Jul 2026· Journal of Pharmaceutical and Biomedical Analysis· Vol 281, pp.
117682
· 0 citations· 21 references
Medicine
TL;DR
The determined impurity profile of baloxavir marboxil and the evaluated impurities' potential safety risks can provide a valuable reference for its impurity control, stability evaluation, and quality risk management.
Abstract
Baloxavir marboxil, a novel RNA polymerase inhibitor for the treatment of influenza, suppresses viral replication at the initiation stage of RNA transcription and has demonstrated significant clinical efficacy in reducing viral load and rapidly alleviating respiratory symptoms. Previous studies have primarily focused on its pharmacodynamics and clinical efficacy, whereas systematic investigations into its related substances (RSs) and their structural characteristics remain limited. In this study, two-dimensional liquid chromatography-quadrupole time-of-flight mass spectrometry (2D-LC-Q-TOF/MS) was employed to detect and characterize process-related impurities in the baloxavir marboxil active pharmaceutical ingredient (API) and degradation products formed in its stressed samples, in accordance with the ICH Q1A(R2) guideline. Based on chromatographic retention behavior and high-resolution MS/MS fragmentation patterns, a total of 14 impurity-related peaks were characterized, including four process-related impurities and ten degradation products. Among them, six impurities, including IMP C and five degradation products (RSs 5, 6, 8, 9 and 12), were not found in the available literature and were characterized in this study. The major degradation pathways were proposed, involving side-chain hydrolysis, oxidation of the sulfur-containing aromatic ring, and the cleavage of heterocyclic linkages. Furthermore, a preliminary genotoxicity assessment of the characterized impurities was conducted in accordance with the ICH M7(R2) guideline using in-silico (Q)SAR prediction approaches, and revealed that these related substances did not exhibit genotoxic structural alerts. The determined impurity profile of baloxavir marboxil and the evaluated impurities' potential safety risks can provide a valuable reference for its impurity control, stability evaluation, and quality risk management.
RATIONALE
Norvancomycin (NVCM) hydrochloride belongs to a group of glycopeptide antibiotics and exhibits strong effects against Gram-positive cocci and bacilli. The safety of patients' lives is directly related to the quality of drugs. The impurities play often an important role in this process. This study aimed to investigate the impurity profile of NVCM hydrochloride.
METHODS
To identify the impurities, a simple and sensitive ultrahigh performance liquid chromatography quadrupole-time-of-flight mass spectrometry (UHPLC-QTOF-MS) method was developed. An ACQUITY UPLC BEH C18 column (2.1 × 100 mm, 1.7 μm) was used. The flow rate was set at 0.2 mL/min. Mobile phase (A) consisted of 10 mmol/L ammonium formate (pH 9.0) and mobile phase (B) was methanol. Gradient elution was performed as follows: 0-5 min, 15% B; 5-25 min, 15% B to 20% B; 25-40 min, 20% B to 70% B. Stress degradation experiments were conducted to check the specificity of the method.
RESULTS
Thirteen impurities and two degradation products were characterized using UHPLC-QTOF-MS.
CONCLUSIONS
Based on MS/MS spectral data and exact mass measurements, the chemical structures of the impurities were elucidated. This study provided a reference method for the quality control of NVCM hydrochloride.
