. The calibration curves for OHP and CUR were linear over the range 0.015-10 µg/mL (determination coefficient r2 = 0.9999) and 0.015-10 µg/mL (r2 = 0.9994), respectively. The drugs were eluted in <12 min and the developed method was applicable for analyzing multiple samples per day. Moreover, it was determined to be robust and was used to quantify OHP and CUR in mice serum/blood. The method could pave the way for quantitative analysis of these drugs during the development of a pharmaceutical preparation for the treatment of colorectal cancer. A simple, cost-effective, eco-friendly HPLC method was developed to simultaneously estimate oxaliplatin and curcumin. The developed method was validated as per the ICH guidelines.

FORMULATION AND IN VITRO EVALUATION OF ETHANOLIC EXTRACT OF POLYHERBAL FACE CREAM

Objective: The aim of the present research work was to formulate and evaluate polyherbal face cream using ethanolic extracts of Aloe Vera gel, Neem, Turmeric and Mint.&#x0D; Methods: Extraction was done by the cold maceration technique. The cream was prepared by using the slab technique for homogeneous mixing of all the excipients and the herbal extracts. Three formulations of Oil in Water (O/W) type herbal cream were developed, namely F1, F2, and F3.&#x0D; Results: All the formulations were evaluated for various physical characteristics viz. pH 6.1-6.3, Spreadability 5-7.5gcm/sec, Viscosity 591-638 Cps, Extrudibility 2564-5102 g/cm2, good appearance and no phase separation was observed. Based on the results, F1 was found to be an optimized formulation with a smooth texture, good spreadability, extrudability and adequate viscosity. Stability studies as per ICH (International Council on Harmonisation) guidelines, revealed that the optimized formulation was stable at 25 °C±1 °C and 6% RH.&#x0D; Conclusion: Based on the results and discussion, the prepared formulations were found to be yellow in colour with a pleasant odour and smooth texture. All the formulations had almost constant pH, homogenous, emollient and non-greasy. As single plant extract possibly cannot increase the extent of efficiency of medicinal and cosmetic property compared to polyherbal plant extract. Considering that, we mixed polyherbal extracts to improve the cosmetic properties of the prepared polyherbal face cream. Hence it can be concluded that the cream was stable and can be safely used topically.

A New and Sensitive HPLC-UV Method for Rapid and Simultaneous Quantification of Curcumin and D-Panthenol: Application to In Vitro Release Studies of Wound Dressings.

Curcumin (CUR) and D-panthenol (DPA) have been widely investigated for wound-healing treatment. In order to analyse these two compounds from a dosage form, such as polymer-based wound dressings or creams, an analytical method that allows the quantification of both drugs simultaneously should be developed. Here, we report for the first time a validated high-performance liquid chromatographic (HPLC) method coupled with UV detection to quantify CUR and DPA based on the standards set by the International Council on Harmonization (ICH) guidelines. The separation of the analytes was performed using a C18 column that utilised a mobile phase consisting of 0.001% v/v phosphoric acid and methanol using a gradient method with a run time of 15 min. The method is linear for drug concentrations within the range of 0.39–12.5 μg mL−1 (R2 = 0.9999) for CUR and 0.39–25 μg mL−1 for DPA (R2 = 1). The validated method was found to be precise and accurate. Moreover, the CUR and DPA solution was found to be stable under specific storage conditions. We, therefore, suggest that the HPLC-UV method developed in this study may be very useful in screening formulations for CUR and DPA within a preclinical setting through in vitro release studies.

Nitrites as precursors of N-nitrosation in pharmaceutical samples – A trace level analysis

The ultra-performance liquid chromatography (UPLC) method, which involves pre-column derivatization of nitrite with 2,3-diaminonaphthalene (DAN) to form 2,3-naphthotriazole (NAT), offers the advantages of easy sample preparation, simple derivatization, stable derivatives, rapid analysis, high sensitivity and specificity and lack of interferences for determining nitrite in pharmaceutical samples. Determination of NAT was performed on a an Acquity UPLC HSS T3 column using a gradient elution of 0.1% formic acid with acetonitrile at flow rate of 0.4 mL/min and temperature at 45 °C. The single-quadrupole mass detector was operated in the positive ion mode. Quadrupole mass analyser was employed in selected ion monitoring mode using a target ion at m/z = 170 as [M+H]+. The UPLC-MS method was validated as per International Council on Harmonization (ICH) guidelines in terms of linearity, limit of detection, limit of quantification, selectivity, accuracy, precision, intermediate precision and stability. The UPLC-MS method was demonstrated to be applicable for the determination of nitrite in various pharmaceutical samples. The proposed UPLC-MS method was used to study the effect of nitrite content in pharmaceutical products on the formation of N-nitrosamines. The high importance of nitrites in relation to the N-nitrosation reaction was discussed. As deduced from theory and justified by the presented results, reducing the nitrite concentration could definitely solve the N-nitrosamine contamination. Nitrites, unlike secondary and tertiary amines, are universal precursors to any N-nitrosamine, so this solution is easily transferable to any relevant pharmaceutical product.

Method Development and Validation of a Specific Stability Indicating RP-HPLC Method for Molnupiravir API

A simple, accurate, isocratic stability indicating RP‐HPLC method was developed for the determination of Molnupiravir in bulk drug. This RP-HPLC was achieved on “Waters 2695 using an Agilent Zorbax Eclipse C18 (250 mm × 4.6 mm × 5 µm)” column with the mobile phase consisting of 30 mM, ammonium phosphate monobasic and Methanol in the ratio of 47:53 %v/v. The stress testing of Molnupiravir was carried out under acidic, alkaline, oxidative, thermal, and photolytic conditions and formed degradation products were well resolved from Molnupiravir API (active pharmaceutical ingredient). The suggested approach was validated according to ICH (International Council on Harmonisation) principles, and all of the validation parameters' findings were within acceptable limits. The method was proven to be appropriate for quality control of Molnupiravir in bulk and pharmaceutical dose forms, as well as stability testing.

Estimating the treatment effect for adherers using multiple imputation

Randomized controlled trials are considered the gold standard to evaluate the treatment effect (estimand) for efficacy and safety. According to the recent International Council on Harmonization (ICH)-E9 addendum (R1), intercurrent events (ICEs) need to be considered when defining an estimand, and principal stratum is one of the five strategies to handle ICEs. Qu et al. (2020, Statistics in Biopharmaceutical Research 12:1-18) proposed estimators for the adherer average causal effect (AdACE) for estimating the treatment difference for those who adhere to one or both treatments based on the causal-inference framework, and demonstrated the consistency of those estimators; however, this method requires complex custom programming related to high-dimensional numeric integrations. In this article, we implemented the AdACE estimators using multiple imputation (MI) and constructed confidence intervals (CIs) through bootstrapping. A simulation study showed that the MI-based estimators provided consistent estimators with the nominal coverage probabilities of CIs for the treatment difference for the adherent populations of interest. As an illustrative example, the new method was applied to data from a real clinical trial comparing two types of basal insulin for patients with type 1 diabetes.