Impurity qualification requirements for drug-linkers related impurities used to generate antibody-drug conjugates.

High-throughput protein A chromatography platform for accurate antibody quantification in IV admixtures.

INTRODUCTION. An effective quality control system for medicinal products is impossible without reference standards used for validation and verification of analytical procedures. It is especially relevant to develop reference standards for quality control of new active pharmaceutical ingredients used to develop medicinal products. AIM. This study aimed to develop a method for obtaining and certifying a primary reference standard of 6,8-dimethyl-2-piperidinomethyl-2,3-dihydrothiazolo[2,3-F] xanthine to be used in quality control of medicinal products. MATERIALS AND METHODS. A reference standard was obtained by recrystallising 6,8-dimethyl-2-piperidinomethyl-2,3-dihydrothiazolo[2,3-F]xanthine from ethanol. The structure of the reference standard was defined by nuclear magnetic resonance and infrared spectroscopy; purity was measured by mass balance, high-performance liquid chromatography (related impurities), and titration (non-aqueous acidimetry). RESULTS. The study assessed physical and chemical properties of a reference standard for 6,8-dimethyl-2-piperidinomethyl-2,3-dihydrothiazolo[2,3-F]xanthine. This included structure elucidation by infrared and nuclear magnetic resonance spectroscopy; loss on drying 0.073±0.015%; sulfate ash 0.080±0.009%; related substances (not found); heavy metal impurities (not more than 0.002%); elemental composition (C — 53.68±0.17%; H — 6.32±0.02%; N — 20.81±0.09%; O — 9.52±0.06%; S — 9.57±0.04%); quantitative determination by non-aqueous titration 99.74±0.12%, and mass balance 99.85±0.01%. INTRODUCTION. An effective quality control system for medicinal products is impossible without reference standards used for validation and verification of analytical procedures. It is especially relevant to develop reference standards for quality control of new active pharmaceutical ingredients used to develop medicinal products. AIM. This study aimed to develop a method for obtaining and certifying a primary reference standard of 6,8-dimethyl-2-piperidinomethyl-2,3-dihydrothiazolo[2,3-F] xanthine to be used in quality control of medicinal products. MATERIALS AND METHODS. A reference standard was obtained by recrystallising 6,8-dimethyl-2-piperidinomethyl-2,3-dihydrothiazolo[2,3-F]xanthine from ethanol. The structure of the reference standard was defined by nuclear magnetic resonance and infrared spectroscopy; purity was measured by mass balance, high-performance liquid chromatography (related impurities), and titration (non-aqueous acidimetry). RESULTS. The study assessed physical and chemical properties of a reference standard for 6,8-dimethyl-2-piperidinomethyl-2,3-dihydrothiazolo[2,3-F]xanthine. This included structure elucidation by infrared and nuclear magnetic resonance spectroscopy; loss on drying 0.073±0.015%; sulfate ash 0.080±0.009%; related substances (not found); heavy metal impurities (not more than 0.002%); elemental composition (C — 53.68±0.17%; H — 6.32±0.02%; N — 20.81±0.09%; O — 9.52±0.06%; S — 9.57±0.04%); quantitative determination by non-aqueous titration 99.74±0.12%, and mass balance 99.85±0.01%. INTRODUCTION. An effective quality control system for medicinal products is impossible without reference standards used for validation and verification of analytical procedures. It is especially relevant to develop reference standards for quality control of new active pharmaceutical ingredients used to develop medicinal products. AIM. This study aimed to develop a method for obtaining and certifying a primary reference standard of 6,8-dimethyl-2-piperidinomethyl-2,3-dihydrothiazolo[2,3-F] xanthine to be used in quality control of medicinal products. MATERIALS AND METHODS. A reference standard was obtained by recrystallising 6,8-dimethyl-2-piperidinomethyl-2,3-dihydrothiazolo[2,3-F]xanthine from ethanol. The structure of the reference standard was defined by nuclear magnetic resonance and infrared spectroscopy; purity was measured by mass balance, high-performance liquid chromatography (related impurities), and titration (non-aqueous acidimetry). RESULTS. The study assessed physical and chemical properties of a reference standard for 6,8-dimethyl-2-piperidinomethyl-2,3-dihydrothiazolo[2,3-F]xanthine. This included structure elucidation by infrared and nuclear magnetic resonance spectroscopy; loss on drying 0.073±0.015%; sulfate ash 0.080±0.009%; related substances (not found); heavy metal impurities (not more than 0.002%); elemental composition (C — 53.68±0.17%; H — 6.32±0.02%; N — 20.81±0.09%; O — 9.52±0.06%; S — 9.57±0.04%); quantitative determination by non-aqueous titration 99.74±0.12%, and mass balance 99.85±0.01%. CONCLUSION. We have developed production process of 6,8-dimethyl-2-piperidinomethyl-2,3-dihydrothiazolo[2,3-F]xanthine reference standard. Physical and chemical properties of the above standard comply with the requirements and allow us to recommend 6,8-dimethyl-2-piperidinomethyl-2,3-dihydrothiazolo[2,3-F]xanthine as a reference in the quality control of medicinal products.

