Background: Counterfeit medicines are an increasing scourge that are difficult to identify and they have become industrialized and widespread through highly organized illegal channels. Objective: This research aims to develop a robust method to determine four phosphodiesterase type-5 inhibitors in counterfeit drugs based on ultra-performance liquid chromatography. Method: Experimental design methodology (DOE) and design space (DS) recommended by ICH Q8 were used side-by-side in the development phase to define the optimal parameters as well as the robustness of the chromatographic method. Moreover, both the uncertainty and risk profile derived from the β-content and γ-confidence tolerance interval were investigated during the validation phase to examine the performance of this method. Results: Successful chromatographic results, in a high resolution between the four active ingredients and an optimal analysis time of less than 1.6 min, were achieved at the end of the optimization phase. In addition, validation results show a low risk of future measurements outside acceptance limits set at 5%. Conclusions: Our procedure was successfully applied in the routine phase to identify 23 illicit formulations of an erectile dysfunction drug. Highlights: An efficient method for the characterization of 4 authorized phosphodiesterase in less than 1.6 min was established. A DS approach was applied to test the performance of this analytical method during analytical development. A risk profile was then carried out to approve the validity of the analytical method through the uncertainty profile approach.

Background: Hypertension is a critical health problem; it is a prevalent risk factor for cardiovascular disease. Many treatments to combat hypertension are available, however many patients are resistant to the standard therapeutic approaches. The association of two or more substances in a fixed-dose combination is effective and tolerated as a substitute for the standard therapeutic approach. Objective: The new ultra performance liquide chromatography method was developed and validated to assay a combination of eight antihypertensive drugs including a diuretic: hydrochlorothiazide, dihydropyridine calcium channel blocker: Amlodipine and angiotensin II type 1 receptor blockers (sartans): valsartan, candesartan, eprosartan, olmesartan, losartan, and irbesartan in the pharmaceutical matrix. Methods: Chromatographic separation was performed on an Acquity® UPLC C18 1.7 µm 2.1 × 100 mm column, with a gradient of buffer solution and acetonitrile, in the proportion of (80:20 v/v). Results: Good resolution was obtained, and an optimal analysis time of less than 5 min was achieved. The method was validated according to the International Conference on Harmonization guidelines following the classical approach and accuracy profile, and it is shown to be suitable for intended applications. The method was successfully used for quality control laboratories and the determination of these drugs combinations in pharmaceutical dosage forms.

Background: Levofloxacin is a third-generation fluoroquinolone that has several advantages over its (R) ofloxacin isomer. It is used to treat different types of infection, including urinary infection and prostatitis. Objective: A new HPLC method for the enantioselective separation of levofloxacin and its chiral impurity was developed and validated to improve the separation of the enantiomers of levofloxacin [impurity(R) and active principle (S)] by increasing the value of the resolution between the eutomer and the distomer. Method: Chromatographic separation was performed on a Prodigy ODS -2, 5 µm 4.6 × 150 mm column, with a gradient of buffer solution and methanol (80:20, v/v). A Box–Behnken design was considered when optimizing the enantioseparation involving the effects of many factors such as the concentration of d-phenylalanine, the pH of the buffer, the percentage of organic modifier in the mobile phase, the flow rate, the temperature of the column, and the type of column. Results: Chiral separation was achieved with an optimal resolution of 3.8. The method was successfully validated following the International Conference on Harmonization Q2 (R1) guideline, fulfilling the acceptance criteria for selectivity [no interference in the retention time of (S) levofloxacin and (R) levofloxacin], linearity (r ≥0.999 in the range 1.25–3.75 µg/mL for all enantiomers), and precision (RSD <2%). Accuracy was assessed by the application of the analytical method to an analyte of known purity, providing evidence for the usefulness of this monitoring system. Conclusions: The method was successfully used for the determination of levofloxacin impurity in raw material and pharmaceutical dosage forms. Highlights: The following method is accurate and robust to quantify and characterize the presence of levofloxacin impurity in raw material for pharmaceutical compounds.