Yanni Liu, Jinqi Zheng, Jia Sun et al.· Rapid Communications in Mass...· 0 citations
Introduction / Objective: The selectivity, potency, and stability of antiviral compounds, including Ritonavir (RTV),
require high-precision analytical testing and impurity profiling for confirmation. As a widely used pharmacokinetic
enhancer in COVID-19 therapy, the regulation and clinic validation of impurities and their toxicity are of the utmost
importance. The main goal of the present work was to develop and confirm a simple, accurate, and stability, indicating
HPLC method for determining the Ritonavir content and its related impurity (Impurity, A Free Base) includes theoretical
and experimental toxicity assessments. Methods: Chromatographic separation was performed on a Phenomenex C18
column (250 4.6 mm, 5 m). The mobile phase was 20:80 of water and acetonitrile, flow rate 1.0 mL/min. The method
validation tested different things using ICH Q2(R1) rules, Accuracy, Precision, Specificity, and Linearity. The toxicity
assessment for Impurity A was done by the insilico prediction tools (PROTOX 3.0) and checked with an in vitro study of
toxicity in PBMC cells. Results: The method demonstrated (R = 0.9999) in a range of 1.64 to 29.94 g/mL, with recovery
rates from 92% to 99% and a relative standard deviation below 2%. Ritonavir and its impurity- A with distinct retention
times is 4.3 min and 2.3 min respectively. In silico studies stated that the drug has a medium acute oral toxicity as low
with a LD50 values of more than 2000 mg/kg with mild hepatotoxic and neurotoxic while MTT results showed an IC50
value >500 µg/mL, suggesting low cytotoxicity. Discussion: The validated HPLC method is a reliable, accurate, and
economical way for the routine analysis of Ritonavir and its contaminants. The use of computational and in vitro toxicity
assessments used to identify impurity hazards and comply with quality control standards of antiviral drugs. Conclusion:
A consistent, precise, and cost-effective HPLC method was successfully developed and validated for Ritonavir and its
related impurity. The integration of in silico and in vitro toxicity studies strengthens impurity risk assessment and supports
the safe and effective quality control of antiviral pharmaceutical products.
Balaji Ramakrishnan, J. S, Esakkimuthukumar Mariappan et al.· International Journal of Dru...· 0 citations
Background: To enhance efficacy and manage dose-limiting toxicities such as hyperglycemia, therapeutic drug monitoring of alpelisib (ALB), a phosphatidylinositol 3-kinase alpha (PI3Kα) inhibitor used in advanced breast cancer, is essential. This study aimed to develop and validate a selective, sensitive, and high-throughput liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantification of ALB in human plasma to support pharmacokinetic studies and therapeutic drug monitoring. Methodology: Alpelisib-D3 was used as a deuterated internal standard (IS). Analytes were extracted from 50 µL of plasma by liquid-liquid extraction using n-hexane and methyl tert-butyl ether (20:80, v/v). Chromatographic separation was performed on an X-Terra RP8 column using an isocratic mobile phase of 5 mM ammonium acetate buffer (pH 5.00) and acetonitrile (10:90, v/v) at 0.4 mL/min. Detection was carried out in positive electrospray ionization mode using multiple reaction monitoring of m/z 442.15→141.07 for ALB and m/z 445.36→144.05 for the IS. Validation included linearity, precision, accuracy, recovery, matrix effect, carryover, dilution integrity, and stability. Results and discussion: The method was linear over 50.0–10,000 ng/mL. Intra- and inter-day precision (CV%) was <4.2%, and accuracy ranged from 94.5% to 106.2%. Mean extraction recovery exceeded 90%, with no significant matrix effects. Carryover, stability, and dilution integrity also met acceptance criteria. Conclusion: A reliable, sensitive, and selective LC-MS/MS method was successfully developed and validated. The method is suitable for high-throughput pharmacokinetic studies and therapeutic drug monitoring of ALB in human plasma.
Venkateswararao Agraharapu, V. Vutla, Vidyadhara Suryadevara· Journal of Applied Pharmaceu...· 0 citations
ABSTRACT Isoniazid (INH) is a key component of tuberculosis treatment regimens but is also associated with hepatotoxicity. This toxicity is mediated in part by its metabolites, but some degrade rapidly in plasma. A rapid freeze/thaw process with a methanol protein precipitation extraction was developed to slow the rapid degradation of acetylisoniazid (AcINH) into isonicotinic acid (INA) and acetylhydrazine (AcHZ) in human plasma. An assay consisting of two UHPLC–MS/MS methods was developed and validated to determine concentrations of INH and four of its metabolites in human plasma: the “polar” method covering AcINH and INA, and the “nonpolar” method covering p‐tolualdehyde derivatized AcHZ, hydrazine (HZ), and INH. Polar and nonpolar method chromatographic separation was accomplished with biphenyl or C18 columns, respectively, mobile phases of 4 mM ammonium formate in water or acetonitrile, and method specific gradient conditions. The validated range for AcINH, INA, and INH was 10.0 to 5000 ng/mL and for AcHZ and HZ was 10.0 to 500 ng/mL from 0.1 mL of plasma, respectively. Though other groups have validated methods to quantitate some or all of these analytes, none have adequately characterized or addressed AcINH degradation. This validated assay will help facilitate a better understanding of INH metabolite disposition and relationships with toxicity.