A robust and stability-indicating Reverse Phase-High Performance Liquid Chromatography (RP-HPLC) method was developed and validated for the quantitative determination of bexagliflozin and its related impurities in accordance with the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH Q2(R1)) guidelines. Chromatographic separation was achieved on a C18 column using a mobile phase of methanol and ammonium acetate buffer (pH 4.2) in a 60:40 (v/v) ratio, with a flow rate of 1.0 mL·min−1 and UV detection at 220 nm. The method was validated for linearity, sensitivity (LOD and LOQ), precision, robustness, and system suitability, all within acceptable limits for low-concentration analysis. Excellent linearity (r2 > 0.999) and precision (%RSD 0.3–4.4%) confirmed its reliability for stability assessment. The assay was performed at 100 µg·mL−1, where all validation parameters showed %RSD values ≤ 2%, demonstrating high precision and robustness. Forced degradation studies under acidic, basic, oxidative, photolytic, and thermal conditions revealed a major degradation product formed under acidic stress. This product was isolated and structurally characterized using LC–MS, 1H NMR, and 13C NMR, and is reported here for the first time. The proposed RP-HPLC method proved to be specific, precise, and reliable for the determination of bexagliflozin and its related impurities, making it suitable for routine stability testing, quality control, and pharmaceutical development applications.

Orodispersible tablets (ODT) constitute an innovative dosage form which provides a patient-friendly option that enhances compliance and therapeutic convenience, particularly for pediatric and geriatric populations with swallowing difficulties. Meclizine ODT (6.25 mg and 12.5 mg) have been developed at F1 Pharma S.A., packaged in blisters and designed for stability studies. This study reports the development and full validation in compliance with ICH Q2(R1) guidelines of two HPLC methods for the quantification of meclizine hydrochloride and its related impurities in the 6.25 mg ODT formulation. Both methods proved highly specific, precise, accurate, and robust, with validated working ranges suitable for routine quality control and stability testing. Forced degradation experiments identified impurities C and H as the primary degradation products. Long-term stability studies conducted under ICH Zone II and III conditions confirmed chemical stability of the formulation, with assay values consistently within ±5% of label claim and impurity levels within acceptable limits. The developed novel ODT formulation meets all regulatory requirements, while validated HPLC methods ensure reliable routine analysis, supporting quality control and stability studies of this innovative dosage form, which enhances patient comfort and treatment effectiveness.

Siponimod fumarate (SIP), a potent selective S1P receptor modulator, has emerged as a critical therapeutic agent in the treatment of multiple sclerosis. Recognizing the increasing regulatory demands for robust impurity profiling and method reliability, this study reports the development and optimization of an HPLC method for the simultaneous determination of SIP and its related impurities in both bulk drug substance and tablet dosage forms. The method was developed within an Analytical Quality by Design (AQbD) framework, guided by ICH Q14 principles, ensuring a systematic and risk-based approach throughout the analytical lifecycle. Chromatographic separation necessary for resolving critical impurities was achieved on an XSelect HSS T3 column (150mm×4.6mm, 3.5µm) using a stepped gradient elution program with 0.1% perchloric acid in water and acetonitrile as the mobile phases. Optimal separation conditions, identified through the AQbD process to meet stringent performance criteria, were determined at a column temperature of 42.5°C, a flow rate of 1.4mLmin-1, and UV detection at 212nm. The method performance was rigorously evaluated through accuracy profiles, confirming both its precision and trueness across the targeted concentration range. In parallel, as part of a holistic method characterization, environmental sustainability was assessed using comprehensive greenness metrics, whereas its practical applicability was further substantiated using the Blue Applicability Grade Index (BAGI) and the Red-Green-Blue 12 (RGB12) algorithms. This approach not only bridges the gap created by the absence of an official pharmacopoeial monograph for SIP but also offers a robust, well-characterized, and sustainable platform for pharmaceutical quality control, aligning method development with both regulatory performance needs and environmental awareness.