Background: Accurate determination of active pharmaceutical ingredients and impurities is essential for ensuring the safety and effectiveness of medications. This study focuses on the validation of a high-performance liquid chromatography (HPLC) method for quantifying atorvastatin and its impurities, addressing a critical aspect of pharmaceutical analysis. Objective: The primary objective is to conduct a comprehensive validation study for the HPLC method, covering specificity assessment, response function establishment, and a detailed analysis of precision, trueness, and tolerance intervals. The emphasis is on demonstrating the method’s precision, accuracy, and stability-indicating capabilities across various concentrations and compounds. Methods: The HPLC method is validated through rigorous assessments, including specificity, response function establishment, and analyses of precision, trueness, and tolerance intervals. Induced degradation experiments are conducted to explore atorvastatin’s behavior under extreme conditions. Insights into the compound’s synthesis and degradation pathways are provided through a proposed mechanism for intramolecular esterification. Results: The results affirm the precision, accuracy, and stability-indicating capabilities of the validated HPLC method. The method effectively differentiates between atorvastatin and its impurities, showcasing its suitability for pharmaceutical quality control. Conclusions: The validated HPLC method emerges as a robust and reliable tool for atorvastatin analysis, contributing significantly to pharmaceutical research and quality control. Its application ensures the safety and efficacy of medications, reinforcing its role in pharmaceutical analysis. Highlights: This study not only validates a crucial HPLC method for atorvastatin analysis but also provides insights into the compound’s behavior under extreme conditions and its synthesis and degradation pathways. The validated method serves as a cornerstone in pharmaceutical research and quality control, ensuring medication safety and efficacy.

Background: Neonicotinoids (NEOs) are used for the phytosanitary treatment of Mentha Spicata.L crops, and this practice requires precise control of these harmful substances at very low concentrations. Objective: The objective of this study is to apply an approach allowing simultaneously validation and evaluation of measurement uncertainty based on total error methodology, in order to accurately quantify the presence of two NEOs in Mentha Spicata.L utilizing a Quick, Easy, Cheap, Effective, Rugged, and Safe (QuEChERS)–LC–MS/MS methodology. Methods: The quantification of imidacloprid and acetamiprid employing a QuEChERS extraction method, coupled with LC–MS/MS, ensuring the accuracy of the analytical method and managing the risks associated with its routine use. A complete and exhaustive validation approach based on the “β-content, γ-confidence” tolerance interval was used for the uncertainty assessment, using the generalized pivot quantity (GPQ) concept and Monte Carlo simulation, which avoids the need for additional data while achieving intermediate precision for each concentration level within predetermined acceptable limits. Results: The validation procedure is based on the choice of a quadratic model for the two NEOs, allowing the validation of acetamiprid and imidacloprid by LC–MS/MS assay within the range of working concentration. The flexibility of the uncertainty profile intervals was demonstrated with a variation in β-content values (66.7, 80, and 90%) and risk values (10 and 5%), which remained within the acceptability limits of 20%, and the relative expanded uncertainty did not exceed 15 and 11%. Conclusion: A QuEChERS–LC–MS/MS method for the analysis of two NEOs has been successfully fully validated using the uncertainty profile strategy. Highlights: Implementation of an overall validation strategy, which involves both the validation and uncertainty assessment known as the uncertainty profile, for the quantification of two important NEOs in Mentha Spicata.L using QuEChERS–LC–MS/MS. This qualimetric approach has been conducted by computing the measurement uncertainty of the method utilizing data from analytical validation under conditions of intermediate precision at each level of concentration without additional effort. After that we have demonstrated the flexibility of this strategy for the LC–MS/MS quantification of acetamiprid and imidacloprid, using a decision tool that enables the choice and modification of β-content and γ-confidence values.

Background/Objectives: The stability of pharmaceutical compounds is a critical quality attribute; it is an essential step in the drug development process. Significant focus is required to understand the variation of quality pharmaceutical compounds under prevailing environmental storage conditions. Simultaneously, many issues arise in understanding updated regulations, knowledge of data sciences, and appreciation of common practices, presenting a challenge for defining a retest period and in predicting a prolongation of the shelf life of drug products. The purpose of this paper is to conduct a statistical study to assess stability and to forecast a prolongation of drugs shelf-life. Methods: A case study is suggested to identify the most appropriate statistical test for assessing stability. The results of physical and chemical tests are considered to detect changes and variability during different conditions (accelerate, intermediate and real). Results: In the stability study, minimal variability in the content of the substance of interest was obtained using the predictive interval approach over a period of 31 months, and an interval of ±1,2%. Conclusion: The example of the statistical study is given to provide different perspectives on statistical approaches for market approval.