David Nerguizian, Brandon Klein, Farah Abdelmawla et al.· Biomedical chromotography· 0 citations
Efonidipine Hydrochloride Ethanolate (EHE) and Chlorthalidone (CTD) are co-formulated for the management of hypertension owing to their complementary pharmacological actions. Ensuring accurate impurity profiling in such combination formulations is vital for maintaining drug stability, safety and regulatory compliance. However, conventional analytical techniques often face challenges related to sustainability and robustness. Reverse Phase High-Performance Liquid Chromatography (RP-HPLC) remains a key analytical approach for impurity determination, yet the integration of Quality by Design (QbD) principles into its development is still evolving. The present study focuses on developing a novel, sustainable RP-HPLC method for impurity profiling of EHE and CTD tablets using a QbD approach and green chemistry. Chromatographic separation was achieved on a Zorbax SB C8 column (150 mm × 4.6 mm, 5 µm). Critical method parameters such as mobile phase composition and pH were systematically optimized through QbD to enhance method robustness. A photolytic degradation product of EHE was identified as Efonidipine Related Compound A, and a plausible photolytic degradation pathway was proposed. A photolytic degradation product was identified in the LC-MS and its structure was confirmed through NMR spectroscopy. The developed green method exhibited excellent resolution, precision and reproducibility across multiple batches, allowing for effective separation and quantification of impurities. This green QbD-optimized, environmentally sustainable RP-HPLC method provides a reliable and scalable solution for impurity profiling in complex pharmaceutical formulations, aligning with modern quality control and regulatory expectations.
Pranav S. Bang, V. Choudhari, V. Gharge· Journal of Chromatography A· 0 citations
BACKGROUND
Bruton's tyrosine kinase inhibitors (BTKis, ibrutinib, zanubrutinib, orelabrutinib) are key targeted therapies for B-cell lymphomas, but interindividual variability and adverse reactions limit their use. Metabolized by CYP3A, co-medications or hepatic dysfunction cause plasma fluctuations, necessitating therapeutic drug monitoring (TDM). Cerebrospinal fluid (CSF) distribution is also critical for CNS lymphoma treatment.
OBJECTIVE
To develop and validate a UHPLC-MS/MS method for simultaneous quantification of three BTKis and one metabolite in human plasma and CSF to support TDM.
METHODS
After protein precipitation with methanol, samples were analyzed on an ACQUITY HSS T3 column with a 3-min gradient at 0.3 mL/min and ESI (+) MRM. Mobile phase: water-acetonitrile-formic acid-ammonium acetate (1 M) (950:50:1:1, A) and (50:950:1:1, B).
RESULTS
The method showed excellent selectivity and linearity (r > 0.997) over 1-200 ng/mL (ibrutinib, zanubrutinib, ibrutinib metabolite) and 5-1000 ng/mL (orelabrutinib) in both matrices. Recoveries were 89.4-103.8%, matrix factors 0.93-1.06, and precision CV ≤8.5%. Stability was confirmed. The method was applied to 34 clinical samples (25 plasma, 9 CSF) from 20 patients.
CONCLUSION
This method detects four BTK-related analytes in plasma/CSF with a 3-min run time, evaluation of hemolytic/lipemic matrices, and clinical validation, increasing throughput, reducing costs, and broadening applicability, providing preliminary feasibility data for clinical TDM.
Sijia Li, Jiameng Xie, F. Xia et al.· Bioanalysis· 0 citations