A stability-indicating and environmentally sustainable UPLC method for simultaneous determination of rizatriptan, meloxicam, and related N-nitrosamine impurities

Comprehensive structural characterization and purity assessment of 1-kestose: quantification of structurally related impurities via an advanced HPLC-CAD platform

ABSTRACT N‐Nitroso impurities have emerged as a critical concern in pharmaceutical development due to their potential genotoxic and carcinogenic risks, often interrupting clinical programs and affecting the quality of commercial drug products. In this study, a microwave‐assisted green synthetic approach was employed to efficiently generate N‐nitroso Tofacitinib, a key nitrosamine impurity associated with the Janus kinase (JAK) inhibitor Tofacitinib. The method enabled rapid conversion under mild conditions, offering a sustainable strategy for impurity synthesis. An extensive structural elucidation of the synthesized N‐nitroso Tofacitinib impurity (TCB Imp 2) namely 1‐((3R,4R)‐4‐methyl‐3‐(methyl(7H‐pyrrolo[2,3‐d]pyrimidin‐4‐yl)amino)piperidin‐1‐yl)‐3‐(nitroso‐l4‐azanylidyne)propan‐1‐one was carried out using advanced spectroscopic and spectrometric techniques 1D, 2D‐ 1 H NMR, 13 C NMR, 15 N NMR, infrared spectroscopy, and mass spectrometry. A key observation from this study is that nitrosation occurred selectively at the nitrile‐derived side chain rather than at the pyrrolic nitrogen of the core heterocycle, providing new insight into the reactivity and formation pathways of N‐nitrosamine drug substance–related impurities (NDSRIs) in Tofacitinib. Additionally, two other potential N‐nitroso impurities of Tofacitinib were synthesized and fully characterized, expanding the impurity knowledge base. This work enhances mechanistic understanding of nitrosation behavior in Tofacitinib, supports regulatory compliance, and provides high‐quality reference standards for impurity profiling within the generic pharmaceutical industry.

Currently, there is a steady increase in the prevalence of metabolic disorders among the population of developed countries. Among them, obesity and type 2 diabetes mellitus are the most important health problems. Tirzepatide is an innovative drug that is a dual agonist of glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) receptors. The medcine is effective for the treatment of type 2 diabetes mellitus and obesity. The first drug with the active substance tirzepatide in Russia was Tirzetta® (manufacturer LLC «PROMOMED RUS»), which is the first in Russia, but not in the world. The reference drug for it is Munjaro® (INN: tirzepatide, Eli Lilly and Company, USA). To date, the question of the equivalence of these drugs has not been fully studied. The aim. To conduct a comprehensive comparative evaluation of the reproduced drug Tirzetta® (INN: tirzepatide, manufacturer LLC “PROMOMED RUS”) and the reference drug Munjaro® (INN: tirzepatide, manufacturer Eli Lilly and Company, USA). Materials and methods. The authenticity and quality of drugs were assessed by physicochemical methods according to the current pharmacopoeia of the EAEU. Spectrophotometry in the UV region, HPLC-MS/OF, and gel filtration chromatography were performed. The analysis of agonism to GIP and GLP-1 receptors was performed in vitro using reporter cell lines. The studies were performed in accordance with EMA, FDA, EAEU guidelines and in accordance with the current EAEU pharmacopoeia. Results. As a result of the evaluation of the physicochemical properties of the studied series of Tirzetta® and the reference drug Munjaro®, it was found out that the absorption spectra in the ultraviolet region, the profile of related impurities and their quantitative content, the profile of high-molecular-weight compounds and their quantitative content, as well as mass spectra in all the studied series were similar. During the evaluation of the biological activity of the Tirzetta® and Munjaro® series, results were obtained that demonstrated the absence of statistically significant differences in the ability to activate GLP-1 and GIP receptors ( p <0.0001). Conclusion. During the studies, the equivalence of the physicochemical properties and biological activity of the Russian drug Tirzetta® to the comparator drug Munjaro® was confirmed.

NNitrosamine formation in pharmaceutical drug products: Development and validation of a biased, conservative, predictive model.

The mutagenicity and bioactivation potential of N-nitrosoparoxetine, a N-nitrosamine drug substance related impurity (NDSRI) of the marketed antidepressant paroxetine, was characterised in vitro. N-Nitrosoparoxetine was not mutagenic in an OECD 471-compliant bacterial reverse mutation assay even under experimental conditions that supported oxidative metabolism by CYP enzymes. The major biotransformation pathways of N-nitrosoparoxetine paralleled the ones previously noted with paroxetine. Virtually all stable metabolites of N-nitrosoparoxetine in human, rat, and hamster liver tissue were derived from phase 2 conjugations on an unstable catechol intermediate, generated from the oxidative 1,3-benzodioxole ring scission by CYP enzymes. The piperidine ring in N-nitrosoparoxetine was resistant to the α-carbon oxidation step leading to piperidine ring scission, and to the eventual formation of a DNA-reactive electrophilic species. CYP reaction phenotyping studies demonstrated that paroxetine was exclusively metabolised by human CYP2D6, whereas N-nitrosoparoxetine was subject to metabolism by multiple human CYP isoforms including CYP2C19, CYP2D6, and CYP3A4. Whether most NDSRIs derived from parent amine precursors that are resistant to α-carbon oxidation by CYPs will also be devoid of a mutagenic response in vitro needs to be scrutinised further with additional examples to bolster structure-genotoxicity relationships of complex NDSRIs.

Background . Capreomycin is a multicomponent natural antibiotic and is a mixture of 4 related compounds (capreomycins IA, IB, IIA, and IIB). Quality control of capreomycin medicinal preparations by active substance is carried out according to the «capreomycin I» indicator, not taking into account the individual contributions of capreomycins IA and IB. With this approach, no correlation is observed between the total content of capreomycin IA and IB components determined by chromatographic methods and capreomycin activity determined by microbiological methods. WHO recommended studying the feasibility of introducing the ratio between capreomycin IA and IB in the specifications for capreomycin preparations. The aim of the study was to develop a technique for quantitative estimation of the capreomycin IA and IB content ratio in the drug preparation Capreomycin sulphate using NMR spectroscopy. Objects of the study . Capreomycin sulphate standard samples, pharmaceutical preparations Capreomycin, powder for preparation of solutions for intravenous and intramuscular administration from various manufacturers. Material and methods: NMR spectroscopy, HPLC. Results. Structural interpretation of 1H and 13C spectra of capreomycin sulfate in D₂O solution was performed. 13C spectra revealed characteristic signals of capreomycin IA and IB, which did not overlap with the signals of minor capreomycin components and related impurities. On the basis of normalized integral intensities, the ratio of mole fractions of capreomycin IA and IB (IA/IB) was calculated. The results of IA/IB measurement using 13C NMR and HPLC practically coincide when the chromatographic conditions allow separating the signals of the major and impurity components of capreomycin on the chromatogram.

ABSTRACT Clozapine is an atypical tricyclic dibenzodiazepine antipsychotic. Both clozapine and its related impurity contain secondary amine moieties that are susceptible to nitrosation, potentially leading to the formation of N‐nitroso Clozapine (N‐CZN) and N‐nitroso clozapine impurity C (N‐CZNC) during manufacturing, storage, and stability testing. This study reports the development and validation of a green and highly sensitive liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for the simultaneous trace‐level quantification of N‐CZN and N‐CZNC in drug substances and pharmaceutical formulations. Chromatographic separation was achieved using an Agilent Poroshell HPH C18 column under gradient elution. Mobile phase A consisted of 0.01 M ammonium bicarbonate in water, while mobile phase B comprised 0.1% formic acid in a 50:50 (v/v) methanol: acetonitrile mixture, with a flow rate of 0.5 mL/min and column temperature maintained at 40°C. Detection was performed by electrospray ionization in multiple reaction monitoring mode, with transitions monitored at m / z 356.0 → 326.0 for N‐CZN and m / z 342.1 → 312.0 for N‐CZNC. The method was validated as per International Council for Harmonization guidelines (ICH) and United States Pharmacopeia (USP) <1225> , demonstrating excellent linearity ( R 2 > 0.999) over the range from the LOQ to 150%. The LOD and LOQ were determined to be 0.058 and 0.177 µg/mL, respectively, for N‐CZN, and 0.014 and 0.044 µg/mL respectively, for N‐CZNC. The method exhibited satisfactory accuracy, with recoveries between 88% and 102%. The validated method was successfully transferred to the quality control laboratory, where it is currently employed for the routine analysis of three batches of drug substances and finished formulations.

Experimental determination and observations of molecular diffusion coefficients of oligonucleotides via the Taylor-Aris method.

Risk (Re)assessment of N-Methyl-N-nitrosophenethylamine for use in computing risk levels of N-Nitrosamine drug substance related impurities.

Abstract Apomorphine, a potent dopamine agonist, plays a significant role in the management of advanced Parkinson's disease, and requires careful monitoring of the drug and its impurities for safety and efficacy. The USP monograph for Apomorphine Hydrochloride currently uses classical methods—titrimetry for assay and Thin-Layer Chromatography (TLC) for impurity testing—which, although effective, are not comparable in accuracy and speed to newer chromatographic techniques like High-Performance Liquid Chromatography (HPLC). For this, an HPLC method was developed using a C18 column, which possesses hydrophobic interaction capacity, and an isocratic elution mode with a mobile phase of 10 mM phosphate buffer (pH 3.0) and methanol (87.5:12.5) containing 0.075% L-tartaric acid. This combination allowed efficient separation of apomorphine from impurities without compromising stability.The method was thoroughly validated according to International Council for Harmonisation (ICH) guidelines and exhibited very good specificity, precision, accuracy, linearity, and robustness. The validation results demonstrated a good linear correlation (r = 0.999) between the peak area and the apomorphine concentration, excellent precision (repeatability of 0.23% and intermediate precision of 0.56%), and accuracy (recoveries of 99.72–100.08%). Robustness was confirmed by minimal variation in percentage recovery (R = 1.3%), and specificity was confirmed by the absence of interfering peaks caused by excipients or formulation components. The method has been successfully employed in the analysis of apomorphine and impurities in commercial products, proving itself reliable from batch to batch. This HPLC method represents a significant improvement over conventional methods, as it provides a precise and quicker approach for the quality control of apomorphine medicinal products.

Development of continuous flow photochemistry-based protocol for the synthesis of Sulindac related impurity

Lipid nanoparticles (LNPs) have gained much attention after the recent launch of mRNA based Covid vaccines. For mRNA encapsulated within LNPs to be successfully translated into target proteins, the mRNA must maintain its integrity and also be free of any unintended chemical modifications. Any process or raw material related impurities—and their degradation products—pose risks of chemically modifying mRNA, thereby affecting the quality of the final product. Given its inherent chemical reactivity and close association with the LNP lipids, encapsulated mRNA is especially susceptible to modifications by reactive impurity species present in the lipids. In our recent efforts to understand mRNA-lipid interactions within LNPs, we observed that the degradants of labile lipid peroxide species in ionizable lipids react with nucleotides in mRNA, resulting in loss of mRNA’s translation efficiency in vitro. Specifically, we identified peroxide species in unsaturated dialkene groups were converted to a variety of reactive aldehyde products in mRNA LNP formulations. These findings enhance the current understanding of the adduct formation between mRNA and aldehyde species, and emphasize the critical role of deep analytical characterization of ionizable lipid stability and purity to enhance LNP product quality and shelf-life.

Synthetic sweeteners have found widespread application in the national economy, but their use has some limitations. Safe intake levels for aspartame and acesulfame potassium are established by the recommendations of the Joint FAO/WHO Expert Committee on Food Additives (JECFA). Compliance with safety requirements can be ensured by controlling the content of sweeteners in food products.In Russia, measurements performed in accordance with GOST standards and certified methods, for which reference materials are essential. The presented research was conducted with the aim of replacing foreign reference materials with domestically produced reference materials to ensure measurement uniformity for the purpose of Russia’s technological sovereignty.The publication contains information on the development of new types of reference materials for the composition of the synthetic sweeteners aspartame and acesulfame potassium, which are metrologically traceable to the State Primary Standard of units of mass (molar) fraction and mass (molar) concentration of organic components in liquid and solid substances and materials based on liquid and gas chromatography-mass spectrometry with isotope dilution and gravimetry GET 208–2024. Identification of the main component in the reference material was performed using high-resolution mass spectrometry. Characterization of the reference materials was carried out using the mass balance method. The content of related compound impurities was measured using high-performance liquid chromatography with a diode-array UV detector; the content of volatile organic compound impurities was measured using gas chromatography with a flame ionization detector; water content was determined by Karl Fischer coulometric titration. The content of non-volatile impurities was measured by gravimetry for aspartame and microwave plasma atomic emission spectrometry for acesulfame potassium. The homogeneity and long-term stability of the reference materials were studied.The metrological characteristics of the new domestically produced reference materials demonstrate parity with foreign analogues: the mass fraction of the main component in the pure aspartame is (96.2 ± 0.3)%; the mass fraction of the main component in the pure acesulfame potassium is (99.98 ± 0.01)%.